Conference organization
Conference chairperson
Christa E. Müller
Conference coordinator
Heidi Zinecker
Scientific coordination
Italian Chapter of Purine Club German Chapter of Purine Club
Maria P. Abbracchio (Milan) Joachim Deitmer (Kaiserslautern)
Pier A. Borea (Ferrara) Peter Illes (Leipzig)
Pier Leopoldo Capecchi (Siena) Karl-Norbert Klotz (Würzburg)
Stefania Ceruti (Milan) Friedrich Koch-Nolte (Hamburg)
Renata Ciccarelli (Chieti) Georg Reiser (Magdeburg)
Patrizia Popoli (Rome) Günther Schmalzing (Aachen)
Anna Maria Pugliese (Florence) Torsten Schöneberg (Leipzig)
Letizia Trincavelli (Pisa) Jürgen Schrader (Düsseldorf)
Rosaria Volpini (Camerino) Norbert Sträter (Leipzig)
Organizing Committee
Ivar von Kügelgen (Bonn)
Christa E. Müller (Bonn)
Karen Nieber (Leipzig)
Alexander Pfeifer (Bonn)
Anke C. Schiedel (Bonn)
Christian Steinhäuser (Bonn)
Herbert Zimmermann (Frankfurt)
Heidi Zinecker (Bonn)
Business Secretariat (University of Bonn)
Michael Hemmersbach
Beate Ponatowski
Anke C. Schiedel
Heidi Zinecker
Contact:
e-mail: purineclub@uni-bonn.de
visit: www.purineclub.uni-bonn.de
call: +49 228 732697
Abstracts—oral presentations
Plenary lectures
Session 1: New insights into P2X7 receptor research
Session 2: Structure and function of purine receptors
Session 3: Purine receptors as emerging targets for drug development
Session 4: Purinergic signalling in neural development
Session 5: Medicinal chemistry
Session 6: Purinergic signalling in inflammation and cancer
Session 7: Purinergic signalling in the CNS I
Session 8: Purinergic signalling in the CNS II
Session 9: P2Y receptors
Session 10: Nucleotide-metabolizing enzymes
Abstracts—poster presentations
Poster session 1: Medicinal chemistry
Poster session 2: P2Y receptors
Poster session 3: P2X receptors
Poster session 4: Nucleoside and nucleotide metabolism and transport
Poster session 5: Novel purine receptors
Poster session 6: Adenosine receptors
Conference program
Friday, July 22
16.00–17.30Registration
(Castle Poppelsdorf, Gartensaal)
17.30–18.00Opening remarks
(Castle Poppelsdorf, lecture hall)
Prof. Dr. Jürgen Fohrmann, Rector, University of Bonn
Jürgen Nimptsch, Mayor of the City of Bonn
Prof. Dr. Christa E. Müller, Congress Chairperson
Presentation of the Giuliana Fassina Award to Peter Illes, University of Leipzig, by Renata Ciccarelli, President of the Italian Purine Club
Plenary lectures
18.00–18.45Geoffrey Burnstock (London, UK)
Purinergic pathophysiology and therapeutic targets
18.45–19.30Maria Teresa Miras-Portugal (Madrid, Spain)
Role of P2X7 in the nervous system: from development to neurodegeneration
19.30Welcome Party
(Castle Poppelsdorf, Botanical Garden)
Saturday, July 23 Oral presentations
(Pharmaceutical Institute, An der Immenburg 4)
Session 1: New insights into P2X7 receptor research
Chair: Pier Andrea Borea
9.00–9.20Francesco Di Virgilio (Ferrara, Italy): (talk had to be cancelled)
Putative physiological role of the “short” P2X7 receptor
9.20–9.40Chiara Parisi (Rome, Italy)
Amyotrophic lateral sclerosis: what is the P2X7 receptor doing there?
9.40–10.00Friedrich Haag (Hamburg, Germany)
Single domain antibodies as tools to block or enhance gating of P2X7
10.00–10.20Michael Schaefer (Leipzig, Germany)
Targeting P2X7 with approved drugs: novel blockers and allosteric modulators
10.20–10.40Beáta Sperlágh (Budapest, Hungary)
Lack of neuroprotection in the absence of P2X7 receptors in toxin-induced animal models of Parkinson’s disease
10.40–11.00Fritz Markwardt (Halle, Germany)
Influence of extracellular anions on purinergic P2X7 receptors
Session 2: Structure and function of purine receptors
Chair: Herbert Zimmermann
11.30–11.50Günther Schmalzing (Aachen, Germany)
P2 receptor expression studied by BAC transgenic mice
11.50–12.10Torsten Schöneberg (Leipzig, Germany): (talk had to be cancelled)
The structural landscape of the ADP receptor P2Y 12
Ralf Hausmann (Aachen, Germany)
Homology docking of suramin derivatives identifies details of ligand recognition by the P2X2 receptor
12.10–12.30Annette Nicke (Göttingen, Germany)
Analysis of conformational changes during P2X1 receptor activation and desensitization by voltage clamp fluorometry
12.30–12.50Carsten Hoffmann (Würzburg, Germany)
Investigation of A 2A -receptor activation by fluorescence resonance energy transfer in living cells
12.50–13.10Adriaan IJzerman (Leiden, The Netherlands)
Mutational analysis of adenosine receptors
13.10–14.10Lunch
Session 3: Purine receptors as emerging targets for drug development
Chair: Karl-Norbert Klotz
14.10–14.30Barbara Albrecht-Küpper (Leverkusen, Germany)
Partial adenosine A 1 receptor agonists: future therapeutic options
14.30–14.50Jürgen Schrader (Düsseldorf, Germany)
The anti-inflammatory activity of a phosphorylated adenosine A 2A receptor agonist (prodrug) in collagen-induced arthritis
14.50–15.10Maria Letizia Trincavelli (Pisa, Italy)
Adenosine A 2A receptors and psychiatric disorders
15.10–15.30David Blum (Lille, France)
What is the impact of A 2A receptor modulation in Huntington’s disease and Tauopathies?
15.30–15.50Gregor Bahrenberg (Aachen, Germany)
In vitro and in vivo characterisation of P2X3/P2X2X3 antagonists for pain
15.50–18.00Coffee break and poster session
Session 4: Purinergic signalling in neural development
Chair: Maria Letizia Trincavelli
18.00–18.20Kristine Gampe (Frankfurt, Germany)
In vivo evidence for purinergic control of adult neurogenesis
18.20–18.40Henning Ulrich (Sáo Paulo, Brazil)
Functions of purinergic receptors in directing neural differentiation and final phenotype specification of pluripotent cells
18.40–19.00Patrizia Rubini (Leipzig, Germany)
Functional P2X7 receptors of mouse adult neural precursor cells
19.30“Beer and pretzel” sponsored by Tocris Bioscience
(Pharmaceutical Institute, An der Immenburg 4)
Sunday, July 24Oral presentations
(Pharmaceutical Institute, An der Immenburg 4)
Session 5: Medicinal chemistry
Chair: Michael Gütschow
9.00–9.20Alexander Flohr (Basel, Switzerland)
Potent and selective, orally active adenosine A 2A receptor antagonists
9.20–9.40Pier G. Baraldi (Ferrara, Italy)
Recent developments in the field of adenosine ligands
9.40–10.00Christa E. Müller (Bonn, Germany)
Purine- and pyrimidine-binding membrane proteins as drug targets
10.00–10.20Piet Herdewijn (Leuven, Belgium)
Stability studies of phosphoramidate nucleosides
10.20–10.40Rosaria Volpini (Camerino, Italy)
A2A Adenosine receptor ligands: effects on alcohol intake in alcohol-preferring rats
10.40–11.15Coffee break
Session 6: Purinergic signalling in inflammation and cancer
Chair: Karen Nieber
11.15–11.35Katia Varani (Ferrara, Italy)
Adenosine receptors in inflammatory disorders
11.35–11.55Pier Capecchi (Siena, Italy)
Adenosine A 2A -dependent modulation of collagen production from human sclerodermic fibroblasts
11.55–12.15Marco Idzko (Freiburg, Germany)
P2R—signalling in the pathogenesis of inflammatory lung diseases
12.15–12.35Ivar von Kügelgen (Bonn, Germany)
A 2B -Receptor-mediated induction of early genes controls proliferation and function of human smooth muscle cells and human mast cells
12.35–14.00Lunch
Session 7: Purinergic signalling in the CNS I
Chair: Christian Steinhäuser
14.00–14.20Patrizia Popoli (Rome, Italy)
Adenosine A 2A receptors modulate cocaine-induced changes of synaptic transmission in the striatum: involvement of tyrosine phosphorylation signalling
14.20–14.40Christian Lohr (Hamburg, Germany)
Purinergic signalling in extrasynaptic axon-glia communication: the how and why
14.40–15.00Helmut Kettenmann (Berlin, Germany)
Purinergic signalling and microglial function
15.00–15.20Joachim Deitmer (Kaiserslautern, Germany)
Purinergic signalling in the cerebellum
15.20–15.40Gabriele Baier-Bitterlich
Hypoxia—adenosinergic neuronal signalling
15.40–17.10Coffee break and poster session
Session 8: Purinergic signalling in the CNS II
Chair: Ralf Hausmann
17.10–17.30Claudia Verderio (Milan, Italy)
Microvesicles shed from microglia represent a new pathway of glia-to-neuron communication
17.30–17.50Stefania Ceruti (Milan, Italy)
Crosstalk between the purinergic system and known algogenic mediators in mouse trigeminal ganglia: implications for basic mechanisms of migraine pain
17.50–18.10Felicita Pedata (Florence, Italy)
Adenosine is released per se under physiological conditions from the rat striatum in vivo; during ischemia it is primarily a product of extracellular ATP
18.10–18.30Renata Ciccarelli (Chieti, Italy)
Involvement of purine receptors in the growth and differentiation of human glioblastoma initiating cells
20.00Social dinner
(Hotel “Königshof”)
Poster prize awards
Monday, July 25Oral presentations
(Pharmaceutical Institute, An der Immenburg 4)
Session 9: P2Y receptors
Chair: Peter Illes
9.00–9.20Maria Pia Abbracchio (Milan, Italy)
Role of the P2Y-like receptor GPR17 in neural precursor cells
9.20–9.40Bilha Fischer (Ramat Gan, Israel)
Can one make a nucleotide-based drug?
Making the impossible possible
9.40–10.00Georg Reiser (Magdeburg, Germany)
Novel aspects of receptor pharmacology and receptor trafficking of the P2Y 11 receptor: insights into physiological significance of the alanine-87-threonine single nucleotide polymorphism of the human P2Y 11 receptor
10.00–10.20Hidetoshi Tozaki-Saitoh (Fukuoka, Japan)
Microglial P2Y 12 receptor as a potential therapeutic target of neuropathic pain
10.20–10.50Coffee break
10.50–11.10Session 10: Nucleotide-metabolizing enzymes
Chair: Anke Schiedel
10.50–11.10Norbert Sträter (Leipzig, Germany)
Structural studies on ectonucleotidases
11.10–11.30Gennady Yegutkin (Turku, Finland)
Purinergic control of coronary blood flow and aortic NTPDase activity are impaired in young pre-atherosclerotic apolipoprotein E- deficient mice
11.30–12.15Plenary lecture
Nicolas Dale (Warwick, UK)
New mechanisms of CO 2 and glucose sensing in the brain
12.15–12.30Closing remarks
Herbert Zimmermann, President of the German Purine Club
Renata Ciccarelli, President of the Italian Purine Club
Christa E. Müller, Congress chairperson
PL1 Plenary lecture
Purinergic pathophysiology and therapeutic targets
Geoff Burnstock
University College, London, UK
The talk will focus on recent studies of the pathophysiology of purinergic signalling and some therapeutic developments. There are several examples where inflammatory and stress conditions appear to lead to an increase in the purinergic component of autonomic cotransmission and in the expression of P2X7 receptors. Current therapeutic explorations of the use of purinergic agents will be considered, for thrombosis and stroke, osteoporosis, cystic fibrosis and dry eye, kidney failure, diabetes, gut motility disorders and colitis, bladder overactivity, visceral pain, disorders of the central nervous system and cancer.
PL2 Plenary lecture
Role of P2X7 in nervous system: from development to neurodegeneration
MT Miras-Portugal, R Gomez-Villafuertes, J Diaz-Hernandez, J Gualix, R Perez-Sen, E Garcia-Delicado, M Diez-Zaera, M Diaz-Hernandez
Biochemistry Department, Veterinary Faculty, University Complutense of Madrid, Madrid, Spain
P2X7 receptors are among the most abundant at isolated presynaptic terminals from the mammalian central nervous system and exhibit increased activity in some neurodegenerative diseases, as occurs in Huntington disease (Diaz-Hernandez et al., FASEB J 23:1893–1906, 2009). In neuronal cells, P2X7R induces exocytotic neurotransmitter release and at the same time cytoskeletal reorganization to favour vesicular dynamics and synaptic plasticity (Gutierrez-Martin, J Biol Chem 286:11370–11381, 2011). P2X7R is also involved in cell proliferation, differentiation and apoptosis. It has been recently reported that extracellular ATP through P2X7R is a key element in axonal growth and branching. P2X7R are located at the end of growing axons in cultured hippocampal neurons, and their inhibition promotes axonal growth; the same result is obtained by small hairpin RNA interference to knockdown the receptor. Besides, P2X7R over-expression significantly reduces axonal length (Diaz-Hernandez et al., J Cell Sci 121:3717–3728, 2008). Whatever the mechanism of ATP release—exocytotic or not—this nucleotide needs to be removed to allow axonal growth. Tissue non-specific alkaline phosphatase (TNAP) co-localizes with P2X7R at the axonal growth cone. Selective inhibition of TNAP activity or specific TNAP knockdown with RNAi inhibits growth of principal axons and branches, whereas addition of alkaline phosphatase (AP) promotes axonal growth. Furthermore, inhibition of the P2X7R reduces TNAP expression whereas addition of AP enhances P2X7R expression (Diez-Zaera, Mol Biol Cell 22:1014–1024, 2011). These results are an example that illustrates the complexity and fine tuning of physiological events that are under the control of purinergic signalling.
PL3 Plenary lecture
New mechanisms of CO 2 and glucose sensing in the brain
Nicholas Dale
School of Life Sciences, University of Warwick, Coventry, UK
The brain measures and regulates important aspects of the body’s internal state that are vital for life. I will consider novel mechanisms underlying the measurement and regulation of two key aspects of the internal state: the partial pressure of CO2 (PCO2) in arterial blood and the concentration of glucose in the brain. As the level of arterial PCO2 determines the pH of all bodily fluids, the homeostatic regulation of arterial PCO2 via adaptive changes in breathing is an important physiological function. While most attention has been focused on brainstem chemosensors that measure pH, we have discovered a novel molecular mechanism by which CO2 can be directly detected via hemichannels comprised of connexin 26 (Cx26). These hemichannels open in a CO2-sensitive manner to allow the release of ATP, which then excites the respiratory network to evoke the necessary adaptive changes in breathing. Our results introduce new principles into the field of respiratory chemosensitivity. As Cx26 is widely distributed throughout the surface of the brain, it may also contribute to further important physiological processes such as the CO2-dependent control of cerebral blood flow and CO2-dependent arousal. In the second part of my talk, I will demonstrate a novel form of ATP-mediated glucose-sensing by specialized glial-like cells called hypothalamic tanycytes. Tanycytes line the fluid-filled third ventricle of the brain, contact the cerebrospinal fluid and send projections into the hypothalamus, towards areas such as the arcuate nucleus and ventromedial hypothalamic nucleus that are central to the regulation of food intake and energy homeostasis. They are ideally placed as chemosensors of the cerebrospinal fluid, and I will argue that tanycytes may be an overlooked contributor to the hypothalamic networks that integrate a variety of signals related to the nutritional and energy status of an individual and control the drive to feed. These two examples of ATP-mediated signaling have conceptual similarities. In both cases, ATP acts at a brain surface: external for CO2 chemosensory transduction and internal for glucose measurement. Furthermore, ATP also acts in both cases as a mediator that transduces external environmental signals to actions within the brain itself.
S1.1 New insights into P2X7 receptor research
Physiological functions of the “short” P2X7 receptor
Francesco Di Virgilio
Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy
P2X7 is the largest and the most intriguing member of the P2X subfamily. Its distinctive feature is the extended carboxyl tail which allows formation of a large, non-selective pore. Recently, a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified (Cheewatrakoolpong et al., Biochem Biophys Res Commun 332:17–27, 2005). We have recently investigated in detail physiological responses of P2X7B in HEK293 cells (HEK293-P2X7B) (Adinolfi et al., FASEB J 24:3393–3404, 2010), a cell type lacking endogenous P2X receptors. We have observed that P2X7B mediated ATP-stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca2+ content, activated the transcription factor NFATc1, increased the cellular ATP content and stimulated growth. In addition, HEK293-P2X7B, as most tumour cells, showed strong soft agar infiltrating ability. When co-expressed with full-length P2X7 (P2X7A), P2X7B co-assembled with P2X7A and potentiated all known responses mediated by this latter receptor. P2X7B was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased upon mitogenic stimulation of peripheral blood lymphocyte. Altogether these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.
S1.2 New insights into P2X7 receptor research
Amyotrophic lateral sclerosis: what is P2X7 receptor doing there?
Parisi C, D’Ambrosi N, Apolloni S, Amadio S, Volonté C
CNR, Cell Biology and Neurobiology Institute and Santa Lucia Foundation, Rome, Italy
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of lower and upper motor neurons. The discovery of superoxyde dismutase (SOD1) mutations in about 20% of familial ALS cases allowed to generate animal models recapitulating the human disease. However, the aetiology of motor neurons death is not clear yet. Evidence indicates that non-neuronal cells contribute to the disease by sustaining inflammatory responses. Analysis of tissues from ALS patients indeed indicates marked microglia-dominated neuroinflammation, and microglial hyperactivity is reported in animal models of ALS. A potent microglial immunomodulator is extracellular ATP acting through P2X and P2Y receptors. We described that P2X4, P2X7 and P2Y6 subtypes are upregulated, while extracellular ATP hydrolyzing activities are downregulated in SOD1 microglia. We now confirm that ectoATPase CD39 protein expression is certainly reduced in ALS microglia. This dual potentiation of purinergic signalling then results in augmented neurotoxicity that we reported to be exerted by ATP mainly through P2X7 receptor. Recently, it was shown that a pathological mechanism downstream to SOD1 in microglial cells is lack of NADPH oxidase (Nox) regulation by Rac1, with persistent ROS production. Considering that Nox is a molecular target of P2X7 receptor, here we investigated if this pathway is involved in BzATP-evoked ALS microglia activation. We find that Rac1 and Nox activation is over-induced by BzATP especially in ALS microglia, but not in P2X7-KO microglia, and these effects are counteracted by the specific antagonist Brilliant Blue G. Our results indicate that the Rac1-Nox pathway is involved in BzATP-induced activation of ALS microglia.
This study was supported by ARISLA RBA50/2009.
S1.3 New insights into P2X7 receptor research
Single domain antibodies as tools to block or enhance gating of P2X7
Welbeck Danquah1, Björn Rissiek1, Catelijne Stortelers2, Toon Laeremans2, Friedrich Haag 1, Friedrich Koch-Nolte1
1 Institute of Immunology, University Medical Center, Hamburg, Germany; 2 Ablynx NV, Ghent, Belgium
P2X7, a heterotrimeric ion channel gated by nucleotides released from injured cells, plays a key role in the activation of the inflammasome. Extracellular ATP gates P2X7 directly by acting as a soluble ligand, extracellular NAD gates P2X7 indirectly via NAD-dependent ADP-ribosylation of P2X7 at R125 by the toxin-related ecto-ADP-ribosyltransferase ART2.2. Tools to activate or block P2X7 are being sought for therapeutic applications in inflammation-mediated clinical conditions. We hypothesized that single domain antibodies (nanobodies) derived from llama heavy-chain antibodies might be particularly suited for this purpose, since these antibodies display a strong propensity to bind to functional crevices on proteins. We have previously shown that NAD-induced cell death can be prevented in vitro and in vivo by lama-derived nanobodies that block the catalytic activity of ART2 (Koch-Nolte et al., FASEB J 21:3490–3498, 2007). We recently also generated P2X7-blocking and P2X7-stimulatory nanobodies from immunized llamas. Intravenous injection of bivalent P2X7-blocking nanobodies into mice efficiently blocked P2X7 on regulatory T cells in blood, spleen, lymph node and liver, as assessed by resistance of these cells to NAD and ATP-induced shedding of CD62L. These nanobodies provide new tools for specifically blocking P2X7 function in vitro and in vivo and pave the way for testing the therapeutic potential of P2X7-specific nanobodies in mouse models of inflammatory diseases.
This study was supported by DFG grant no. 310/8-1.
S1.4 New insights into P2X7 receptor research
Targeting P2X7 with approved drugs: novel blockers and allosteric modulators
Michael Schaefer, Christoph Hempel, Nicole Urban, Helga Sobottka, Wolfgang Nörenberg
Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Härtelstr. 16–18, 04107 Leipzig, Germany
The ATP-gated, poorly selective cation channel P2X7 is expressed in many cell types, including glia and immune cells as well as in various tumour cell lineages. P2X7 confers a lasting sodium and calcium influx, mediates potassium efflux and, presumably by dilating its pore, allows organic molecules to permeate across the cell membrane. P2X7 activity in turn triggers multiple functions, such as cytokine release, control of growth, migration and secretion. By systematically assessing the impact of approved or clinically tested drugs on P2X7 receptor activity, we screened a compound library comprising 1,040 pharmaceuticals and 800 natural products in HEK cells stably expressing human P2X7. The experiments revealed that P2X7 is modulated by several small molecules. Potentiation of ATP-induced calcium entry and ionic currents was found in the presence of the H1 antihistaminic drug clemastine (5–100 μM) but also with ivermectin (0.01–3 μM), an antibiotic that has formerly been claimed to act in a P2X4-specific manner. Comparison between ivermectin effects on human and rat P2X7 revealed a species-selective activity, explaining why ivermectin effects on human P2X7 have been overlooked so far. Among the compounds that inhibited or abrogated P2X7 activity, perazine-type neuroleptic drugs displayed the highest potency. For example, prochlorperazine inhibited the ATP-induced Ca2+ entry (IC50 ~ 0.5 μM), whereas the chemically related prochlorpromazine was inefficient. Astonishingly, rat P2X7 is not inhibited, but potentiated by perazines. In human P2X7, the electrophysiological properties showed an initial potentiation that was followed by a time- and concentration-dependent suppression of P2X7 currents. Since the reported peak plasma concentrations of prochlorperazine are in the low micromolar range, P2X7 inhibition appears to be tolerable in humans. Potentially beneficial co-analgetic effects of perazines in diseased states such as neuropathic pain or osteoarthritis may be tested in comparison to the P2X7-sparing promazines.
This project is supported by the Deutsche Forschungsgemeinschaft (DFG) within the collaborative research group FOR-748.
S1.5 New insights into P2X7 receptor research
Lack of neuroprotection in the absence of P2X7 receptors in toxin-induced animal models of Parkinson’s disease
B. Sperlágh 1, Zs. Hracskó1, M. Baranyi1, C. Csölle1, F. Gölöncsér1, Á. Kittel1, E. Madarász2
1 Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary; 2 Laboratory of Cellular and Developmental Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary
The main objective of our study was to examine whether genetic deletion or pharmacological blockade of P2X7 receptors influences dopaminergic cell death in various models of Parkinson’s disease (PD). mRNA encoding P2X7 and P2X4 receptors was upregulated after treatment of PC12 cells with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). P2X7 antagonists protected against MPTP and rotenone induced toxicity in the LDH assay but failed to protect after rotenone treatment in the MTT assay in PC12 cells and in primary midbrain culture. In vivo MPTP and in vitro rotenone pretreatments increased the mRNA expression of P2X7 receptors in the striatum and substantia nigra of wild-type mice. Genetic deletion or pharmacological inhibition of P2X7 receptors did not change survival rate or depletion of striatal endogenous dopamine content after in vivo MPTP or in vitro rotenone treatment. The basal ATP content was higher in the substantia nigra of wild-type mice, but the ADP level was lower. Rotenone treatment elicited a similar reduction in ATP content in the substantia nigra of both genotypes, whereas reduction of ATP was more pronounced after rotenone treatment in striatal slices of P2X7-deficient mice. Although the endogenous amino acid content remained unchanged, the level of the neuroprotective endocannabinoid, 2-AG, was elevated by rotenone in the striatum of wild-type mice, an effect that was absent in mice deficient in P2X7 receptors. We conclude that P2X7 receptor deficiency or inhibition does not protect but rather aggravates dopaminergic cell death in an in vivo or in vitro model of PD.
This study was supported by grants of the Hungarian Research and Development Fund (NN79957) and the Hungarian Medical Research Council (ETT 05–102).
S1.6 New insights into P2X7 receptor research
Influence of extracellular anions on purinergic P2X7 receptors
Christoph Kubick1, Günther Schmalzing2 and Fritz Markwardt 1
1 Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle, Halle, Germany; 2 Molecular Pharmacology, RWTH Aachen University, Aachen, Germany
P2X7 receptors are nonselective cation channels opened by binding of extracellular ATP. They are involved in the modulation of the immune system, inflammation and nociception. Here, we investigated the effect of extracellular anions on P2X7 receptor channel gating and permeation. Human P2X7 receptors were expressed in Xenopus laevis oocytes. Cell membrane currents were measured by the two microelectrode voltage clamp technique. If extracellular Cl− was substituted by organic anions like glutamate or aspartate, the ATP-induced P2X7 receptor-mediated currents were increased. In contrast, if Cl− was replaced by inorganic anions like nitrate, sulfate or iodide, the P2X7 receptor-dependent currents were inhibited. The main Cl− substitution effect on the ATP concentration response was that glutamate increased and iodide decreased the agonist efficacy of high ATP concentrations. However, at low ATP concentrations, Cl− substitution was without effect on P2X7 receptor activation. For further analysis of the anion effect on P2X7 receptors, we performed single channel current measurements by the patch clamp technique in the outside-out configuration. While Cl− substitution did not affect the single channel conductance, P2X7 receptor channel open probability was increased or decreased if Cl− was replaced by glutamate or iodide, respectively. We conclude that anions affect ion channel opening after binding of all three ATP4− molecules on the P2X7 receptor.
This study was supported by DFG (Ma1581/15-1 and Schm536/9-1) and by Roux Programme of MLU (22/18).
S2.1 Structure and function of purine receptors
P2 receptor expression studied by BAC transgenic mice
Marcus Grohmann2, Janka Günther1, Michaela Schumacher1, Tanja Nussbaum1, Heike Franke2, Ralf Hausmann1, Günther Schmalzing 1
1 Department of Molecular Pharmacology, RWTH Aachen University, Aachen, Germany; 2 Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany
Various P2 receptors including P2X3, P2X2/3 and P2Y1 are promising targets for new drugs against chronic inflammatory and neuropathic pain. To allow for a comprehensive mapping of the expression of these receptors in the central and peripheral nervous system in health and disease, we generated various BAC transgenic mice expressing either fluorescent receptor subunits (P2X2-StrepII-His-TagRFP, P2X3-StrepII-His-EGFP) or soluble fluorescent reporter proteins (EGFP for P2X3 and TagRFP for P2Y1) under the control of their appropriate endogenous promoters. Transgenic mice expressing P2X2-His-StrepII-TagRFP or P2X3-His-StrepII-EGFP fusion proteins showed weak membrane-bound fluorescence in isolated DRG neurons and in the auditory cortex, but not in other cells. In contrast, mice expressing soluble EGFP under control of the P2X3 subunit gene promoter showed a bright GFP fluorescence in the cytoplasm of cultured small- and medium-diameter DRG neurons including the axonal processes. Immunostaining demonstrated that EGFP colocalizes with isolectin-B4 and the neuronal marker MAP2, indicating that the GFP expression of the BAC mice mirrors the known neuronal localization of the P2X3 receptor in DRGs. Treatment of primary cultured DRG neurons with NGF or GDNF increased the P2X3 receptor expression in agreement with current literature. Also, nine transgenic founder mice expressing TagRFP as a fluorescent reporter protein under control of the P2RY1 gene promoter have been identified and await characterization.
This study was supported by the DFG (FOR748).
S2.2a Structure and function of purine receptors
The structural landscape of the ADP receptor P2Y 12
T. Schöneberg
University of Leipzig, Leipzig, Germany
Evaluating the functional relevance of natural occurring gene variants usually requires experimental testing or is even impossible because of the lack of appropriated functional assays. Functional characterization of P2Y receptors and their mutants in mammalian expression systems is problematic because of the endogenous abundance of nucleotide receptors, nucleosidases, and nucleotide release. We tested whether comparative sequence data from orthologs are suitable to predict the functional relevance of mutations in a model protein, a G protein-coupled receptor (GPCR) for ADP (P2Y12). Thus, the functional effect of every possible substitution at each amino acid position within a portion of P2Y12 (1,254 mutants) was individually determined in a yeast expression system. Comparison with sequence data of over 70 P2Y12 vertebrate orthologs revealed that the amino acid variability which assures proper receptor function highly correlates between in vitro experimental and ortholog sequence data. This yeast expression system is therefore suitable to identify new agonists for the human P2Y12 and inverse agonists for constitutively active mutants. In sum, ortholog sequence data are sufficient to predict the functional relevance of every position and mutations in P2Y12 and are therefore helpful in picturing the structural landscape of this and probably other GPCR.
S2.2b Structure and function of purine receptors
Homology docking of suramin derivatives identifies details of ligand recognition by the P2X2 receptor
Ralf Hausmann 1, Matthias U Kassack2, Achim Kless3, Gregor Bahrenberg3, Günther Schmalzing1
1 Molecular Pharmacology, RWTH Aachen University, Aachen, Germany; 2 Institute of Pharmaceutical & Medicinal Chemistry, University of Düsseldorf, Düsseldorf, Germany; 3 Grünenthal GmbH, Global Drug Discovery, Aachen, Germany
Within a series of 139 suramin derivatives, we identified NF770 as a nanomolar-potent competitive P2X2 receptor antagonist and found that the inhibitory potency of NF770 at the rat P2X2 receptor is defined by three structural determinants: (a) a “large urea” structure with two symmetric phenylenecarbonylimino groups, (b) attachment of the naphthalene moiety in position 7,7′, and (c) the specific position of two sulfonic acid groups and of one methoxy group at the naphthalene moiety. The SAR was interpreted using a P2X2 homology model based on the crystal structure of the zfP2X4 receptor. Docking of the suramin derivatives into the modeled ATP-binding pocket provides a uniform explanation for the differences in inhibitory potencies. Changes in the chemical structure that increase the inhibitory potency of the suramin derivatives improved the spatial orientation within the ATP-binding pocket to allow for stronger polar interactions of functional groups with Gly72, Glu167, or Arg290. Mutational analysis confirmed that residues Gly72 and Glu167 are as important for ATP binding as Arg290, whose ATP-binding role has been shown in previous studies. Our data strongly confirm the location of the ATP-binding pocket and show molecular details of ligand recognition by the P2X2 receptor.
This study was supported by the DFG (FOR748).
S2.3 Structure and function of purine receptors
Analysis of conformational changes during P2X1 receptor activation and desensitization by voltage clamp fluorometry
Annette Nicke 1, Eva Lörinczi1, Sebastien Dutertre2, Jürgen Rettinger3
1 Max Planck Institute for Experimental Medicine, Göttingen, Germany; 2 Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia; 3 Multi Channel Systems GmbH, Aspenhaustrasse 21, 72770 Reutlingen, Germany
P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Recently, the first crystal structure of a P2X receptor has been resolved confirming many predictions that were based on the interpretation of mutagenesis studies. However, the conformational changes governing channel opening, desensitization and recovery from desensitization remain unknown, as does the exact mode of ATP binding. Here, we used voltage clamp fluorometry to determine amino acid residues in the fast desensitizing P2X1 subtype that undergo conformational changes during channel opening and desensitization. We identified a range of single cysteine mutants that were specifically labelled by the fluorophore TMRM and revealed significant changes in the emission of this fluorescence probe upon activation by the agonists ATP and Bz-ATP. Depending on their time courses, fluorescence changes in different mutants appeared to correlate with either ligand binding and/or channel opening or desensitization of the receptor. Using the competitive antagonist TNP-ATP, we further discriminated between fluorescence changes that were caused by ligand-induced fluorophore quenching or conformational changes associated with channel opening or desensitization. In summary, we identified a range of mutants with specific fluorescence changes that are most likely associated with distinct processes during channel activation. Our study provides useful tools and a basis to further decipher the molecular function of the P2X receptor.
This project was funded by the DFG (FOR 748, RE2711/1, NI592/5).
S2.4 Structure and function of purine receptors
Investigation of A 2A -adenosine receptor activation by fluorescence resonance energy transfer in living cells
Dang Ngoc Tu1,2, Nicole Ziegler1, Ulrike Zabel1, Christa E. Müller2, Rob Lane3,4, Martin J. Lohse1, Ad IJzerman3, Carsten Hoffmann 1
1 Institute of Pharmacology and Toxicology, University of Wuerzburg, 97078 Wuerzburg, Germany; 2 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, 53121 Bonn, Germany; 3 Division of Medicinal Chemistry, Leiden/Amsterdam Centre for Drug Research, Leiden University, PO Box 9502, 2300 RA Leiden, the Netherlands; 4 Current address: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, 3052 Parkville, Australia
The publication of the agonist- or antagonist-bound structures of the adenosine A2A receptor has revealed details about the conformational changes associated with the activation process. To study receptor activation in living cells, we used dynamic fluorescence resonance energy transfer (FRET) measurements of a modified A2A-receptor construct. The receptor contained the amino acid motif CCPGCC in the third intracellular loop and was fused to the cyan fluorescent protein (CFP) at the C terminus. The CCPGCC motif can selectively bind the small soluble fluorophore FlAsH, which together with CFP is suitable for FRET measurements. Additional mutations were inserted in 11 different positions that were predicted from the crystal structure to interact with agonist or antagonists. These constructs were expressed in HEK293 cells, and due to conformational changes upon receptor activation, we could observe ligand binding of adenosine or inhibition thereof by theophylline as dynamic changes in the FRET signal. Mutation of F168A fully prevented changes in FRET, most likely by loss of adenosine binding. Mutation of S227A led to an 80-fold rightward shift in adenosine concentrations needed for receptor activation, but had no influence on theophylline. Mutation of W246A led to smaller amplitude of the FRET signal, but the affinity of adenosine was not affected. Mutations N253A and H250A had stronger influence on the effect of theophylline than adenosine. All other mutants had intermediate effects on both ligands. In summary, we could demonstrate that this novel technology can be used to study the endogenous ligand- and mutation-dependent effects in living cells.
This work was supported by the German Research Foundation grant SFB-487 TPA1.
S2.5 Structure and function of purine receptors
Mutational analysis of adenosine receptors
MC Peeters, AP IJzerman
Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, PO Box 9502, 2300RA Leiden, the Netherlands
G protein-coupled receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Despite the progress made in the last decade in molecular and structural biology, their exact activation mechanism is still unknown. Here we describe new insights in specific regions essential in adenosine receptor activation. As an example, we applied unbiased random mutagenesis on the middle part of the human adenosine A2B receptor, consisting of transmembrane domains 4 and 5 linked by extracellular loop 2. We subsequently screened in a medium-throughput manner for constitutively active (CAMs) and inactive mutants (CIMs). For that purpose, we used a genetically engineered yeast strain (Saccharomyces cerevisiae MMY24) with growth as a read-out for activation. Both the CAM and the CIM screen revealed many informative amino acid residues that would not have been identified by any other, more rational approach. This new screening strategy could be applied to all GPCRs that can be functionally expressed in yeast.
This work was supported by the Dutch Top Institute Pharma, project The GPCR Forum (D1-105).
S3.1 Purine receptors as emerging targets for drug development
Partial adenosine A 1 receptor agonists: future therapeutic options
Barbara Albrecht-Küpper
Bayer HealthCare AG, Leverkusen, Germany
Adenosine A1 receptor agonists have the potential to be the base for several new therapies in pathologies of different tissues. In the heart, adenosine is an important component of the intrinsic cardioprotective arsenal of the heart. Adenosine inhibits a variety of pathologies associated with ischemia and reperfusion, including stunning, arrhythmogenesis, coronary dysfunction, infarction, apoptosis and heart failure. Many of the adenosine-induced protective effects are due to A1 receptor activation. The various cardiovascular (CV) effects addressed by A1 receptors open up several options for new therapies. The main issue of using A1 receptor agonists in CV therapies is the broad spectrum of physiological A1 effects. Desired CV effects such as cardioprotection can be counter-regulated by undesired cardiac effects, like pronounced bradycardia (AV-block) as seen with tecadenoson or selodenoson or systemic effects, like decreased GFR and reduction of diuresis. This restriction for the use of A1 receptor agonists as therapeutic target can be overcome by tailoring compounds only to the desired pharmacological efficacy by the development of partial adenosine A1 receptor agonists. Adenosine-like partial A1 agonists like CVT 3619 and VCP-28 have been published in the past. Bayer Healthcare Pharmaceuticals developed the first non-adenosine like A1 agonists which cover the whole spectrum of full A1 agonists, shades of partial A1 agonists and antagonists. Capadenoson is a first example of a non-adenosine like partial A1 agonist with reduced bradycardia and no induction of an AV block, but pronounced cardioprotective effects in preclinical models and patients.
S3.2 Purine receptors as emerging targets for drug development
The anti-inflammatory activity of a phosphorylated adenosine A 2A receptor agonist (prodrug) in collagen-induced arthritis
J Schrader 1, U Flögel1, S Burghoff1, S Temme1, CE Müller2, A El-Tayeb2, P van Lent3, S Huels1, F Bönner1, N Borg1, L Galbarz1
1 Institute of Molecular Cardiology, University Düsseldorf, Düsseldorf, Germany; 2 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany; 3 Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Nijmegen Medical Care Center, Nijmegen, the Netherlands
Adenosine A2A receptor agonists are potent anti-inflammatory drugs; however, they are equally potent vasodilators and are used clinically as pharmacologic stress agent for myocardial perfusion imaging. To separate the anti-inflammatory from the vasodilatory action, phosphorylated A2A agonists were synthesized which are well dephosphorylated by CD73 present on immune cells and thus may provide local immune-modulatory activity (El-Tayeb et al., J Med Chem 52(23):7669–7677, 2009). To test this concept, we have used the model of collagen-induced arthritis in mice which received the AMP-analog 2-cyclohexylethylthioadenosine-monophosphate (chet-AMP) by intravenous infusion via osmotic minipumps. The extent of inflammation was measured by visual scoring and 19F-MRI (Flögel et al., Circulation 118(2):140–148, 2008). Infusion of chet-AMP at 0.25 and 0.5 μg/kg BW/min reduced the inflammatory response by ~50% and 75%, respectively. The A2A agonist, chet-adenosine, when applied at the same infusion rate, did not alter the inflammatory state. Measurement of the plasma concentration of chet-adenosine after chet-AMP infusion by mass spectrometry revealed that vasodilatation (drop in blood pressure) occurred at 10−6–10−5 M while the anti-inflammatory effect described above was reached at 10−10–10−9 M. FACS analysis of immune cells present in inflamed joints revealed significant upregulation of CD73 on granulocytes. Together these results demonstrate that Chet-AMP is a potent anti-inflammatory purine derivative with negligible vasodilatory activity which preferentially acts locally on immune cells invading inflamed tissue.
S3.3 Purine receptors as emerging targets for drug development
Adenosine A 2A receptors and psychiatric disorders
ML Trincavelli 1, S Daniele2, C Martini1
1 Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy; 2 Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Genoa, Italy
The lack of clear end-points and of animal models of psychiatric diseases has seriously hampers the ability to critically evaluate the potential of any particular molecule as a relevant target to develop novel drugs to manage psychiatric disorders. The interest in the adenosine system mostly stems from the recognition that its main function is to assist maintaining homeostasis in biological systems and it should be considered a system of choice to manipulate brain circuits to restore their proper function. The targeting of adenosine A2A receptors (A2A ARs) in the realm of psychiatric diseases first arose based on its tight physical and functional interaction with dopamine D2 receptors (D2 DRs). D2 DRs and A2A ARs are expressed in the same striatal GABAergic neurons and, through the formation of heteromers, antagonize each other. On this basis, A2A AR agonists have been suggested as possible drugs, in association with D2 DR antagonists, for the treatment of psychiatric diseases associated with dopamine hypertone, including schizophrenia and bipolar disorders. Investigation of the molecular mechanisms involved in the cross-talk between A2AARs and D2 DRs may represent a crucial starting point to clarify the role of the adenosine system in several psychiatric disorders. In this context, we investigated the structural and functional modulation of A2A AR induced by D2 DR agonists and antagonists (neuroleptic drugs) in transfected cell lines. Then, we investigate A2A AR expression and functioning in platelets of patients affected by bipolar disorders and pathological gambling. Our results demonstrated A2A ARs are functionally regulated by dopaminergic system and may be considered a useful pharmacological target in several psychiatric diseases.
S3.4 Purine receptors as emerging targets for drug development
What is the impact of A 2A receptor modulation in Huntington’s disease and tauopathies?
D Blum 1,2,3, S Mievis4, S Burnouf1,2,3, C Duru5, C Simonin6, Julia Salleron7, C Laurent1,2,3, A Leboucher1,2,3, M Hamdane1,2,3, FJ Fernandez Gomez1,2,3, F Richard7, C Ledent4,P Krystowiak5, L Buée1,2,3
1 Inserm U837, Lille, France; 2 UDSL, IMPRT, Jean-Pierre Aubert Rsearch Centre, Lille, France; 3 CHRU-Lille, Lille, France; 4 IRIBHM, ULB, Brussels, Belgium; 5 Department of Neurology, Amiens University Hospital, EA4559, Amiens, France; 6 Department of Neurology, EA 2683, IMPRT, CHRU, Lille, France; 7 Unité d’épidémiologie et de Biostatistiques, Pôle de Santé Publique, CHRU, Lille, France
A2A receptors are G protein-coupled receptors largely expressed in the central nervous system by neurons and glial cells. They are seen as a potential target in neurodegenerative disorders through their particular ability to modulate synaptic function and neuro-inflammation. However, whether they may represent a valuable target in Huntington’s (HD) and Alzheimer’s (AD) diseases, two proteinopathies affecting, respectively, the striatum and the hippocampus remain unclear so far. In this context, we have obtained data supporting that A2A receptor blockade would lead to an opposite outcomes in both disorders. Pharmacological and genetic blockade of A2A receptor were found deleterious in phenotypic and transgenic mouse models of HD. We particularly observed that A2A knockout worsened survival and motor function in the N171-82Q transgenic model. In accordance, in a retrospective study realized in 80 HD patients from the Huntington French Speaking Network, we found—after adjustment on CAG repeat length, tobacco and alcohol consumptions—that patients exhibiting higher consumption of caffeine (>190 mg/days)—a non-selective A2A receptor antagonist—had an earlier age at onset. Conversely, we obtained experimental data supporting a beneficial effect of A2A receptor blockade against AD-like tau pathology. Specifically, we found that A2A receptor knockout prevented from memory defects, tau hyperphosphorylation and hippocampal inflammation in the THY-tau22 transgenic model mimicking the tau-side of AD. This appears particularly relevant since cognitive deficits in AD are well correlated with the spatiotemporal progression of tau pathology in the brain of AD patients. In conclusion, depending on the neurodegenerative context, the potential of A2A receptor blockade is different. Complexity of relationships between underlying pathophysiological mechanisms and A2A receptors makes its targeting questionable in HD while this approach deserves further investigation in AD field.
S3.5 Purine receptors as emerging targets for drug development
In vitro and in vivo characterisation of P2X3/P2X2X3 antagonists for pain
Gregor Bahrenberg 1, Thomas Christoph2, Klaus Schiene2, Achim Kless3, Peter Illes4
1 Department of Molecular Pharmacology, Global Preclinical Drug Discovery, Grünenthal Innovation, Zieglerstrasse 6, 52078 Aachen, Germany; 2 Department of Pharmacology, Global Preclinical Drug Development, Grünenthal Innovation, Zieglerstrasse 6, 52078 Aachen, Germany; 3 Department of Discovery Informatics, Global Preclinical Drug Discovery, Grünenthal Innovation, Zieglerstrasse 6, 52078 Aachen, Germany; 4 Rudolf-Boehm-Institute of Pharmacology and Toxicology, Härtelstrasse 16-18, 04107 Leipzig, Germany
Purinergic P2X3 and P2X2/3 heteromultimers mediate ATP-evoked responses in dorsal root, nodose, and trigeminal ganglia as well as in sensory afferents in visceral organs. Therefore, besides other indications, genetic and pharmacological studies have revealed important roles for these receptors in inflammatory, neuropathic, and cancer pain, but not acute nociception. Therefore, several pharmaceutical companies have started programs searching for P2X3 or/and P2X2/3 inhibitors, which led to the discovery of interesting dual P2X3 inhibitors like A-317491 (Abbott) or RO-4 (Roche). In our hands, the P2X3,2/3 reference antagonists were effective in neuropathic pain models, and we identified central availability to be crucial for full efficacy in the formalin test. Following a luminescence and fluorescence screening campaign of 170 k test substances on CHO cells expressing human heteromultimeric P2X2/3 receptors, hit series from four different structural classes with moderate activity were identified (three-digit nanomolars to one-digit micromolars), and their inhibition of αβ-meATP-induced currents was further characterised by means of manual patch clamping on rat dorsal root ganglia cells (DRG). Surprisingly, some hits and their analogues showed unproportionally higher potencies and efficacies in the rat DRG experiments, Cmpd A even in the potency and efficacy range of the reference Cmpds RO-4 and A-317491. Cmpd A reduced cold allodynia in a neuropathic pain model after intravenous and intrathecal application by a maximum of 60%. These experiments suggest to include different screening technologies from human and non-human species into the characterisation of P2X3 antagonists.
S4.1 Purinergic signalling in neural development
In vivo evidence for purinergic control of adult neurogenesis
Kristine Gampe 1, Simon C. Robson2, Herbert Zimmermann1
1 Institute of Cell Biology and Neuroscience, Biocenter, Goethe-University, Frankfurt, Germany; 2 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
In the adult mammalian central nervous system, two regions retain the capacity to generate new neurons, the subependymal zone (SEZ) of the lateral ventricles and the subgranular layer (SGL) of the hippocampus. Adult neurogenesis includes progenitor cell proliferation, fate specification, survival and integration of young neurons. Each step is controlled by the interplay of a variety of signalling factors. We have previously shown that ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), an enzyme that predominantly hydrolyzes nucleoside triphosphates, is specifically expressed by neural progenitors of the two neurogenic regions in situ and that functional P2Y receptors are expressed in cultured adult neural stem cells. Deletion of NTPDase2 should increase the extracellular concentrations of P2 receptor agonists in the vicinity of the NTPDase2-depleted cells and thus enhance any nucleotide-mediated effect on neurogenesis. We investigated short-term progenitor cell proliferation and young neuron survival in NTPDase2−/− mice. Cell proliferation was doubled both in isolated neurospheres and in the SEZ and SGL in vivo. However, long-term survival of newly formed cells remained unaltered, suggesting that additional factors are required for cell survival. Our data provide first in vivo evidence for an involvement of purinergic signalling in the control of adult neurogenesis.
This work was supported by the Deutsche Forschungsgemeinschaft (140/17-4 to H.Z.).
S4.2 Purinergic signalling in neural development
Functions of purinergic receptors in directing neural differentiation and final phenotype specification of pluripotent cells
Henning Ulrich
Depto de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
Purinergic signaling is one of the first functional neurotransmitter systems in the developing brain. ATP and other nucleotides acting through ionotropic P2X and metabotropic P2Y receptors trigger intracellular calcium transients at defined time points and control proliferation and differentiation of stem cells into neurons and glial cells. We have used the murine ES-E14TG2a embryonic (ES) stem and P19 embryonal carcinoma (EC) cell lines as in vitro models for studying purinergic receptor expression and activity and their participation in regulating proliferation and differentiation of pluripotent cells. Activation of purinergic ionotropic receptors by Bz-ATP enhanced gene expression of stage-specific antigen-1 in ES cells, indicating that P2X7 and possibly other ionotropic purinergic receptor activities were involved in the maintenance of the pluripotent state. In agreement, P2X7 receptor expression was reduced when ES cells were induced to differentiation by retinoic acid. Mechanisms of proliferation and differentiation induction were studied in detail in P19 EC cells. Metabotropic P2Y1 and P2Y2 receptors triggered proliferation of P19 embryonic and progenitor cells. Increased expression of neuronal marker proteins in the presence of ATP provided evidence for the involvement of purinergic receptors in the progress of differentiation. Downregulation of P2X2 or P2X7 receptor expression along differentiation by stable RNA interference resulted in inhibition of neurogenesis or gliogenesis, respectively. Moreover, P2Y1, P2Y2, and P2X2 receptor activities participated in final phenotype specification of differentiating neurons. As overall result, metabotropic and ionotropic P2 receptor subtypes were involved in all stages of differentiation providing mechanisms for purinergic signaling in processes occurring during neuroectodermal development.
This study was supported by grants awarded by FAPESP and CNPq (Brazil).
S4.3 Purinergic signalling in neural development
Functional P2X7 receptors of mouse adult neural precursor cells
Patrizia Rubini 1, Nanette Messemer1, Christin Kunert1, Marcus Grohmann1, Heike Franke1, Karen Nieber1, Herbert Zimmermann2, Peter Illes1
1 Rudolph-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, 04107 Leipzig, Germany; 2 Zoologisches Institut, Biozentrum der Universität, 60439 Frankfurt/Main, Germany
Both RT-PCR and immunocytochemistry demonstrated the presence of P2X7 receptors in cultured adult neural precursor cells (NPCs) isolated from the subventricular zone of the mouse brain. Membrane currents and intracellular Ca2+ ([Ca2+]i) transients were recorded by the whole-cell patch clamp technique and the fura-2 method, respectively. Whereas NPCs exhibited only very small current responses to ATP or the more selective P2X7 receptor agonist dibenzoyl-ATP (BzATP) in a normal superfusion medium, the current amplitudes largely increased in a low Ca2+-containing, Mg2+-free medium. Although both P2 receptor agonists had to be applied in rather high concentrations to establish concentration–response relationships, Bz-ATP had a higher potency than ATP itself. The P2X7 selective antagonists Brilliant Blue G and A 438079 blocked the ATP effects. Ivermectin, an allosteric modulator of P2X4 receptors, increased the BzATP current. NPCs prepared from P2X7−/− mice did not respond to ATP and BzATP. Measurements of Ca2+ transients in a normal Ca2+-containing Mg2+-deficient medium, where the omission of external Ca2+ abolished the effect of BzATP, fully confirmed the electrophysiological results. In addition, it was found that in NPCs of P2X7−/− mice, a small residual [Ca2+]i transient could be evoked by Bz-ATP, which was abolished by MRS 2179 indicating that it was due to P2Y1 receptor activation. In conclusion, the use of agonists, antagonists, and allosteric modulators bears out the presence of P2X7 and probably also P2X4 receptors in conjunction with the previously described P2Y1 receptors at mouse NPCs, all increasing the concentration of [Ca2+]i and thereby possibly regulating cell proliferation.
S5.1 Medicinal chemistry
Potent and selective, orally active adenosine A 2A antagonists
A Flohr 1, A Alanine1, J-L Moreau2, S Poli3, C Riemer1
1 Discovery Chemistry, F. Hoffmann-La Roche, Basel, Switzerland; 2 CNS Discovery, F. Hoffmann-La Roche, Basel, Switzerland; 3 ADDEX Pharmaceuticals, Plan-les-Ouates, Geneva, Switzerland
Adenosine is a ubiquitous modulator of a variety of physiological processes and belongs to the group of purinergic messengers. Adenosine A2A receptors are enriched in the basal ganglia (in particular the striatum), where they are highly co-expressed together with the dopamine D2 receptor. This is suggesting a potential role in a variety of neurodegenerative diseases associated with severe movement impairment. This hypothesis is supported by epidemiological data indicating a beneficial role of coffee consumption towards the development of, e.g. Parkinson’s disease. Starting from a screening hit, highly potent and subtype-selective antagonists have been developed with good oral bioavailability. Their physicochemical and ADME properties will be discussed. Representatives from this series have demonstrated robust in vivo effects in a variety of CNS models. They might be useful as therapeutics for the treatment of neurodegenerative diseases (esp. Parkinson’s), depression and other mood disorders.
S5.2 Medicinal chemistry
Recent development in the adenosine ligands field
Pier Giovanni Baraldi 1, Mojgan Aghazadeh Tabrizi1, Delia Preti1, Giulia Saponaro1, Stefania Baraldi1, Romeo Romagnoli1, Pier Andrea Borea2
1 Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44121 Ferrara, Italy; 2 Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, Università di Ferrara, 44121 Ferrara, Italy
Adenosine is released from metabolically active cells and is generated by the degradation of released ATP. The action of adenosine occurs through the stimulation of the purinergic receptors that were classified as P1 receptors, whereas the receptors activated by nucleotides like adenosine 5′-triphosphate were classified as P2 receptors (Fredholm, Pharmacol Rev 53:527–552, 2001).
P1 receptors: A 1 adenosine receptors In this lecture, we will describe the synthesis and biological evaluation of a novel series of 2-amino-3-aroyl-thiophenes, with variable modifications at the 4- and 5-position (Baraldi et al., Curr Med Chem, 17:3488–3502, 2010).
A 2A adenosine receptors: from the initial work on SCH 58261, we have prepared several compounds series, bearing substitution at the pyrazole nitrogen, such as SCH 63390 and their oxygenated derivatives (Baraldi et al., Curr Med Chem 2:707–722, 1995).
A 3 adenosine receptors: in the past years, we have synthesized an extensive series of pyrazolotriazolopyrimidines bearing different substitutions on the phenylcarbamoyl moieties at the N5 position, being highly potent and selective human A3 adenosine receptor antagonists (Baraldi et al., J Med Chem 45:115–126, 2002); we obtained [3H]MRE3008, a new selective and potent human A3 receptor antagonist radioligand.
A 2B adenosine receptors: herein we report our studies on the synthesis and biological evaluation of A2B adenosine agonists and antagonists (Baraldi et al., J Med Chem 47:1434–1447, 2004; J Med Chem 50:374–380, 2007; Bioorg Med Chem 15:2514–2527, 2007). In this lecture will be presented an overview of our recent achievements on medicinal chemistry studies on adenosine receptor ligands (Baraldi et al., Chem Rev 108:238–263, 2008).
S5.3 Medicinal chemistry
Purine- and pyrimidine-binding membrane proteins as drug targets
Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Membrane receptors for purines and pyrimidines are subdivided into three different families: P0 receptors or adenine receptors (activated by the nucleobase adenine; receptor nomenclature not yet officially acknowledged), P1 receptors (adenosine receptors, AR), and P2 receptors (nucleotide receptors; subfamilies: P2Y and P2X). While P0, P1, and P2Y receptors are G protein-coupled receptors, P2X receptors represent ATP-gated ion channels. In addition, a variety of mostly membrane-bound ecto-enzymes exist, which can metabolize the physiological ligands of purine and pyrimidine receptors. For example, ecto-nucleotidases hydrolyze phosphate ester bonds of nucleotides thus being able to catalyze the conversion of P2 receptor ligands into the P1 agonist adenosine. The families of purine and pyrimidine receptors (Müller and Jacobson, Biochim Biophys Acta 1808:1290–1308, 2011; Brunschweiger and Müller, Curr Med Chem 13:289–312, 2006) as well as ecto-nucleotidases (Brunschweiger et al., J Med Chem 51:4518–4528, 2008; Baqi et al., J Med Chem 53:2076–2086, 2010) are in the focus of intensive drug development efforts. This talk will present new results (a) on the binding site of adenine receptors, which constitute potential targets for neuropathic pain (Borrmann et al., J Med Chem 52:5974–5989, 2009); (b) the development of new potent and selective A2A AR antagonists, which are of interest for the treatment of Parkinson’s disease and other neurodegenerative processes (Müller and Ferré, Front CNS Drug Discov 1:304–341, 2010); and (c) the identification and characterization of new ecto-nucleotidase inhibitors.
Acknowledgement: We are grateful to the German Federal Ministry of Education and Research (BMBF, BioPharma Neuroallianz project), the European Commission (ERANET Neuron project), and the Deutsche Forschungsgemeinschaft (GRK804) for financial support.
S5.4 Medicinal chemistry
Stability studies of phosphoramidate nucleosides
Munmun Maiti, Piet Herdewijn
Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
Phosphoramidate nucleotides (as conjugates between nucleoside monophosphate and amino acids) have been developed as potential antiviral/antitumoral prodrugs and, recently, have been evaluated as substrate for polymerases. Despite of these intensive chemical and biological investigations, carried out with phosphoramidate nucleotides, little is known about their chemical stability and the influence of the nucleobase on the kinetics of the hydrolytic reactions leading to degradation of these compounds. We have observed that the stability and mechanism of hydrolysis (P–N versus P–O bond cleavage) is base dependent and influenced by pH. For example, compounds with a 1-deazaadenine base are very labile and exclusively hydrolyzed by P–O bond cleavage, while compounds with a thymine base are very stable and preferentially hydrolyzed by P–N bond cleavage. These stability studies are important in view of the further development of this chemistry for biological purposes.
S5.5 Medicinal chemistry
A 2A adenosine receptor ligands: effects on alcohol intake in alcohol-preferring rats
Rosaria Volpini 1, Michela Buccioni1, Diego Dal Ben1, Catia Lambertucci1, Gabriella Marucci1, Maria Vittoria Micioni Di B2, Carlo Cifani2, Maurizio Massi2, Gloria Cristalli1
1 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, 62032 Camerino, Italy; 2 Pharmacology Unit, University of Camerino, 62032 Camerino, Italy
Increasing evidence relates A2A adenosine receptors (ARs) with several pathological conditions such as neurodegenerative disorders, drug addition, inflammation, and pharmacological stress. SARs of ligands at the A2AAR indicate that substitution of adenosine at the 2-position, especially with (thio)ethers, secondary amines, and alkynes combined with a 5′-N-ethyluronamide led to selective agonists. These two modifications are present in the widely used A2AAR agonist CGS 21680 and in another agonist named VT 7, synthesized in our lab. On the other hand, substitution of the adenosine ribose moiety with alkyl chains combined with the introduction of suitable substituents at the adenine core resulted in A2AAR antagonists, as in the case of ANR 94. Here, the effect of CGS 21680, VT 7, and of ANR 94 on voluntary drinking and operant self-administration of 10% ethanol in Marchigian Sardinian alcohol-preferring (msP) rats was evaluated. Results provide evidence that A2AAR agonists markedly attenuate both voluntary drinking and operant ethanol self-administration in msP rats. In particular, the A2AAR agonist VT 7, differently from CGS 21680, was able to evoke an effect on alcohol consumption, which does not significantly affect locomotor activity, so proving to be behaviourally selective. Thus, A2AARs may represent a potential target for the alcohol abuse and alcoholism treatment.
S6.1 Purinergic signalling in inflammation and cancer
Adenosine receptors in inflammatory disorders
Katia Varani and Pier Andrea Borea
Department of Clinical and Experimental Medicine, Institute of Pharmacology, University of Ferrara, Ferrara, Italy
Adenosine and adenosine receptors (ARs) exert modulatory effects on many body functions and primarily regulate the cardiovascular and central systems, pain modulation, cancer and inflammation. In Parkinson’s disease (PD), we have found a specific A2AARs alteration in human lymphocytes and neutrophils from PD patients if compared with healthy subjects. A statistically linear correlation among A2AARs density or functionality and the Unified Parkinson’s Disease Rating Scale motor score was reported. The interaction of ARs and inflammatory joint diseases as osteoarthritis (OA) and rheumatoid arthritis (RA) was observed. In OA human synoviocytes, the activation of A2A and A3ARs mediates a decrease of the release of proinflammatory cytokines. In human blood cells from RA patients was present an A2A and A3ARs upregulation closely associated with a reduction of the inflammatory state. The effect of adenosine on lung pathologic states was also investigated. A2BARs are significantly decreased in broncholaveolar lavage macrophages from patients with chronic obstructive pulmonary disease compared to healthy subjects. The A2BARs reduction could be partially due to the oxidative/nitrosative stress but not through the increase of proinflammatory mediators. We have also verified the involvement of ARs in pleural malignant mesothelioma (MM). A3ARs are overexpressed in pleura from MM patients and mediate the decrease of proliferation and exert cytotoxic and pro-apoptotic effects in MM cells related to the deregulation of Akt/NFkB pathway. In conclusion, these new findings demonstrate a pivotal role of ARs in different human pathologies suggesting novel pharmacological treatments based on ARs modulation in the inflammatory disorders.
S6.2 Purinergic signalling in inflammation and cancer
Adenosine A 2A -dependent modulation of collagen production from human sclerodermic fibroblasts
Capecchi PL, Lazzerini PE, Natale M, Gianchecchi E, Montilli C, Castrichini M, Balistreri E, Ricci G, Selvi E, Garcia-Gonzalez E, Galeazzi M, Laghi Pasini F
Department of Clinical Medicine and Immunological Sciences, University of Siena, Siena, Italy
Systemic sclerosis (SSc) is a connective tissue disease characterized by exaggerated collagen deposition in the skin and visceral organs. Adenosine A2A receptor stimulation (A2Ar) promotes dermal fibrosis, while the cannabinoid system modulates fibrogenesis in vitro and in animal models of SSc. Moreover, evidence in central nervous system suggests that the A2A and the cannabinoid (CB1) receptors may physically and functionally interact. On this basis, we investigated the A2Ar expression and function in modulating collagen biosynthesis from SSc dermal fibroblasts, also analyzing the cross-talk with the cannabinoid receptors. In sclerodermic cells, A2Ar expression (RT-PCR, Western blotting) and the effects of A2A agonists and/or antagonists on collagen biosynthesis (EIA, Western blotting) were evaluated. The putative physical and functional interactions between the A2A and the cannabinoid receptors were respectively assessed by co-immunoprecipitation and co-incubating the cells with the unselective cannabinoid agonist WIN55,212-2 and the selective A2A antagonist ZM-241385. In SSc fibroblasts, (a) A2Ar is overexpressed and its occupancy with the selective agonist CGS-21680 increases collagen production, myofibroblast trans-differentiation, and ERK-1/2 phosphorylation; (b) the A2Ar forms an heteromer with the cannabinoid CB1 receptor; and (c) the unselective cannabinoid receptor stimulation with a per se ineffective dose of WIN55,212-2 results in a marked anti-fibrotic effect after the A2Ar blockage. In conclusion, A2Ar stimulation induces a pro-fibrotic phenotype in SSc dermal fibroblasts, either directly or indirectly by activating the CB1 cannabinoid receptor. These findings increase our knowledge of the pathophysiology of sclerodermic fibrosis also further suggesting a new therapeutic approach to the disease.
S6.3 Purinergic signalling in inflammation and cancer
P2 receptor signalling in the pathogenesis of inflammatory lung diseases
Marco Idzko
Department of Pneumology, University Hospital Freiburg, Freiburg, Germany
Asthma and chronic obstructive pulmonary disease (COPD) are the most common inflammatory lung disease and are responsible for more than a fourth of death in the developed countries. The exact mediators responsible for the development and chronification of lung inflammation in asthma and COPD are still unknown. Increasing evidences point out to an important role of extracellular nucleotides and their P2 receptors in the modulation of immune responses in the lung. Indeed previously we demonstrated that BALF ATP levels are increased after allergen challenge in humans and mice and that neutralizing intrapulmonary ATP levels or the application of unselective P2 receptor antagonists can abrogate all cardinal features of experimental asthma in mice. Interestingly also in the airways of patient with COPD and mice with smoke-induced lung inflammation increased ATP-levels could be detected, which were correlating with of BALF neutrophils and air flow limitation. These increased ATP levels are of pathophysiological relevance as the development of smoke-induced lung inflammation and emphysema in mice could be markedly reduced by neutralization of ATP or broad P2R inhibition. Next to elucidated the precise P2R subtypes involved in both diseases experiments with specific P2R-subtype antagonist, knockout animals and bone marrow chimeras were performed. Thereby we could demonstrate that P2Y2R and P2X7R signalling on haematopoietic system and P2Y6R signalling on the non-haematopoietic system contributes to development of chronic allergic airway inflammation/asthma and smoke induced lung inflammation/COPD. In summary, our data suggest that targeting of P2Y2, P2Y6 and P2X7R might be a new therapeutic option for the treatment of asthma and COPD.
S6.4 Purinergic signalling in inflammation and cancer
A 2B receptor-mediated changes in gene expression, mediator synthesis and proliferation in human mast cells and coronary smooth muscle cells
Ivar von Kügelgen 1, Kristina Hoffmann1, Annette Viktoria Hinze1,2, Peter Mayer2
1 Department of Pharmacology and Toxicology, University of Bonn, Bonn, Germany; 2 Federal Institute for Drugs and Medical Devices, Bonn, Germany
Activation of membrane-bound adenosine receptors induces long-lasting changes in cellular function via the induction of transcription factors. We examined the involvement of transcription factors in the A2B-receptor-mediated synthesis of interleukin-8 (IL-8) in human mast cells (HMC1) and the A2B-receptor-mediated inhibition of proliferation of primary human coronary smooth muscle cells. In HMC1 cells, the adenosine analogue N-ethyl-carboxamido-adenosine (NECA) increased IL-8 in the supernatant after an incubation time of at least 2 h. This effect was markedly attenuated by the A2B receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamide; 1 μM). NECA also markedly upregulated the transcription factors activator protein-1 (AP1) and NFkappaB measured by reporter gene assays. These responses to NECA were blocked or markedly diminished by MRS1754 in agreement with a coupling of A2B receptors to AP1 and NFkappaB in human mast cells. In human coronary smooth muscle cells, 2-chloroadenosine decreased cell proliferation in the presence of platelet-derived growth factor when applied over 5 days. This effect was attenuated by the A2B receptor antagonist PSB-601 (8-[4-(4-benzylpiperazide-1-sulfonyl)phenyl]-1-propylxanthine; 4 μM). Adenosine, 2-chloroadenosine and NECA induced the transcription factor nuclear receptor subfamily 4, group A, member 1 (NR4A1) 0.5 to 2 h after addition of the agonists. The induction of NR4A1 was blocked by the A2B receptor antagonists MRS1754 and PSB-601. Forskolin and the exchange protein activated by cAMP agonist 8-(4-chlorophenylthio)-2′-O-methyl-cAMP also increased the expression of NR4A1. Moreover, treatment with siRNA against NR4A1 abolished the effect of 2-chloroadenosine on cell proliferation indicating the involvement of the transcription factor NR4A1 in the A2B-receptor-mediated decrease in proliferation of human coronary smooth muscle cells.
S7.1 Purinergic signalling in the CNS I
Adenosine A 2A receptors modulate cocaine-induced changes of synaptic transmission in the striatum: involvement of tyrosine phosphorylation signalling
MR Domenici1, V Chiodi1, A Ferrante1, A Martire1, MT Tebano1, C Mallozzi2, AMM Di Stasi2, JF Chen3, P Popoli 1
1 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy; 2 Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy; 3 Boston University School of Medicine, Boston, MA, USA
Adenosine A2A receptors (A2ARs) are highly expressed in the striatum, a brain area implicated in the pharmacological action of drugs of abuse. In vivo studies demonstrated that cocaine effects are, at least in part, mediated by A2ARs. The aim of the present study was to explore the influence of striatal A2ARs on the cellular and synaptic mechanisms implicated in the pharmacological action of cocaine. Glutamatergic synaptic transmission was studied in corticostriatal slices from wild type (wt) and striatum-specific postsynaptic A2AR knockout mice (stA2ARko) by recording extracellular field potentials (FPs) in the dorsal striatum after stimulation of cortical glutamatergic afferents. In wt mice, slice perfusion with cocaine 10 μM induced a decrease of FP amplitude that was significantly reduced by the A2AR antagonist ZM241385. In stA2ARko mice, cocaine-induced reduction of synaptic transmission was significantly attenuated with respect to wt mice and was unaffected by ZM241385, thus demonstrating the involvement of postsynaptic A2ARs in the modulation of cocaine-induced effects. Since tyrosine phosphorylation signalling has been implicated in cocaine-mediated synaptic effects, the role of src family protein tyrosine kinases (src-PTKs) and phosphatases (PTPs), as well as the involvement of A2ARs, has been also investigated. We found that the activity of both src-PTKs and PTPs was increased by cocaine and was dependent on postsynaptic A2AR activation. In particular, we found that cocaine stimulated the activity of striatal-enriched tyrosine phosphatase (STEP), an effect prevented by ZM241385 and absent in stA2ARko mice. These data demonstrate that A2ARs modulate cocaine-induced synaptic effects in the striatum and identify post-synaptic A2ARs receptors as the major players. This finding, as well as the observation that A2ARs are necessary for cocaine to modulate STEP activity, may have important implications in the pathophysiology of drug abuse.
S7.2 Purinergic signalling in the CNS I
Purinergic signalling in extrasynaptic axon-glia communication: the how and why
Christian Lohr
University of Hamburg, Hamburg, Germany
Release of neurotransmitters such as glutamate occurs mainly at synapses but has also been demonstrated extrasynaptically along axons. ATP is often co-released from vesicles with classical neurotransmitters at central synapses, but the mechanism of axonal release of ATP is still unclear. In the nerve layer of the olfactory bulb, olfactory ensheathings cells (OECs), a specialized glial cell-type enwrapping bundles of olfactory receptor axons, respond to glutamate and ATP with a cytosolic Ca2+ increase mediated by mGluR1 and P2Y1 receptors. Here, we used OECs as a monitor to detect glutamate and ATP co-release from olfactory receptor axons evoked by electrical stimulation. ATP and glutamate release was dependent on Ca2+ influx into receptor axons and could be inhibited when vesicular release was suppressed by bafilomycin A1 and botulinum toxin A. In contrast, blocking gap junction hemichannels and P2X7 purinoceptors, respectively, had no effect on stimulation-induced ATP and glutamate release. Antibody staining revealed the presence of synaptophysin and bassoon, two proteins involved in synaptic vesicle fusion, as well as the vesicular glutamate uptake transporter VGLUT2, in the olfactory nerve. Ca2+ signalling in OECs evoked by ATP or nerve stimulation resulted in constriction of adjacent blood vessels. Our results indicate that in the olfactory nerve, ATP as well as glutamate are released from vesicles and mediate neurovascular coupling via glial Ca2+ signalling.
This study was supported by the DFG (LO779/3).
S7.3 Purinergic signalling in the CNS I
Purinergic signaling and microglial function
Helmut Kettenmann
Max Delbrueck Center for Molecular Medicine (MDC) Berlin, Robert Roessle Str. 10, 13092 Berlin, Germany
kettenmann@mdc-berlin.de
Microglia are the immunocompetent cells of the central nervous system. They respond with a process termed ‘microglial activation’ to any type of pathologic event. But also under normal conditions microglial cells are highly active by constantly screening their environment with their processes. Studies over the last years have established that microglial cells express a variety of different purinergic receptors and we and others have established that these receptors are functional both in cell culture as well as in situ. Purinergic receptors control a variety of different microglial functions such as cytokine release, phagocytosis and migration. Interestingly, microglial cells express selectively defined ATP degrading enzymes, namely CD39 and CD73 which were originally used as microglial cell markers. We could establish that these enzymes modulate purinergic signaling and have an impact on microglial migration and phagocytosis. Thus, purinergic signaling is complex and ATP has emerged as important signal to control microglial function.
S7.4 Purinergic signalling in the CNS I
Purinergic signalling in the cerebellum
Joachim W. Deitmer
FB Biology, TU Kaiserslautern, Kaiserslautern, Germany
Purinergic receptors have been found in most brain areas, and the diversity of P1 (A1), P2X and P2Y receptor family appears to be a major part of the complex information processing, in particular where glia–neuron communication is involved. The rodent cerebellum is well suited to study neuronal circuitry, synaptic plasticity and glia–neuron interactions. Modulation of synaptic transmission is one target of purinergic signalling in the cerebellar cortex, mediated, e.g. by presynaptic P1/A1 adenosine receptors on granule cell terminals. These receptors can also be addressed by ATP and ADP, which is degraded to adenosine by ectonucleotidases in the tissue. Expression of P2Y receptors has been found in Bergmann glial (BG) cells, which are closely associated with the Purkinje neurons (PN) in the cerebellum. BG cells respond to ATP and ADP via P2Y1 receptors with cytosolic calcium transients. These purinergic receptors/calcium responses in BG cells are developmentally regulated and can be activated by 1–10 μM ATP/ADP. No calcium responses to purinergic stimulation could be found in identified neurons of the cerebellar cortex. Synaptic activity in the cerebellum (PN) is modulated by endogenously released as well as by exogenously applied ATP and ADP during the first postnatal weeks, when the wiring of the cerebellar cortex is under construction. Thus, purinergic signalling may influence the development and is involved in the information processing of the cerebellar cortex via different purinergic receptors. The full impact of purines for the development and function of the cerebellum, however, has only started to be recognized. In particular, the location and mechanisms of purine release and the role of enzymatic degradation of ATP/ADP to adenosine need to be studied in more detail.
This study was supported by the Deutsche Forschungsgemeinschaft, SFB 530 and SPP 1176.
S7.5 Purinergic signalling in the CNS I
Hypoxia—adenosinergic neuronal signalling
G. Baier-Bitterlich
Medical University of Innsbruck, Innsbruck, Austria
Hypoxic–ischemic brain injury is an evolving process that begins during the insult and extends into a recovery–reperfusion period. Various mechanisms have evolved that allow cells to adapt to hypoxia. In response to hypoxia/ischemia, purine nucleosides are formed (Sitkovsky, Trends Immunol 30(3):102–108, 2009; Fredholm, Exp Cell Res 316(8):1284–1288, 2010) and act as powerful endogenous neuroprotectants. A number of signaling pathways have been identified that regulate gene expression during hypoxia (Seta et al., Sci STKE 2002(146):RE11, 2002). In our work, using two neuronal cell models (A2A receptor positive PC12 cells and A1 receptor positive primary cerebellar granule neurons) and a low oxygen incubator, we have identified the importance of two key signaling modules. We found that viability and neurite formation critically depend on functional activity of the mitogen-activated protein kinase and the stabilization of the hypoxia-inducible factor-1 alpha. Furthermore, employing siRNA knockdown techniques, pioneer studies of our group showed the involvement of the protein-C-related kinase in purine-mediated neuroprotection (Thauerer et al., J Neurochem 113(2):432–446, 2010). The talk will address purine nucleoside-mediated mechanisms following hypoxic neuronal injury, with an emphasis on the identification of potential targets for pharmacological intervention in humans.
S8.1 Purinergic signalling in the CNS II
Microvesicles shed from microglia represent a new pathway of glia-to-neuron communication
Flavia Antonucci1, Elena Turola1, Loredana Riganti1, Matteo Caleo2, Cristiana Perrotta3 , Luisa Novellino1, Emilio Clementi3,4, Paola Giussani5, Paola Viani5, Michela Matteoli1, Claudia Verderio 1
1 CNR Institute of Neuroscience and Department of Medical Pharmacology, Università di Milano, Via Vanvitelli 32, 20129 Milan, Italy. 2 CNR Institute of Neuroscience, via Moruzzi 1 56124 Pisa, Italy. 3 Unit of Clinical Pharmacology, Department of Clinical Sciences, University Hospital Luigi Sacco, Università di Milano, Milano, Italy. 4 E-Medea Scientific Institute, 23842 Bosisio Parini (LC), Italy. 5 Department of Medical Chemistry, Biochemistry and Biotechnology, Università di Milano, Laboratorio Interdisciplinare di Tecnologie Avanzate, via Fratelli Cervi 93, 20090 Segrate, Milan, Italy
Microvesicles (MVs) are released into the extracellular space by most cell types and are emerging as a novel way of intercellular communication. We have recently shown that microglia, upon P2X7 receptor activation, shed MVs containing pro-inflammatory cytokines. However, the role of microglia-derived MVs in intercellular communication is still completely unexplored. Aim of the present study was to investigate whether MVs shed from microglia upon P2X7 activation affect neurotransmission. Analysis of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal cultures exposed to MVs revealed an increase in mEPSCs frequency without changes in mEPSC amplitude. Paired recording analysis of evoked transmission in vitro showed that microglial MVs increase the amplitude of EPSCs and reduce paired pulse ratio in synaptically connected cells, suggesting an increase in probability of release. Consistent with this finding, microglial MVs also increased sucrose-evoked exocytosis. Injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli, validating in vivo the enhancement of excitatory transmission induced by MVs.
Since sphingolipids regulate neurotransmitter release and localize at MVs surface, we explored the possibility that sphingolipids (sphingosine and sphingosine-1P) could mediate the MV-induced enhancement of excitatory neurotransmission. Direct measurements of sphingolipid metabolism revealed a clear increase in ceramide and sphingosine production from sphingomyelin in neurons exposed to MVs. Pre-treatment of cultured neurons with the inhibitor of sphingosine synthesis N-oleoylethanolamine strongly inhibited the increase in mEPSCs frequency induced by MVs. Conversely, no inhibition was observed when sphingosine-1P production was prevented by neuron incubation with the inhibitor of sphingosine kinase 1. These data identify microglia-derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microglia-to-neuron signalling pathway.
S8.2 Purinergic signalling in the CNS II
Crosstalk between the purinergic system and known algogenic mediators in mouse trigeminal ganglia: implications for basic mechanisms of migraine pain
Stefania Ceruti 1, Giovanni Villa1, Marta Fumagalli1, Laura Colombo1, Giulia Magni1, Matteo Zanardelli1, Elsa Fabbretti2,3, Claudia Verderio4, Arn M. J. M. van den Maagdenberg5,6, Andrea Nistri2, Maria P. Abbracchio1
1 Department of Pharmacological Sciences, Università degli Studi di Milano, Milan, Italy. 2 Neurobiology Sector and Italian Institute of Technology Unit, International School for Advanced Studies (SISSA), Trieste, Italy. 3 University of Nova Gorica, Nova Gorica, Slovenia. 4 CNR Institute of Neuroscience, Department of Medical Pharmacology, Università degli Studi di Milano, Milan, Italy. 5 Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands; 6 Department of Neurology, Leiden University Medical Centre, Leiden, The Netherlands
In the present study, we focused on the role of the purinergic system in neuron-to-satellite glial cell (SGCs) communication within the trigeminal ganglion and on its crosstalk with known pro-algogenic mediators (e.g., bradykinin (BK) and calcitonin gene-related peptide (CGRP)). BK potentiates ATP- and UTP-sensitive P2Y receptors on SGCs in primary trigeminal cultures (Ceruti et al., Cell Calcium 43:576, 2008). Here, we investigated the molecular basis of this effect in wild-type mice and CaV2.1 α1 R192Q mutant knock-in (KI) mice expressing a human mutation causing familial hemiplegic migraine type 1. Exposure to BK led to the activation of its neuronal receptors, followed by the release of CGRP acting paracrinally on surrounding SGCs. In fact, both the CGRP antagonist CGRP8-37 and the anti-migraine drug sumatriptan (which targets neurons) inhibited BK actions. Moreover, unlike CGRP, BK was ineffective in neuron-free SGCs cultures. CGRP-mediated glial P2Y receptor potentiation was mediated by ERK1/2 pathway, and the release of several cytokines was also detected, which could further contribute to cell-to-cell communication within the TG. Interestingly, both basal and BK-stimulated CGRP release was higher in KI mouse cultures. Indeed, BK significantly up-regulated the number of SGCs showing functional P2Y receptors, particularly the UTP-sensitive subtypes, only in cultures from KI mice (Ceruti et al.. J Neurosci 31:3638, 2011). Our findings suggest, for the first time, that P2Y receptors on glial cells act as novel players in the cellular processes underlying migraine pathophysiology and might represent new targets for the development of innovative therapeutic agents against migraine pain.
This study was sponsored by the Italian Comitato Telethon, grants no. GGP07032 and GGP10082.
S8.3 Purinergic signalling in the CNS II
Adenosine is released per se under physiological conditions from the rat striatum in vivo; during ischemia it is primarily a product of extracellular ATP
Pedata Felicita 1, Melani Alessia1, Corti Francesca1, Stephan Holger2, Christa E. Müller3, Vannucchi Maria Giuliana4
1 Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy, felicita.pedata@unifi.it. 2 Helmholtz-Zentrum Dresden-Rossendorf, Institut für Radiopharmazie, 01314 Dresden, Germany. 3 Pharmaceutical Institute, Pharmaceutical Sciences Bonn (PSB), University of Bonn, Bonn, Germany. 4 Department of Anatomy, Histology and Forensic Medicine, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy
In this study, extracellular concentrations of adenosine and ATP from the rat striatum were estimated by the microdialysis technique under physiological conditions and during focal cerebral ischemia induced by middle cerebral artery occlusion (MCAo). Under physiological conditions, adenosine and ATP concentrations were in the range of 130 and 30 nM, respectively. Blocking the ecto-ATPases, with the novel inhibitor polyanion [TiW11CoO40]8−, PV4 (100 μM), recently synthesized and characterized by Stephan and Müller, we demonstrated that the extracellular concentration of ATP increased 12-fold and that adenosine concentration was not modified. This result indicates that under physiological conditions, adenosine is released per se from cells. In the presence of PV4 and of the adenosine equilibrative transporter inhibitor dipyridamole (100 μM), adenosine extracellular concentration was increased by threefold. This result excludes the possibility that adenosine is carried out of cells by a carrier mediated efflux. By using immunolabeling and electron microscopy, we showed the presence of the CNT2 on plasma membrane of synaptic terminals and on vesicle membranes. Results suggest that under in vivo physiological conditions, adenosine is transported in vesicles and is released in an excitation–secretion manner. In the first 4 h after in vivo ischemia induced by MCAo, adenosine increased to ~690 nM and ATP to ~50 nM. In the presence of PV4, the extracellular concentration of ATP increased to ~440 nM and extracellular adenosine decreased to ~270 nM. An upregulation of ecto-nucleotidases after ischemia might represent an important mechanism in hydrolysis of ATP and formation of extracellular adenosine in the first hours after ischemia.
Acknowledgments: The work was supported by grants from PRIN (Ministero dell’Istruzione, dell’Università e della Ricerca, Italy), University of Florence, and Ente Cassa di Risparmio of Florence, Italy.
S8.4 Purinergic signalling in the CNS II
Involvement of purine receptors in the growth and differentiation of human glioblastoma initiating cells
I. D’Alimonte1,2*, E. Nargi1,2*, A. Lannutti1,2*, P. Di Iorio1, P. Ballerini1, P. Giuliani1, L. Ricci-Vitiani3, R. De Maria3, F. Caciagli1, R. Ciccarelli 1,2
1 Department of Biomedical Sciences, University of Chieti, Chieti, Italy. 2 Stem TeCh group, University of Chieti, Chieti, Italy. 3 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
*Equally contributed to the study
Since novel therapies need to treat malignant brain tumors arising from stem cells with high self-renewal degree and resistance to chemotherapy, we investigated the antitumor potential of purines that may exert cytotoxic effects interacting with purinoceptors, in glioblastoma-initiating cells (GICs) derived from human glioblastoma multiforme grade IV. Cultured GICs showed a major expression of P2Y1-P2Y2-P2Y4-P2Y13 and P2X7 subtypes of ATP/P2 receptors. Their exposure for 48 h to ATP (ligand of all P2 receptors) caused a dose-dependent increase (10–100 μM) or decrease (250–1,000 μM) of cell survival measured after further 24 h. Cell death was induced also by 48-h treatment with 500–1,000 μM UTP that more selectively binds P2Y2/4 receptors or by 24-h treatment with 500–1,000 μM benzoyl-ATP (Bz-ATP), P2X7 receptor agonist. A renewal of cell duplication was found after further 96 h only in cells exposed to 100–500 μM ATP. In contrast, 48-h treatment with 10–200 μM ADPβS (rather selective agonist for P2Y1/11/13 receptors) dose-dependently stimulated GIC proliferation even after further 96 h. The effects of ATP-UTP-ADPβS and Bz-ATP were counteracted by suramin and oxidized ATP, which are non-selective P2 receptor and P2X7 receptor antagonists, respectively. We are currently evaluating whether P2 receptor ligands may affect GIC differentiation towards an astrocytic phenotype and the eventual synergism with agents used for brain cancer chemotherapy. At present, our results indicating that stimulation of some ATP/P2 receptors causes cytotoxic effects in cells originating malignant brain tumors hopefully contribute to open a new exciting avenue of investigation.
This study was supported by a grant to R.C. from the Italian Government (MIUR).
S9.1 P2Y receptors
Role of the P2Y-like receptor GPR17 in neural precursor cells
Abbracchio Maria P 1, Daniele Simona2, Lecca Davide1, Parravicini Chiara1, Trincavelli Maria Letizia2, Boccazzi Marta1, Ceruti Stefania1, Martini Claudia2, Fumagalli Marta1
1 Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological Sciences, University of Milan, Milan, Italy. 2 Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy
Three main findings have recently opened new opportunities for repairing the adult brain in both acute (trauma, stroke) and chronic disorders, as Alzheimer’s and Parkinson’s diseases and multiple sclerosis. First, the generation of new neurons and new glia (neurogenesis) continues throughout life. Second, adult neurogenesis does not only occur in “neurogenic niches,” but the entire brain’s parenchyma is full of quiescent progenitors that are activated after injury. Two main types of such cells have been identified: (1) proliferating reactive astrocytes and (2) NG2-positive polydendrocytes that, under some conditions, can generate oligodendrocytes, neurons, and astrocytes (Nishiyama et al., Nat Rev Neurosci 10:9–22, 2009). Third, various types of P2Y receptors are present on polydendrocytes, including the P2Y-like receptor GPR17. Specifically, GPR17 decorates two subsets of slowly proliferating NG2-expressing cells: (1) morphologically immature cells expressing early proteins like Olig2 and PDGF receptor-α, and (2) ramified pre-oligodendrocytes already expressing more mature factors, like O4 and O1. Activation of GPR17 by some of its endogenous ligands such as uracil nucleotides (Ciana et al., EMBO J 25:4615–4627, 2006; Benned-Jensen and Rosenkilde, Br J Pharmacol 159:1092–1105, 2010) promotes cell differentiation to mature myelinating oligodendrocytes, while its inhibition by receptor antagonists or small interfering RNAs retain cells in an undifferentiated state (Fumagalli et al., J Biol Chem 286:10593–10604, 2011; see also Ceruti et al., Glia 59:363–378, 2011). GPR17 is thus a new key player in polydendrocytes maturation. We are currently assessing if GPR17 manipulation can also instruct NG2-expressing cells to generate new neurons.
S9.2 P2Y receptors
Can one make a nucleotide-based drug? Making the impossible possible
Bilha Fischer
Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel
Nucleotides are potential drug candidates targeting proteins such as nucleotide receptors (P2-Rs), DNA/RNA polymerases, metabolic enzymes (e.g. NTPDases and NPPs) and metalo-matrix proteinases for the treatment of various health disorders including cystic fibrosis, diabetes, thrombosis, herpes, hepatitis B, HIV and cancer. Yet, nucleotides suffer from limitations due to their chemical and metabolic instability, the need for bio-target selectivity and GI absorption.
In my talk, I will describe drug candidates we have recently developed based on nucleotide/dinucleotide scaffolds. By suitable chemical modifications, we have conferred hydrolytic and metabolic stability to the nucleotides as well as bio-target selectivity. In this way, we have developed promising agents for the treatment of glaucoma (targeting P2Y-Rs). These drugs, evaluated in rabbits, were found superior to current drugs in terms of potency, efficacy, and/or duration of effect, and present a promising alternative to Timolol maleate, which cannot be used for the treatment of patients suffering from cardiovascular problems, asthma or diabetes. In addition, we have developed nucleotide- and dinucleotide- based insulin secretagogues for the treatment of type 2 diabetes via P2Y-Rs activation. One of the analogues administered in rats increased blood insulin levels fourfold, as compared to basal levels, and decreased blood glucose load from 155 mg/dL to ca. 100 mg/dL, unlike the commonly prescribed glibenclamide which reduced glucose levels below normal values (60 mg/dL). These findings suggest that this analogue may prove to be an effective and safe treatment for type 2 diabetes.
S9.3 P2Y receptors
Novel aspects of receptor pharmacology and receptor trafficking of the P2Y 11 receptor: insights into physiological significance of the Alanine-87-Threonine single nucleotide polymorphism of the human P2Y 11 receptor
Michael Haas, Ahmed Shaaban, Georg Reiser
Institut für Neurobiochemie, Medizinische Fakultät der Otto-von-Guericke Universität Magdeburg Leipziger Str., 44 39120 Magdeburg, Germany
The human P2Y11 nucleotide receptor is the only P2Y receptor subtype dually signaling via IP3 and cAMP formation. Moreover, the P2Y11 receptor is connected to cAPDR formation and β-arrestin-2 recruitment. Synthesis of P2Y11 receptor mRNA in human tissues is widespread, including brain, spleen, and lymphocytes, but it can also be detected in macrophages, platelets, neutrophils, dendritic cells, heart, and, to some extent, in all other human tissues. In dendritic cells, the P2Y11 receptor was found to regulate ATP-dependent maturation, cell migration, and interleukin-8 release. While there are indications for a strong association of the human P2Y11 receptor with immune response-related processes, it was found also to play a role in fluid and electrolyte secretion in pancreatic duct epithelial cells. The P2Y11 receptor activity in human therefore continues to be in the focus as potential drug target. The P2Y11 receptor initially has been investigated in 1321 N1 astrocytoma cells lacking endogenous P2Y receptors. Later, our studies with HEK293 cells revealed hetero-oligomerization of the P2Y11 receptor with the endogenous P2Y1 receptor, resulting in a functional and pharmacological profile, which reflected neither that of the P2Y1 nor that of the P2Y11 receptor (Ecke et al., Biochem J 409:107–116, 2008). In view of this finding, P2Y1-P2Y11 hetero-oligomers might represent the active receptor in cells co-expressing both P2Y receptor subtypes. For acute myocardial infarction (AMI), one of the major causes for death in the western world, a statistical link to the Ala87Thr single nucleotide polymorphism (SNP) was reported (Amisten et al., Eur Heart J 28:13–18, 2007). The functional impact of the mutation on receptor function, however, remains unknown until now.
In 1321 N1 astrocytoma cells, a difference in ATP potency regarding the rise of intracellular calcium concentration between P2Y11 wild-type and P2Y11A87T receptors cannot be demonstrated in our experiments. In HEK293 cells, however, a remarkably decreased potency for ATP and the P2Y11 receptor-specific agonist BzATP could be shown. Thus, we hypothesized an effect of the Ala87Thr SNP within the receptor hetero-oligomer. To investigate further the consequences of the amino acid switch, the mutant Ala87Ser was also generated and functionally investigated. We found that this mutation completely restored the ability of the receptors to be activated by ATP and to generate an intracellular calcium response. The calcium response to BzATP could be restored partially. This indicates that the change of amino acid polarity, from nonpolar alanine to polar threonine and serine, might not be the reason for its altered function. In addition, we could not show nucleotide-induced internalization for both mutated receptors in HEK293 cells, as known for P2Y11 wild-type receptors and important for cell desensitization. The functional and physiological consequences of these differences in receptor characteristics still need to be elucidated.
Atherosclerosis is a risk factor for AMI. P2Y1 and P2Y11 receptors are co-expressed in dendritic cells and macrophages, both relevant for the development of atherosclerosis. Thus, a P2Y1-P2Y11A87T hetero-oligomer in those cells could contribute to the development of the disease process. These data provide first functional evidence in line with the initial statistical connection of the Ala87Thr SNP of the P2Y11 receptor with increased AMI risk. However, understanding the precise mechanism of how Ala87Thr interferes with P2Y1-P2Y11 hetero-oligomer function as well as its connection to AMI remains major tasks.
S9.4 P2Y receptors
Microglial P2Y 12 receptor as a potential therapeutic target of neuropathic pain
Hidetoshi Tozaki-Saitoh 1, Makoto Tsuda1, Hiroshi Kiyama2, Keiko Ohsawa1, Kazuhide Inoue1
1 Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi, Fukuoka 812–8582, Japan. 2 Department of Functional Anatomy and Neuroscience, Graduate School of Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464–8601, Japan
Extracellular nucleotides have been implicated as signaling molecules used by microglia to sense adverse physiological conditions. They act through purinoceptors, especially P2Y12 receptor (P2Y12R) which is closely linked to microglial chemotactic function. Emerging evidence has indicated that activated spinal microglia are key cellular intermediaries in the neuropathic pain. However, the role of microglial P2Y12Rs in neuropathic pain remains unclear. Here, we show that the level of P2Y12R expression was markedly increased in the spinal cord and that this expression was highly restricted to spinal microglia. Mice lacking P2ry 12 displayed impaired tactile allodynia after nerve injury, and the intrathecal administration of P2Y12R antagonist prevented the development of tactile allodynia. Using electron microscopy, we found an increased number of microglial cells adhering to and engulfing both injured and uninjured myelinated axons after nerve injury. These events were also significantly suppressed by the P2Y12R antagonist. Together, our findings indicate that activation of P2Y12Rs in spinal microglia may be a critical for physical interaction between microglia and neurons, and therefore in the pathogenesis of neuropathic pain.
This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and also grants from Core Research for Evolutional Science and Technology (CREST) program supported by the Japan Science and Technology Agency (JST).
S10.1 Nucleotide-metabolizing enzymes
Structural studies on ectonucleotidases
Norbert Sträter, Matthias Zebisch, Karen Yates
Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany
A number of extracellular nucleotidases are involved in the conversion of nucleotides in purinergic signalling. The NTPDases (ecto-nucleoside triphosphate diphosphohydrolases) dephosphorylate ATP via ADP to AMP. There are eight different types (NTPDase1-8) in humans. Ecto-5′-nucleotidase (eN, CD73) catalyses the hydrolysis of AMP to adenosine. In addition to the receptors for extracellular nucleotides and nucleosides, the ecto-nucleotidases have also been recognized as pharmaceutical targets to interfere with purinergic signalling pathways. Specific inhibitors would not only be valuable tools in biopharmaceutical fundamental research, they would also constitute potential clinical therapeutics (e.g. in the treatment of chronic pain, immune system diseases and cancer) as they would prolong the physiological effects of extracellular nucleotides or simultaneously administered nucleotide analogues.
With the aim to characterize its catalytic mechanism and in order to rationally develop specific inhibitors for pharmaceutical and biological studies, we have overexpressed the ectodomains of NTPDases and CD73 in Escherichia coli in inclusion bodies and developed efficient refolding protocols to produce milligram amounts of pure and highly active enzyme. Thus, preparations of NTPDases as well as CD73 are stable in soluble form without glycosylation and are suitable for structural studies (Zebisch and Sträter, Biochemistry 46:11945–11956, 2007; Proc Natl Acad Sci USA 105:6882–6887, 2008). For the NTPDases, the kinetic properties of the ectodomains resemble that of the membrane-bound full-length enzymes, although differences exist in detail, e.g. the processivity of NTPDase1 is lost partially in the ectodomain alone (Zebisch and Sträter, Biochemistry 46:11945–11956, 2007). It has been proposed that the coupling of the transmembrane helices to the two domains of the protein influences the catalytic properties by a control of a domain motion. Structural studies on rat NTPDase1 in a crystal containing four independent copies of the molecule revealed first structural details on the nature of this domain movement.
S10.2 Nucleotide-metabolizing enzymes
Purinergic control of coronary blood flow and aortic NTPDase activity are impaired in young pre-atherosclerotic apolipoprotein E-deficient mice
Gennady G. Yegutkin 1, Nathalie Mercier1, Tuomas O. Kiviniemi2,3, Antti Saraste3, Sirpa Jalkanen1
1 Medicity Research Laboratory and Department of Medical Microbiology, University of Turku, Turku, Finland. 2 Department of Clinical Physiology, Turku University Hospital, Turku, Finland. 3 Department of Medicine and Turku PET Centre, Turku University Hospital, Turku, Finland
Disturbed vasodilation predisposes pathological vascular remodeling during atherosclerosis. Given the important role of intravascular ATP and ADP in control of inflammation, thrombosis, vascular tone, and angiogenesis, we aimed to evaluate the contribution of purinergic signaling to atherosclerosis progression. Transthoracic Doppler echocardiography was employed for non-invasive imaging of blood flow velocity in the left coronary artery of apolipoprotein E-deficient (ApoE−/−) and wild-type C57BL/6 mice. Young (10–15-week-old) plaque-free ApoE−/− mice displayed diminished coronary reactivity in response to ATP but not adenosine. The impaired vasodilatory response to ATP persisted in older 20–30-week-old ApoE−/− mice, which were additionally characterized by mild signs of atherosclerosis (as ascertained by Oil Red-O staining) and systemic increase in plasma ATP and ADP levels. Concurrent thin-layer chromatographic analysis of nucleoside triphosphate diphosphohydrolase (NTPDase) and ecto-5′-nucleotidase/CD73 activities in murine thoracic aortas, peripheral lymph nodes, spleen, and serum revealed significant tissue-specific down-regulation of aortic NTPDase by 40–50% both in young and mature aged ApoE−/− mice. Collectively, disordered purinergic signaling in young ApoE−/− mice may serve as important prerequisite for impaired coronary blood flow, local accumulation of ATP and ADP at sites of atherogenesis, and, eventually, the exacerbation of atherosclerosis.
PS1.1 Medicinal Chemistry
N -(5-Substituted-4-phenylthiazol-2-yl)carboxamide derivatives: potent adenosine A 1 receptor antagonists
Aliaa Abdelrahman 1, Swapnil G. Yerande2, Ali El-Tayeb1, Sunil D. More2, Kiran M. Newase2, Christa E. Müller1
1 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany. 2 Progressive Education Society’s Modern College of Pharmacy Sector 21, Yamunanagar, Nigdi, Pune 411044, India
Adenosine A1 receptors (AdoA1R) are expressed in the central nervous system as well as in peripheral tissues. Activation of central AdoA1R leads to sedation and anticonvulsive and anxiolytic effects, and therefore, A1 antagonists have been suggested for the treatment of cognitive deficits. In the lung, adenosine mediates bronchoconstriction, inflammation, increased endothelial cell permeability, and mucin production. AdoA1R antagonists may therefore be useful for the treatment of asthma. In the heart, AdoA1R antagonists could be used to treat cardiac arrhythmia and congestive heart failure. In the kidneys, AdoA1R antagonist may be helpful for the treatment of renal failure, renal dysfunction, nephritis, hypertension, and edema (Fredholm et al., Pharmacol Rev 63:1–34, 2011). In our previous work, we discovered 2-amino-5-benzoyl-phenylthiazoles as potent and selective AdoA1R antagonists (Scheiff et al., Bioorg Med Chem 18:2195–2203, 2010). In the present study, we have further modified the thiazole core at the 2-amino group by amide formation, and furthermore, we have introduced various substituents into the 5-position of the 4-phenylthiazole core structure. The obtained series of thiazole derivatives was screened in radioligand binding studies at AdoA1R. Subnanomolar A1 affinities were observed for many of the investigated derivatives. Selectivity versus the other adenosine receptor subtypes was assessed in radioligand binding assays. For example, 4-methoxy-N-(5-(4-methylbenzoyl)-4-phenylthiazol-2-yl)benzamide showed a K i value of 0.529 nM at rat and 9.12 nM at human AdoA1R with high selectivity against the other AdoR subtypes.
PS1.2 Medicinal Chemistry
[1,2,4]Triazolo[1,5- c ]quinazolin-2-amines—potent adenosine A 1 and A 2A receptor antagonists
Wadih Ghattas, Joachim C. Burbiel, Jörg Hockemeyer, Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Considerable evidence has been accumulated from animal studies indicating that adenosine A2A receptor antagonists may have potential for the treatment of Parkinson’s disease (PD), Alzheimer’s disease, depression, and drug addiction. Several A2A antagonists are currently evaluated in clinical trials for PD (Müller and Ferré, Frontiers in CNS Drug Discovery 1:304–341, 2010). Antagonists at A1 receptors exhibit diuretic and kidney-protective activity and may therefore be useful for the treatment of congestive heart failure (Vallon et al., European Journal of Heart Failure 10:176–187, 2008). However, a phase III clinical study for the xanthine A1 antagonist rolofylline was unsuccessful. However, it is not clear whether this would also apply to structurally different, non-xanthine A1 antagonists. Our goal was to develop non-xanthine adenosine receptor antagonists with selectivity either for A2A or A1 receptors. Initial screening of a small proprietary compound library led to the discovery that certain [1,2,4]triazolo[1,5-c]quinazoline derivatives showed high affinity for A2A and A1 receptors. Subsequently, we explored the structure–activity relationships of this family by a two-stage strategy. At the first stage, position 2 was investigated and various substituents were introduced, including R′ = H, OH, SH, NH2, and Ph, while R was kept constant being either Ph or H. We observed that [1,2,4]triazolo[1,5-c]quinazolin-2-amines, i.e., R′ = NH2, showed the highest affinity for adenosine receptors. At the second stage, position 5 was investigated by varying R using diverse substituents such as aromatic, heteroaromatic, and substituted amino and thio functions, as well as aliphatic groups, while position 2 was kept constant and substituted with NH2. Selected [1,2,4]triazolo[1,5-c]quinazoline-2-amines with R′ = NH2 and their affinity constants for A1 and A2A receptors are shown below.
Affinity for both A1 and A2A receptors was obtained with K i values in the nanomolar range. Furthermore, selectivity for either A1 or A2A could be achieved for some of the compounds. Data obtained from studies at human receptors were similar to that at rat receptors. These results suggest that [1,2,4]triazolo[1,5-c]quinazolin-2-amines constitute a family of potent adenosine receptor antagonists. Further analyses will be carried out in order to investigate their selectivity towards the other adenosine receptor subtypes.
Acknowledgments: We are grateful to the German Federal Ministry of Education and Research (BMBF) and UCB Pharma for financial support (BIOPHARMA Neuroallianz project).
PS1.3 Medicinal Chemistry
N -Amino-substituted xanthine derivatives: affinity at adenosine receptors
Nikolay T. Tzvetkov, Petra Küppers, Jörg Hockemeyer, Christa E. Müller
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Xanthine derivatives related to the alkaloids caffeine and theophylline constitute an important class of adenosine receptor (AR) antagonists. Adenosine receptors are class A G-protein-coupled receptors subdivided into A1, A2A, A2B, and A3 subtypes. 1,3-Diethyl-8-(3,4-dimethoxystyryl)xanthine (KW-6002, istradefylline), an A2A-selective antagonist, is being evaluated in phase III clinical trials for the treatment of Parkinson’s disease. Another xanthine derivate that is currently undergoing clinical trials (phase I) is 3-ethyl-1-propyl-8-(1-(3-(trifluoromethyl)benzyl)-1H-pyrazol-4-yl)xanthine (GS 6201, formerly CVT-6883), an A2B-selective antagonist targeting pulmonary diseases. A major drawback of xanthine derivatives is their low water solubility, which may limit their bioavailability. In the present study, we introduced amino groups directly attached to the nitrogen atoms N3 and/or N7 of the xanthine core. Our goal was to investigate (1) whether such a modification was tolerated by AR and (2) how it would change the physicochemical properties, especially water solubility, of the compounds by allowing salt, e.g., hydrochloride, formation.
7-Amino-8-cyclopentyl-1,3-dipropylxanthine, the 7-amino derivative of the potent A1 antagonist DPCPX, was obtained. Furthermore, several N1-, N3-, and N7-(dimethyl)amino-substituted derivatives of A2A-selective xanthine derivatives were prepared. In contrast to the A1 AR, which did not tolerate 7-amino substitution, several 7-aminoxanthine derivatives were found to be potent antagonists at A2A AR. Thus, 7-amino-1,3-diethyl-8-(3,4-dimethoxystyryl)xanthine, the N7-amino-substituted analog of istradefylline was a potent and selective A2A AR antagonist (K i = 57.5 nM).
Acknowledgments: We are grateful to the German Federal Ministry of Education and Research (BMBF) and UCB Pharma for financial support (BIOPHARMA Neuroallianz project).
PS1.4 Medicinal Chemistry
Development of PSB-0777: a highly polar, not perorally absorbable A 2A -selective agonist with potential for the treatment of inflammatory bowel diseases
Ali El-Tayeb 1, Sebastian Michael2, Aliaa Abdelrahman1, Andrea Behrenswerth1, Sabrina Gollos1, Karen Nieber2, Christa E. Müller1
1 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany. 2 Institute of Pharmacy, University of Leipzig, Talstr. 33, 04109 Leipzig, Germany
A2A adenosine receptors (A2AAR) are expressed in intestinal mucosa, enteric neurons, hepatocytes, and a variety of immune cells. In the intestine, the A2AAR are expressed in the jejunum, ileum, and cecum. Activation of the A2AAR on a variety of inflammatory cell types leads to anti-inflammatory effects. Therefore, A2AAR agonists have been suggested as novel anti-inflammatory drugs. However, the systemic use of A2A adenosine receptor agonists as anti-inflammatory drugs is limited by their potent hypotensive activity (Fredholm et al., Pharmacol Rev 63:1–34, 2011; El-Tayeb et al., J Med Chem 52:7669–7677, 2009).
In the present study, we developed highly polar and therefore not perorally absorbable A2A-selective agonists. Such compounds would be useful for local anti-inflammatory therapy, e.g., of inflammatory bowel disease and gastrointestinal ulcers avoiding the hypotensive effect of centrally acting anti-inflammatory A2A agonists. Therefore, we introduce acidic functions, such as sulfonate and carboxylate moieties, into 2-thioadenosine derivatives and evaluate their A2A agonistic activity as well as their ability to reduce inflammation in an inflamed rat ileum/jejunum preparation. PSB-0777 was identified as a potent (K i rat A2A = 44.4 nM) and subtype-selective (K i rat A1, human A2B, and human A3 ≥ 10,000 nM) A2AAR agonist. In cAMP accumulation studies using recombinant CHO cells expressing the human A2A receptor, it was found that PSB-0777 behaved as a full agonist (EC50 = 117 nM). Further evaluation in untreated and inflamed rat ileum/jejunum preparations in ex vivo experiments indicated that PSB-0777 may be useful for the treatment of inflammatory bowel diseases.
PS1.5 Medicinal Chemistry
Pharmacological characterization of novel adenosine A 2A receptor antagonists: N -cycloalkyl-substituted imidazo-, pyrimido-, and 1,3-diazepino-[2,1- f ]purinediones
Anna Drabczyńska1, Meryem Köse2, Minka Paskaleva2, Anke C. Schiedel2, Tadeusz Karcz 1, Christa E. Müller2, Katarzyna Kieć-Kononowicz1
1 Department of Technology and Biotechnology of Drugs, Medical College, Jagiellonian University, Medyczna 9, 30–688 Kraków, Poland. 2 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Adenosine A2A receptors (A2A ARs) belong to the GPCR superfamily and play an important role in regulating smooth and well-coordinated movement. There is now vast evidence that A2A AR antagonists may provide a novel therapy for the treatment of Parkinson’s disease with a reduced risk of dyskinesias as compared to current therapies and with potential neuroprotective effects (Yuzlenko and Kieć-Kononowicz, Curr Med Chem 13:3609–3625, 2006; Simola et al., Curr Pharm Des 14:1475–1489, 2008; Müller and Jacobson, Biochim Biophys Acta 1808:1290–1308, 2011).
Taking into account the importance of A2A ARs, a series of cycloalkyl derivatives of annelated purinediones were tested for their affinities to rat A1 and A2A adenosine receptors. For selected derivatives, the affinity to the other subtypes of human adenosine receptors was determined, and they were additionally evaluated in functional studies.
Most of the compounds showed moderate to high A2A AR selectivity. The results of the radioligand binding assays revealed that the majority of investigated compounds exhibited A2A AR affinity at micromolar or submicromolar concentrations, with the best compound exhibiting a K i value of 0.33 μM at rat A2A AR and 0.31 μM at human A2A AR. Interestingly, some of the compounds showed higher affinity to human rather than rat A2A AR, while the opposite species preference has typically been found for other xanthine derivatives. Based on the results of cAMP accumulation assays, all investigated derivatives were found to be antagonists at A2A AR.
Acknowledgments: This study was partly supported by Polish Ministry of Science and Higher Education grant no. NN 405 297 836.
PS1.6 Medicinal Chemistry
8-Phenylethynylxanthines—highly potent and selective adenosine A 2A receptor antagonists
Amelie Zech, Jörg Hockemeyer, Nikolay Tzvetkov, Meryem Köse, Hamid Radjainia, Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Adenosine A2A receptors are highly expressed in the brain striatum and are known to interact with colocalized dopamine D2 receptors. A2A antagonists have been demonstrated to be effective in animal models of Parkinson’s disease, and several A2A antagonists are currently undergoing clinical evaluation (Müller and Ferré, Frontiers in CNS Drug Discovery 1:304–341, 2010). A2A antagonists do not only show positive effects on motor symptoms, without causing dyskinesia, but in addition, they have been shown to mediate neuroprotective effects and therefore may be true disease-modifying agents suitable for the treatment of a neurodegenerative diseases in general, including Alzheimer’s and Parkinson’s diseases (Yu et al., Ann Neurol 63:338–346, 2008). The first class of nonselective adenosine receptor antagonists with moderate affinity was the natural xanthine derivatives theophylline and caffeine (Fredholm et al., Pharmacol Rev 63:1–34, 2011). The introduction of a styryl group at the C8 position of xanthines was essential for obtaining compounds with enhanced A2A receptor affinity and selectivity by reducing A1 affinity. However, the presence of the double bond at the 8-position in 8-styrylxanthines led to photosensitive compounds (Hockemeyer et al., J Org Chem 69:3308-3318, 2004). The replacement of the styryl double bond by a triple bond yielded the photostable 8-phenylethynylxanthines (Müller et al., Eur J Med Chem 32:709–719, 1997).
In order to study the structure–activity relationships of this new class of A2A-selective antagonists, we introduced a variety of substituents in different positions, in particular at the ring nitrogen atoms N1, N3, and N7, as well as differently substituted phenylethynyl residues at C8. Thus, we obtained derivatives showing high affinity at the A2A receptor in the low nanomolar range combined with excellent selectivity. Optimized substituents with regard to A2A affinity are a propargyl residue at N1 and a methyl group at the N7 position; small non-polar, unbranched alkyl chains at N3 led to the best results at that position, and a 3,4-dimethoxyphenylethynyl residue proved to be one of the best substituents at C8 with regard to A2A affinity and selectivity.
Acknowledgments: We are grateful to the German Federal Ministry of Education and Research (BMBF) and UCB Pharma for financial support (BIOPHARMA Neuroallianz project).
PS1.7 Medicinal Chemistry
[ 3 H]PSB-1010—a new, optimized antagonist radioligand for adenosine A 2A receptors
Petra Küppers, Amelie Zech, Simone Siebers, Judith Paschkowiak, Jörg Hockemeyer, Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Adenosine A2A receptors are of considerable interest as novel drug targets: A2A antagonists are clinically evaluated for the therapy of Parkinson’s disease and may be useful for the treatment of other neurodegenerative diseases as well, including Alzheimer’s disease (Müller and Ferré, Frontiers in CNS Drug Discovery 1:304–341, 2010). 3H-labeled radioligands for A2A receptors are widely used in drug screening, including high-throughput screening approaches. Important properties of a radioligand for A2A receptors include high affinity (low nanomolar range), high specific radioactivity, low non-specific binding, and chemical and enzymatic stability. Several antagonist radioligands for A2A receptors have been described, but none of them is perfect. One of the best radioligands (Sihver et al., Eur J Pharmacol 616:107–114, 2009), the 8-styrylxanthine derivative [3H]MSX-2 (Müller et al., Eur J Pharm Sci 10:259–265, 2000), fulfills all the criteria, but one: it is unstable when exposed to light showing fast, light-induced E/Z isomerization in dilute solutions. In solid form, styrylxanthines may undergo light-induced dimerization forming cyclobutane derivatives (Hockemeyer et al., J Org Chem 69:3308-3318, 2004). In order to stabilize the structure of styrylxanthines, we replaced the double bond by a triple bond yielding stable 8-(phenylethynyl)xanthines (Müller et al., Eur J Med Chem 32:709–719, 1997). Careful optimization led to PSB-1010 (3-cyclopropyl-8-(3,4-dimethoxyphenylethynyl)-7-methyl-1-(2-propynyl)xanthine, which was subsequently obtained in 3H-labeled form from its 7-demethyl precursor.
[3H]PSB-1010 is an improved antagonist radioligand highly suitable for the specific labeling of human as well as rodent adenosine A2A receptors.
Acknowledgments: We are grateful to the German Federal Ministry of Education and Research (BMBF) and UCB Pharma for financial support (BIOPHARMA Neuroallianz project).
PS1.8 Medicinal Chemistry
3-Aryl-[1,2,4]triazino[4,3- a ]benzimidazol-4(10 H )-one: a novel template for the design of highly selective A 2B adenosine receptor antagonists
S Taliani 1, I Pugliesi1, E Barresi1, S Salerno1, F Simorini1, C La Motta1, AM Marini1, F Da Settimo1, S Cosconati2, L Marinelli2, B Cosimelli3, G Greco2, E Novellino2, S Daniele4, ML Trincavelli4, C Martini4
1 Dipartimento di Scienze Farmaceutiche, Università di Napoli “Federico II”, 80131 Napoli, Italy. 2 Dipartimento di Chimica Farmaceutica e Tossicologia, Universita di Napoli “Federico II”, Napoli, Italy. 3 Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologia, Pisa, Italy. 4 Dipartimento di Scienze Ambientali, Seconda Universita di Napoli, Caserta, Italy.
Adenosine modulates numerous physiological processes through interaction with specific cell-surface G-protein-coupled receptors, which are classified into four subtypes, namely A1, A2A, A2B, and A3 adenosine receptors (ARs) (Fredholm et al., Pharmacol Rev 53:527, 2001). Many evidences show that the “low-affinity” A2B ARs (for adenosine EC50 = 24 μM) play a crucial role in the regulation of a wide range of physiopathological events, including cardiovascular functions, genesis of inflammatory processes, angiogenesis induction, glucose metabolism, and growth and development of certain tumours (Ortore et al., Curr Top Med Chem 10:923, 2010).
In view of these findings, highly affine and selective A2B AR antagonists may be considered valuable agents for the therapeutic treatment of inflammatory diseases such as asthma, bowel inflammatory diseases, and angiogenic diseases like diabetic retinopathy and cancer (Kolachala et al., Br J Pharmacol 55:127, 2008).
In recent years, our research group developed a series of 3-aryl-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones (ATBIs) I as potent ARs antagonists (Da Settimo et al., J Med Chem 44:316, 2001; Drug Development Research 63:1, 2004). To identify novel ligands possessing high affinity and selectivity for the A2B AR, in this study we synthesize a novel series of ligands from class I featuring modified substituents at the 10-position, combined with an appropriate aromatic ring at the 3-position. Synthesis, biological evaluation, structure–activity relationships, and molecular modeling studies for these new derivatives will be presented.
This study was supported by a grant from MIUR (PRIN 2008).
PS1.9 Medicinal Chemistry
Synthesis of new fluorescent molecular sensors for studying adenosine receptors
Sabrina Gollos, Ali El-Tayeb, Fabian Heisig, Andrea Behrenswerth, Sven Jan Freudenthal, Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, 53121 Bonn, Germany
After several decades of intensive research on adenosine receptors, they are still of considerable interest due to their high potential as novel drug targets (Fredholm et al. Pharmacol Rev 63:1–34, 2011; Müller and Jacobson, Biochim Biophys Acta Biomembranes 1808:1290–1308, 2011). In the present study, we developed fluorescent adenosine receptor ligands containing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-s-indacene (BODIPY) as a fluorescent dye. Such fluorophores are useful tools to image processes in living cells due to their high photochemical stability, their exceptional spectral properties, their high absorption coefficients and their small size. BODIPY dyes show high fluorescence quantum yields (Φ>0.5) and high peak intensities (Loudet and Burgess, Chem Rev 107:4891–4932, 2007). Hence, they are highly suitable for biological investigations (Middleton et al., J Med Chem 50:782–793, 2007). In the present study, we synthesized new, functionalized BODIPY derivatives, which fluoresce at approximately 500 nm and therefore do not interfere with biological fluorophores. Various functional groups (Br, I, NH2, SH, OH) were attached via alkyl spacers of different lengths to the fluorophore in order to allow for its attachment to receptor ligands or its integration into the pharmacophore of adenosine receptor ligands. All new, functionalized BODIPY derivatives showed high fluorescence quantum yields and absorption and emission wavelengths near 500 nm. In a subsequent step, we coupled the functionalized BODIPY dyes with different alkyl spacer lengths to 2-thioadenosine. The affinities of the obtained derivatives were determined in radioligand binding studies at A1, A2A, A2B, and A3 receptors.
A series of fluorescent-labeled 2-thioadenosine derivatives with different spacer lengths were obtained, some of which show high affinities at A1, A2A, and A3 receptors (K i values in a nanomolar range). The compounds were also tested for receptor subtype selectivity: The derivative with a short linker (n = 1) shows receptor subtype selectivity for A3, while a longer alkyl chain (n = 9) leads to a preference for A1 receptors. The new fluorescent adenosine receptor ligands will be useful tools for studying adenosine receptor binding and function.
This study was supported by the DFG (GRK 804).
PS1.10 Medicinal Chemistry
ADP- and ATP-mimetics derived from 2′(3′)-C-methyladenosine as human P2Y 1 and P2Y 2 receptor ligands
Loredana Cappellacci 1, Riccardo Petrelli1, Patrizia Vita1, Ilaria Torquati1, Kenneth A. Jacobson2, Matthew O. Barrett3, Derek Franklin3 , Kendall T. Harden3 , Palmarisa Franchetti1, Mario Grifantini1
1 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, 62032 Camerino (MC), Italy. 2 Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, MD 20892-0810, USA. 3 Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, NC 27599-7365, USA.
Nucleotides and their hydrolytic products act through specific nucleotide receptors which are divided into ionotropic P2X and metabotropic P2Y receptors coupled to G proteins. The P2Y receptor family consists of at least eight human subtypes (P2Y1,2,4,6,11–14) that are activated by either or both adenine and uracil nucleotides [1]. Activation of P2Y receptors generally results in the stimulation of phospholipase C (PLC), which generates inositol phosphates and diacylglycerol from phosphatidyl inositol(4,5)bisphosphate, leading to a rise in intracellular calcium and activation of protein kinase C. According to a dendrogram relating sequence homology, the P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors form a cluster of preferentially Gq-coupled receptors, and the P2Y12, P2Y13 and P2Y14 form a cluster of preferentially Gi-coupled receptors.
The P2Y1 and P2Y2 receptors are expressed in most human tissues, including brain, heart, placenta, lungs, liver, skeletal muscle, kidneys, pancreas and various blood cells. P2Y1 receptor is activated by ADP, while P2Y2 is activated equipotently by both ATP and UTP. Several ADP and ATP analogues have been investigated to explore structure–activity relationships for P2Y1 and P2Y2 receptors. It was reported that constraining the ribose-like ring as methanocarba analogues of ADP and ATP, the Northern (N, 2′-exo) conformation was associated with increased agonist potency at recombinant human P2Y1 and P2Y2 receptors, while the Southern (S, 2′-endo) conformer was less potent. A similar conformational preference of the ribose moiety in binding to uridine nucleotide-activated hP2Y2 receptor was detected with the methanocarba analogues of UTP [2].
In order to further investigate the influence of conformation of ribose-modified ADP and ATP analogues on affinity and efficacy at hP2Y1 and hP2Y2 receptors, we have synthesized the 2′- and 3′-C-methyl derivatives of ADP and ATP. These nucleotides were evaluated for their capacity to promote hP2Y1 and hP2Y2 receptor-mediated activation of PLC at recombinant human receptors expressed in astrocytoma cells. The results of the functional assay will be discussed.
References
1. Abbracchio MP et al (2006). International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 58:281–341.
2. Kim HS et al. (2002) Methanocarba modification of uracil and adenine nucleotides: high potency of northern ring conformation at P2Y1, P2Y2, P2Y4 and P2Y11 but not P2Y6 receptors. J Med Chem 45:208–218
PS1.11 Medicinal Chemistry
Anthraquinone as a privileged structure in drug discovery targeting P2Y receptors
Younis Baqi, Anja B. Scheiff, Christa E. Müller
Pharma-Zentrum Bonn, Pharmazeutisches Institut, Pharmazeutische Chemie I, Universität Bonn, Bonn, Germany
The P2 purinergic receptors are a major class of receptors in the human body activated by nucleotides, such as ATP, ADP, UTP, or UDP (Burnstock, Physiol Rev 87:659–797, 2007). They are subdivided into G protein-coupled or metabotropic P2 receptors (GPCRs), designated P2Y, and ligand-gated ion channels or ionotropic receptors, termed P2X (LGICs) (Khakh et al., Pharmacol Rev 53:107–118, 2001). Both families, GPCRs as well as LGICs constitute important drug targets. In recent years, it was estimated that up to 50% of available drugs act directly via stimulating or blocking GPCRs (Hancock, Biochem Pharmacol 71:1103–1113, 2006).
The anthraquinone dye Reactive Blue 2 (RB-2) has been reported to act as a non-selective antagonist of nucleotide receptors (P2X1, P2X2, P2X4, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12 receptors) (Weyler et al., Bioorg Med Chem Lett 18:223–227, 2008; Baqi et al., J Med Chem 52:3784–3793, 2009; Baqi et al., J Med Chem 54:817–830, 2011; Hoffmann et al., J Pharmacol Exp Ther 331:648–655, 2009) and also as a non-selective inhibitor of ectonucleotidases (Baqi et al., J Med Chem 53:2076–2086, 2010; Baqi et al., Purinergic Signal 5:91–106, 2009). RB-2 was used as a lead structure for developing ligands with enhanced potency and selectivity for particular nucleotide-binding P2Y receptor subtypes. New synthetic protocols have been developed for accessing this very important new class of compounds (Baqi and Müller, Org Lett 9:1271–1274, 2007; Baqi and Müller, J Org Chem 72:5908–5911, 2007; Baqi and Müller, Nat Protoc 5:945–953, 2010); most of the synthesized compounds are new, not previously described in literature. The novel compounds were investigated for their potency to inhibit agonist-induced calcium mobilization in stably transfected 1321 N1 astrocytoma cells, expressing either human P2Y2, human P2Y4, or rat or human P2Y6 receptors.
Compound 1 showed the highest selectivity for P2Y2 (IC50 = 3.93 μM, P2Y4 and P2Y6: IC50 >> 10 μM), while 2 exhibited the highest affinity and the highest selectivity for P2Y4 with an IC50 value of 2.32 μM, while the IC50 values at P2Y2 and P2Y6 were greater than 10 μM. Compound 3 showed the highest affinity for the P2Y6 receptor (IC50 = 4.86 μM) and selective versus P2Y2 and P2Y4 (IC50 > 10 μM).
Compound | IC50 (μM ± SEM) | ||
|---|---|---|---|
P2Y2 | P2Y4 | P2Y6 | |
1 | 3.93 ± 0.47 | >>10 | >>10 |
2 | >10 | 2.32 ± 0.29 | >10 |
3 | >10 | >10 | 4.86 ± 0.79 |
PS1.12 Medicinal Chemistry
Synthesis and biological evaluation of novel P2Y 11 receptor antagonists
D Esser and MU Kassack
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University of Duesseldorf, Düsseldorf, Germany
The P2Y11 receptor is expressed in the spleen, intestine, on granulocytes, neutrophils, and dendritic cells, among others (Communi et al., J Biol Chem 272:31969–31973, 1997; Moreschi et al., J Biol Chem 281:31419–31429, 2006; Vaughan et al., J Immunol 179:8544–8553, 2007; Wilkin et al., J Immunol 166:7172–7177, 2001). ATP, ATPγS, BzATP, and 2-MeSATP are potent agonists at P2Y11. The first selective and potent P2Y11 antagonist was NF157, a non-nucleotide symmetrical urea closely related to suramin, which is a non-selective P2 antagonist (Ullmann et al., J Med Chem 48:7040–7048, 2005). Systematic variation led to the nanomolar potency antagonist NF340 with an apparent K i of 19.2 nM (Meis et al., J Pharmacol Exp Ther 332: 237–247, 2010).
These antagonists turned out very useful for in vitro and ex vivo tests (Moreschi et al., J Biol Chem 281:31419–31429, 2006; Vaughan et al., J Immunol 179:8544–8553, 2007) but cannot be used for in vivo experiments due to their polyanionic character. In a first attempt to develop bioavailable ligands, carboxylic acid derivatives were synthesized and tested for inhibition of P2Y11 using a functional calcium assay in 1321N1 P2Y11 astrocytoma cells. Low nanomolar potency antagonists were discovered among a series of di- and tetracarboxylic compounds. Structure–activity relationship will be discussed.
PS1.13 Medicinal Chemistry
Screening of natural compounds for P2X7 receptor modulation
Wolfgang Fischer, Nicole Urban, Michael Schaefer
Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany
The ligand-gated P2X7 receptor (P2X7R) is a membrane-bound, non-selective cation channel, expressed in a variety of cell types. Activation of this subtype requires elevated levels of extracellular ATP. The channel activity is accompanied by slow or no desensitization and prolonged or repeated exposure to high concentrations of ATP may induce the formation of transmembrane pores and trigger apoptosis. P2X7R activation affects a wide range of cellular functions frequently linked with pathophysiological processes, including immune responses and inflammation, neuropathic pain, release of gliotransmitters, cancer cell growth, or neurodegenerative diseases. Consequently, the P2X7R has attracted considerable interest as a therapeutic target (Skaper et al., FASEB J 24:337–345, 2010). Our screening of a compound library using HEK293 cells with stably transfected human P2X7R revealed that several natural compounds can block or sensitize the ATP (1 mM)-induced P2X7R activation. For instance, tanshinone and teniposide, occurring in the roots of red sage and American mayapple, respectively, and reported to have anti-inflammatory or antitumor properties, showed potent and selective blocking of the human P2X7R (IC50 ≤ 0.5 μM) but no effects in HEK293 cells stably transfected with human P2X4 or rat P2X2 receptors. A pronounced blocking was also observed in mouse microglial cells and human A375 melanoma cells endogenously expressing P2X7R. On the other hand, garcinolic acid and agelasine, derived from garcinia trees and marine sponges, potentiate the ATP (1 mM)-induced P2X7R response. In agreement with findings of other groups, most prominently tanshinone or related compounds may have potential for further development as neuroprotective or antineoplastic drugs.
This study was supported by the DFG: FG 748.
PS1.14 Medicinal Chemistry
New purine derivatives as P2X3 receptor antagonists
Rosaria Volpini 1, Michela Buccioni1, Diego Dal Ben1, Catia Lambertucci1, Gabriella Marucci1, Maya Sundukova2, Andrea Nistri2, Gloria Cristalli1
1 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, 62032 Camerino, Italy. 2 Sector of Neurobiology, International School for Advanced Studies, I-34014 Trieste, Italy.
P2X3 receptors (P2X3Rs) play an important role in neuropathic and chronic pain pathways; hence, novel P2X3R antagonists are potential agents for the treatment of chronic pain conditions and migraine. Based on molecular modeling studies, a new series of 2,6-diaminopurine derivatives bearing a substituted benzyl chain in 9-position and lipophilic substituents in N 6-position were synthesized. The new derivatives were evaluated on rat P2X3Rs by using patch clamp recording from HEK-transfected cells, and some selected ones were also tested at human and at native P2X3Rs expressed by trigeminal ganglion (TG) sensory neurons in culture.
Furthermore, since P2X3Rs expressing sensory neurons such as TG neurons are known to express also 5-HT and GABA receptors, this property was used to evaluate the selectivity of the new molecules.
Biological results showed that the new compounds behave as antagonists of P2X3Rs; one of them appears to be potent on both recombinant and native P2X3Rs and, besides that, to substantially inhibit the 5-HT3 receptor subtype. Since studies with 5-HT3 receptor antagonists and knockout mice revealed that these receptors are positively involved in nociception and pain transduction, the novel antagonist might produce an analgesic effect due to the block of not only P2X3 but also 5-HT3 receptors of sensory neurons.
PS1.15 Medicinal Chemistry
Peptidonucleosides as inhibitors of ecto-5′-nucleotidase—a promising target for anti-cancer-drugs
Wenjin Li 1, Andreas Brunschweiger1, Sangyong Lee1, Herbert Zimmermann2, Christa E. Müller1
1 Pharma-Zentrum Bonn, Pharmazeutische Chemie I, Universität Bonn, An der Immenburg 4, 53121 Bonn, Germany. 2 AK Neurochemie, Biozentrum der J.W. Goethe-Universität, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Ecto-5′-nucleotidase (eN, CD73) catalyzes the hydrolysis of the phosphoric acid ester bond of AMP to the corresponding nucleoside adenosine and inorganic phosphate. The enzyme has been found to be overexpressed on many cancer cells. Therefore, treatment with a eN inhibitors may be a promising novel strategy for cancer therapy. Thus, we investigated nucleotide mimetics which consisted of a nucleoside scaffold substituted in the 5′-position with a dipeptide moiety. The compounds were investigated at rat eN using a capillary electrophoresis-based assay (Iqbal et al., Anal Biochem 373:129–140, 2008). Potent inhibitors of eN could be identified. The test results showed that the inhibitory potency of the compounds appeared to be pH dependent: when the buffer pH was decreased, the potency of the compounds increased. Since tumor tissues typically show low extracellular pH values, the new inhibitors might act as tumor-selective eN inhibitors without affecting physiologically important functions of eN, e.g., the production of adenosine in blood vessels mediating vasodilation.
PS2.1 P2Y receptors
Myeloid derived suppressor cells express P2 receptors
Marta Vuerich 1, Francesca Papalini2, Francesca Simonato2, Sara Gulinelli1, Francesco Di Virgilio1, Vincenzo Bronte3, Davide Ferrari1
1 Section of General Pathology, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy. 2 Istituto Oncologico Veneto and Venetian Institute for Molecular Medicine, Padova, Italy. 3 Immunology Section, Department of Pathology and Diagnostic, Verona University, Verona, Italy
Myeloid-derived suppressor cells (MDSC) are a recently identified cell population that accumulates in cancer. MDSC induce a potent immune suppression (both innate and adaptative responses) and are considered as a major contributor to the failure of immunotherapy against tumors. MDSC production is driven by multiple factors secreted by tumors, among which VEGF, GM-CSF, IL-1β, and IL-6 play an important role. Extracellular ATP is highly concentrated at tumor sites, and it may be crucial not only by stimulating tumor growth but also as a stimulus to modulate the immune response against neoplasia. Since MDSC infiltrate tumors, we asked whether MDSC were responsive to extracelluar ATP. Therefore, expression and function of P2 receptors (P2R) were examined in two mouse cell lines (MSC-1 and MSC-2).
Stimulation of MDSC with extracellular ATP, UTP, or BzATP induced an increase in the intracellular calcium concentration. ATP and BzATP induced plasma membrane depolarization and permeabilization to the extracellular dyes ethidium bromide and lucifer yellow. These responses were followed by contraction of cell volume and membrane blebbing. Expression of mRNA and protein of different P2R subtypes has also been investigated. Since MDSC population is involved in tumor growth, it is urgent to develop novel therapies. Our investigation offers a new opportunity to fight tumor by modulating MDSC “purinome.”
PS2.2 P2Y receptors
P2Y 1 receptor-mediated fibre outgrowth in organotypic slice co-cultures
Claudia Heine1,2, Nico Scherf1, Annett Wegner2, Heike Franke 2
1 Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig, Germany; 2 Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany
Purines have the characteristic to act as signalling molecules, growth factors and toxic agents by stimulating P2X/Y receptors. In own previous studies, using dopaminergic organotypic slice co-cultures including the ventral tegmental area/substantia nigra and the prefrontal cortex, the expression of several P2X/YRs and a growth promoting effect of selected P2R agonists have been shown. The aim of the present study was to characterise the outgrowing fibres in the border region and to confirm the involvement of the P2Y1R subtype in process formation in rat and mice. Multiple fluorescence labelling studies indicated the expression of several neuronal and glial markers as well as receptor subtypes on the developing fibres. Moreover, substance application studies using the P2YR agonist ADPβS in combination with biocytin tracing and a quantification procedure showed the significant increase in axonal fibre outgrowth in the border region. This effect could be inhibited by pre-treatment with the antagonist PPADS. To verify these results, dopaminergic and hippocampal co-cultures were investigated using P2Y −/−1 mice and the appropriate wild-type mice. In both ex vivo systems, only the co-cultures prepared from the wild-type mice showed a significant enhancement of fibre outgrowth after ADPβS treatment, whereas in P2Y −/−1 mice only a minor stimulating capacity was observed. In conclusion, our results indicate the stimulating capacity of ADPβS on fibre outgrowth, suggesting a trophic role of purinergic signalling in the developing brain and supporting a participation of the P2Y1R in fibre outgrowth.
The study was funded by the German Federal Ministry of Education and Research (BMBF), PtJ-Bio, 0315883.
PS2.3 P2Y receptors
Human traumatic brain injury—involvement of P2Y 1 receptors in astrogliosis
Kristina Bremicker1, Marcus Grohmann1, Jan Dreßler2, Marco Weber3, Heike Franke 1
1 Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany. 2 Institute of Legal Medicine, University of Leipzig, Leipzig, Germany. 3 Institute of Legal Medicine, University of Halle (Saale), Halle (Saale), Germany
Traumatic brain injury (TBI) is an important cause of death and disability in humans. TBI is highly correlated with the induction of astrogliosis, and the knowledge of specific receptors and transduction mechanisms involved in this process are crucial for diagnosis and treatment. In previous studies in rats, we have demonstrated the participation of purinergic receptors (e.g., the P2Y1R) in the induction and maintenance of reactive gliosis and anti-apoptotic processes. The aim of the present study was to verify a possible influence of this subtype in astrogliosis in human brain.
Based on human post-mortem autopsy material of TBI patients, astroglial reaction around the traumatic area in the prefrontal cortex was investigated. The results indicate that 3TBI in human is associated with an elevated expression of the number of GFAP-positive cells, GFAP-immunoreactivity, and protein content in relation to the post-traumatic period as well as with a time-dependent upregulation of the P2Y1R expression. The subtype was observed on astrocytes and MAP2-positve neurons. Furthermore, after TBI, the expression of other P2X/YRs on glial cells as well as of different apoptotic marker was found.
In conclusion, present data show for the first time the involvement of the P2Y1R in injury-induced astrologlial reactions in humans, suggesting specific roles of purinergic receptors in glial cell pathophysiology in neurodegenerative diseases.
PS2.4 P2Y receptors
Nucleotide-induced relaxations in monkey cerebral arteries isolated from various regions
Ayman Geddawy 1,2, Takashi Shimosato1, Masashi Tawa1, Takeshi Imamura1, Tomio Okamura1
1 Department of Pharmacology, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan. 2 Department of Pharmacology, Faculty of Medicine, El-Minia University, El-Minia 61519, Egypt
Cerebral circulation must be properly regulated and coupled with metabolic needs to maintain steady function of different brain regions. Extracellular nucleotides are reported to play a role in the endothelial regulation of vascular tone via P2 receptor-mediated pathway. Less is known about this role in primate cerebral vessels. We previously reported that endothelium-derived hyperpolarizing factor (EDHF) contributes to endothelium-dependent relaxations induced by 2-methylthio-ADP (2MeSADP) via stimulation of P2Y1 receptor in the non-human primate cerebral artery (J Pharmacol Sci 114:180–188, 2010). Here, we examined the responses of monkey cerebral arteries isolated from various brain regions to the nucleotides ATP, UTP, and 2MeSADP. Mechanical responses of endothelium-intact and endothelium-denuded strips to the agents were isometrically recorded, and concentration–response curves were obtained. In endothelium-intact preparations treated with indomethacin (to inhibit prostacyclin production), the nucleotide-induced relaxations in arteries from posterior brain region tended to be greater than those in arteries from anterior brain region, but the difference being statistically insignificant. However, when endothelium-intact arteries were treated with indomethacin plus NG-nitro-L-arginine (to inhibit NO production), EDHF-type relaxations by nucleotides were significantly greater in arteries from posterior than those in arteries from anterior brain regions, whereas endothelium-independent relaxations by forskolin or by nitoglycerin did not significantly differ. From these results, it appears that nucleotide-induced EDHF-type relaxations display regional variability in primate cerebral artery.
PS2.5 P2Y receptors
On the signal transduction pathway of UTP in cardiac myocytes
U Gergs, D Rothkirch, J Neumann
Institute for Pharmacology and Toxicology, Medical Faculty, Martin-Luther-University Halle-Wittenberg, 06097 Halle (Saale), Germany
Extracellular UTP can be released from the heart during pathological conditions such as ischemia or hypoxia. In humans, UTP levels are increased during myocardial infarction. UTP can act via P2Y-purinoceptors which are further divided in P2Y1–14 receptors. In electrically driven (1 Hz) mouse left atrial preparations and atrial preparations from patients undergoing cardiac bypass surgery, we noted a positive inotropic effect of UTP (cumulatively applied from 10 to 100 μM). The positive inotropic effect of UTP in human preparations was not attenuated by the adenylate cyclase inhibitor SQ22536 (10 μM) or the phospholipase C inhibitor U73122 (10 μM). Therefore, the inotropic effects of UTP on force of contraction in the human atrium probably occur independently of cAMP and IP3. To investigate the involved signal transduction pathway, we studied the effects of UTP on MAPK phosphorylation in isolated neonatal rat cardiac myocytes as well as in adult mouse cardiac myocytes using phosphorylation-specific antibodies. UTP of 100 μM transiently increased phosphorylation of ERK1/2 and p38 MAPK with a maximum effect at 5 to 10 min after application of UTP in both neonatal and adult cardiac myocytes (n = 3 preparations each). After 20 min, pre-drug values of MAPK phosphorylation were reached again. In summary, we noted a UTP-induced phosphorylation of ERK1/2 and p38 MAPK in isolated rat and mouse cardiac myocytes. The involved receptor subtype(s) and the link between MAPK phosphorylation and inotropic effect of UTP need to be elucidated.
PS2.6 P2Y receptors
The metabolic relevance of the UDP-glucose receptor P2Y 14
Jaroslawna Meister, Torsten Schöneberg, Angela Schulz
Molecular Biochemistry, Institute of Biochemistry, Medical Faculty, University of Leipzig, 04103 Leipzig, Germany
The G protein-coupled receptor P2Y14 is activated by UDP, UDP-glucose, and other UDP-sugars. Northern blot and qPCR analyses showed that P2Y14 is widely expressed in human and mouse tissues. Most studies focus on P2Y14 presence in immune cells, suggesting a physiological relevance in immune and inflammatory responses. Although highest expression is found in the gastrointestinal (GI) tract and adipose tissue, little is known about the metabolic relevance of P2Y14.
To clarify the physiological role of P2Y14 with specific focus on energy homeostasis we use a P2Y14-deficient mouse model. This P2Y14-knockout (KO) mouse strain expresses the bacterial LacZ reporter gene to monitor the physiological expression pattern of P2Y14. KO and wild-type (WT) mice were compared in numerous functional tests including glucose and insulin tolerance tests under standard and western-type diets.
We found that P2Y14 is mainly expressed in a subpopulation of smooth muscle cells of the GI tract, pancreas, salivary glands, blood vessels, lung, and uterus. Among other phenotypical differences, KO mice showed a significantly impaired glucose tolerance following oral and intraperitoneal glucose application. Because insulin tolerance was not different between KO and WT mice, P2Y14 is most probably involved in blood glucose regulation distal of glucose uptake from the GI and proximal of glucose uptake into peripheral tissues. Our study revealed P2Y14 as an important regulator of smooth muscle function and glucose homeostasis.
PS2.7 P2Y receptors
LPS-activated microglia overexpress P2Y 6 receptors that mediate an inhibition of astroglial proliferation by inducing no mediated apoptosis
Glória Queiroz, Clara Quintas, Jorge Gonçalves
Laboratory of Pharmacology, REQUIMTE, Department of Drug Sciences, Faculty of Pharmacy, University of Porto, 4050–047 Porto, Portugal13
Microglia P2Y pyrimidine receptors trigger phagocytosis, the release of cytokines and of other messengers that modulate astrogliosis (Koizumi et al., Nature 446:1091–1095, 2007; Di Virgilio et al., Trends Neurosci 32(2):79–87, 2009). Using cultures of astrocytes with activated microglia, we investigated the influence of P2Y pyrimidine on the astroglial proliferation and the mechanisms involved. In co-cultures of astrocytes and LPS (0.1 μg/mL)-activated microglia, the effects of several pyrimidine nucleotides in cell proliferation were investigated by measuring methyl-[3H]-thymidine incorporation. P2Y2,4,6 receptor expression was evaluated by Western blot and P2Y6 receptor cellular localization by double immunofluorescence: microglia were labelled with anti-OX42 antibody followed by incubation with Alexa Fluor, and P2Y6 receptors were labelled with a rabbit anti-P2Y6 antibody and revealed by FICT. The release of nitrates plus nitrites was measured and used as an index of NO release. Cell death was evaluated by LDH release and Tunnel assays.
UDP-glucose caused no effect, but PSB-0470 (0.01–10 μM), UTP and UDP (0.01–1 mM) inhibited astroglial proliferation up to 43 ± 2% (n = 10, P < 0.05). The inhibitory effects of UTP, UDP and PSB-0474 were prevented by the selective P2Y6 antagonist MRS2578 (1 μM).
Western blot revealed that P2Y2,4,6 receptors were expressed in these cultures, and the experiments of double immunofluorescence revealed that P2Y6 receptors were mainly co-localized with microglia. The inhibition caused by (UTP 0.1 mM; 40 ± 2%; n = 15, P < 0.05) was reversed into a proliferative effect of 53 ± 5% (n = 7, P < 0.05), in the presence ARL67156 (0.1 mM), an effect mimicked by UTPγS (0.3 mM; 51 ± 9%; n = 4, P < 0.05), and partially antagonized by suramin (0.1 mM) and by RB2 (10 μM). The inhibition caused by UDP (1 mM; 44 ± 2%, n = 15, P < 0.05) was mimicked by the NO donor, SNP (0.1 mM) and abolished by the NOS inhibitor L-NAME (0.1 mM), the guanyl cyclase inhibitor ODQ (10 μM) and the PKG inhibitor KT 5823 (1 μM). UTP, UDP and PSB 0474 all caused an increase in NO release and induced cell dead by apoptosis.
P2Y2/4 receptor-induced astroglial proliferation is prevented by a fast UTP metabolism with UDP formation and activation of P2Y6 receptors which are highly expressed in activated microglia and increase NO production, thereby inducing cell death by apoptosis, thus contributing to the inhibition of astroglial proliferation.
This study was supported by FCT Projects (PTDC/SAU-TOX/115597/2009 and REQUIMTE/CEQUP).
PS2.8 P2Y receptors
Analysis of P2Y receptors by blue native polyacrylamide gel electrophoresis
M Schumacher, U Braam, S Detro-Dassen, R Hausmann, G Schmalzing
Molecular Pharmacology, RWTH Aachen University, Aachen, Germany
P2Y receptors are seven transmembrane domain proteins that couple to G proteins after activation by ADP or ATP. Various seven transmembrane domain receptors have been found to assemble to higher order structures, mostly homo- and heterodimers. To examine whether blue native polyacrylamide gel electrophoresis (BN-PAGE) might be suitable for the detection of the assembly state of P2Y receptors, we expressed the C-terminally His-tagged mouse P2Y1 (His-mP2Y1) receptor or the His-hP2Y12 receptor in Xenopus laevis oocytes. The membrane proteins were solubilized with digitonin or dodecyl maltoside, purified by non-denaturing Ni-NTA affinity chromatography and resolved in their 35S-methionine-labeled total form and their Cy5-labeled plasma membrane-bound form by SDS-urea-PAGE and BN-PAGE. Deglycosylation and mutational experiments revealed that the His-mP2Y1 receptor carries two N-glycans and exists in two different forms at the plasma membrane, a complex-glycosylated ~50-kDa form and a non-glycosylated ~32-kDa form. The ~32-kDa form may arise from proteolytical cleavage in the N-terminal domain. The His-hP2Y12 receptor behaved exclusively as a single complex glycosylated protein. BN-PAGE provided no evidence for the assembly of the non-activated mP2Y1 receptor to higher order structures. In contrast and consistent with previous studies, the hP2Y12 receptor migrated mostly in the constitutively homodimeric form in the BN-PAGE gel. Overall, our data suggest that BN-PAGE is suited to detect oligomeric forms of seven transmembrane domain proteins.
This project was supported by grants of the DFG (FOR748).
PS2.9 P2Y receptors
Identification of an inverse agonist at the ADP receptor P2Y 12
Philipp Schmidt, Maxi Cöster, Kay-Uwe Simon, Torsten Schöneberg
Institute of Biochemistry, Medical Faculty, University of Leipzig, 04103 Leipzig, Germany
The ADP receptor P2Y12 is important for ADP-induced platelet aggregation and plays a pivotal role in thrombosis and hemostasis. Therefore, potent antagonists, e.g., Clopidogrel, are of clinical relevance in prophylaxis and treatment of thromboembolic events. At least in vitro P2Y12 displays a high basal activity, and inverse agonist may be therapeutically beneficial compared to antagonists. Only a few inverse agonists of P2Y12 are described, and we, therefore, screened for compounds reducing the basal activity of constitutively active P2Y12 mutants. Several activating mutants of the human P2Y12 were identified using mutant libraries, in which each amino acid position is replaced with all other possible amino acids. Constitutively active mutants were expressed in a heterologous yeast expression system to efficiently screen an adenosine nucleotide library (80 structurally categorized and chemically defined compounds). We detected a potent inverse agonist for the human P2Y12. The inverse agonist is a competitive ligand for the ADP-binding site. Structure–function relationship studies revealed specific substitutions at the ribose mediate inverse agonistic activity.
PS2.10 P2Y receptors
Characterisation of functionally important amino acid residues in the human purinergic 12 receptor using yeast Saccharomyces cerevisiae expression system
Vita Ignatovica 1, Kaspars Megnis1, Maris Lapins2, Helgi B. Schioth3, Janis Klovins1
1 Latvian Biomedical Research and Study Centre, Riga, Latvia. 2 Department of Pharmaceutical Pharmacology, Uppsala University, Uppsala, Sweden. 3 Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden
The purinergic P2Y12 receptor (P2Y12R) serves as a major drug target for anticoagulant therapies, but so far, little is known about the regions that are involved in ligand binding and activation of this receptor. We generated four randomised P2Y12R libraries and investigated their ligand binding characteristics.
The P2Y12R was expressed in Saccharomyces cerevisiae model system. Four libraries were generated consisting of randomised amino acids at positions 181, 256, 265 and 280. The mutant variants were screened for functional activity in yeast using the natural P2Y12R ligand ADP. The activation results were explored using quantitative structure–activity relationship and ligand-receptor docking.
The results of this study demonstrate that positions 181, 256, 265 and 280 of P2Y12R are important for the functional integrity of the receptor. Moreover, K280 appears to be crucial feature of the P2Y12R ligand binding pocket.
Acknowledgements: The study was supported by Latvian Council of Science Grant LZPSP10.0010.10.04, Latvian Research Program 4VPP-2010-2/2.1, ESF grant 1DP/1.1.1.2.0/09/APIA/VIAA/150, Society for Medical Research Sweden and Åke Wiberg Foundation and the Swedish Research Council.
PS2.11 P2Y receptors
The structural and functional landscape of the human ADP receptor P2Y 12
Maxi Cöster 1, Doreen Wittkopf1, Kay-Uwe Simon1, Doreen Thor1, Annika Kreuchwig2, Gunnar Kleinau2, Gerd Krause2, Torsten Schöneberg1
1 Institute of Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany. 2 Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany
P2Y12-like receptors belong to the family of rhodopsin-like G protein-coupled receptors (GPCR). In spite of considerable sequence similarity within the P2Y12-like receptor group, members display high specificity towards distinct nucleotides. The structural basis of ligand specificity is still unknown although recent crystal structures of GPCR represent a first step in defining the molecular landscape of these receptors. However, these homology models have only a limited predictive value and need additional refinement based on structure–function studies. Here, we combine the information from naturally occurring P2Y12 variants (orthologs) together with high-throughput saturation mutagenesis, in vitro pharmacological testing, and bioinformatic analysis to generate a dynamic GPCR model. We focused on the ADP receptor P2Y12 as a model system because it is the best characterized prototype of P2Y12-like receptors. Saturating mutagenesis of a receptor portion spanning transmembrane helix 6 to 7, in which each amino acid position in the human P2Y12 was replaced with all other possible amino acids (1,254 mutants), and functional in vitro testing provided a detailed picture of the functional effects of every amino acid position. Analyses revealed that ~10% of the mutations present wild-type function and ~87% showed partial or complete loss of function. Regions where mutations activate the receptor were identified as well. The structural conservation found by comparing P2Y12 orthologs strongly correlated with the functional importance of the respective positions in the in vitro tests. Our saturating mutagenesis data set together with the ortholog data will be used to compile a “dynamic” molecular model of P2Y12.
PS2.12 P2Y receptors
Involvement of Cys194 in the interaction of the human platelet P2Y 12 receptor with reactive blue 2
Kristina Hoffmann 1, Dominique Lutz1, Younis Baqi2, Christa E. Müller2, Ivar von Kügelgen1
1 Department of Pharmacology and Toxicology, University of Bonn, Bonn, Germany. 2 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Germany
Background: The P2Y12 receptor plays a crucial role in platelet aggregation. In previous studies, we identified amino acid residues contributing to the ligand binding site. Now, we analyzed the roles of amino acid residues in close proximity.
Methods: Wild-type or mutant receptor proteins were stably expressed in CHO Flp-In cells. Receptor expression was analyzed by immunofluorescence staining with a FITC-coupled antibody against a receptor epitope. Receptor function was assessed by measuring agonist-induced changes in the cAMP-response-element (CRE)-directed luciferase expression in cells stimulated by forskolin [1 μM].
Results: In cells expressing the wild-type receptor, 2-methylthio-ADP [0.01 nM–1 μM] inhibited the CRE-directed luciferase expression concentration-dependently with an IC50 value of about 1 nM. When the amino acid residue C194 in transmembrane region 5 (TM5) was replaced by alanine, no change in the IC50 concentration was observed indicating that this amino acid residue is not involved in the interaction with the agonist. In cells expressing C194A-mutant receptors, the antagonistic effect of reactive blue 2 was markedly increased (about 30-fold). The same was true for PSB-0739 and its analogue PSB-0826 (missing sulphonic acid residue at ring D). In contrast, there was a decrease of the antagonistic potency of PSB-0801 (missing sulphonic acid residue at the antrachinon backbone) at C194A-mutant receptors.
Conclusions: These results show that the residue C194 (TM5) is involved in the interaction with reactive blue 2 and its analogues.
PS3.1 P2X receptors
Investigation of the activation and desensitization of P2X1 receptors by voltage clamp fluorometry
Eva Lörinczi 1, Stephen F. Marino2, Sebastien Dutertre3, Yogesh Bhargava2, Jürgen Rettinger2,4 and Annette Nicke1
1 Department of Molecular Biology of Neuronal Signals, MPI of Experimental Medicine, Hermann-Rein-Str 3, Göttingen, Germany, 2 MPI of Biophysics, Department of Biophysical Chemistry, Max-von-Laue-Str. 3, Frankfurt aM, Germany, 3 Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia, 4 Present address: Multi Channel Systems MCS GmbH, Aspenhaustrasse 21, Reutlingen, Germany
P2X receptors are trimeric cation channels that are activated upon binding of extracellular ATP. Although residues involved in ATP binding have been localized at the interfaces of the neighboring subunits and confirmed by the recently solved crystal structure of a P2X receptor, the exact mode of ATP binding and the subsequent receptor activation are still not understood. Here, we used voltage clamp fluorometry to investigate the conformational changes of a conserved cysteine-rich extracellular domain (118–125) during ligand binding, activation, and desensitization of the P2X1 receptor. We have identified six amino acid residues that were specifically labeled by TMRM when substituted with cysteine residues. Upon ligand application (the agonists ATP and BzATP, the antagonist TNP-ATP), five of these mutants showed significant changes in the emission of the fluorescence probe. In response to ATP, two mutants showed decreasing fluorescence with a fast time course, which appears to correlate with ATP binding and/or channel opening. Three mutants showed increasing fluorescence changes with slower time courses, more likely associated with the desensitization of the receptor. The competitive antagonist TNP-ATP induced decreasing fluorescence changes of various amplitudes in all of the studied mutants, suggesting an interaction between the fluorophore and the ligand.
In conclusion, our observations provide details about the orientation of residues in the cysteine-rich domain that projects over the supposed ATP binding site and demonstrate that movements in this domain are involved in P2X receptor desensitization.
This project was funded by the DFG (FOR 748, RE2711/1, NI592/5).
PS3.2 P2X receptors
P2X1 receptor-mediated inhibition of the proliferation of human coronary smooth muscle cells via induction of NR4A1
Annette Viktoria Hinze 1,2, Anja Harst 1, Peter Mayer 1, Ivar von Kügelgen 2
1 Federal Institute for Drugs and Medical Devices, Bonn, Germany, 2 Department of Pharmacology and Toxicology, University of Bonn, Bonn, Germany
Extracellular adenosine and adenine nucleotides play important roles in the cardiovascular system. Recently, we have shown that adenosine A2B receptors mediate the anti-proliferative effects of adenosine on vascular smooth muscle cells by the induction of the transcription factor NR4A1 (nuclear receptor subfamily 4, group A, member 1, Nur77, TR3; Mayer P, Hinze AV, Harst A, von Kügelgen I. Cardiovasc Res; 90:148–156, 2011). In the present study, we searched for the effects of P2X1 receptor agonists on early gene expression and changes in cell proliferation in cultured primary human coronary smooth muscle cells.
The P2X1 receptor agonists α,β-methylene-ATP and β,γ-methylene-l-ATP (0.3–30 μM) inhibited proliferation by about 40% when applied for 5 days in the absence of platelet-derived growth factor. The effects of both agonists were blocked by the P2X1 antagonist NF 449 (4,4′,4″,4′″-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbony-limino)))tetrakis-benzene-1,3-disulfonic acid, 100–1,000 nM). Moreover, both nucleotides significantly increased the expression of NR4A1 after 1 h; these effects were blocked or attenuated by the antagonist NF 449. Treatment with siRNA against NR4A1 (10 μM) reduced the inhibitory effect of α,β-methylene-ATP and β,γ-methylene-l-ATP on cell proliferation when agonists and siRNA were added for 5 days. The A2B receptor antagonist MRS 1754 (0.3–3 μM) attenuated the effects of α,β-methylene-ATP and β,γ-methylene-l-ATP on cell proliferation, suggesting that a P2X1-mediated release of adenosine contributes to the effects on cell proliferation. In summary, our results demonstrate that the activation of vascular P2X1 receptors mediates an inhibition of smooth muscle cell proliferation with an involvement of the transcription factor NR4A1.
PS3.3 P2X receptors
No functional interaction between purinergic P2X7 receptors and pannexin-1
Manuela Klapperstück 1, Ronja Woltersdorf2, Günther Schmalzing2 and Fritz Markwardt1
1 Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle, Magdeburger Straße 6, D-06097 Halle/Saale, Germany, 2 Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany
The P2X7 receptor is a member of the P2X receptor family containing ion channels which are opened by binding of extracellular ATP and are permeable to small cations with a diameter of <9 Å. A marked feature of sustained P2X7 receptor activation by high ATP concentrations is the induction of a large cytolytic pore permeable to cations up to 900 Da. Because the view has been strengthened that the cytolytic pore is not formed by the P2X7 receptor itself, pannexin-1 is considered as an attractive candidate as a P2X7 receptor-associated pore-forming channel protein. Here, we investigated whether a functional interaction exists between pannexin-1 and P2X7 receptors.
P2X7 receptors and pannexin-1 were co-expressed in Xenopus laevis oocytes. Cell membrane currents were measured by the two-microelectrode voltage clamp technique. Verification of functional pannexin-1 expression was performed by measuring the ion currents induced by a voltage ramp going from −100 to +100 mV within 60 s. For investigating P2X7 receptor-dependent ion conductances, 0.1 mM free ATP4− was applied for about 1 min and the ATP-induced conductance measured by periodically depolarizing the membrane potential from −60 to +40 mV within 1 s in a ramp-like fashion. The co-expression of pannexin-1 with P2X7 receptors did not change the ATP-induced conductance.
The results obtained for the functional interaction measurements between human P2X7–human pannexin-1 and mouse P2X7–mouse pannexin-1 were similar. We therefore conclude that pannexin-1 is not involved in the P2X7 receptor-mediated induction of pores for large cations.
PS3.4 P2X receptors
IL-18 is highly concentrated in microvesicles shed from human macrophages in response to P2X7 receptor stimulation
Sara Gulinelli,1 Dania Bozzato,1 Cinzia Pizzirani,1 Giorgio Bolognesi,1 Erica Salaro,1 Marco Idzko,2 Francesco Di Virgilio,1 and Davide Ferrari1
1 Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, I-44100 Ferrara, Italy, 2 Department of Pneumology, University Hospital Freiburg, Freiburg, Germany
Extracellular ATP, released upon microbial infection, cell damage or inflammation, acts as an alert signal towards immune cells by activating P2 plasma membrane receptors. In this report, we show that extracellular ATP induced in human macrophages the release of membrane microvesicles in which the inflammatory cytokine IL-18 was highly concentrated, while on the contrary, it was almost absent from the culture medium. ATP and BzATP, but not UTP or UDP, evoked microvesicle shedding. The phenomenon was completely abrogated by pretreatment of macrophages with the P2X inhibitor oxidized ATP and partially prevented by the non-covalent P2X7 inhibitor KN-62. Microvesicle release and IL-18 content were greatly decreased in the absence of extracellular Ca2+, while the caspase inhibitor Ac-YVAD-CMK reduced vesicle shedding without interfering with IL-18 maturation. Disassembly of the cytoskeletal microtubules or actin filaments did not impair the formation and release of the microvesicles, while inhibition of iPLA2 or cPLA2 delayed it. Interestingly, differently from IL-1β, accumulation of IL-18 did not require pretreatment of cells with bacterial endotoxin (LPS). NALP-3 inflammasome (NALP-3, casp-1, ASC, Cardinal) was also associated with microvesicles, although its inhibition did not change the amount of IL-18, nor its elaboration. The presented data point to a P2X-mediated release of IL-18-loaded membrane vesicles from human macrophage.
PS3.5 P2X receptors
Enhanced expression and function of the P2X7 purinergic receptor in peripheral blood mononuclear cells from patients with Behçet’s disease
Castrichini M 1, Lazzerini PE1, Capecchi PL1, Franceschini R2, Gamberucci A3, Hammoud M4, Moramarco A2, Natale M1, Gianchecchi E1, Montilli C1, Ricci G1, Zimbone S1, Galeazzi M4, Laghi-Pasini F1.
1 Department of Clinical Medicine and Immunological Sciences, Section of Clinical Immunology, University of Siena, Siena, Italy, 2 Department of Ophthalmology and NeuroSurgery, University of Siena, Siena, Italy, 3 Department of Pathophysiology, Experimental medicine, and Public Health, University of Siena, Siena, Italy, 4 Department of Clinical Medicine and Immunological Sciences, Section of Rheumatology, University of Siena, Siena, Italy
Background: The P2X7 receptor is a nucleotide-gated ion channel involved in the inflammatory response triggered by the passive release of ATP from damaged cells. It is expressed in monocytes and plays a key role in promoting the release of IL-1β. Behçet’s disease (BD) is a systemic immune-inflammatory disorder of unknown origin whose clinical manifestations include oral and genital ulcers, skin lesions, uveitis, and arthritis. Since innate immunity activation and IL-1β release seem to play a relevant role in BD, we hypothesized a P2X7 involvement in the pathogenesis of the disease.
Methods: PBMC, or isolated monocytes, were prepared from 18 BD patients and 17 healthy controls matched for age and sex. In these cells, we evaluated: (1) P2X7 expression (by flow cytometry) and (2) function, induced by P2X7 receptor stimulation, as determined by cytosolic free Ca2+ flux measurements (by single-cell fluorescent microscopy), IL-1β release (by ELISA test), and apoptosis induction (by flow cytometry).
Results: In BD monocytes, P2X7 expression and the amount of Ca2+ influx, induced by the selective P2X7 receptor agonist 2′-3′-O-(4-benzoylbenzoyl)ATP (BzATP), were higher than in controls. Moreover, in BD patients, BzATP stimulation significantly enhanced the release of IL-1β from lipopolysaccharide-primed monocytes and promoted apoptosis in PBMC.
Conclusions: Our results provide evidence that in PBMCs from BD patients, both the expression and the function of the purinergic P2X7 receptor are increased with respect to healthy controls. These data, suggesting the putative involvement of this pathway in the immuno-inflammatory activation underlying BD, may designate the P2X7 receptor as a new potential therapeutic target of the disease.
PS3.6 P2X receptors
P2X7 expression increases tumoral growth, acting at proliferation and neovascularization
Elena Adinolfi1, Lizzia Raffaghello2, Anna Lisa Giuliani1, Francesca Amoroso 1, Luigi Cavazzini3, Giovanna Bianchi2, Vito Pistoia2, Francesco Di Virgilio1
1 Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Ferrara, Italy, 2 Laboratory of Oncology, Giannina Gaslini Institute, Genoa, Italy, 3 Section of Pathological Anatomy, University of Ferrara, Ferrara, Italy
Several malignancies overexpress the P2X7 receptor, including neuroblastoma, chronic and acute lymphocytic leukaemia, and prostatic, pancreatic, breast and thyroid carcinomas. The increased expression of the receptor was related to its trophic activity in different in vitro models, but an in vivo proof of the P2X7 transforming activity was lacking. Here, we show that HEK293 cells transfected with human P2X7 (HEK293-P2X7) are more tumorigenic in a nude/nude mice model than HEK293 cells transfected with the empty vector (HEK293-mock). HEK293-P2X7 cells generated increased numbers of faster growing tumours. HEK293-P2X7 neoplasms appeared more anaplastic and showed a higher number of blood vessels, detected by haematoxilin–eosin, smooth muscle actin and murine CD34 staining. According to the neovascularization data, HEK293-P2X7 secreted increased levels of vascular endothelial growth factor both in vitro and in vivo. Accelerated tumour growth was due to increased proliferation and reduced apoptosis, as shown by augmented Ki67 labelling and reduced TUNEL staining. In addition, the transcription factor NFAT2/c1 was consistently more activated in HEK293-P2X7 than in HEK293-mock tumours. This study provides a direct in vivo evidence of the P2X7-dependent growth-promoting activity and points to a role for this receptor in carcinogenesis.
PS3.7 P2X receptors
Expression of the P2X7 receptor and metabolic adaptation in serum and glucose deprivation
Amoroso F 1, Falzoni S1, Adinolfi E1 and Di Virgilio F1
1 Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Ferrara, Italy
One of the main features in cancers is the adaptation to stressing conditions. This adaptation confers them the ability to grow under reduced nutrient availability shape. Several findings point to the PI3K/Akt pathway as the main pathway involved. Lately, adaptation to glucose deprivation, as well as to serum starvation, is considered a crucial event in tumour progression. Recently, in our laboratory, we have demonstrated a close relationship between expression of the P2X7 receptor and increased proliferation in the absence of serum (Adinolfi et al. 2009). Here, we analyze the adaptation of HEK293 cells stably expressing the P2X7 receptor (HEK293-P2X7) to two key metabolic stress factors: glucose deprivation and serum starvation. In the absence of glucose (4 mM), HEK293-P2X7 have a higher growth rate compared with HEK293 cells transfected with the empty vector (HEK293-mock). In addition, HEK293-P2X7 are shown to produce twofold more ATP and release twice as much lactate (p < 0.05) than HEK293-mock. Moreover, in the absence of serum, PKM2 and PDHK1 are overexpressed in HEK293-P2X7 and are hypermodulated by the glucose deprivation. GLUT1 is overexpressed in HEK293-P2X7. Akt is activated in HEK293-P2X7. These observations suggest a higher efficiency in glucose uptake and employment by HEK293-P2X7, explaining a possible role for the P2X7 receptor in cancer cell survival.
Adinolfi et al. (2009) J Biol Chem 284(15):10120–10128
PS3.8 P2X receptors
Upregulation of P2X receptor expression and activity during maintenance of pluripotent state of embryonic stem cells
Talita Glaser, Claudiana Lameu, Maynara Fornazari, Henning Ulrich
1 Instituto de Química/Universidade de São Paulo, São Paulo, Brazil
In the last decades, stem cells have been intensively studied regarding their differentiation capacity and their therapeutic applications. Embryonic stem (ES) cells are distinguished by their ability to proliferate indefinitely and yield all cell types of an adult organism. Purinergic P2Y receptors have been implicated to participate in the regulation of ES cell proliferation and differentiation; however, little is known about the functions of ionotropic P2X receptors which are also expressed in undifferentiated cells. In this work, we have investigated whether purinergic receptors are required for maintaining ES cells in their undifferentiated pluripotent state. We show that P2X2, P2X5, and P2X7 are highly expressed in ES cells and that the activation of purinergic ionotropic receptors by α,β-methylene-ATP and Bz-ATP enhanced the gene expression of the pluripotency marker protein stage-specific antigene (SSEA)-1 during LIF removal and induction with retinoic acid, which accompany a decrease in these receptor subunits. Our work defines applications of purinergic agonists for the maintenance of undifferentiated stem cell cultures for future therapeutic applications.
Supported by FAPESP and CNPq, Brazil.
PS3.9 P2X receptors
Activation of P2X7 causes depletion of intracellular ATP in T lymphoma cells
Sorush Nader, Friedrich Koch-Nolte, and Friedrich Haag
1 Institute of Immunology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
Stimulation of T lymphocytes via their T cell receptor induces them to produce and accumulate intracellular ATP. Measurement of intracellular ATP (iATP) levels in T cells is used to assess the status of cell-mediated immunity in patients undergoing immunosuppressive therapy following transplantation. In these patients, high iATP levels herald an increased risk of rejection, whereas low iATP levels indicate an increased risk of infection. Activation of the P2X7 purinoreceptor has been reported to induce the release of ATP by cells. We thus investigated whether P2X7 activation influences iATP levels in T cells.
We chose Yac mouse T lymphoma cells as a model since these cells constitutively express P2X7 and ADP-ribosyltransferase Art2.2, permitting the activation of P2X7 via NAD-dependent ADP-ribosylation in the absence of exogenously added ATP. Incubation with NAD markedly decreased iATP levels. This was not due to the induction of cell death as FACS analysis showed no increase in the number of dead cells in NAD-treated vs. untreated cells. To assess whether NAD-induced iATP depletion is dependent on the P2X7 pathway, cells were pretreated with inhibitors Art2.2 or P2X7. Indeed, NAD-induced depletion of iATP was completely blocked by the inhibition of either Art2.2 or P2X7, demonstrating that it was dependent on the Art2/P2X7 pathway. Our results show that P2X7 activation can profoundly influence intracellular ATP levels, likely by inducing ATP release, without causing cell death.
PS3.10 P2X receptors
Functional P2X7 receptors of mouse adult neural precursor cells
Christin Kunert 1, Nanette Messemer1, Markus Grohmann1, Heike Franke1, Karen Nieber2, Herbert Zimmermann3, Peter Illes1 and Patrizia Rubini1
1 Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Leipzig, Germany , 2 Institut für Pharmazie, Universität Leipzig, 04103 Leipzig, Germany, 3 Institut für Zellbiologie und Neurowissenschaft, Biozentrum der Universität, Frankfurt/Main, Germany
Both RT-PCR and immunocytochemistry demonstrated the presence of P2X7 receptors in cultured adult neural precursor cells (NPCs) isolated from the subventricular zone of the mouse brain. Intracellular Ca2+ ([Ca2+]i) transients and membrane currents were recorded by the fura-2 method and the whole-cell patch clamp technique, respectively. First of all, we generated conditions under which the preferential P2X7 receptor agonist dibenzoyl-ATP (BzATP) induced [Ca2+]i transients absolutely dependent on the extracellular Ca2+ concentration. For this purpose, a normal Ca2+-containing medium was used with no added Mg2+. The P2X7-selective antagonists Brilliant Blue G and A438079 concentration-dependently blocked the BzATP effect; the non-selective P2X receptor antagonists PPADS and NF449 acted in a similar manner. The P2X1,3 preferential antagonist TNP-ATP had no effect, excluding the presence of the respective receptors. Interestingly, ivermectin, an allosteric modulator of P2X4 receptors, potentiated the BzATP-induced increase of [Ca2+]i, suggesting that P2X4 and P2X7 receptors are co-localized at the NPC membrane. In accordance with our expectations, acidification inhibited whereas alkalization facilitated the BzATP effect. NPCs prepared from P2X7−/− mice responded to BzATP only with a small residual [Ca2+]i transient, which was further depressed, but not abolished, either in a Ca2+-free medium or in the presence of MRS 2179, indicating that it was due to P2X4/P2Y1 receptor activation. The measurements of membrane currents caused by BzATP and ATP confirmed our data obtained with Ca2+ imaging. In conclusion, the application of agonists, antagonists and allosteric modulators bears out the presence of P2X7 and probably also P2X4 receptors in conjunction with the previously described P2Y receptors in mouse NPCs, all increasing the concentration of [Ca2+]i and thereby possibly regulating cell proliferation.
PS3.11 P2X receptors
Complement-induced haemolysis and purinergic signalling
Julie L. Hejl, Marianne Skals, Jens Leipziger and Helle A. Praetorius
1 Department of Physiology and Biophysics, Aarhus University, Aarhus, Denmark
The complement system is a key element of the innate immune system. The system elicits efficient responses against cells identified as non-self via the insertion of membrane attack complexes and is associated with many types of haemolytic anaemia including typical [1] and atypical haemolytic–uremic syndrome [2] as well as paroxysmal nocturnal haemoglobinuria [3]. Recently, we found that haemolysis caused by other types of membrane pore formers such as α-haemolysin (HlyA) from Escherichia coli and α-toxin from Staphylococcus aureus inflict their cytotoxic effects through P2 receptor activation (4–6).
Here, we show that similarly to HlyA-induced haemolysis, red cell lysis caused by complement activation is amplified through ATP release and subsequent P2 receptor activation. Ovine and human erythrocytes were incubated with anti-sheep erythrocyte antibodies or anti-Rh (D) antibodies, respectively, with either human plasma or guinea pig serum as complement donors. Non-selective P2 antagonists (PPADS and suramin) concentration-dependently inhibit complement-induced haemolysis. More specific P2 receptor antagonists imply that P2X7 and P2X1 are the main receptors involved in this response. Moreover, complement activation produces a sustained increase in [Ca2+]i, which triggers significant erythrocyte shrinkage that precedes swelling and lysis. This early volume reduction is likely to result from the activation of the K+ channel Kca3.1 as TRAM34 and clotrimazole augment the complement-induced haemolysis. These results indicate that complement, similar to HlyA, requires purinergic signalling for full haemolysis and that activation of the erythrocyte volume regulation protracts the lysis. This finding points several new pathways to interfere with haemolytic diseases and implies that P2 receptor antagonists potentially can be used in more broad terms to prevent intravascular haemolysis.
References
1. Orth D, Wurzner R (2010) Semin Thromb Hemost 36:620
2. Roumenina LT et al. (2011) J Immunol Methods 365:8
3. Wiedmer Tet al. (1993) Blood 82:1192
4. Skals M, Jorgensen NR, Leipziger J, Praetorius HA (2009) Proc Natl Acad Sci U S A 106:4030
5. Skals M et al. (2010) J Biol Chem 285:15557
6. Skals M, Leipziger J, Praetorius HA (2011) (submitted)
PS3.12 P2X receptors
Functional role of amino acids in the second transmembrane domain of human P2X7 receptors
Anja Pippel 1, Michaela Stolz2, Ronja Woltersdorf2, Günther Schmalzing2 and Fritz Markwardt1
1 Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle, Magdeburger Straße 6, D-06097 Halle/Saale, Germany, 2 Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany
The human P2X7 receptor is an ATP-dependent cation channel expressed in immune cells. It has a large extracellular domain, two transmembrane domains per subunit, and intracellularly located N- and C-termini. The second transmembrane domain (TM2) has been shown to be involved in the ion permeation and the pore opening of P2X receptor channels. We tested this assumption for the P2X7 receptor by single-cysteine scanning mutagenesis of TM2 residues near the putative intracellular vestibule of P2X receptors, which is involved in the formation of the P2X2 receptor channel pore. Xenopus laevis oocytes were injected with cRNAs for the various His-hP2X7 constructs, and ATP-dependent currents were measured using the two-microelectrode voltage clamp technique.
P2X7 receptor mutants with single-cysteine substitutions of residues F344–Y358 were found to be insensitive to the extracellularly applied sulfhydryl group-modifying reagent MTSEA. As a result of the exchange of G345, A348 or D352 by cysteine, the ATP-dependent currents became smaller compared with the wt P2X7 receptor. These constructs were insensitive to the extracellular application of the small thiol-reactive ion Ag2+ or the reducing substance TCEP. The latter effect rules out that aberrant cysteine bridges are responsible for the smaller currents mediated by these constructs. None of the mutations affected cell surface expression, as assessed by protein labelling with a lysine-reactive fluorescent dye combined with hP2X7R purification and SDS-PAGE.
We conclude that the TM2 region from F344 to Y358 does not contribute to the formation of the ion selectivity filter of the human P2X7 receptor.
PS3.13 P2X receptors
Preparation and validation of constructs for the generation of P2X7 BAC transgenic mice
Karina Kaczmarek 1, Conny Neblung1, Volker Eulenburg2, Annette Nicke1
1 Department of Molecular Biology of Neuronal Signals, Max Planck Institute for Experimental Medicine, Göttingen, Germany, 2 Institute for Biochemistry and Molecular Medicine, University of Erlangen, Erlangen, Germany
The P2X7 receptor (P2X7R) is an unusual member of the ATP-gated P2X channel family. Its prolonged stimulation induces the formation of a large, non-selective membrane pore, which consequently may cause cell death. Analyses of P2X7-deficient mice demonstrated an important role of the receptor in inflammation and neuropathic pain. P2X7R may therefore provide a useful therapeutic target for the treatment of a wide range of conditions including neurodegenerative diseases. Nevertheless, the precise localization of P2X7R, its molecular and physiological functions, and its regulation under physiological and pathophysiological conditions remain poorly understood. In particular, the presence and function of P2X7R in neurons and the molecular mechanism of pore formation are a matter of ongoing debate. In order to address these issues, we use bacterial artificial chromosome recombineering methods to establish mouse models that express reporter genes and/or tagged P2X7 receptors under the control of P2X7R-specific regulatory sequences. Here, we describe the strategies used for the generation of three different P2X7 transgenes and the completed recombination steps. In addition, we demonstrate the biochemical and functional characterization of a GFP-tagged P2X7 construct. These transgenic mice lines should provide useful models to investigate P2X7R expression and function in vivo.
This project was funded by the DFG (FOR 748, NI592/4; NI592/5).
PS3.14 P2X receptors
Generation and characterisation of BAC transgenic P2X3 reporter mice
Janka Günther 1, Marcus Grohmann2, Heike Franke2, Ralf Hausmann1, Günther Schmalzing1
1 Department of Molecular Pharmacology, RWTH Aachen University, Aachen, Germany, 2 Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany
Studies combining pharmacological, electrophysiological and genetic approaches have demonstrated a crucial role of the P2X3 receptor in pain transmission. Indeed, P2X3 receptor-specific antagonists are in the early clinical development used against chronic inflammatory and neuropathic pain. To allow for a comprehensive prediction of the possible side effects of these antagonists, knowledge of the precise expression pattern of the P2X3 receptor is of significant interest, but hampered by the low specificity of the available antibodies.
To facilitate the identification of cells carrying the P2X3 receptor, we generated BAC transgenic mice expressing the P2X3 subunit as a GFP fusion protein under the control of its endogenous promoter. Among a total of five founder mice, there was only one expressing weak membrane-bound fluorescence in isolated DRG neurons, but not in other cells. To enhance detection by microscopy, we generated additional BAC transgenic reporter mice expressing soluble GFP instead of membrane-bound P2X3-GFP under the control of the P2X3 promoter. Four soluble GFP-expressing founder mice were obtained. Initial experiments show a bright GFP fluorescence in the cytoplasm of cultured small- and medium-diameter DRG neurons including the axonal processes. Immunostaining demonstrated that GFP co-localizes with isolectin-B4 and the neuronal marker MAP2, indicating that the GFP expression of the BAC mice mirrors the known neuronal localization of the P2X3 receptor in DRGs.
Supported by the DFG (FOR748).
PS3.15 P2X receptors
Transgenic mice expressing affinity-tagged fluorescent P2X2 receptors
Marcus Grohmann 1, Tanja Nußbaum2, Ralf Hausmann2, HaiHong Wang3, Heike Franke1, Günther Schmalzing2
1 Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany, 2 Molecular Pharmacology, RWTH Aachen University, Aachen, Germany, 3 Department of Physiology Medical School, Tongji University, Shanghai, China
Chronic pain greatly impairs the quality of life. For developing new effective analgesics with less adverse effects, knowledge of the precise tissue distribution of receptors regulating pain processing can be a crucial factor. The involvement of purinergic receptors in nociception has attracted prominent attention, and extensive evidence supports in particular an important role for P2X2-containing receptors in sensory neurotransmission.
To facilitate the morphological and functional identification of neurons carrying P2X2R, the aim of the study was to create a transgenic mouse strain expressing a red fluorescent P2X2R subunit as a fusion protein including two affinity tags under the control of their own promoter. This mouse model was generated by a recombination-mediated genetic engineering using the suitability of bacterial artificial chromosomes, an efficient method to construct vectors for manipulation of the mouse genome. We obtained eight mice lines which inherit the transgene. An intensive screening indicated one mice line containing the red fluorescence in DRG neurons. Immunostaining of the red fluorescent protein co-localizes with the neuron-specific protein MAP2 and with isolectin-B4, indicating small- and medium-sized non-peptidergic DRG neurons.
In summary, these transgenic mice should provide a rich resource to enable new insights into the contribution of P2X2-containing receptors in pain transmission under physiological and pathological conditions.
Supported by the DFG (FOR748).
PS3.16 P2X receptors
Importance of N-terminal tyrosine and threonin in the regulation of human P2X4 receptor activity
Arquimedes Cheffer and Henning Ulrich
Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil
The P2X4 receptor is a member of a family of ATP-gated cation channels that are composed of three subunits. Each subunit has two transmembrane domains linked by a large extracellular loop and intracellularly located N- and C-termini. The receptors are expressed in excitable and non-excitable cells and have been implicated in the modulation of membrane excitability, calcium signaling, neurotransmitter and hormone release, and pain physiology. We have studied the participation of highly conserved amino acid residues located intracellularly in the N-terminus of P2X4 subunits as a critical determinant for receptor activation. These studies were done using site-directed mutagenesis and electrophysiological characterization of the recombinant human P2X4 receptors transiently expressed in HEK-293T cells. As results, we have found that the mutant receptors P2X4E14A and D16A exhibited properties not different from wild-type P2X4 receptors. However, in contrast, substitution of alanine for Tyr15 and Thr17 produced non-functional receptors expressed at the plasma membrane. From these results, we conclude that Tyr15 and Thr17 residues play a key role for P2X4 receptor activity. Thr17 is part of a site phosphorylated by PKC, suggesting that P2X4 activity may be regulated by PKC.
Acknowledgements: We acknowledge financial support from FAPESP and CNPq, Brazil.
PS3.17 P2X receptors
Expression of P2X receptor ectodomains
Christoph Döhler 1, M. Zebisch1, N. Sträter1
Centre for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Deutscher Platz 5, 04103 Leipzig, Germany
christoph.doehler@bbz.uni-leipzig.de
P2X receptors are trimeric cation-selective ion channels regulated by extracellular ATP. Their involvement in a lot of physiological diseases including chronic pain makes them an attractive drug target. Protein crystallography in combination with structure-based drug design is a helpful tool to discover new subtype-specific and highly affine agonists and antagonists. In 2009, the 3.1-Å structure of the zebrafish P2X4 was determined. Despite high functional insight, the binding site of the natural agonist ATP remains speculative. High-resolution complex structures of ideally all seven human P2X receptor subtypes are required for investigations of the subtype-specific binding modes and the design of novel lead molecules.
Since the expression, purification and crystallization of full-length P2X receptors is very difficult, we are testing strategies to express the ectodomains (ECDs) of the receptors. Human P2X ECDs could not be expressed in functional, i.e. folded, form in Escherichia coli in our hands. We are therefore currently testing eukaryotic expression systems for the preparation of functional P2X ectodomains.
To enhance the folding properties, the P2X ECDs of the trimeric receptor could be cloned as a concatameric construct. This is possible due to the close distance between the N- and C-termini of different monomers. The fusion of three P2X ECDs into a concatameric protein leads to a decrease of the intermolecular distances and to a higher chance of protein folding. Specific heteromeric P2X receptors are attractive drug targets as well and may be prepared using this strategy. A hydrophilic and flexible linker fuses the subunits to a trimeric arrangement.
PS3.18 P2X receptors
Transient up-regulation of P2 receptors influence differentiation of human mesenchymal stem cells
Nina Zippel, Christian Limbach, Andreas Pansky, Edda Tobiasch
Department of Natural Sciences, University of Applied Sciences Bonn-Rhein-Sieg, D-53359 Rheinbach, Germany
All known P2 receptors have been investigated and a variety of P2X and P2Y receptor subtypes were detected on MSCs. Interestingly, during adipogenic and osteogenic differentiation, several P2 receptor subtypes were found to be up- or down-regulated on the gene and protein levels, and the application of agonists and antagonists or nucleotide cleavage by apyrase had a direct influence on these differentiations.
Here, we show for the first time that the combination of several P2 receptors plays a role in the differentiation of adult stem cells. The pattern of the P2 receptors, as well as their fate in differentiation, varies in stem cells of mesenchymal when compared with ectomesenchymal origin. The subtypes P2X6, P2Y4 and P2Y14 seem to be pivotal regulators in mesenchymal stem cell commitment since they are regulated in both adipogenic and osteogenic differentiation of ATSCs and DFCs. In addition, a transient up-regulation of these receptors directly influences stem cell fate. These findings provide new insights into the differentiation processes and might reveal novel options to control stem cell fate in the future.
PS4.1 Nucleoside and nucleotide metabolism and transport
Effect of hypoxia on ecto-nucleotidase activities and barrier function in arterial vasa vasorum endothelial cells: implications for angiogenesis
G.G. Yegutkin 1, M.H. Helenius1, S. Jalkanen1 K.R. Stenmark2, and E.V. Gerasimovskaya2
1 MediCity Research Laboratory, Turku University, Turku, 20520, Finland, 2 Department of Pediatrics, UCD, Aurora, CO 80045, USA
Vascular remodeling plays a pivotal role in a variety of (patho)physiological conditions where hypoxia and inflammation are prominent features. Extracellular ATP and ADP have long been known as important regulators of vascular tone, permeability and homeostasis; however, the contribution of purinergic signalling to endothelial cell growth and proliferation remains poorly understood. Using vasa vasorum endothelial cells (VVEC) isolated from pulmonary artery adventitial compartments of control and chronically hypoxic neonatal calves, this study aimed to characterize the biochemical and functional properties of the microvascular endothelial network at sites of active angiogenesis. In comparison with normoxic endothelial cells, VVEC from hypoxic animals are characterized by (1) enhanced leakiness and disordered purinergic control of vascular barrier function and (2) drastically impaired nucleoside triphosphate diphosphohydrolase (NTPDase) and ecto-5′-nucleotidase activities with a respective constitutive increase in extracellular ATP and ADP levels. In contrast, in vitro challenging of cultured VVEC to acute hypoxia (4–24 h, 1% O2) was accompanied by a moderate 15–20% up-regulation of ectonucleotidase activities. Together, these results suggest that a unique pattern of nucleotide-mediated angiogenic activation of VVEC from chronically hypoxic vessels may be defined by disordered nucleotide homeostasis and impaired endothelial barrier function at sites of active neovascularization. Furthermore, different molecular mechanisms are presumably involved in the regulation of purinergic signalling cascade during acute and chronic hypoxia.
PS4.2 Nucleoside and nucleotide metabolism and transport
Kinetics of the extracellular catabolism of adenine nucleotides by fibroblasts of the human subcutaneous connective tissue
Ana Rita Pinheiro 1, Cristina Costa1, Célia Soares1,Teresa Magalhães-Cardoso1, Maria Adelina Costa1,2 & Paulo Correia-de-Sá1
1 Laboratório de Farmacologia e Neurobiologia, Instituto de Ciências Biomédicas Abel Salazar –Universidade do Porto (ICBAS-UP), L. Prof. Abel Salazar, 2 4099–003 Porto, Portugal, 2 Departamento de Química, UMIB, Instituto de Ciências Biomédicas Abel Salazar–Universidade do Porto (ICBAS-UP), L. Prof. Abel Salazar, 2 4099-003 Porto, Portugal
Fibroblasts of the subcutaneous connective tissue (SCT) may play a role in the pathogenesis of intractable chronic pain, yet the mechanism by which these cells interplay with sensory neurons remains unknown. Endogenous ATP and its metabolites (via P2 purinoceptors) are involved in pain perception. There is, however, a lack of information regarding the catabolism of extracellular adenine nucleotides in the connective tissue. Here, we evaluated the kinetics of ATP, ADP, AMP and adenosine hydrolysis in fibroblast cell cultures from the rat and human SCT by HPLC. We found no difference in the kinetics of nucleotides hydrolysis in the two species. ATP (3–30 μM) was catabolised into ADP, adenosine and inosine; surprisingly, low levels of AMP were detected in the cultures. The catabolism of ADP (3–30 μM) was slower (t 1/2 ~ 37 min) than that of ATP (t 1/2 ~ 20 min). Extracellular AMP (3–30 μM) was quickly (t 1/2 ~ 3 min) hydrolysed into adenosine, with vestigial formation of inosine and hypoxanthine; the half-degradation time of AMP was threefold increased in the presence of the ecto-5′-nucleotidase inhibitor, concanavalin A (0.1 mg/mL). Fast dephosphorylation of AMP into adenosine due to high ecto-5′-nucleotidase activity might explain why AMP accumulation was almost undetectable in fibroblasts incubated with ATP. ATP seems to be preferably hydrolyzed into AMP, but further studies are required to determine the ectoenzymes involved. A better understanding of extracellular nucleotide signalling mechanisms by fibroblasts may represent an opportunity to clarify their involvement in the pathogenesis of chronic pain of musculoskeletal origin.
Work supported by FCT. (FEDER funding, PTDC/SAU-FCF/108263/2008, UMIB-215/94, SFRH/BD/47373/2008, BII/UMIB-ICBAS/2009-1).
PS4.3 Nucleoside and nucleotide metabolism and transport
CD39 overexpression prevents autoimmune diabetes through adenosine 2 receptor (A 2 R)-dependent mechanisms
Joanne Chia1,2, Jennifer McRae1,2, Stacy Fynch3, Holger Eltzschig4, Simon Robson5, Peter Cowan1,2 and Karen Dwyer 1,2
1 Immunology Research Centre, St. Vincent’s Hospital, Melbourne, 2 Department of Medicine, The University of Melbourne, Melbourne, Australia, 3 St. Vincent’s Institute of Medical Research, Melbourne, Melbourne, Australia, 4 Department of Anesthesiology and Perioperative Medicine, University of Colorado Health Science Center, Denver, CO, USA, 5 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
Background: Type 1 diabetes results from the autoimmune-mediated destruction of islet cells. Although the transplantation of islet cells offers a potential cure, graft survival is compromised by a number of factors including recurrent autoimmune disease.
Aim: The aim of this study was to examine the effect of CD39 overexpression on the development of autoimmune diabetes.
Methods: C57BL/6 male mice overexpressing CD39 (CD39tg), CD39 knockout (KO), A2ARKO, A2BRKO and wild type (WT) were treated with multiple low-dose streptozotocin (MLDS, 50 mg/kg, i.p., for 5 days). Some mice were treated with the A2BR inhibitor (A2BRi, 0.5 mg/kg, i.p., bd). A blood glucose level (BGL) >20 mM was deemed diabetic. Adoptive transfer experiments were performed to isolate the effect of CD39 overexpression.
Results: WT mice became diabetic 42 days following MLDS (BGL = 21.2 ± 2.4 mM). The onset of diabetes was faster in CD39KO, A2ARKO (days 12 and 10 respectively), and A2BRKO mice (day 30). CD39tg mice remained normoglycemic throughout the 90-day follow-up period (BGL = 8.1 ± 0.2 mM). The protective effect of CD39 overexpression was lost by crossing CD39tg with A2ARKO mice (BGL = 21.2 ± 1.2 mM at day 30) or by treating the CD39tg with A2BRi (BGL = 17.6 ± 1.5 mM at day 22). The overexpression of CD39 on tissues alone prevented diabetes (BGL = 10.3 ± 1.1 mM at day 30).
Conclusion: The overexpression of CD39 mitigates MLDS-induced autoimmune diabetes through A2A and A2BR mechanisms. CD39 overexpression on the tissues alone was sufficient to confer this protection. Modification of human islets to overexpress CD39 may improve islet graft survival by inhibiting recurrent autoimmune disease.
PS4.4 Nucleoside and nucleotide metabolism and transport
Resident cardiac immune cells and expression of CD39 and CD73
Borg N, Bönner F, Bongardt S, Schrader J
Institut für molekulare Kardiologie, Düsseldorf, Germany
CD39 and CD73 are ectoenzymes expressed on immune cells catalysing the extracellular degradation of ATP to adenosine. We analysed the basal expression of these both ectoenzymes on immune cells present in mouse cardiac tissue and blood.
Leukocytes of mouse myocardial tissue were extracted in a multistep procedure including Langendorff perfusion, enzymatic digestion and centrifugation. Blood immune cells were isolated from retro-orbital blood after lysis of erythrocytes. Expression analysis of CD39 and CD73 was performed by flow cytometry (BD FACS Canto II).
We found that already the unstressed heart contains 3.21 ± 1.60 × 105 leukocytes consisting of T cells (4.1%), B cells (6%), natural killer cells (0.5%), granulocytes (1.6%), monocytes (7%) and antigen-presenting cells (78%). T cells are the dominant cell population expressing CD73, whereas the expression of CD73 on myeloid cells is generally low. An opposite expression pattern was found for CD39, with the highest expression on myeloid cells. The expression pattern of CD39 and CD73 on leukocytes in the heart was similar to that in blood. However, the percentage of T cells expressing CD73 and their expression level was higher in blood as compared with cardiac tissue.
We conclude that the normal heart already contains significant amounts of immune cells, the most prominent fraction being antigen-presenting cells. Because of the inverse expression of CD73 and CD39 on lymphatic and myeloid cells, it appears that both cell types operate together in the extracellular degradation of ATP to adenosine.
PS4.5 Nucleoside and nucleotide metabolism and transport
Modulation of immune response by CD73-derived adenosine during myocardial remodeling after ischemia/reperfusion
Bönner F, Borg N, Jacoby C, Bongardt S, Flögel U, Schrader J
Department of Molecular Cardiology, University of Duesseldorf, Düsseldorf, Germany
CD 73, an ecto-enzyme catalysing the extracellular degradation from AMP to adenosine, is present on immune cells, particularly T cells, and adenosine is well known for its potent anti-inflammatory action. We therefore explored whether CD73-derived adenosine plays a role in cardiac remodelling after ischaemia/reperfusion (I/R). CD73−/− and WT C57Bl/6 mice (n = 9 and n = 8, respectively) underwent 50 min of coronary ligation with subsequent reperfusion. Functional and contrast-enhanced MRI (DRX 9.4-T NMR) was performed at baseline, 1, 7 and 14 days after I/R for functional analysis, measurement of infarct size (enhanced/non-enhanced myocardial signal intensity) and T2 times. Cardiac immune cell infiltration was analysed by flow cytometry (BD, FACS Canto II) at indicated time points in parallel experiments. One day after I/R, the infarct size was comparable between CD73−/− (29.3 ± 5.3%) and WT animals (32.9 ± 8.2%). However, 14 days after I/R, CD73−/− mice showed a significantly (p < 0.01) lower ejection fraction (CD73−/−, 41.0 ± 3.7%; WT, 61.1 ± 2.9%) and a significant (p < 0.05) rise in myocardial area with increased T2 time (51.3 ± 3.8% vs. 27.7 ± 9.1%). FACS analysis at day 14 after I/R revealed that hearts from CD73−/− mice contained more T cells and a higher percentage of ly6chigh monocytes as compared with WT controls. In summary, CD73-derived adenosine promotes cardiac remodelling and recovery of ventricular performance after I/R, most likely by acting on T cells. Thus, adenosine appears to be an important modulator of cardiac healing.
PS4.6 Nucleoside and nucleotide metabolism and transport
CD73-derived adenosine modulates lipolysis in vivo
Sandra Burghoff 1, Sabine Bongardt1, Volker Burkart2, Michael Roden2,3, Ulrich Flögel1, Jürgen Schrader1
1 Institute of Molecular Cardiology, Heinrich Heine University, Düsseldorf, Germany, 2 German Diabetes-Center, Heinrich Heine University, Düsseldorf, Germany, 3 Clinic for Metabolic Diseases, Heinrich Heine University, Düsseldorf, Germany
Adenosine is well known to inhibit lipolysis, which is mediated by adenosine A1 receptors. It is unknown, however, whether this effect is functionally relevant under in vivo conditions. It is also unknown whether adenosine formed intracellularly or extracellularly by CD73 is involved in this anti-lipolytic action. We therefore aimed to identify the players of the anti-lipolytic adenosine action by using CD73 knockout mice at the age of 6–8 months. As measured with 1H MRI, we found a significantly decreased superficial fat content in CD73−/− mice (WT, 2.6 ± 0.9 AU; CD73−/−, 1.4 ± 0.5 AU). At the same point of time, the concentrations of serum-free fatty acids (WT, 203 ± 65 μM; CD73−/−, 354 ± 141 μM) and triglycerides (WT, 4.2 ± 4.9 mg/dl; CD73−/−, 15.4 ± 12.2 mg/dl) were found to be significantly increased, while animal weight was not significantly different. We also found significant changes in the serum level of glucose (WT, 111 ± 14 mg/dl; CD73−/−, 146 ± 24 mg/dl), GLP-1 (WT, 25.4 ± 9.6 pg/ml; CD73−/−, 10.6 ± 5.4 pg/ml) and resistin (WT, 252.6 ± 102.1 ng/ml; CD73−/−, 111.7 ± 48.4 ng/ml). The serum levels of glucagon, ghrelin, GIP, leptin and PAI-1 remained unchanged. Despite the increased glucose in CD73−/− mice, insulin levels (WT, 1.09 ± 0.33 ng/ml; CD73−/−, 1.33 ± 0.59 ng/ml) were unaltered. Separate studies on isolated islets revealed that secreted insulin upon glucose stimulation was not different between WT and CD73−/− mice. This is in line with our finding that no CD73 mRNA, protein and activity could be detected in WT islets. Taken together, our findings suggest that CD73-derived adenosine is an important insulin-independent modulator of lipolysis in fat tissue under in vivo conditions.
Grant: This study was supported by the SFB612.
PS4.7 Nucleoside and nucleotide metabolism and transport
Domain motion of 5’-nucleotidase
Ulrike Krug 1, Matthias Zebisch1, Rica Patzschke2, Jochen Balbach2, Norbert Sträter1
1 Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany, 2 Institute for Physics, Martin-Luther-University Halle-Wittenberg, Betty-Heimann-Str. 7, 06120 Halle/Saale, Germany
In vertebrates, ecto-5′-nucleotidase (CD73, 5NT) hydrolyzes extracellular AMP to adenosine as part of the extracellular purinergic signaling pathways. The structure of a related nucleotidase from Escherichia coli has been characterized in open and closed conformations, which differ in the relative orientation of the two domains by a rotation of up to 96°. The domain movement can be described as a rotation of the C-terminal domain around an axis, which passes through the center of the C-terminal domain. The resulting hinge-bending domain movement is unique in that the cleft between the domains does not open up, but the residues of the domain interface slide along the interface. The conformational change is necessary for the catalytic action of the enzyme, presumably to allow for substrate binding and product release [1, 2].
The domain motion of E. coli 5NT is studied by NMR, EPR and FRET spectroscopy. Due to the independent motion of the two domains, the NMR spectra show relatively sharp peaks for a 58-kDa protein. In order to study the domain orientation in solution via residual dipolar couplings, the two domains were expressed independently and the resonances are assigned via HNCACB and HNCOCACB spectra.
[1] Knöfel, Sträter (2001) J Mol Biol 309:255–266
[2] Schultz-Heienbrok et al. (2005) Biochemistry 44:2244–2252
PS4.8 Nucleoside and nucleotide metabolism and transport
Rational design of inhibitors of human ecto-5′-nucleotidase
J. Pippel 1, K. Yates1, M. Zebisch1, C.E. Müller2, N. Sträter1
1 Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Deutscher Platz 5, 04103 Leipzig, Germany, 2 Pharmazeutisches Institut, An der Immenburg 4, D-53121 Bonn, Germany
Jan.Pippel@bbz.uni-leipzig.de
The eukaryotic ecto-5′-nucleotidase (e5NT) is an extracellular enzyme which catalyzes the hydrolysis of AMP to adenosine and phosphate [1]. Since the effects of adenosine are often opposite to those of ATP, e5NT might be an appealing new drug target with various fields of application.
For the production of recombinant e5NT, an Escherichia coli expression system was established including refolding procedures. Different variants were expressed and tested for their suitability for crystallization. The structural characterization revealed that e5NT is composed of two domains. Via a large (~100°) rotation of the N-terminal domain, the enzyme is able to switch between an open and a closed conformation.
Based on these results, we hope to support rational inhibitor design by determining complex structures. Therefore, we identified several potential e5NT inhibitors via high-throughput inhibitor screening which are now used in co-crystallization and soaking experiments. Also inhibitors that mimic the natural substrate are of interest as such complex structures could provide further insights into the substrate binding mode and control of substrate specificity. In contrast to the bacterial 5′-nucleotidases, e5NT is specific for AMP and does not hydrolyze ADP or ATP.
[1] Sträter N (2006) Ecto-5′-nucleotidase: structure function relationships. Purinergic Signalling 2:343–350
We gratefully thank the “Deutsche Forschungsgemeinschaft” (DFG) for funding.
PS4.9 Nucleoside and nucleotide metabolism and transport
High-sensitivity capillary electrophoresis method for monitoring purine nucleoside phosphorylase and adenosine deaminase reactions by a reversed electrode polarity switching mode
Jamshed Iqbal 1,2 and Christa E. Müller1
1 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany, 2 Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology Abbottabad, Pakistan
Purine nucleoside phosphorylase (PNP; E.C. 2.4.2.1) catalyzes the cleavage of the glycosidic bond of ribo- and deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate to generate the purine base and (deoxy)ribose-1-phosphate [1]. The enzymatic function of human PNP has important medical implications. T cell leukemias and lymphomas can be impaired by designing efficient inhibitors that target PNP. This will lead to drug inhibition targeting of PNP for chemotherapeutic intervention in the treatment of cancer ). PNP inhibitors are also in the center of interest for the potentiation of anti-tumor and antiviral activities of nucleoside analogues. Adenosine deaminase (ADA; EC 3.5.4.4) is involved in purine nucleoside metabolism, catalyzing the deamination of adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respectively [2]. To investigate the inhibitory potential of a library of compounds in order to identify lead structures, fast and easy enzyme screening methods are required. Several methods have been developed for the quantitative determination of nucleosides and nucleobases. All of these methods are time-consuming and suffer from further serious drawbacks. Capillary electrophoresis (CE) has several advantages: high separation efficiency, minimum or no sample pretreatment required, short analysis time, low operational costs, and low sample consumption. Capillary electrophoresis is one of the most powerful separation tools, but it suffers from a relatively poor sensitivity. Therefore, in this current work, we have improved the sensitivity of capillary electrophoresis by integrating an online preconcentration process for sample analytes using large-volume sample stacking with polarity switching.
A simple, efficient, and highly sensitive in-line CE method was developed [3] for the characterization and for inhibition studies of the nucleoside-metabolizing enzymes PNP and ADA present in membrane preparations of human 1539 melanoma cells. An about 10-fold improvement of sensitivity for the five investigated analytes (adenosine, inosine, adenine, hypoxanthine, xanthine) was achieved by large-volume stacking with polarity switching when compared with CE without stacking. Using a reversed electrode polarity switching mode with UV detection at 260 nm, detection limits of 60 nM were achieved for inosine and adenosine. To the best of our knowledge, this represents the highest sensitivity for nucleoside and nucleobase analyses using CE with UV detection reported so far. The Michaelis–Menten constants (K m) for PNP and ADA and the inhibition constants (K i) for standard inhibitors determined with the new method were consistent with literature data.
[1] Bzowska A, Kulikowska E, Shugar D (2000) Pharmacol Ther 88:349–425
[2] Ajloo D, Saboury AA, Haghi-Asli N, Ataei-Jafarai G, Moosavi-Movahedi AA, Ahmadi M, Mahnam K, Namaki S (2007) J Enzyme Inhib Med Chem 22:395–406
[3] Iqbal J, Müller CE (2011) J Chromatogr A 1218:4764–4771
PS4.10 Nucleoside and nucleotide metabolism and transport
High-throughput screening of tissue-nonspecific alkaline phosphatase: search for novel inhibitors
Amelie Zech and Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Alkaline phosphatases are dimeric enzymes that are present in most organisms. In humans, three isozymes are tissue-specific, while a fourth one is tissue-nonspecific (TNAP) and is expressed in the bone, liver and kidneys. TNAP catalyzes the hydrolysis of many phosphorylated compounds in vitro, but so far, only inorganic pyrophosphate (K M = 0.48 mM) [1] and pyridoxal-5′-phosphate (K M = 0.13 mM) [1] have been confirmed as physiological substrates. TNAP plays an important role in the regulation of extracellular matrix calcification during bone formation and growth. However, the functions of TNAP in other organs are less well understood. Therefore, potent and selective inhibitors are required as pharmacological tools to investigate the roles of TNAP in diverse (patho)physiological conditions as well as its potential as a drug target [1–4].
For the identification of inhibitors, we established a luminescent assay based on the method described by Sergienko and Millán [2] using CDP-Star, a 1,2-dioxetane-based synthetic substrate of TNAP. This new method is more sensitive and faster for screening TNAP than the conventional colorimetric assay [2, 3]. After kinetic characterization of the enzymatic reaction and validation of the assay, we tested >200 test compounds as well as plant and bacterial extracts. The assay was found to be highly robust and reproducible, as shown by an average Z′ factor value of >0.7. We identified five hit compounds and several extracts, respectively, with IC50 values in the lower micromolar or micrograms per millilitre range, which can now serve as starting points for optimization by medicinal chemistry approaches.
[1] Di Mauro S, Manes T, Hessle L, Kozlenkov A, Pizauro JM, Hoylaerts MF, Millán JL (2002) Kinetic characterization of hypophosphatasia mutations with physiological substrates. J Bone Miner Res 17:1383–1391
[2] Sergienko EA, Millán JL (2010) High-throughput screening of tissue-nonspecific alkaline phosphatase for identification of effectors with diverse modes of action. Nat Protoc 5:1431–1439
[3] Sergienko EA, Su Y, Garcia X, Brown B, Hurder A, Narisawa S, Millán JL (2009) Identification and characterization of novel tissue-nonspecific alkaline phosphatase inhibitors with diverse modes of action. J Biomol Screen 14:824–837
[4] Dahl R, Sergienko EA, Su Y, Mostofi YS, Yang L, Simao AM, Narisawa S, Brown B, Mangravita-Novo A, Vicchiarelli M, Smith LH, O’Neill WC, Millán JL, Cosford NDP (2009) Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP). J Med Chem 52:6919–6925
PS4.11 Nucleoside and nucleotide metabolism and transport
Inhibition of adenosine deaminase (ADA) overactivity failed to restore neuromuscular transmission failure in rats with toxin-induced Mysthenia gravis
C. Costa, D. Meireles, M. Viegas, D. Paramos, M.A. Timóteo, M.T. Magalhães-Cardoso, L. Oliveira & P. Correia-de-Sá
Laboratório de Farmacologia e Neurobiologia, UMIB, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Portugal.
Normally, neuromuscular tetanic fade is overcome by tonic activation of facilitatory adenosine A2A receptors on motor nerve terminals, but this mechanism seems to be impaired in myasthenics (Noronha-Matos et al. J Neurochem, 2011, in press). Using rats with α-bungarotoxin-induced Myasthenia gravis (TIMG; Plomp et al., Physiology 458:487–99, 1992), we showed that the motor endplate region of these animals exhibits higher adenosine deaminase (ADA) activity (t 1/2 = 32 ± 11 min, n = 5) than controls (t 1/2 = 59 ± 13 min, n = 3). The data also showed that phrenic nerve stimulation accumulated smaller amounts of adenosine in TIMG animals as compared with controls. In this study, we investigated whether regulation of endogenous adenosine clearance by inhibiting ADA overactivity with EHNA (50 μM) could restore the facilitatory A2A receptor tonus in TIMG rats. EHNA, applied in a concentration (50 μM) that favours tonic A2A receptor activation in healthy rats (Correia-de-Sá and Ribeiro, Neuroscience 73:85–92, 1996), failed to modify [3H]ACh release evoked by phrenic nerve stimulation (5 Hz, 750 pulses, 0 ± 8%, n = 6) in myasthenics. Inhibition of ecto-AMP deaminase with deoxycoformicin (200 μM), a pathway that blunts ATP-derived adenosine A2A receptor facilitation, significantly reduced (21 ± 7%, n = 5) transmitter exocytosis in TIMG rats. This indicates that myasthenic transmission failure results from the decreased amounts of adenosine formed from released ATP. The characteristic rise in ADA activity detected in myasthenic patients may contribute to decrease endogenous adenosine to levels lower than the threshold required to activate facilitatory A2A receptors, but this could not be overcome by inhibiting ADA activity.
Work supported by FCT (FEDER funding, PTDC/SAU-FCF/108462/2008 and UMIB-215/94).
PS4.12 Nucleoside and nucleotide metabolism and transport
Gliotransmitter release from retinal (Müller) glia cells
Wurm A 1, Pannicke T1, Brückner E1, Hirrlinger J2, Bringmann A3, Münch T4, Haydon PG5, Pfrieger FW6, Reichenbach A1
1 Paul Flechsig Institute of Brain Research, University of Leipzig, Jahnallee 59, 04109 Leipzig, Germany, 2 Carl Ludwig Institute, University of Leipzig, Liebigstraße 27, 04103 Leipzig, Germany, 3 Department of Ophthalmology and Eye Clinic, University of Leipzig, Liebigstr. 10–14, 04103 Leipzig, Germany, 4 Center for Integrative Neurosciences (CIN), University of Tübingen, 72076 Tübingen, Germany, 5 Department of Neuroscience, Tufts University School of Medicine, Boston, MA, USA, 6 CNRS UPR 3212, Institute of Cellular and Integrative Neurosciences (INCI), University of Strasbourg, 67084 Strasbourg, France
There is an ongoing debate whether glial cells release neuroactive transmitters via regulated exocytosis and thereby fuel the neuron–glia cross talk. Here, we studied transmitter release involved in the volume regulatory signalling cascade of retinal (Müller) glial cells. Growth factor-stimulated release of glutamate, activation of metabotropic glutamate receptors and subsequent release of adenosine-5′-triphosphate (ATP) are key steps of this pathway. Although the action of these transmitters on Müller cells is well described, little is known about their release mechanisms.
Using a pharmacological approach, we found that glutamate release is mediated by exocytosis, while ATP is liberated via connexin hemichannels. Now, we studied two transgenic mouse lines which allow for glia-specific block of exocytosis in vivo: (1) mice expressing botulinum neurotoxin type B (BoNT/B) under the control of the glutamate–aspartate transporter–promoter which targets Müller cells (the toxin cleaves vesicle-associated membrane proteins 1–3 (VAMP1–3) and thereby disrupts exocytosis) and (2) dnSNARE mice expressing a dominant negative mutant of synaptobrevin (also known as VAMP2) controlled by the human GFAP promoter. Consequently, Müller cells should lack vesicular release. In both mouse lines, we found a disturbed volume regulation resulting in cell swelling under hypotonic conditions, which was not observed in control mice. Nonetheless, there are no substantial morphological or functional alterations in the retina of these mice.
These data support the assumption that Müller cells release glutamate via regulated exocytosis, while ATP is set free via opening of hemichannels.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (FOR 748; GRK 1097/1).
P5.1 Novel purine receptors
Agonist-induced desensitisation/resensitisation of human GPR17: a functional cross talk between purinergic and cysteinyl–leukotriene ligands
Trincavelli M.L. 1, Daniele S. 1, Gabelloni P. 1, Lecca D. 2, Rosa P. 3, Abbracchio M.P. 2, Martini C1.
1 Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Italy, 2 Department of Pharmacological Sciences, University of Milan, Milan, Italy, 3 CNR–Institute of Neuroscience, Department of Medical Pharmacology University of Milan, Milan, Italy
GPR17 is a P2Y-like receptor that responds to both uracil nucleotides (as UDP-glucose) and cysteinyl–leukotrienes (cysLTs, as LTD4). By bioinformatic analysis, two distinct binding sites have been hypothesized to be present on GPR17, but little is known on their putative cross-regulation and on GPR17 desensitisation/resensitisation upon agonist exposure. In this study, we investigated the cross-regulation between purinergic- and cysLT-mediated responses and analysed GPR17 regulation after prolonged agonist exposure. The GPR17 functional activity was evaluated in GPR17-expressing 1321N1 cells by both [35S]GTPγS binding and the cAMP assay. UDP-glucose was found to enhance LTD4 potency in mediating the activation of G proteins, and vice versa, likely through an allosteric mechanism. Both agonists induced a time- and concentration-dependent GPR17 loss of response (homologous desensitisation) with similar kinetics. Upon removal, receptor resensitisation occurred with typical kinetics of G protein-coupled receptors. Finally, activation of GPR17 by UDP-glucose (but not vice versa) induced a partial heterologous desensitisation of LTD4-mediated responses, suggesting that nucleotides have a hierarchy in producing desensitising signals. GPR17 homologous desensitisation was accompanied by the internalisation of receptors inside the cells, which occurred in a time-dependent manner with similar kinetics for both agonists.
These findings suggest a functional cross talk between purinergic and cys-LT ligands at GPR17. Because of the recently suggested key role of GPR17 in brain oligodendrogliogenesis and myelination, this cross talk may have profound implications in fine-tuning cell responses to demyelinating and inflammatory conditions when these ligands accumulate at lesion sites.
P5.2 Novel purine receptors
Innovative functional cAMP assay: application to the pharmacological characterization of GPR17
Gabriella Marucci 1, Michela Buccioni1, Stefania Ceruti2, Diego Dal Ben1, Dania Giacobbe1, Catia Lambertucci1, Davide Lecca1, Ajiroghene Thomas1, Rosaria Volpini1, Maria Pia Abbracchio2, Gloria Cristalli1
1 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Camerino, Italy, 2 Department of Pharmacological Sciences, Laboratory of Cellular and Molecular Pharmacology of Purinergic Transmission, University of Milan, Milan, Italy
Although several assays already exist for monitoring G protein-coupled receptor (GPCR) functions and for pharmacologically characterizing their ligands, the development of new non-radioactive technologies will undoubtedly be an advantage in drug discovery. In this work, an innovative and non-radioactive functional cAMP assay was validated at GPR17, a dual uracil nucleotide/cysteinyl leukotrine receptor. The new assay monitors GPCR activity through a change in the intracellular cAMP concentration using a genetically modified form of firefly luciferase containing a cAMP-binding protein moiety. Binding of cAMP induces a conformational change leading to increased light output, which allows evaluating the activity of ligands at the receptor under study. The results, expressed as EC50 or IC50 values for agonists and antagonists, respectively, showed a strong correlation with those obtained with [35S]GTPγS binding assay, thus confirming the validity of this approach in the study of new ligands for GPR17. Moreover, this method allowed confirming that GPR17 is coupled with a Gαi.
P5.3 Novel purine receptors
GPR17 ligands: synthesis and characterization of new potent agonists
Rosaria Volpini 1, Michela Buccioni1, Stefania Ceruti2, Diego Dal Ben1, Simona Daniele3, Catia Lambertucci1, Davide Lecca2, Gabriella Marucci1, Maria Letizia Trincavelli3, Claudia Martini3, *Maria Pia Abbracchio2, Gloria Cristalli1
1 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Camerino, Italy, 2 Department of Pharmacological Sciences, Laboratory of Cellular and Molecular Pharmacology of Purinergic Transmission, University of Milan, Milan, Italy, 3 Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy
The G protein-coupled receptor GPR17, a dualistic receptor that responds to nucleotides and cysteinyl leukotrienes, is believed to represent a novel target for the development of new relevant therapeutic approaches to human stroke and ischemic damage.
[35S]GTPγS binding assay, performed on transfected 1321N1 cells, demonstrated that 2-phenylethynylATP behaves as a strong agonist (EC50 = 36 pM) of the receptor [1]. On the other hand, N 6-methylATP showed antagonist activity as well as some purine nucleotide bisphosphates. On these bases, in the search for potent GPR17 ligands, 2-phenylethynyl ATP derivatives bearing alkyl groups in the N 6 position, a new bisphosphate derivative, and a stable analogue bearing the aforementioned 2-phenylethynyl chain were synthesized. Biological studies showed that the new compounds behave as strong agonists of GPR17 with EC50 ranging from low nanomolar to subnanomolar values; hence, they could be efficacious tools for the further characterization of the receptor and to study its role in neurodegeneration processes.
[1] Volpini R et al. (2010) Frontal affinity chromatography–mass spectrometry useful for characterization of new ligands for GPR17 receptor. J Med Chem 53:3489–3501
P5.4 Novel purine receptors
First Ade for Chinese hamsters—exploring a new adenine receptor
Dominik Thimm, Miguel Moutinho, Anke C. Schiedel, Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Adenine receptors (AdeR) are GPCRs that represent a new class of recently discovered purinergic receptors [1]. Only three adenine receptors have been identified so far: one in rats (rAdeR1) [2] and two in mice (mAdeR1, mAdeR2) [2, 3]. Several mammalian cell lines were evaluated as expression systems for adenine receptors, one of which was the Chinese hamster ovary (CHO) cell. However, these cells themselves show high-affinity adenine binding and thus did not prove to be a suitable expression system. To clarify whether the detected adenine binding site in CHO cells is a hamster orthologue of the rat and mouse AdeR, we decided to clone the DNA coding for this putative receptor. Although the genomic sequences of the Chinese hamster are not available, we were able to clone different fragments of the gene and were able to combine the fragments to yield a DNA sequence highly homologous to the cDNAs of known AdeR. We subsequently expressed the new putative adenine receptor in Sf9 insect cells and CHO cells. The membranes of infected Sf9 cells were used to perform radioligand binding assays to test several adenine-derived compounds. We could show that the expressed receptor binds adenine with high affinity (IC50 = 71.1 vs. 10 nM [3H]adenine). Any alteration of the adenine molecule, however, led to a decrease in affinity, an observation that had already been made at the other rodent AdeR [4]. Using transfected CHO cells, we could show that the examined receptor is activated by adenine and is Gi (EC50 = 13.4 nM) as well as Gq protein-coupled (EC50 = 108 nM). In conclusion, we could identify a new adenine receptor expressed in the Chinese hamster (Cricetulus griseus) sharing 86%, 74% and 73% identity with rAdeR1, mAdeR1 and mAdeR2, respectively. We propose to name this receptor cAdeR.
D.T., A.C.S. and C.E.M. are grateful for support by the NRW International Graduate Research School BIOTECH-PHARMA.
[1] Brunschweiger A, Müller CE (2006) P2 receptors activated by uracil nucleotides—an update. Curr Med Chem 13:289–312
[2] Bender E, Buist A, Jurzak M, Langlois X, Baggerman G, Verhasselt P, Ercken M, Guo HQ, Wintmolders C, Van den Wyngaert I, Van Oers I, Schoofs L, Luyten W (2002) Characterization of an orphan G protein-coupled receptor localized in the dorsal root ganglia reveals adenine as a signaling molecule. Proc Natl Acad Sci U S A 99:8573–8578
[3] von Kügelgen I, Schiedel AC, Hoffmann K, Alsdorf BB, Abdelrahman A, Müller CE (2008) Cloning and functional expression of a novel Gi protein-coupled receptor for adenine from mouse brain. Mol Pharmacol 73:469–477
[4] Borrmann T, Abdelrahman A, Volpini R, Lambertucci C, Alksnis E, Gorzalka S, Knospe M, Schiedel AC, Cristalli G, Müller CE (2009) Structure–activity relationships of adenine and deazaadenine derivatives as ligands for adenine receptors, a new purinergic receptor family. J Med Chem 52:5974–5989
P5.5 Novel purine receptors
The predicted putative agonist binding site of the rat adenine receptor investigated by mutagenesis studies
Melanie Knospe, Anke C. Schiedel, Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, 53121 Bonn, Germany
In 2002, an orphan G protein-coupled receptor (GPCR) from rat (rAde1R) has been demonstrated to act as a receptor for the nucleobase adenine [1]. A homology model was generated using a homology model of the related mMrgC11 receptor as a template, which was based on the frog rhodpsin X-ray structure [2, 3]. The predicted agonist binding site was predicted to be located within the transmembrane domains 3, 4, 5, and 6 (see Fig. A) [2]. Asn88 and Asn146 were identified as key residues for adenine binding. Also, Phe83, Leu174, and His225 were assumed to be involved in adenine binding [2].
In the present study, all five amino acid residues were exchanged for alanine by site-directed mutagenesis, and the mutated receptors, each carrying a single mutation, were expressed in Spodoptera frugiperda insect cells using the Baculovirus expression system. Cell membranes prepared from the infected cells were used for subsequent radioligand binding studies with [3H]adenine. All of the mutants exhibited high-affinity binding for [3H]adenine. The IC50 values were not significantly different from that of the wild-type receptor, except for the Asn88Ala mutant which showed a moderate threefold decrease in affinity for adenine. In conclusion, the adenine binding site of the rAde1R predicted by Heo et al. on the basis of a GPCR homology model cannot be correct. Predictions of binding sites for very small ligands, such as adenine, are highly speculative when they are based on the X-ray structures of distantly related proteins.
Supported by the NRW International Graduate Research School Chemical Biology.
[1] Bender E (2002) Proc Natl Acad Sci U S A 99:8573–8578
[2] Heo J (2007) J Mol Graph Model 26:800–812
[3] Heo J (2007) ChemBioChem 8:1527–1539
P5.6 Novel purine receptors
Adenine receptors in rat and human cells: signalling pathways and pathophysiological relevance
F.Siegert1, A. Obst1, B. Bumnaran1, M. Bloßfeld1, T. Borrmann2, C.E. Müller2 and K. Nieber 1
1 Institute of Pharmacy, University of Leipzig, Leipzig, Germany, 2 PharmaCenter Bonn, Pharmaceutical Institute, University of Bonn, Bonn, Germany
Adenine was recently identified as an endogenous ligand of an orphan rat G protein-coupled receptor. Radioligand binding studies using membrane preparations provided further evidence for the expression of the adenine receptor in rat neuronal tissues. The aim of the present study was to detect the adenine receptor on rat (B-104) and human (SH-SY5Y) neuronal cell lines and to investigate signalling pathways. Additionally, intracellular recordings were made on cortical pyramidal cells in rat brain slices. Adenine (10 μM–1 mM) as well as the receptor ligand TB-74 (100 μM–1 mM) depressed concentration-dependently the electrical-evoked synaptic potential in rat brain slices and inhibited the ATP-induced increase of intracellular Ca2+ concentration in B-104 cells. Inhibiting key signalling pathway steps indicated different coupling in rat B-104 and human SH-SY5Y cells. It is suggested that in B-104 cells, the receptor couples to a Gq protein, whereas in SH-SY5Y cells adenine activates a Gi protein. The coupling may be responsible for the different effects of adenine on rat and human neuronal cells during hypoxia. Adenine enhanced the hypoxia-induced cell death in SH-SY5Y cells, whereas PSB-08162, described as an adenine receptor antagonist, was without effect. Contrary to human cells, adenine was without effect on hypoxia-induced cell death and PSB-08162 diminished hypoxia-induced cell death in rat neuronal cells. Our study represents pharmacological evidence of an adenine receptor in human cell lines of neuronal origin. This receptor differed in its functionality from the receptor identified in rat neuronal tissues or cell lines. We postulate that species-dependent receptor subtypes occur.
PS6.1 Adenosine receptors
The biological function of adenosine receptors in brown adipose tissue
Samet Mutlu 1, Thorsten Gnad1, Christa E. Müller2, Alexander Pfeifer1
1 PharmaCenter Bonn, Institute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany, 2 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany
Brown adipose tissue (BAT) is responsible for basal and inducible energy expenditure in mammals. BAT contains large amounts of mitochondria and small lipid droplets and is highly vascularized. BAT lipolysis and thermogenesis are stimulated by sympathetic neurons. Importantly, recent findings indicate that adult humans possess metabolically active BAT [1]. Here, we analyzed the expression of adenosine receptors in BAT. Adenosine receptors (AdoR) are members of the superfamily of G protein-coupled receptors. There are four subtypes of AdoRs in humans referred to as AdoRA1, A2A, A2B, and A3. They are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of cAMP levels within cells. Interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through AdoRA1 or A2A, respectively [2]. However, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in BAT function is not clear. To analyze the role of AdoRs in BAT, we use preadipocytes isolated from BAT of newborn mice and subject them to a differentiation protocol. Cells are grown in differentiation medium containing the hormones insulin and T3 until confluence and are then induced for 2 days with IBMX and dexamethasone followed by five more days culturing in differentiation medium [3].
Abundance of AdoRA1, A2A, A2B, and A3 mRNA was measured using RT-PCR. All four receptor subtypes are present in preadipocytes and all are transcriptionally upregulated—albeit at varying degrees—during differentiation. AdoRA1 is upregulated 1.7- and 1.6-fold at days 4 and 7, respectively, as compared with preadipocytes (n = 3). AdoRA2A is 2-fold upregulated at day 4 and 1.8-fold at day 7, respectively (n = 2). Similar results were obtained for AdoRA2B, which is two times stronger expressed at days 4 and 7 compared with preadipocytes (n = 2). The A3 receptor is the most strongly upregulated subtype showing a more than 10-fold higher expression level at days 4 and 7 (n = 2). Presently, we are investigating the functional role of AdoRs in mature brown adipocytes, e.g., in lipolysis.
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1
Virtanen KA et al. (2009) Functional brown adipose tissue in healthy adults. N Engl J Med 360
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2
Borglum JD et al. (1996) Changes in adenosine A1- and A2-receptor expression during adipose cell differentiation. Mol Cell Endocrinol 117
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3
Haas B et al. (2009) Protein kinase G controls brown fat cell differentiation and mitochondrial biogenesis. Sci Signal 2(99)
PS6.2 Adenosine receptors
Adenosine A 1 R inhibition of calcium channels in retinal ganglion cells
Steven Barnes, Melanie Lalonde, Xiaolu Sun, William Baldridge
Department of Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia Canada
Adenosine A1Rs and A2Rs have been identified in the retina, but their role is not yet fully understood. We investigated the action of adenosine and adenosine receptor agonists and antagonists on voltage-gated Ca channel-mediated calcium influx in retinal ganglion cells (RGCs) isolated from rat retina and in salamander retinal slices. Purified RGC cultures were generated from neonatal rats with a two-step panning procedure. Calcium channel currents were studied with patch clamp techniques. Adenosine produced a concentration-dependent decrease in the amplitude of calcium channel current with maximum inhibition of ~40%. Adenosine caused an irreversible inhibition of calcium channel current in cells dialyzed with GTP-γ-s. The inhibitory effect of adenosine on calcium channel current was blocked by the A1 antagonist 8-cyclopentyltheophylline (1–10 μM), but not by the A2 antagonist 3-7-dimethyl-1-propargylxanthine (10 μM), and was mimicked by the A1 agonist N 6-cyclohexyladenosine (1 μm) but not by the A2 agonist 5′-(N-cyclopropyl) carbox-amidoadenosine (1 μm). Adenosine inhibition of calcium channel current was not affected by the L-type calcium channel blocker nifedipine (5 μM). Adenosine’s inhibition of calcium channel current was reduced to ~10% in ω-conotoxin GVIA (1 μM), suggesting that adenosine inhibits N-type calcium channels. These results show that adenosine acts on an A1 adenosine receptor subtype via a G protein-coupled pathway to inhibit calcium channel currents carried in N-type calcium channels. This work supports a role for adenosine as a neuroprotective agent and modulator of RGCs.
PS6.3 Adenosine receptors
Ca v 1 (L) channel blockade uncovers adenosine negative inotropism leading to a loss of A 1 “chronoselectivity” in the spontaneously beating rat atria
Nádia Oliveira-Monteiro, Ana Patrícia Fontes-Sousa, Bruno Bragança, Sílvia Marques, Miguel Faria & Paulo Correia-de-Sá
Laboratório de Farmacologia e Neurobiologia/UMIB, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, L. Prof. Abel Salazar, 2, 4099-003 Porto, Portugal
Cardiac activation of adenosine A1 receptors (A1R) mediates negative chronotropic, dromotropic and inotropic responses, similarly to muscarinic M2 receptors (M2R). Both receptors couple to GIRK/KIR 3.1/3.4 channels via βγ-subunits. Receptor activation reduces action potential duration, thereby decreasing the time available for Ca2+ influx and, thus, heart rate and muscle contraction. However, the negative chronotropic effect of A1R is evidenced at lower concentrations than the negative inotropic action (unpublished data). In this context, we explored the rationale for this “chronoselectivity” by testing the influence of Ca2+ and K+ channel blockers in the effect of the A1R agonist, R-PIA, as compared with oxotremorine (Oxo, a M2R agonist). Experiments were performed in isolated atria from Wistar rats continuously superfused with gassed Tyrode’s solution (37°C). In some experiments, the atria were paced at a constant rate (4 Hz; 2 ms, 60 V). The parameters reported include rate and force of contraction. The data indicate that adenosine acting via A1R is a “chronoselective” atrial depressant as compared with Oxo. While both A1R and M2R promote the opening of tertiapin Q-sensitive GIRK/KIR 3.1/3.4 channels modulating SA node automatism, adenosine A1R (via a G protein α-subunit) may prevent KCa2/SK activation, increasing the time available for Ca2+ influx through Cav1 (L-type) channels. Thus, blockade of Cav1 (L-type) channels by verapamil (often used to normalize SV tachycardia rhythms) predisposes the atria to the negative inotropic action of adenosine by uncoupling A1 receptor-mediated KCa2/SK channel closure from its effector system.
Work supported by FCT (FEDER funding). B.B. acknowledges the Young Investigator Fellowship (BII/UMIB-ICBAS/2009-2).
PS6.4 Adenosine receptors
Functional cAMP assay optimization at CHO cells expressing A 2A adenosine receptors and stably transfected with firefly luciferase biosensor
Michela Buccioni 1, Diego Dal Ben1, Catia Lambertucci1, Gabriella Marucci1, Ajiroghene Thomas1, Dania Giacobbe1, Rosaria Volpini1, Karl-Norbert Klotz2, Gloria Cristalli1
1 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, I-62032 Camerino, Italy, 2 Institut für Pharmakologie und Toxikologie, Universität Würzburg, D-97078 Würzburg, Germany
A2A adenosine receptor (A2AAR) ligands have been proposed as an attractive pharmacological tool for the treatment of several diseases, including neurodegenerative disorders. In this work, we perform and validate a new functional cAMP assay suitable for the evaluation of A2AAR ligand activity. This functional assay uses a biosensor expressing a mutant form of Photinus pyralis luciferase into which a cAMP-binding protein moiety has been inserted. When cAMP binds to the biosensor, there is an increased light output proportional to the A2AAR ligand activities. Hence, a new CHO cell line expressing A2AARs and stably transfected with firefly luciferase biosensor was generated. A2AAR agonists and antagonists were tested using cAMP assay in the new cell line, which allowed speeding up and facilitating the screening of the ligands. The results achieved are in agreement with those obtained with binding studies.
PS6.5 Adenosine receptors
A role of cAMP extrusion and A 2A receptors in activation of adenylyl cyclase
Thomas Pleli 1, Oliver Waidmann1, Karel Dvorak1, Stefan Zeuzem1, Herbert Zimmermann2, Ricardo M. Biondi1, and Albrecht Piiper2
1 Department of Medicine I, University of Frankfurt/M., D-60590 Frankfurt, Germany, 2 Department of Neurochemistry, Biocenter of the Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany
Adenosine, produced locally at the cell surface from released adenine nucleotides, modulates important biological events through the activation of cell surface adenosine receptors (ARs). Under hypoxic or inflammatory conditions, released ATP serves as a major precursor of extracellular adenosine. Upon stimulation of Gs protein-coupled receptors (GsPCR) and adenylyl cyclase, large amounts of cAMP are extruded, presumably through multidrug resistance-associated proteins. To date, extruded cAMP and ARs have not been recognized as signaling intermediates of GsPCR and adenylyl cyclase. Here, we provide evidence for a role of cAMP efflux and Gs protein-coupled A2A adenosine receptors in the activation of adenylyl cyclase and protein kinase A following the application of pituitary adenylyl cyclase-activating peptide or cholera toxin to PC12 cells. Under these conditions, extracellular ATP levels were not significantly altered. Exogenous cAMP was hydrolyzed to AMP and adenosine by PC12 cells. In human renal mesangial cells, an A2B rather than A2A adenosine receptor antagonist inhibited the activation of adenylyl cyclase by glucagon or cholera toxin. Our study indicates that the activation of non-AR GsPCRs can elicit the activation of cell surface ARs by a signaling pathway involving cAMP extrusion and conversion to adenosine at the cell surface.
PS6.6 Adenosine receptors
Caffeine and other A 2A ligands modulate the binding of raclopride to D2 receptors
Jordi Bonaventura 1, Marc Brugarolas1, Gemma Navarro1, Josefa Mallol1, Enric I Canela1, Carme Lluís1, Rafael Franco1,2, Javier Meana3,4, Joan Sallés4,5, Antoni Cortés1, Vicent Casadó1, Sergi Ferré6
1 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain, 2 Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Avda. Pio XII 55, 31008 Pamplona, Spain, 3 Department of Pharmacology, University of the Basque Country, Leioa, Bizkaia, Spain, 4 Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain, 5 Department of Pharmacology (Facultad de Farmacia), University of the Basque Country, Vitoria-Gasteiz, Spain, 6 National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Department of Health and Human Services, 251 Bayview Blvd., Baltimore, MD, 21224, USA
Raclopride is a common dopamine D2 and D3 receptor antagonist. In this study, we show how caffeine and other A2A ligands (both agonists and antagonists) can modulate the binding properties of raclopride in transfected CHO cells and in human striatum. Moreover, agonist and antagonist binding to A2A receptors modulates the agonist binding to dopamine D2 receptors. The A2A receptor ligand-induced modulation of ligand binding to dopamine D2 receptors is mediated by A2A–D2 receptor interaction, and changes in the quaternary structure of A2A–D2 receptor heteromers involving intracellular domains do not affect that modulation.
This study was supported by Grants from Spanish Ministerio de Ciencia y Tecnología (SAF2008-00146, SAF2008-03229-E and SAF2009-07276).
PS6.7 Adenosine receptors
The relationship between binding kinetics and functional efficacy of adenosine A 2A receptor agonists
D. Guo, L. H. Heitman, A.P. IJzerman
Division of Medicinal Chemistry, LACDR, Leiden University, Leiden, the Netherlands
The adenosine A2A receptor (A2AR) belongs to the superfamily of G protein-coupled receptors and is a promising therapeutic target. Traditionally, the discovery of novel A2AR agents started from assessments performed in equilibrium, largely ignoring the kinetic aspects of the ligand–receptor interaction. The aim of the study was to test the binding kinetics of A2AR agonists and explore a possible relationship with their functional efficacy.
We used a radioligand competition association assay to determine the binding kinetics of unlabelled A2AR ligands. The functional efficacies of these agonists were determined in a cAMP assay.
A correlation was observed between the dissociation rate of agonists (i.e. residence time) and their functional efficacy. In other words, the longer the agonist stayed on the receptor, the higher the efficacy. Notably, the affinity of A2AR agonists was not correlated to their functional efficacy.
This study shows that the residence time of A2AR agonists is linked to their efficacy, unlike their affinity. Hence, more attention should be paid to drug target residence time to improve the efficacy of new generations of A2AR ligands.
PS6.8 Adenosine receptors
A 2A -receptor induced reorganization of the actin cytoskeleton in growth cones of regenerating sensory neurons
J.-C. Eilert, M. Hartmann, S. Bürge, H. Harz,
Max Planck Institute of Neurobiology, D-82152 Martinsried, Germany
Growth cones are dynamic structures at the tips of growing axons responsible for elongation and steering. The intricate signal transduction network of growth cones is influenced by a multiplicity of extracellular molecules. In a recent publication, we have shown that extracellular concentration gradients of adenosine can induce growth cone turning of regenerating sensory neurons from chicken embryos [1].
Here, we show that A2A receptor (A2A-R) stimulation changes the F-actin content of growth cones from chicken sensory neurons. Whereas 100 nM of the A2A agonist CGS21680 reduces the total amount of growth cone F-actin by 11%, a stimulus of 1 μM increases the F-actin content by 13%. This dose-dependent effect can be reproduced in a permanent cell line. HEK293 cells that have a low native A2A-R expression show a decrease in F-actin content after stimulation with 100 nM of CGS, whereas this stimulus increased the F-actin content in cells transiently overexpressing the A2A-R. Obviously, A2A-R takes part in the regulation of the F-actin content in neuronal and non-neuronal cells.
It is known that an F-actin rearrangement is involved in neuronal growth and guidance. The data shown here suggest a role of A2A-R in the regulation of these processes, particularly as the outcome of A2A-R stimulation can be switched from F-actin increase to a decrease, depending on the stimulus level.
[1] Grau B et al. (2008) Adenosine induces growth-cone turning of sensory neurons. Purinergic Signal 4(4):357–364
PS6.10 Adenosine receptors
A 2A and A 2B receptor subtypes modulate contractility in untreated and inflamed rat colon preparations
Voß, U 1, Müller, C2, Nieber, K1
1 University of Leipzig, Institute of Pharmacy, 04103 Leipzig, Germany, 2 Rheinische Friedrich-Wilhems Universität Bonn, Institute of Pharmacy, 53121 Bonn, Germany
The purine nucleoside adenosine, which is involved in several physiological functions, regulates a wide variety of immune and inflammatory responses and acts as a modulator of gut functions. Although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to the micromolar range. Recent evidence suggests a prominent role of A2A receptors (A2AR) and A2B receptors (A2BR) in the pathophysiology of inflammation (Sitkovsky and Lukashev 2005; Palmer and Trevethick 2008). In the current study, we investigated the role of A2AR and A2BR to regulate contractility in untreated and inflamed rat colon preparations using specific receptor agonists and antagonists.
Inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS; 0.01/0.1 M, 30 min). mRNA expression was determined using RT-PCR. Contractions were measured isometrically in an organ bath setup. All four adenosine receptor subtypes were expressed in untreated colon preparations. Activation of A1, A2B, and A3 receptors with specific agonists reduced the acetylcholine (ACh, 10 μM)-induced contractions, while activation of A2BR was enhanced. After incubation with TNBS morphologically, damages in the colonic mucosa and muscle walls were detectable followed by reduced ACh contractions. The TNBS-mediated decrease of ACh contractions as well as the morphological damages were partially normalized by co-incubation of TNBS with the A2AR agonist 2-p-[carboxyethyl]phenethylamino-5′-N-ethylcarboxamido-adenosine (CGS 21680, 10 μM) or the A2BR antagonist 4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl-benzenesulfonic acid (PSB 1115, 100 μM). In this study, using an in vitro inflammatory model, we demonstrate that the A2AR agonist CGS 21680 or the A2BR antagonist PSB 1115 effectively counteracted the development of TNBS-induced disturbances in colon preparations.
PS6.11 Adenosine receptors
One is all it takes: disulfide bonds and cysteine residues in the extracellular loop 2 of the adenosine A 2B receptor
Sonja Hinz1, Anke C. Schiedel 1, Dominik Thimm1,3, Farag Sherbiny2, Thomas Borrmann1, Astrid Maaß2, and Christa E. Müller1
1 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany, 2 Fraunhofer Institute SCAI, Schloss Birlinghoven, 53754 Sankt Augustin, Germany, 3 NRW International Graduate Research School Biotech-Pharma, Bonn, Germany
The P1 family of purinergic receptors consists of four subtypes of adenosine receptors: A1, A2A, A2B, and A3. The A2B receptor is the least investigated subtype; it has been shown to be involved in inflammatory diseases including asthma, pain, and cancer, which makes the A2B receptor an interesting new drug target.
The human A2B receptor contains the most cysteine residues in the extracellular loop 2 found in any class A GPCR. Therefore, we were interested in investigating the role and function of those cysteine residues, in particular their potential involvement in the formation of disulfide bonds by using a combination of mutagenesis, molecular modeling, and chemical and pharmacological experiments. Pretreatment of wt A2B receptors expressed in CHO cells with dithiothreitol resulted in a 74-fold increase in the EC50 value of NECA in cAMP accumulation studies, suggesting that at least one disulfide bond is essential for ligand binding and/or receptor function. High-affinity binding of the A2B antagonist radioligand [3H]PSB-603 was abolished and agonists were inactive in cAMP assays in the C783.25S and the C17145.50S mutants. The C16645.45S and the C16745.46S, as well as the C16645.45S–C16745.46S double mutant, behaved like the wt receptor, while the C15445.33S mutant led to significant, although more subtle, effects on cAMP accumulation—either increase or decrease—depending on the structure of the agonist used. Our data indicate that in the A2B receptor, only the C3.25-C45.50 disulfide bond is essential for ligand binding and receptor activation [1].
[1] Schiedel et al. (2011) Biochem Pharmacol. 82:389–399
PS6.12 Adenosine receptors
Dissecting the individual roles of the second extracellular loop of adenosine A 2A and A 2B receptors
Seibt BF, Schiedel AC, Müller CE
1 PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
The human adenosine A2B receptor, which belongs to the family of class A G protein-coupled receptors (GPCRs), appears to play an important role in inflammatory processes. In the respiratory system, A2B receptor activation results in pro-inflammatory effects. In contrast, the closely related A2A receptor subtype mediates anti-inflammatory and immunosuppressive effects. Adenosine and most of its derivatives, e.g. NECA and CGS-21680, show a considerably higher affinity for the A2A than for the A2B receptor despite high sequence identity of 56% and a similarity of 70% (for the human receptors).
In many GPCRs, the second extracellular loop (EL2) is known to participate in ligand binding. In the present study, the complete EL2 of the A2B receptor was replaced by the EL2 of the A2A receptor by overlap extension mutagenesis. Furthermore, single amino acid residues Asp148, Ser149, Thr151, Glu164, and Ser165 in the EL2 were exchanged for alanine by site-directed mutagenesis. The resulting mutant receptors were stably expressed in CHO cells using a retroviral expression system and characterized by radioligand binding studies using [3H]PSB-603 as a radioligand and by cAMP accumulation assays. All agonists investigated (adenosine, NECA, and the non-nucleosidic A2B agonist BAY60-6583) showed increased efficacy at the mutant A2B(EL2-A2A) receptor as compared with the wild-type (wt) A2B receptor (using forskolin as a control). In contrast to the wt A2B receptor, at which CGS-21680 is inactive, the A2B(EL2-A2A) mutant could be activated by the A2A-selective agonist CGS-21680, exhibiting an EC50 value of 47 μM. Interestingly, the agonists were more potent at the E164A mutant than at the wild-type receptor, indicating that this amino acid might contribute to the relatively low affinity of agonists at the A2B receptor subtype. The EL2 of the adenosine A2 receptor subtypes appears to play an important role in receptor activation.
Financial support by the DFG (GRK804) is gratefully acknowledged.
PS6.13 Adenosine receptors
Adenosine modulates the network activity in the olfactory bulb
Natalie Rotermund, Christian Lohr, Daniela Hirnet
1 University of Hamburg, Hamburg, Germany
The purinergic signalling system is unique with regard to its complexity and specificity, which is achieved by tissue-specific combinations of transmitters—ATP, ADP and adenosine—with a variety of receptor subtypes, second messenger systems, transporters and enzymes. ATP and its metabolites, in particular adenosine, are ubiquitous co-transmitters and neuromodulators, participating in synaptic transmission as well as in neuron–glia interactions. Members of the purinergic signalling machinery are highly expressed in the olfactory bulb of rodents, suggesting purinergic modulation of the olfactory information processing. Recently, we found functional evidence for purinergic cell–cell communication in the olfactory bulb: In addition to the principal neurotransmitter, glutamate, the olfactory receptor neurons (ORN) release ATP, which elicits calcium signalling in astrocytes in the glomerular layer. In addition, ATP is degraded to adenosine, which stimulates glial A2A receptors. In the present study, we focus on the effect of adenosine on the neuronal network activity in the olfactory bulb. We monitored the activity of olfactory bulb neurons by recording postsynaptic whole-cell currents of mitral cells, the output neurons of the olfactory bulb. Bath application of adenosine reversibly reduced the frequency of spontaneous synaptic inputs in mitral cells. DPCPX, a specific antagonist of the A1 receptor subtype, blocked the effect of adenosine. We used paired-pulse stimulation of receptor axons and imaging of vesicle fusion in OMP-synapto-pHluorin mice to test the effect of adenosine on synapses between receptor axons and mitral cells. Adenosine neither changed the amplitude of the EPSC response nor the paired-pulse ratio as compared with control stimulations. Similarly, vesicle fusion in presynaptic structures of ORNs did not change in the presence of adenosine. After pharmacological isolation of glutamatergic inputs by gabazine, adenosine failed to decrease the frequency of spontaneous (glutamatergic) inputs in mitral cells. In contrast, adenosine still exerts its depressing effect on the spontaneous GABAergic synaptic inputs in mitral cells after blocking the glutamatergic transmission by D-APV and NBQX. This indicates that in the olfactory bulb, purinergic modulation of olfactory information processing is mediated by adenosine aiming at GABAergic interneurons via A1 receptors.
Financial support for this project is permitted by the DFG (Lo779/6).
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Abstracts of the Fourth Joint German-Italian Purine Club Meeting 2011. Purinergic Signalling 8, 107–175 (2012). https://doi.org/10.1007/s11302-011-9257-4
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DOI: https://doi.org/10.1007/s11302-011-9257-4