Skip to main content

Advertisement

SpringerLink
Log in

PACAP-Induced PAC1 Receptor Internalization and Recruitment of Endosomal Signaling Regulate Cardiac Neuron Excitability

  • Published:
Journal of Molecular Neuroscience Aims and scope Submit manuscript

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors (Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades, as well as by endosomal signaling mechanisms. PACAP/PAC1 receptor-mediated activation of plasma membrane adenylyl cyclase (AC) and the resulting increase in cellular cAMP enhances a hyperpolarization-induced nonselective cationic current Ih, which contributes to the PACAP-induced increase in cardiac neuron excitability. Further, PACAP-mediated AC/cAMP/PKA downstream signaling also appears to enhance cardiac neuron IT to facilitate the excitatory responses. PACAP activation of PAC1 receptors rapidly stimulates receptor internalization, and reducing ambient temperature or treatments with the clathrin inhibitor Pitstop2 or the dynamin I/II inhibitor dynasore to block endocytic events can suppress PACAP-enhanced neuronal excitability. Thus, endocytosis inhibitors essentially eliminate PACAP-enhanced excitability suggesting that endosomal platforms represent a primary signaling mechanism. Endosomal signaling is associated canonically with ERK activation and in accord, PACAP-enhanced cardiac neuron excitability is reduced by MEK inhibitor pretreatments. PACAP activation of MEK/ERK signaling can enhance currents through voltage-dependent Nav1.7 channels. Hence, PACAP-induced PAC1 receptor internalization/endosomal signaling, recruitment of MEK/ERK signaling, and modulation of Nav1.7 are implicated as key mechanisms contributing to the PACAP-enhanced neuronal excitability. PACAP/PAC1 receptor-mediated endosomal ERK signaling in central circuits can play key roles in development of chronic pain and anxiety-related responses; thus, PAC1 endosomal signaling likely participates in a variety of homeostatic responses within neuronal circuits in the CNS.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Arimura A (1998) Perspectives on pituitary adenylate cyclase activating polypeptide (PACAP) in the neuroendocrine, endocrine, and nervous systems. Jap J Physiol 48:301–331

    Article  CAS  Google Scholar 

  • Auciello G, Cunningham DL, Tatar T, Heath JK, Rappoport JZ (2013) Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8. J Cell Science 126:613–624

    Article  CAS  PubMed  Google Scholar 

  • Barrie AP, Clohessy AM, Buensuceso CS, Rogers MV, Allen JM (1997) Pituitary adenylyl cyclase-activating peptide stimulates extracellular signal-regulated kinase 1 or 2 (ERK1/2) activity in a Ras-independent, mitogen-activated protein kinase/ERK kinase 1 or 2-dependent manner in PC12 cells. J Biol Chem 272:19666–19671

    Article  CAS  PubMed  Google Scholar 

  • Bouschet T, Perez V, Fernandez C, Bockaert J, Eychene A, Journot L (2003) Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras, protein kinase C, and protein kinase A in neuronal cells. J Biol Chem 278:4778–4785

    Article  CAS  PubMed  Google Scholar 

  • Braas KM, May V (1999) Pituitary adenylate cyclase-activating polypeptides directly stimulate sympathetic neuron neuropeptide Y release through PAC1 receptor isoform activation of specific intracellular signaling pathways. J Biol Chem 274:27702–27710

    Article  CAS  PubMed  Google Scholar 

  • Braas KM, May V, Harakall SA, Hardwick JC, Parsons RL (1998) Pituitary adenylate cyclase-activating polypeptide expression and modulation of neuronal excitability in guinea pig cardiac ganglia. J Neurosci 18:9766–9779

    Article  CAS  PubMed  Google Scholar 

  • Calebiro D, Nikolaev VO, Gagliani MC, de Filippis T, Dees C, Tacchetti C, Persani L, Lohse MJ (2009) Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol 7:e1000172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Calebiro D, Nikolaev VO, Persani L, Lohse MJ (2010) Signaling by internalized G-protein-coupled receptors. Trends Pharmacol Sci 31:221–228

    Article  CAS  PubMed  Google Scholar 

  • Calupca MA, Vizzard MA, Parsons RL (2000) Origin of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive fibers innervating guinea pig parasympathetic cardiac ganglia. J Comp Neurol 423:26–39

    Article  CAS  PubMed  Google Scholar 

  • Chemin J, Mezghrani A, Bidaud I, Dupasquier S, Marger F, Barrère C, Nargeot J, Lory P (2007) Temperature-dependent modulation of CaV3 T-type calcium channels by protein kinases C and A in mammalian cells. J Biol Chem 282:32710–32718

    Article  CAS  PubMed  Google Scholar 

  • Cho J-H, Zushida K, Shumyatsky GP, Carlezon WA, Meloni EG, Bolshakov VY (2012) Pituitary adenylate cyclase-activating polypeptide induces postsynaptically expressed potentiation in the intra-amygdala circuit. J Neurosci 32:14165–141177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Clason TA, Girard BM, May V, Parsons RL (2016) Activation of MEK/ERK signaling by PACAP in guinea pig cardiac neurons. J Mol Neurosci 59:309–316

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Delom F, Fessart D (2011) Role of phosphorylation in the control of clathrin-mediated internalization of GPCR. Int J Cell Biol 2011:246954

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Deutsch PJ, Sun Y (1992) The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth. J Biol Chem 267:5108–5113

    CAS  PubMed  Google Scholar 

  • Di Fiore PP, von Zastrow M (2016) Endocytosis, signaling and beyond. Cold Spring Harb Perspect Biol 6:a016865

    Article  CAS  Google Scholar 

  • Edwards FR, Hirst GDS, Klemm MF, Steele PA (1995) Different types of ganglion cell in the cardiac plexus of guinea-pigs. J Physiol 486:453–471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ferrandon S, Feinstein TN, Castro M, Wang B, Bouley R, Potts JT, Gardella TJ, Vilardaga JP (2009) Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat Chem Biol 5:734–742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fitzgerald EM, Dolphin AC (1997) Regulation of rat neuronal voltage-dependent calcium channels by endogenous p21-ras. Eur J Neurosci 9:1252–1261

    Article  CAS  PubMed  Google Scholar 

  • Gupte RP, Kadunganattil S, Shepard AJ, Merrill R, Planer W, Bruchas MR, Strack S, Mphapatra DP (2016) Convergent phosphomodulation of the major neuronal dendritic potassium channel KV4.2 by pituitary adenylate cyclase-activating polypeptide. Neuropharm 101:291–308

    Article  CAS  Google Scholar 

  • Hammack SE, May V (2014) Pituitary adenylate cyclase activating polypeptide in stress-related disorders: data convergence from animal and human studies. Biol Psychiatry 78(3):167–177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hammack SE, Chung J, Rhoades KM, Schutz KC, Falls WA, Braas KM, May V (2009) Chronic stress increases pituitary adenylate cyclase-activating polypeptide (PACAP) and brain-derived neurotrophic factor (BDNF) mRNA expression in the bed nucleus of the stria terminalis (BNST); roles for PACAP in anxiety-like behavior. Psychoneuroendrocrinology 34:833–843

    Article  CAS  Google Scholar 

  • Harmar T, Lutz E (1994) Multiple receptors for PACAP and VIP. Trends Pharmacol Sci 15:97–99

    Article  CAS  PubMed  Google Scholar 

  • Hill J, Chan S-A, Kuri B, Smith C (2011) Pituitary adenylate cyclase-activating peptide (PACAP) recruits low voltage-activated T-type calcium influx under acute sympathetic stimulation in mouse adrenal chromaffin cells. J Biol Chem 286:42459–42469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hoover DB, Tompkins JD, Parsons RL (2009) Differential activation of guinea pig intrinsic cardiac neurons by the PAC1 agonists maxadilan and pituitary adenylate cyclase-activating polypeptide 27 (PACAP27). J Pharmacol Exp Ther 331:197–203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hu HJ, Glauner KS, Gereau RW 4th (2003) ERK integrates PKA and PKC signaling in superficial dorsal horn neurons. I. Modulation of A-type K+ currents. J Neurophysiol 90:1671–1679

    Article  CAS  PubMed  Google Scholar 

  • Iftinca MC, Zamponi GW (2008) Regulation of neuronal T-type calcium channels. Trends Pharmacol Sci 30:32–40

    Article  CAS  PubMed  Google Scholar 

  • Irannejad R, Tomshine JC, Tomshine JR, Chevalier M, Mahoney JP, Steyaert J, Rasmussen SG, Sunahara RK, El-Samad H, Huang B, von Zastrow M (2013) Conformational biosensors reveal GPCR signalling from endosomes. Nature 495:534–538

    Article  CAS  PubMed  Google Scholar 

  • Jalink K, Moolenaar WH (2010) G protein-coupled receptors: the inside story. Bioessays 32:13–16

    Article  CAS  PubMed  Google Scholar 

  • von Kleist L, Stahlschmidt W, Bulut H, Gromova K, Puchkov D, Robertson MJ, MacGregor KA, Tomilin N, Pechstein A, Chau N, Chircop M, Sakoff J, von Kries JP, Saenger W, Kräusslich HG, Shupliakov O, Robinson PJ, McCluskey A, Haucke V (2011) Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell 146:471–484

    Article  CAS  Google Scholar 

  • Legradi G, Das M, Giunta B, Hirani K, Mitchell EA, Diamond DM (2007) Microinjection of pituitary adenylate cyclase-activating polypeptide into the central nucleus of amygdale of the rat produces a shift from an active to passive mode of coping in the shock-probe fear/defensive burying test. Neural Plast 2007:79102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Luttrell LM, Ferguson SS, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, Lin F, Kawakatsu H, Owada K, Luttrell DK, Caron MG, Lefkowitz RJ (1999) Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science 283:655–661

    Article  CAS  PubMed  Google Scholar 

  • Lyu RM, Germano PM, Choi JK, Le SV, Pisegna JR (2000) Identification of an essential amino acid motif within the C terminus of the pituitary adenylate cyclase-activating polypeptide type I receptor that is critical for signal transduction but not for receptor internalization. J Biol Chem 275:36134–36142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Maca E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T (2006) Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 10:839–850

    Article  CAS  Google Scholar 

  • Magalhaes A, Dunn H, Ferguson S (2012) Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins. Br J Pharmacol 165:1717–1736

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • May V, Parsons RL (2016) G protein-coupled receptor endosomal signaling and regulation of neuronal excitability and stress responses: signaling options and lessons from the PAC1 receptor. J Cell Physiol 232:698–706

    Article  CAS  PubMed  Google Scholar 

  • May V, Lutz E, MacKenzie C, Schutz KC, Dozark K, Braas KM (2010) Pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP1HOP1receptor activation coordinates multiple neurotrophic signaling pathways: Akt activation through phosphatidylinositol 3-kinase gamma and vesicle endocytosis for neuronal survival. J Biol Chem 285:9749–9761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • May V, Buttolph TR, Girard BM, Clason TA, Parsons RL (2014) PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling. Am J Physiol Cell Physiol 306:C1068–C1079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McCormack K, Santos S, Chapman ML, Krafte DS, Marron BE, West CW, Krambis MJ, Antonio BM, Zellmer SG, Printzenhoff D, Padilla KM, Lin Z, Wagoner PK, Swain NA, Stupple PA, de Groot M, Butt RP, Castle NA (2013) Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels. PNAS 110:E2724–E2732

    Article  PubMed  Google Scholar 

  • McMahon HT, Boucrot E (2011) Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 12:517–533

    Article  CAS  PubMed  Google Scholar 

  • Merriam LA, Barstow KL, Parsons RL (2004) Pituitary adenylate cyclase-activating polypeptide enhances the hyperpolarization-activated nonselective cationic conductance, I h, in dissociated guinea pig intracardiac neurons. Regul Pept 123:123–133

    Article  CAS  PubMed  Google Scholar 

  • Merriam LA, Baran CN, Girard BM, Hardwick JC, May V, Parsons RL (2013) Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability. J Neurosci 33:4614–4622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Missig G, Mei L, Vizzard MA, Braas KM, Waschek JA, Ressler KJ, Hammack SE, May V (2017) Biol Psychiatry 81:671–682. Parabrachial pituitary adenylate cyclase-activating polypeptide activation of amygdala endosomal extracellular signal-regulated kinase signaling regulates the emotional component of pain

    Article  CAS  PubMed  Google Scholar 

  • Parsons RL, Tompkins JD, Merriam LA (2008) Source and action of pituitary adenylate cyclase-activating polypeptide in guinea pig intrinsic cardiac ganglia. Tzu Chi Med J 20:11–18

    Article  Google Scholar 

  • Parsons RL, Tompkins JD, Hardwick JC, Merriam LA, Girard BM, May V (2016) Multiple mechanisms contribute to the PAC1 modulation of parasympathetic cardiac neuron excitability. In: Reglodi D, Tamas A (eds) Pituitary adenylate cyclase activating polypeptide – PACAP. New York, Springer Nature, pp 205–225

    Chapter  Google Scholar 

  • Pisegna JR, Wank SA (1996) Cloning and characterization of the signal transduction of four splice variants of the human pituitary adenylate cyclase activating polypeptide receptor. Evidence for dual coupling to adenylate cyclase and phospholipase C. J Biol Chem 271:17267–17274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reinecke J, Caplain S (2014) Endocytosis and the Src family of non-receptor tyrosine kinases. Biomol Concepts 5:143–155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ressler KJ, Mercer KB, Bradley B, Javovanovic T, Mahan A, Kerley K, Norrholm SD, Kilaru V, Smith AK, Myers AJ, Ramirez M, Engel A, Hammack SE, Toufexis D, Braas KM, Binder EB, May V (2011) Post-traumatic stress disorder is associated with PACAP and the PAC1 receptor. Nature 470:492–497

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scita G, Di Fiore PP (2010) The endocytic matrix. Nature 463:464–473

    Article  CAS  PubMed  Google Scholar 

  • Simms BA, Zamponi GW (2014) Neuronal voltage-gated calcium channels: structure, function and dysfunction. Neuron 82:24–45

    Article  CAS  PubMed  Google Scholar 

  • Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH, Journot L (1993) Differential signal transduction by five splice variants of the PACAP receptor. Nature 365:170–175

    Article  CAS  PubMed  Google Scholar 

  • Stamboulian S, Choi J-S, Ahn H-S, Chang Y-W, Tyrrell L, Black JA, Waxman SC, Dib-Hajj SD (2010) ERK1/2 mitogen-activated protein kinase phosphorylates sodium channel NaV1.7 and alters its gating properties. J Neurosci 30:1637–1647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stroth N, Kuri BA, Mustafa T, Chan SA, Smith CB, Eiden LE (2012) PACAP controls adrenomedullary catecholamine secretion and expression of catecholamine biosynthetic enzymes at high splanchnic nerve firing rates characteristic of stress transduction in male mice. Endocrinology 154:330–339

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sweatt JD (2004) Mitogen-activated protein kinases in synaptic plasticity and memory. Curr Opin Neurobiol 14:311–317

    Article  CAS  PubMed  Google Scholar 

  • Talavera K, Nilius B (2006) Biophysics and structure-function relationships of T-type Ca2+ channels. Cell Calcium 40:97–114

    Article  CAS  PubMed  Google Scholar 

  • Toledo-Aral JJ, Brehm P, Halegoua S, Mandel G (1995) A single pulse of nerve growth factor triggers long-term neuronal excitability through sodium channel gene induction. Neuron 14:607–611

    Article  CAS  PubMed  Google Scholar 

  • Tompkins JD, Hardwick JC, Locknar SA, Merriam LA, Parsons RL (2006) Ca2+ influx, but not Ca2+ release from internal stores, is required for the PACAP-induced increase in excitability in guinea pig intracardiac neurons. J Neurophysiol 95:2134–2142

    Article  CAS  PubMed  Google Scholar 

  • Tompkins JD, Ardell JL, Hoover DB, Parsons RL (2007) Neurally released pituitary adenylate cyclase-activating polypeptide enhances guinea pig intrinsic cardiac neurone excitability. J Physiol 582:87–93

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tompkins JD, Lawrence YT, Parsons RL (2009) Enhancement of I h, but not inhibition of I M, is a key mechanism underlying the PACAP-induced increase in excitability of guinea pig intrinsic cardiac neurons. Am J Physiol Regul Integr Comp Physiol 297:R52–R59

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tompkins JD, Merriam LA, Girard BM, May V, Parsons RL (2015) Nickel suppresses the PACAP-induced increase in guinea pig cardiac neuron excitability. Am J Physiol Cell Physiol. 308:C857–C866

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tompkins JD, Clason TA, Hardwick JC, Girard BM, Merriam LA, May V, Parsons RL (2016) Activation of MEK/ERK signalling contributes to the PACAP-induced increase in guinea pig cardiac neuron excitability. Am J Physiol Cell Physiol 311:C643–C651

    Article  PubMed  PubMed Central  Google Scholar 

  • Tompkins JD, Clason TA, Buttolph TR, Girard BM, Linden AK, Hardwick JC, Merriam LA, May V, Parsons RL (2018) Src family inhibitors blunt the PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK and increase in cardiac neuron excitability. Am J Physiol Cell Physiol 314:C233–C241

    Article  CAS  PubMed  Google Scholar 

  • Vaudry D, Falluel-Morel A, Bourgault S, Basille M, Burel D, Wurtz O, Fournier A, Chow BKC, Hashimoto H, Galas L, Vaudry H (2009) Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery. Pharmacol Rev 61:283–357

    Article  CAS  PubMed  Google Scholar 

  • Vilardaga J-P, Jean-Alphonse FG, Gardella T (2014) Endosomal generation of cAMP in GPCR signalling. Nat Chem Biol 10:700–706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Q, Lu R, Zhao J, Limbird LE (2006) Arrestin serves as a molecular switch, linking endogenous α2-adrenergic receptor to SRC-dependent, but not SRC-independent, ERK activation. J Biol Chem 281:25948–25955

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This work was financially supported in part by National Institutes of Health (NIH) grant National Institute of General Medical Sciences (NIGMS) P30 GM103498/National Center for Research Resources (NCRR) P30 RR032135 (RLP) and Office of the Director grant S10 OD017969-01 (RLP).

Author information

Authors and Affiliations

  1. Departmental of Neurological Sciences, Robert Larner College of Medicine, University of Vermont, Burlington, VT, USA

    Rodney L. Parsons & Victor May

Authors
  1. Rodney L. Parsons
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Victor May
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RLP and VM wrote the paper and approved the final version.

Corresponding author

Correspondence to Rodney L. Parsons.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Parsons, R.L., May, V. PACAP-Induced PAC1 Receptor Internalization and Recruitment of Endosomal Signaling Regulate Cardiac Neuron Excitability. J Mol Neurosci 68, 340–347 (2019). https://doi.org/10.1007/s12031-018-1127-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12031-018-1127-x

Keywords

Search

Navigation