Purinergic P2Y2 receptors mediate rapid Ca2+ mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells
Introduction
Nucleotides, such as ATP, ADP, UTP and UDP, serve as functionally important agonists for P2-type purinergic receptors, and are released into the extracellular space from active nerve endings, the degranulation of aggregrating platelets, cellular efflux via membrane transport processes, and cell death. Once released, these compounds may act in a paracrine or autocrine to modulate local tissue function via two pharmacologically and structurally distinct classes of P2-type receptors denoted P2Y and P2X. P2Y receptors belong to the superfamily of metabotropic, G-protein coupled receptors, whereas P2X receptors represent ligand-gated ion channel/receptor complexes analogous to the ionotropic glutamate receptors found in the CNS. Molecular cloning has identified eight distinct cDNAs encoding P2Y receptors in mammals (i.e. P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, 12, 13 and 14), along with seven cDNAs for homomeric P2X receptors, denoted P2X1-7 [1], [2]. A common functional feature of P2X and P2Y receptors is that activation of either one often leads to elevation of intracellular free Ca2+ in the stimulated cell. P2X receptors mediate this response by directly conducting the entry of external Ca2+, whereas P2Y receptors initiate the enzymatic breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) via the stimulation of phospholipase Cβ, leading to inositol 1,4,5-trisphosphate (IP3) generation and the release of intracellular Ca2+ stores. In vascular endothelium, Ca2+ mobilizing agonists typically stimulate membrane hyperpolarizing responses, and the production of vasodilatory compounds, such as prostacyclin and nitric oxide (NO) [3], [4].
Recently, we have demonstrated that the ATP-stimulated NO production in human vascular endothelial cells is tightly linked to elevations in cytosolic free Ca2+ and membrane hyperpolarization [5], [6]. As endothelium is reported to express both P2X and P2Y receptors, we sought to identify the major purinergic receptor sub-type underlying these important functional responses. The results of our study strongly suggest that G-protein coupled, P2Y2 receptors mediate the observed ATP-evoked cellular events in isolated human endothelial cells.
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Materials and methods
The human umbilical vein endothelial cell line EA.hy926 (1) was loaded with either DAF-FM or Fluo-4 AM, as previously described [5], [7], and agonist-evoked increases in cellular fluorescence were monitored in groups of 1–3 isolated cells. A manually adjustable optical diaphragm was used to restrict the emitted fluorescent signal to the only the cell(s) of interest, thereby eliminating potential stray fluorescence from neighboring cells on the glass cover slip.
Results
We had previously reported that acute exposure of cultured human vascular endothelial cells to ATP evoked rapid Ca2+ mobilization, membrane hyperpolarization and de novo synthesis of NO [5], [6]. Pharmacologically, ATP is known to activate both metabotropic P2Y receptors (i.e. G-protein coupled) and ionotropic P2X receptors [2], [11]. In order to distinguish the purinergic receptor type primarily responsible for these functional responses observed in human endothelial cells, we examined the
Discussion
In peripheral vascular beds, the release of ATP from autonomic nerve endings and aggregating platelets may act on endothelial cells to produce transient vasodilatations that affect local blood flow patterns within a tissue [16]. We have previously reported that stimulation of vascular endothelial cells by purinergic agonist ATP leads to elevation of intracellular free Ca2+, and the subsequent activation of small and intermediate conduction, Ca-activated K+ channels, membrane hyperpolarization,
Conflict of interest statement
The authors declare no conflict of interest.
Acknowledgements
This work was supported by research funding awarded to A.P. Braun by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, N.W.T. and Nunavut.
References (23)
Pharmacological profiles of cloned mammalian P2Y-receptor subtypes
Pharmacol. Therap.
(2006)- et al.
EDHF: bringing the concepts together
TIPS
(2002) - et al.
Pharmacology of cloned P2X receptors
Annu. Rev. Pharmacol. Toxicol.
(2000) - et al.
Signal transduction in endothelium-dependent vasodilatation
Eur. Heart J.
(1993) - et al.
Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells
Am. J. Physiol. Cell Physiol.
(2007) - et al.
Estrogen- and the Ca2+-mobilizing agonist ATP evoke acute nitric oxide synthesis via distinct pathways in an individual human vascular endothelial cell
Am. J. Physiol. Cell Physiol.
(2008) - et al.
Openers of SKCa and IKCa channels enhance agonist-evoked endothelial nitric oxide synthesis and arteriolar dilation
FASEB J.
(2009) - et al.
DAF-FM (4-amino-5-methylamino-2′,7′-difluorofluorescein) diacetate detects impairment of agonist-stimulated nitric oxide synthesis by elevated glucose in human vascular endothelial cells: reversal by vitamin C and l-sepiapterin
J. Pharmacol. Exp. Ther.
(2005) Cleavage of structural proteins during the assembly of the head of bacteriophage T4
Nature
(1970)- et al.
Expression of both P1 and P2 purine receptor genes by human articular chondrocytes and profile of ligand-mediated prostaglandin E2 release
Arthritis Rheum.
(1999)
Receptors for purines and pyrimidines
Pharmacol. Rev.
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Present address: iCAPTURE Centre, St. Paul's Hospital, University of British Columbia, Vancouver, Canada V6Z 1Y6.
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Present address: Department of Reproductive Endocrinology, School of Medicine, Zhejiang University, Zhejiang Province 310006, China.