Elsevier

Cell Calcium

Volume 49, Issue 4, April 2011, Pages 240-248
Cell Calcium

Purinergic P2Y2 receptors mediate rapid Ca2+ mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells

https://doi.org/10.1016/j.ceca.2011.02.008Get rights and content

Abstract

In blood vessels, stimulation of the vascular endothelium by the Ca2+-mobilizing agonist ATP initiates a number of cellular events that cause relaxation of the adjacent smooth muscle layer. Although vascular endothelial cells are reported to express several subtypes of purinergic P2Y and P2X receptors, the major isoform(s) responsible for the ATP-induced generation of vasorelaxant signals in human endothelium has not been well characterized. To address this issue, ATP-evoked changes in cytosolic Ca2+, membrane potential and acute nitric oxide production were measured in isolated human umbilical vein endothelial cells (HUVECs) and profiled using established P2X and P2Y receptor probes. Whereas selective P2X agonist (i.e. α,β-methyl ATP) and antagonists (i.e. TNP-ATP and PPADS) could neither mimic nor block the observed ATP-evoked cellular responses, the specific P2Y receptor agonist UTP functionally reproduced all the ATP-stimulated effects. Furthermore, both ATP and UTP induced intracellular Ca2+ mobilization with comparable EC50 values (i.e. 1–3 μM). Collectively, these functional and pharmacological profiles strongly suggest that ATP acts primarily via a P2Y2 receptor sub-type in human endothelial cells. In support, P2Y2 receptor mRNA and protein were readily detected in isolated HUVECs, and siRNA-mediated knockdown of endogenous P2Y2 receptor protein significantly blunted the cytosolic Ca2+ elevations in response to ATP and UTP, but did not affect the histamine-evoked response. In summary, these results identify the P2Y2 isoform as the major purinergic receptor in human vascular endothelial cells that mediates the cellular actions of ATP linked to vasorelaxation.

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.

Section snippets

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.

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