Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries

In rat mesenteric arteries, the ability of ACh to evoke hyperpolarization of smooth muscle cells and consummate dilatation relies on an increase in endothelial cell cytosolic free [Ca2+] and activation of Ca2+-activated K+ channels (K-Ca). The time course of average and spatially organized rises in endothelial cell [Ca2+](i) and concomitant effects on membrane potential were investigated in individual cells of pressurized arteries and isolated sheets of native cells stimulated with ACh. In both cases, ACh stimulated a sustained and oscillating rise in endothelial cell [Ca2+](i). Overall, the oscillations remained asynchronous between cells, yet occasionally localized intercellular coordination became evident. In pressurized arteries, repetitive waves of Ca2+ moved longitudinally across endothelial cells, and depended on Ca2+-store refilling. The rise in endothelial cell Ca2+ was associated with sustained hyperpolarization of endothelial cells in both preparations. This hyperpolarization was also evident when recording from smooth muscle cells in pressurized arteries, and from resting membrane potential, selective inhibition of small-conductance K-Ca (SKCa) with apamin (50 nM) was sufficient to inhibit this response. In the presence of phenylephrine-tone, both aparnin and the selective inhibitor of intermediate conductance K-Ca (IKCa) TRAM-34 (1 mu M) were required to inhibit the non-nitric oxide-mediated dilatation to ACh. When hyperpolarization of endothelial cells was fully prevented either with inhibitors of K-Ca or in KCl (35 mM)-depolarized cells, both the time course and frequency of oscillations in endothelial cell [Ca2+](i) to ACh were unaffected. Together, these data show that although a rise in endothelial cell [Ca2+](i) stimulates hyperpolarization, depletion of intracellular stores with ACh stimulates Ca2+-influx which is not significantly influenced by the increase in cellular electrochemical gradient for Ca2+ caused by that hyperpolarization. (c) 2005 Elsevier Ltd. All rights reserved.