Elsevier

Nitric Oxide

Volume 16, Issue 1, February 2007, Pages 104-109
Nitric Oxide

Effect of endogenous and exogenous nitric oxide on calcium sparks as targets for vasodilation in rat cerebral artery

https://doi.org/10.1016/j.niox.2006.06.007Get rights and content

Abstract

The potent vasodilator nitric oxide (NO), produced mainly by the endothelium, acts through a BKCa-dependent mechanism to increase the frequency of calcium sparks (Ca2+ sparks) in myocyte isolated from rat cerebral arteries. Our present aim has been to assess the role of endogenous and exogenous NO on the Ca2+ sparks through ryanodine-sensitive channels in the sarcoplasmic reticulum of an intact artery. Calcium sparks, detected with fluo-4 and laser scanning confocal microscopy, were examined in isolated pressurized rat posterior cerebral arteries with (intact) and without endothelium (denuded). Addition of the NO donor, DEA-NONOate (N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine), did not change the amplitude and frequency of Ca2+ sparks in the intact artery. However, inhibition of nitric oxide synthase with N-ω-nitro-l-arginine or removal of endothelium reduced Ca2+ sparks frequency by about 50%. Under these conditions (i.e., absence of endogenous NO production), DEA-NONOate, increased Ca2+ spark frequency 3- to 4-fold. These results suggest that endothelial NO modulates local Ca2+ release events in the arterial smooth muscle and that this mechanism may contribute to the actions of nitrovasodilators.

Introduction

Ca2+ sparks represent the local, rapid and transient Ca2+ release from sarcoplasmic reticulum (SR) through ryanodine-sensitive (RyR) channels [1] and are thought to be elementary calcium signals resulting from the opening of a cluster of RyR channels. Ca2+ sparks in smooth muscle cells were first described in myocytes from rat cerebral arteries [2], and have subsequently been measured in smooth muscle cells from several different arteries [3], [4]. In arterial smooth muscle cells, RyR channels are mostly located in the subsarcolemmal region close to the cell membrane. The close spatial localization allows a special communication between the RyR channels and the sarcolemmal large-conductance Ca2+-sensitive K+ channels (BKCa) [5]. A single Ca2+ spark is capable of producing a high subsarcolemmal increase in Ca2+ which activates 20–30 nearby BKCa channels [6]. A single Ca2+ spark through activation of BKCa channels hyperpolarizes the membrane potential by 20–30 mV, which closes voltage-dependent Ca2+ channels and leads to smooth muscle relaxation [7]. The triumvirate of dihydropyridine-sensitive voltage-dependent Ca2+ channels, RyR channels, and BKCa act as a functional unit to regulate vascular tone by controlling the level of smooth muscle cell [Ca2+]i [8]. Several studies have shown that different vasodilators, as well as the potent vasodilator Nitric oxide (NO), act through a BKCa-dependent mechanism. Further, recent evidence suggests that sodium nitroprusside, a donor of NO, increases Ca2+ spark frequency in myocytes isolated from rat cerebral arteries [9]. Since NO, produced mainly by the endothelium, is an important endogenous vasodilator, we investigated the effect of NO on Ca2+ sparks in an intact artery. Intact arteries are a more physiological model that allows us to distinguish between the effects of endogenous and exogenous NO on Ca2+ sparks. Our data show that in intact arteries endogenous NO modulates Ca2+ spark frequency while exogenous NO increases Ca2+ sparks only in arteries pre-treated with N-ω-nitro-l-arginine (l-NNA) and in arteries without endothelium (denuded). Given that NO plays an important role in the autoregulation of blood flow, this finding provides insight into the cellular signalling mechanisms of NO vasodilatation that can lead to a better understanding of the regulation of vascular tone.

Section snippets

Tissue preparation

Adult female rats (∼250 g) were euthanized with sodium pentobarbital followed by thoracotomy as approved by the Office of Animal Care Management at the University of Vermont. Posterior cerebral arteries were removed from the brain and placed in cold Hepes-buffered saline. After removing the connective tissue, segments of the artery (2–4 mm in length; 50–120 μm) were placed in a chamber specially designed to measure Ca2+ responses in pressurized arteries. Arteries were tied to glass cannulae

Nitric oxide increases open probability of RyR channels (Ca2+ spark frequency)

To investigate the effect of nitric oxide (NO) on Ca2+ release from the SR, Ca2+ sparks were measured in pressurized, intact isolated posterior cerebral arteries (rPCA), in the presence and absence of DEA-NONOate (10 μM). This NO donor generates a controlled release of NO in solution, dilating pre-constricted rat middle cerebral artery in a dose-dependent manner by 79% at 10 μM [11]. Ca2+ spark frequency (Fig. 1) and amplitude (Table 1) were unchanged by this concentration of DEA-NONOate in rPCA.

Discussion

The present study reveals, for the first time, an effect of NO on Ca2+ spark frequency in isolated, pressurized rat posterior cerebral arteries. The inhibition of vascular NO by l-NNA or by removing the endothelium significantly reduced Ca2+ spark frequency. While, exogenous NO released from the nitrovasodilator, NONOate, did not alter Ca2+ spark frequency in intact arteries unless endogenous NO was inhibited. The results of this study, on pressurized intact isolated posterior cerebral

Acknowledgments

This work was supported by National Institute of Health Grants HL63722 and HL44455 and the Totman Trust for Medical Research to M.T. Nelson.

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