NADPH OXIDASE‐MEDIATED O 2 −· PRODUCTION AMPLIFIES VASOCONSTRICTOR RESPONSE OF SMALL CORONARY ARTERIES TO M 1 ‐MUSCARINIC RECEPTOR ACTIVATION

Abstract

The present study test the hypothesis that NADPH oxidase (Nox) mediated O2−· production amplifies coronary arterial constrictor response to M1-receptor (M1R) agonists. Fluorescence imaging analysis showed that M1R agonist, acetylcholine (Ach), simultaneously increased intracellular Ca2+ and O2−· levels in bovine coronary arterial smooth muscle cells (CASMCs) with a maximal increase of 821 + 67 nM in Ca2+ levels accompanied by 75% in O2−· production. However, when cells were transfected with siRNA of p47phox(a key subunit for Nox activation), Ach-induced increases in Ca2+ and O2−· were significantly attenuated. Similarly, p47phoxsiRNA significantly reduced oxotremorine (OXO)-induced O2−· production in CASMCs by 67%. Functionally, OXO-induced constrictor response of endothelium-denuded small bovine coronary arteries was significantly reduced by Nox inhibitor, DPI or O2−· scavenger cocktail (tempol + catalase). Moreover, introduction of siRNA for p47phox, NOX1 and NOX4 into arterial smooth muscle by ultrasound microbubble technology significantly blocked this OXO-induced contraction. We concluded that Nox-derived O2−· is involved in M1R-mediated intracellular Ca2+ release in CASMCs and thereby importantly contributes to amplification of vasoconstrictor response of coronary arteries to M1R activation (Supported by NIH grants HL057244, HL075316 and AHA predoctoral fellowship 0410061Z).