Autocrine/paracrine pattern of superoxide production through NAD(P)H oxidase in coronary arterial myocytes

Autocrine/ paracrine pattern of superoxide production through NAD(P)H oxidase in coronary arterial myocytes. Am J Physiol Heart Circ Physiol 292: H483-H495, 2007. First published September 8, 2006; doi:10.1152/ajpheart. 00632.2006.-The present study tested the hypothesis that membrane-bound NAD(P) H oxidase in coronary arterial myocytes (CAMs) is capable of producing superoxide (O-2(center dot-)) toward extracellular space to exert an autocrine- or paracrine-like action in these cells. Using a high-speed wavelength-switching fluorescent microscopic imaging technique, we simultaneously monitored the binding of dihydroethidium-oxidizing product to exogenous salmon testes DNA trapped outside CAMs and to nuclear DNA as indicators of extra- and intracellular O2(center dot-) production. It was found that a muscarinic agonist oxotremorine (OXO; 80 mu M) increased O2(center dot-) levels more rapidly outside than inside CAMs. In the presence of superoxide dismutase (500 U/ml) plus catalase (400 U/ml) and NAD(P) H oxidase inhibitor diphenylene iodonium (50 mu M) or apocynin (100 mu M), these increases in extra- and intracellular O2(center dot-) levels were substantially abolished or attenuated. The O2(center dot-) increase outside CAMs was also confirmed by detecting oxidation of nitro blue tetrazolium and confocal microscopic localization of Matrigel-trapped OxyBURST H2HFF Green BSA staining around these cells. By electron spin resonance spectrometry, the extracellular accumulation of O-2(center dot-) was demonstrated as a superoxide dismutase-sensitive component outside CAMs. Furthermore, RNA interference of NAD(P) H oxidase subunits Nox1 or p47 markedly blocked OXO-induced increases in both extra- and intracellular O-2(center dot-) levels, whereas small inhibitory RNA of Nox4 only attenuated intracellular O-2(center dot-) accumulation. These results suggest that Nox1 represents a major NAD(P) H oxidase isoform responsible for extracellular O-2(center dot-) production. This rapid extracellular production of O-2(center dot-) seems to be unique to OXO-induced M-1-receptor activation, since ANG II-induced intra-and extracellular O-2(center dot-) increases in parallel. It is concluded that the outward production of O-2(center dot-) via NAD(P) H oxidase in CAMs may represent an important producing pattern for its autocrine or paracrine actions.