OBJECTIVE: To determine whether endothelin-1 production is involved in the sustained superoxide production induced by angiotensin II.

DESIGN AND METHODS : Cultured vascular smooth muscle cells (VSMC) from Sprague-Dawley rats were treated with angiotensin II (10^–6M) or endothelin-1 (10^–7M) for 6 hours simultaneously with LU302872 (10^–7M), BQ-123 (10^–7M), actinomycin D (1 µg/ml) and apocynin (10^–4M). The production of superoxide anion and the NAD(P)H oxidase activity were evaluated by a chemiluminescence methods using lucigenin. The expression of the p47 NAD(P)H subunit was measured by western blot. In addition, aortas from rats with and without endothelium were isolated and incubated in culture media in the presence of angiotensin II . Endothelin-1 production was measured from the culture media of the tissues and the cells with an ELISA kit.

RESULTS: The non-selective blockade of endothelin receptors by LU302872 or the ETA blockade by BQ-123 prevented angiotensin II-induced superoxide production after 6 hours by 89% and 56% respectively. Actinomycin D, an inhibitor of protein synthesis, and apocynin, an NAD(P)H oxidase inhibitor, also prevented this production by 54% and 95% respectively. The NAD(P)H oxidase subunit p47 was also increased after an angiotensin II treatment and that increase was blunted with the treatment with LU302872 and BQ123. The production of endothelin by isolated aortas was found to be increased after chronic stimulation with angiotensin II even in the absence of endothelium.

CONCLUSIONS: Our results thus suggest that endothelin-1 synthesis is important in the sustained production of oxidative stress induced by angiotensin II presumably through NAD(P)H oxidase activation and expression. Moreover, the endothelin-1 production can be stimulated in smooth muscle cells by angiotensin II in the absence of endothelial cells.

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