Effect of Ang II on the generation of superoxide anion via NAD(P)H Oxidase enzyme complex in primary cultured vascular smooth muscle cells isolated from spontaneously hypertensive and normotensive rat aortas, and role of Src in this metabolic pathway

It is known that superoxide anion has an important role in progressing hypertension. Ang II contributes to altered vascular tone, endothelial dysfunction, structural remodeling and vascular inflammation, characteristic features of vascular damage in hypertension, atherosclerosis and diabetes. Ang II induces its effects via at least two G-protein-coupled transmembrane receptors, Ang II receptor 1 (AT1R) and Ang II receptor 2 (AT2R), of which the former is considered to be more physiologically important. The best characterized system in vascular cells is Ang II-stimulated NAD(P)H oxidase-mediated generation of superoxide anion, which appears to be upregulated in hypertension.In our present study, vascular smooth muscle cells (VSMC) were isolated from thoracic aortas of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Vascular smooth muscle cells, to determine the AT1R-mediated Src phosphorylation and superoxide anion production via NADPH oxidase, were stimulated with Ang II with or without Losartan (AT1 receptor blocker), diphenyl iodonium (DPI; NAD(P)H oxidase inhibitor), PP1 (a selective Src inhibitor) and GF109203X (PKC inhibitor). Src phosphorylation was determined by western blot method. Superoxide anion production was measured by cytochrom c reduction method.Src phosphorylation increased both SHR and WKY groups after Ang II stimulation compared with controls. On the other hand, Src phosphorylations were decreased both SHR and WKY groups after Ang II stimulation with inhibitors except DPI. Superoxide anion production was enhanced in both two groups in Ang II-stimulated cells. However superoxide anion production in SHR was significantly greater than WKY. All inhibitors reduced superoxide anion production both SHR and WKY groups in spite of Ang II stimulation.According to our results, we found that Src and PKC phosphorylation may occur before the NAD(P)H activation. We showed that Ang II induced NADPH oxidase via Src and this mechanism may be important in developing drugs and medicine against hypertension.