Involvement of NADPH oxidase and protein kinase C in endothelin-1-induced superoxide production in retinal microvessels

Redox signaling has been implicated in pathophysiological changes in the vascular system. We examined whether endothelin-1 (ET-1) increases the formation of superoxide anions in retinal microvessels. Freshly isolated retinal microvessels from rats were exposed to ET-1 (100 nM), and the intracellular superoxide formation in the retinal pericytes was assessed semi-quantitatively by time-lapse fluorometric analyses using hydroethidine. The receptor mechanisms were determined by BQ-123 and BQ-788, receptor antagonists for ET_A and ET_B receptors, respectively, and also by IRL-1620, a selective agonist for ET_B receptors. In addition, the changes induced by adding apocynin (10 μM), myr-PKC (1.0 μM), allopurinol (100 μM), rotenone (10 μM), or L-NAME (100 μM) with ET-1 were evaluated. Microvessels were incubated with phorbol 12-myristate 13-acetate (PMA, 10 nM), a protein kinase C (PKC) activator. Fluorometric analyses showed ethidium fluorescence-positive regions that coincided well with the location of retinal pericytes. The intracellular superoxide levels were significantly increased after addition of ET-1 (100 nM), and this elevation was suppressed by apocynin or myr-PKC. Other enzyme inhibitors including L-NAME had no effect. The ET-1-induced increase of superoxide was significantly suppressed by BQ-123 (1.0 μM), while effects of adding BQ-788 (1.0 μM) were insignificant. IRL-1620 (100 nM) did not increase superoxide formation significantly. PMA (10 nM) mimicked the effect of ET-1. These results suggest that ET-1 increases the formation of superoxides in the retinal microvascular pericytes most likely by activating NADPH oxidase through ET_A receptors. The activation of PKC may be involved in the mechanism. Thus, ET-1 may augment its vasoconstrictive effects through the formation of superoxide, which may impair the bioavailability of nitric oxide in the retinal microvasculature.