The small G-protein Rac mediates depolarization-induced superoxide formation in human endothelial cells

Superoxide anions impair nitric oxide-mediated responses and are involved i n the development of hypertensive vascular hypertrophy, The regulation of t heir production in the vascular system is, however, poorly understood. We i nvestigated whether changes in membrane potential that occur in hypertensiv e vessels modulate endothelial superoxide production, In cultured human umb ilical vein endothelial cells, changes in membrane potential were induced b y high potassium buffer, the non-selective potassium channel blocker tetrab utylammonium chloride (1 mM), and the non-selective cat ion ionophore grami cidin (1 mu M). Superoxide formation was significantly elevated to a simila r degree by all three treatments (by similar to 60%, n = 23, p < 0.01), whe reas hyperpolarization by the K-ATP channel activator Hoe234 (1 mu M) signi ficantly decreased superoxide formation. Depolarization also induced an inc reased tyrosine phosphorylation of several not yet identified proteins (90- 110 kDa) and resulted in a significant increase in membrane association of the small G-protein Rac. Accordingly, the Rac inhibitor Clostridium diffici le toxin B blocked the effects of depolarization on superoxide formation. T he tyrosine kinase inhibitor genistein (30 mu M, n = 15) abolished depolari zation-induced superoxide formation and also prevented depolarization-induc ed Rac translocation associated with it. It is concluded that depolarizatio n is an important stimulus of endothelial superoxide production, which invo lves a tyrosine phosphorylation-dependent translocation of the small G-prot ein Rac.