The small G-protein Rac mediates depolarization-induced superoxide formation in human endothelial cells.
Superoxide anions impair nitric oxide-mediated responses and are involved in the development of hypertensive vascular hypertrophy. The regulation of their production in the vascular system is, however, poorly understood. We investigated whether changes in membrane potential that occur in hypertensive vessels modulate endothelial superoxide production. In cultured human umbilical vein endothelial cells, changes in membrane potential were induced by high potassium buffer, the non-selective potassium channel blocker tetrabutylammonium chloride (1 mm), and the non-selective cation ionophore gramicidin (1 micrometer). Superoxide formation was significantly elevated to a similar degree by all three treatments (by approximately 60%, n = 23, p < 0.01), whereas hyperpolarization by the K(ATP) channel activator Hoe234 (1 micrometer) significantly decreased superoxide formation. Depolarization also induced an increased 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 difficile toxin B blocked the effects of depolarization on superoxide formation. The tyrosine kinase inhibitor genistein (30 micrometer, n = 15) abolished depolarization-induced superoxide formation and also prevented depolarization-induced Rac translocation associated with it. It is concluded that depolarization is an important stimulus of endothelial superoxide production, which involves a tyrosine phosphorylation-dependent translocation of the small G-protein Rac.
Sohn
H Y
HY
Institute of Physiology, Ludwig Maximilians University Munich, 80336 Munich, Germany. sohn@lrz.uni-muenchen.de
Keller
M
M
Gloe
T
T
Morawietz
H
H
Rueckschloss
U
U
Pohl
U
U
D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology*; D019255:NADPH Oxidases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000502:physiology*
D013481:Superoxides; D014443:Tyrosine; D019255:NADPH Oxidases; D011493:Protein Kinase C; D020744:rac GTP-Binding Proteins