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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2045.)
© 2000 American Heart Association, Inc.

Vascular Biology

Stimulation of Receptor-Mediated Nitric Oxide Production by Vanadate

Gary R. Hellermann; Brenda R. Flam; Duane C. Eichler; Larry P. Solomonson

From the Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa.

Correspondence to Larry P. Solomonson, Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, 12901 Bruce B. Downs Blvd, MDC Box 7, Tampa, FL 33612-4799. E-mail lsolomon{at}hsc.usf.edu

Abstract—Nitric oxide (NO) production by endothelial cells in response to bradykinin (Bk) treatment was markedly and synergistically enhanced by cotreatment with sodium orthovanadate (vanadate), a phosphotyrosine phosphatase inhibitor. This enhancement was blocked by tyrosine kinase inhibitors. Calcium ionophore– (A23187) activated production of NO was also enhanced by cotreatment with vanadate. No significant changes were found in total endothelial NO synthase (eNOS) protein or in eNOS distribution between membrane (caveolae) and cytosolic fractions in response to the various treatments. Vanadate had no direct effect on eNOS activity, and lysates prepared from cells treated with vanadate showed little change in specific activity of eNOS. Western blots of immunoprecipitated eNOS showed the presence of a major tyrosine-phosphorylated protein band at a mass corresponding to 125 kDa and 2 minor bands corresponding to 105 and 75 kDa after treatment with vanadate/Bk. No tyrosine phosphorylation of eNOS after treatment with vanadate/Bk was observed. Geldanamycin, an inhibitor of heat shock protein 90, also inhibited the enhancement of NO production by vanadate/Bk or vanadate/A23187, and there was an increase in the amount of heat shock protein 90 that coimmunoprecipitated with eNOS after treatment with vanadate/Bk. These results show that there is a clear link between tyrosine phosphorylation and stimulation of eNO production, which does not appear to involve direct modification of eNOS, changes in eNOS levels, or compartmentation, but rather appears to be due to changes in proteins associating with eNOS, thereby enhancing the state of activation of eNOS.

Key Words: nitric oxide • endothelial • vanadate • bradykinin • tyrosine phosphorylation

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