ASSOCIATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE AND CAVEOLIN IS REGULATED BY AGONIST-STIMULATED TYROSINE PHOSPHORYLATION. † 1090

Abstract

An impairment in production of nitric oxide (NO) by the endothelial nitric oxide synthase (eNOS) appears to be an underlying cause of persistent pulmonary hypertension of the newborn (PPHN). A detailed understanding of eNOS regulation may therefore provide insights into the molecular defects associated with PPHN and other diseases characterized by abnormalities in eNOS activity or expression. In the present study, we have examined the role of tyrosine phosphorylation in agonist-activation and regulation of eNOS in cultured endothelial cells. Treatment of bovine aortic endothelial cells(BAEC) or bovine lung microvascular endothelial cells (BLMVEC) with bradykinin(10 μM), histamine (10 μM), or ATP (1 mM) produces a transient increase in detergent insolubilities of both eNOS and the plasmalemmal caveolae marker protein, caveolin. Agonist-stimulated changes in detergent solubilities are blocked by the tyrosine kinase inhibitor, geldanamycin and are mimicked by the tyrosine phosphatase inhibitors, phenylarsine oxide and sodium orthovanadate. Increases in detergent insolubilities of eNOS and caveolin are temporally correlated with increases in the amount of eNOS associated with caveolin. Bradykinin and phenylarsine oxide increase the association by 82% and 380%, respectively. Conversely, geldanamycin decreases association by 86%. Taken together, these results suggest that agonist-activation of eNOS in endothelial cells involves tyrosine phosphorylation-dependent formation of a detergent-insoluble complex of eNOS and caveolin. Complex formation of eNOS and caveolin in plasma membrane caveolae may have a role in regulation of eNOS activity or Ca2+-sensitivity or in increasing the efficiency of extracellular NO release.