Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation

Endothelial nitric-oxide synthase (eNOS) is phosphorylated at Ser-1179 (bov ine sequence) by Akt after growth factor or shear stress stimulation of end othelial cells, resulting in increased eNOS activity. Purified eNOS is also phosphorylated at Thr-497 by purified AMP-activated protein kinase, result ing in decreased eNOS activity, We investigated whether bradykinin (BK) sti mulation of bovine aortic endothelial cells (BAECs) regulates eNOS through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortm annin and LY294002, inhibitors of phosphatidylinositol 3-kinase, suggesting that Akt activation occurs downstream from phosphatidylinositol 3-kinase, BK stimulates a transient phosphorylation of eNOS at Ser-1179 that is corre lated temporally with a transient dephosphorylation of eNOS at Thr-497. Pho sphorylation at Ser-1179, but not dephosphorylation at Thr-497, is blocked by wortmannin and LY294002. BK also stimulates a transient nitric oxide (NO ) release from BAECs with a time-course similar to Ser-1179 phosphorylation and Thr-497 dephosphorylation, NO release is not altered by wortmannin. BK -stimulated dephosphorylation of Thr-497 and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily involves deinhibition of the enzyme through cal cineurin-mediated dephosphorylation at Thr-497.