Abstract-Thrombin has been shown to activate endothelial NO synthase (eNOS) leading to endothelium-dependent vasorelaxation. In addition to its activation by Ca 2ϩ /calmodulin, eNOS has several regulatory sites. Ser 1179 phosphorylation of eNOS by the phosphatidylinositol 3-kinase-dependent Akt stimulates its catalytic activity. In this study, we have elucidated the signaling mechanism of thrombin-induced phosphorylation of eNOS in the regulation of NO production. Immunoblot analysis showed that thrombin rapidly phosphorylates eNOS at Ser 1179 in cultured bovine aortic endothelial cells. Also, thrombin was unable to stimulate eNOS if the Ser 1179 was mutated to Ala. Akt is phosphorylated in response to thrombin at Ser 473 at a later time point than eNOS. In this regard, a phosphatidylinositol 3-kinase inhibitor, LY294002, blocked Akt phosphorylation without affecting eNOS phosphorylation and cGMP production by thrombin. The Ca 2ϩ ionophore A23187 stimulated eNOS phosphorylation, as well as cGMP production, and pretreatment with intracellular or extracellular Ca 2ϩ chelators inhibited thrombin-induced eNOS phosphorylation and cGMP production. Moreover, infection of bovine aortic endothelial cell with adenovirus encoding dominant-negative mutants of protein kinase C (PKC)␣ and PKC␦ or pretreatment of bovine aortic endothelial cells with PKC inhibitors revealed that PKC␦ is indispensable for thrombin-induced eNOS phosphorylation and activation. From these data, we concluded that thrombin induces the Ser 1179 phosphorylation-dependent eNOS activation through a Ca 2ϩ -dependent, PKC␦-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway. (Hypertension. 2007;49:577-583.)

Abstract-Insulin resistance is an important risk factor in the development of cardiovascular diseases such as hypertension and atherosclerosis. However, the specific role of insulin resistance in the etiology of these diseases is poorly understood. Angiotensin (Ang) II is a potent vasculotrophic and vasoconstricting factor. We hypothesize that in vascular smooth muscle cells (VSMCs), Ang II interferes with insulin action by inhibiting Akt, a major signaling molecule implicated in the biological actions of insulin. By immunoblotting with a phospho-specific antibody for Akt, we found that Ang II inhibits insulin-induced Akt phosphorylation in a time-and concentration-dependent manner. The inhibitory effect of Ang II was blocked by a Ang II type 1 receptor antagonist, RNH6270. A protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, also inhibited insulin-induced Akt phosphorylation. PKC inhibitors, including Go6976 (specific for ␣-and ␤-isoforms), blocked the Ang II-and PMA-induced inhibition of Akt phosphorylation by insulin. Moreover, overexpression of PKC-␣ but not PKC-␤ isoform by adenovirus inhibited insulin-induced Akt phosphorylation. By contrast, an epidermal growth factor receptor inhibitor (AG1478), a p42/44 mitogen-activated protein kinase (MAPK) kinase inhibitor (PD 598,059), and a p38 MAPK inhibitor (SB 203,580) did not block the Ang II-induced inhibition of Akt phosphorylation. From these data, we conclude that Ang II negatively regulates the insulin signal, Akt, in the vasculature specifically through PKC-␣ activation, providing an alternative molecular mechanism that may explain the association of hyperinsulinemia with cardiovascular diseases.