Summary

Little is known about the signaling cascades that eventuallyregulate the activity of the endothelial nitric oxide synthase(eNOS) in platelets. Here, we investigated the effects of insulinon the phosphorylation and activation of eNOS in washedhuman platelets and in endothelial cells.Insulin activated the protein kinase Akt in cultured endothelialcells and increased the phosphorylation of eNOS on Ser¹¹⁷⁷ butfailed to increase endothelial cyclic GMP levels or to elicit therelaxation of endothelium-intact porcine coronary arteries. Inplatelets, insulin also elicited the activation of Akt as well as thephosphorylation of eNOS and initiated NO production whichwas associated with increased cyclic GMP levels and the inhibitionof thrombin-induced aggregation.The insulin-induced inhi-bitionof aggregation was accompanied by a decreased Ca²⁺response to thrombin and was also prevented by N^®. nitro-L-arginine. In platelets, but not in endothelial cells, insulin inducedthe activation of the AMP-activated protein kinase (AMPK), ametabolic stress-sensing kinase which was sensitive to thephosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin andthe AMPK inhibitor iodotubercidin. Moreover, the insulin-medi-atedinhibition of thrombin-induced aggregation was preventedby iodotubercidin. Insulin-independent activation of theAMPK using 5-aminoimidazole-4-carboxamide ribonucleoside,increased platelet eNOS phosphorylation, increased cyclicGMP levels and attenuated platelet aggregation.These results highlight the differences in the signal transductioncascade activated by insulin in endothelial cells and platelets,and demonstrate that insulin stimulates the formation of NO inhuman platelets, in the absence of an increase in Ca²⁺, by acti-vatingPI3-K and AMPK which phosphorylates eNOS on Ser¹¹⁷⁷.