Phosphatidylinositol 3-kinase (PI3-K) has been shown to mediate insulin and insulin-like growth factor-1 (IGF-1)-induced nitric oxide (NO) generation and, thus, vascular tone. A role for P13-K in G-protein-coupled receptor signal transduction has been reported. As beta (beta (2))-adrenergic vascular actions are partly dependent on NO, we have investigated the role of P13-K in isoproterenol. (Iso) and IGF-1 induced endothelial NO synthase (ecNOS) activity in rat aortic endothelial cells (RAEC). Cell lysates of RAEC, exposed to Iso (10 mu mol/L) for 5 min and 6 h, and to IGF-1 (100 nM) for 10 min and 6 h, or pretreated with P13-K inhibitor Wortmannin (WT17), were used for measuring P13-K activity, p85kDa regulatory protein, and citrulline production. Results show that Iso and IGF-1 increased a p85 subunit and citrulline production, and also enhanced P-32 incorporation into PIP,. Pretreatment with WT inhibited Iso-stimulated ecNOS, as well as, P13-K activity. Iso enhanced association of ecNOS with the triton X-100-insoluble fraction of RAEC. These data indicate that the endothelial cell P13-K pathway mediates, in part, the release of NO and subsequent vasorelaxation in response to this beta -agonist, as well as, IGF-1.