Phosphatidylinositol 3-kinase may mediate isoproterenol-induced vascular relaxation in part through nitric oxide production

Abstract

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 PI3-K in G-protein-coupled receptor signal transduction has also been reported. As beta2 -adrenergic vascular actions are partly dependent on NO, this study the role of PI3-K on in vitro isoproterenol (Iso)-induced endothelial cell (EC) nitric oxide synthase (NOS) activation and rat aortic vascular relaxation. Cell lysates of rat aortic EC (RAEC), exposed to Iso (10 micromol/L) for 5 minutes, were immunoprecipitated with an antiphosphotyrosine antibody prior to assay for Western blot for the p85-kd regulatory subunit of PI3-K. Endothelial NOS activity was determined by measuring nitrite production. Endothelium-intact aortic rings from male Wistar rats were preincubated with the PI3-K inhibitors, wortmannin (WT), or LY294002 (LY), precontracted with phenylepinephrine (PE), and relaxation to graded doses of Iso was measured. NO contribution to vascular relaxation was assessed by L-N(G)-nitroarginine methyl ester (L-NAME), a NOS inhibitor. Both Iso and IGF-1 induced an increase in p85 subunit phosphorylation as demonstrated by Western analysis, effects inhibited by preincubation with WT. Iso also enhanced association of p85 with the Triton X-100-insoluble fraction of RAEC, reflecting translocation of this enzyme to a cytoskeletal fraction. In addition, Iso as well as IGF-1 significantly increased eNOS activity measured by nitrite production. Both WT and LY markedly inhibited relaxation to Iso, while L-NAME nearly abolished this beta-adrenergic-mediated vasorelaxation. These data indicate that both Iso and IGF-1 activate the EC PI3-K pathway which mediates, in part, the release of NO and subsequent vasorelaxation in response to this beta-agonist Iso as well as to IGF-1.