Involvement Of Phosphatidylinositol 3-kinase Signaling In Angiotensin II-and Insulin-induced Hypertrophy Of Vascular Smooth Muscle Cells

Objective: To investigate the role of phosphatidylinositol 3-kinase ( PI3K ) pathway in angiotensin II ( Ang II ) - and insulin-induced protein synthesis, and to study the mediation of this pathway in the phosphorylat ion of 4E-binding protein 1 ( 4E-BP1 ) and p70 S6 kinase in vascular smooth muscle cells.Methods: Vascular smooth muscle cells from 8-week-old Sprague-Dawley rats were cultured. ( 1 ) Protein synthesis was measured by [3H]leucine incorporation. ( 2 ) The phosphorylation of PKB, 4E-BP1 and p70 S6 kinase was detected by Western blotting.Results: 1. 100 nmol/L Ang II and 100 nmol/L insulin increased [3H]leucine incorporation in vascular smooth muscle cells when compared with control ( Ang II: 8.00 ?0.54 vs 5. 15 ?0. 26 X 103 cpm, P < 0.05; insulin: 6.13 ?0.31 vs 5.05 ?0.35 X 103 cpm, P < 0.05 ) . Inclusion of PI3K inhibitor, LY294002, at theconcentrations of 0. 1 u mol/L, 1 u raol/L and 10 u. mol/L, decreased Ang Il-stimulated [3H]leucine incorporation by 38 % ( P< 0. 05 ), 62 % ( P< 0. 05 ) and 92 % ( P< 0. 05 ) respectively. LY294002 at similar concentrations suppressed insulin-stimulated [3H]leucine incorporation by 43 % ( P < 0. 05 ), 57 % ( P < 0. 05 ) and 97 % ( P < 0. 05 ) respectively.2. Addition of Ang II or insulin to quiescent vascular smooth muscle cells significantly increased the phosphorylation of PKB. The stimulation of PKB phosphorylation by Ang II and by insulin was maximal at 5 min and 30 min, respectively. LY294002 exhibited inhibitory effect on PKB phosphorylation in a dose ( 0. 1 n mol/L-10 u mol/L ) -dependent manner.3. The basal level of the phosphorylation of 4E-BP1 and p70 S6 kinase was low. Ang II and Insulin induced the increase of 4E-BP1 and p70 S6 kinase phosphorylation. The change of 4E-BP1 phosphorylation was rapid and transitory, which peaked at 10 min ( Ang II ) and 30 min ( insulin ) , and then decreased quickly. The increase of p70 S6 kinase phosphorylation was rapid and lasting, with a peak occurring at 30 min ( Ang II ) and 10 min ( insulin ) , and then declined slowly. LY294002 suppressed both 4E-BP1 and p70 S6 kinase phosphorylation induced by Ang II and insulin in a dose ( 0. 1 u mol/L-10 u mol/L ) -dependent manner.Conclusion: ( 1 ) Ang II and insulin stimulated protein synthesis in vascular smooth muscle cells. ( 2 ) Ang II and insulin induced the phosphorylation of 4E-BP1 and p70 S6 kinase. ( 3 ) PI3K was involved in Ang II-induced and insulin-induced protein synthesis and the phosphorylation of 4E-BP1 and p70 S6 kinase in vascular smooth muscle cells, indicating PI3K pathway mediated the hypertrophic effect of Ang II and insulin.