α₁ Adrenergic receptors activate phosphatidylinositol 3-kinase in human vascular smooth muscle cells: Role in mitogenesis

Activation of α₁ adrenergic receptors stimulates mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine signaling pathways by which activation of α₁ receptors may induce mitogenesis in HVSMCs, we have found that α₁ receptor stimulated-DNA synthesis and activation of mitogen- activated protein (MAP) kinase are blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). To determine directly if activation of α₁ receptors stimulated PI 3-kinase, in vitro assays of kinase activity were performed in immunocomplexes precipitated by an antibody against the p85α subunit of PI 3-kinase. Noradrenaline stimulated a time- and concentration-dependent activation of PI 3-kinase in the presence of a β adrenergic receptor antagonist. Noradrenaline-stimulated PI 3-kinase activation was blocked by antagonists of α₁ receptors and by pertussis toxin, suggesting that α₁ receptors activate PI 3-kinase via a pertussis toxin-sensitive G protein. Direct activation of protein kinase C by a phorbol ester did not stimulate PI 3-kinase; also, a Ca²⁺ L-channel blocker did not inhibit noradrenaline-stimulated PI 3-kinase activity. Increased PI 3-kinase activity was detected in both anti-Ras and anti-phosphotyrosine immunoprecipitates from noradrenaline-stimulated HVSMCs. Moreover, noradrenaline stimulated formation of active Ras-GTP complexes. Because blockade of PI 3-kinase by wortmannin inhibited formation of this complex, this result suggests that Ras might be a target of PI 3-kinase. Noradrenaline stimulated tyrosine phosphorylation of the p85 subunit of PI 3-kinase, and a phosphorylated tyrosine protein could be co-immunoprecipitated with anti-p85 of PI 3-kinase. These results demonstrate that stimulation of α₁ receptors activates PI 3-kinase in HVSMCs and that α₁ receptor-activated PI 3-kinase is associated with an increase in active Ras-GTP and activation of tyrosine protein phosphorylation. These pathways may contribute to α₁ receptor- stimulated mitogenic responses including activation of MAP kinase and DNA synthesis in HVSMCs.