Abstract
Activation of alpha1 adrenergic receptors stimulates mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine signaling pathways by which activation of alpha1 receptors may induce mitogenesis in HVSMCs, we have found that alpha1 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 alpha1 receptors stimulated PI 3-kinase, in vitro assays of kinase activity were performed in immunocomplexes precipitated by an antibody against the p85 alpha subunit of PI 3-kinase. Noradrenaline stimulated a time- and concentration-dependent activation of PI 3-kinase in the presence of a beta adrenergic receptor antagonist. Noradrenaline-stimulated PI 3-kinase activation was blocked by antagonists of alpha1 receptors and by pertussis toxin, suggesting that alpha1 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 alpha1 receptors activates PI 3-kinase in HVSMCs and that alpha1 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 alpha1 receptor-stimulated mitogenic responses including activation of MAP kinase and DNA synthesis in HVSMCs.
Highlights
To examine signaling pathways by which activation of ␣1 receptors may induce mitogenesis in human vascular smooth muscle cells (HVSMCs), we have found that ␣1 receptor stimulatedDNA synthesis and activation of mitogen-activated protein (MAP) kinase are blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase)
To determine whether the ␣1 receptor-mediated increase in [3H]thymidine incorporation was mediated via pertussis toxin-sensitive G proteins, cells were preincubated with pertussis toxin (100 ng/ml) for 12 h before stimulation with noradrenaline
Since MAP kinase has been postulated to play a key role in mediating mitogenic responses of many receptors, including ␣1 adrenergic receptors in myocytes, we examined ␣1 adrenergic receptor-mediated activation of MAP kinase in HVSMCs (Fig. 1B)
Summary
(Received for publication, December 7, 1995, and in revised form, January 29, 1996). Department of Medicine, Stanford University School of Medicine, Stanford, California 94305 and Veterans Affairs Medical Center, Palo Alto, California 94304. Recent evidence demonstrates that ␣1 receptor-stimulated mitogenic responses in myocytes may be due to activation of tyrosine protein kinases (TPKs) and MAP kinases (Thorburn et al, 1994), suggesting that ␣1 adrenergic receptors may share common signal pathways with tyrosine kinase receptors in the stimulation of mitogenesis. We have found that ␣1 adrenergic receptor-stimulated mitogenic responses, such as DNA synthesis and activation of MAP kinase in HVSMCs, are inhibited by wortmannin, a specific inhibitor of PI 3-kinase. This result suggests that activation of PI 3-kinase is associated with ␣1 adrenergic receptor-stimulated responses in HVSMCs. We further demonstrate directly that stimulation of ␣1 receptors activates PI 3-kinase via a pertussis toxin-sensitive G protein pathway. Noradrenaline-stimulated PI 3-kinase is associated with activation of Ras proteins and tyrosine protein kinases
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