α₁-Adrenergic receptor stimulation of mitogenesis in human vascular smooth muscle cells: Role of tyrosine protein kinases and calcium in activation of mitogen-activated protein kinase

Signaling pathways of many G protein-coupled receptors overlap with those of receptor tyrosine kinases. We have found previously that α₁- adrenergic receptors stimulate DNA synthesis and cell proliferation in human vascular smooth muscle cells; these effects were attenuated by the tyrosine protein kinase (TPK) inhibitor genistein and the mitogen-activated protein kinase (MAPK) antagonist 2-aminopurine. Experiments were designed to determine if activation of α₁ receptors directly stimulated TPKs and MAPKs in human vascular smooth muscle cells. Norepinephrine stimulated time- and concentration-dependent tyrosine phosphorylation of multiple proteins, including p52-, 75-, 85-, 120-, and 145-kDa proteins. Increased TPK activity was demonstrated in proteins precipitated by an antiphosphotyrosine antibody, both in autophosphorylation assays and with a peptide substrate. These effects of norepinephrine were completely blocked by α₁ receptor antagonists. A membrane-permeable Ca²⁺ chelator [1,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester], completely blocked norepinephrine stimulation of phosphorylation of tyrosine proteins, suggesting that intracellular Ca²⁺ plays a critical role in α₁ receptor stimulation phosphorylation of tyrosine proteins. Of the tyrosine- phosphorylated proteins, the results suggest that two of them are PLCγ1 and adapter protein Shc. Also, α₁ receptor stimulation caused a time-dependent increase in MAPK activity due to increased phosphorylation of p42/44(ERK1/2). The α₁ receptor-mediated activation of MAPK was also attenuated by TPK inhibitors and intracellular Ca²⁺ chelator [1,2-bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester]. These results suggest that phosphorylation of tyrosine proteins and intracellular Ca²⁺ plays a critical role in α₁ receptor-stimulated MAPK signaling pathways, potentially contributing to increased DNA synthesis and cell proliferation.