alpha(1)-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 o f receptor tyrosine kinases. We have found previously that alpha(1)-adrener gic receptors stimulate DNA synthesis and cell proliferation in human vascu lar smooth muscle cells; these effects were attenuated by the tyrosine prot ein kinase (TPK) inhibitor genistein and the mitogen-activated protein kina se (MAPK) antagonist 2-aminopurine. Experiments were designed to determine if activation of ct, receptors directly stimulated TPKs and MAPKs in human vascular smooth muscle cells. Norepinephrine stimulated time- and concentra tion-dependent tyrosine phosphorylation of multiple proteins, including p52 -, 75-, 85-, 120-, and 145-kDa proteins. Increased TPK activity was demonst rated in proteins precipitated by an antiphosphotyrosine antibody, both in autophosphorylation assays and with a peptide substrate. These effects of n orepinephrine were completely blocked by alpha(1) receptor antagonists. A m embrane-permeable Ca2+ chelator [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-te traacetic acid tetra(acetoxymethyl)ester], completely blocked norepinephrin e stimulation of phosphorylation of tyrosine proteins, suggesting that intr acellular Ca2+ plays a critical role in alpha(1) receptor stimulation phosp horylation of tyrosine proteins. Of the tyrosine-phosphorylated proteins, t he results suggest that two of them are PLC gamma 1 and adapter protein Shc . Also, alpha(1) receptor stimulation caused a time-dependent increase in M APK activity due to increased phosphorylation of p42/44(ERK1/2) The alpha(1 ) receptor-mediated activation of MARK was also attenuated by TPK inhibitor s and intracellular Ca2+ chelator [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid tetra(acetoxymethyl)ester]. These results suggest that pho sphorylation of tyrosine proteins and intracellular Ca2+ plays a critical r ole in alpha(1) receptor-stimulated MARK signaling pathways, potentially co ntributing to increased DNA synthesis and cell proliferation.