alpha1-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 alpha1-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 alpha1 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 alpha1 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 alpha1 receptor stimulation phosphorylation of tyrosine proteins. Of the tyrosine-phosphorylated proteins, the results suggest that two of them are PLCgamma1 and adapter protein Shc. Also, alpha1 receptor stimulation caused a time-dependent increase in MAPK activity due to increased phosphorylation of p42/44(ERK1/2). The alpha1 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 alpha1 receptor-stimulated MAPK signaling pathways, potentially contributing to increased DNA synthesis and cell proliferation.
Hu
Z W
ZW
Department of Medicine, Stanford University School of Medicine, and Veterans Affairs Palo Alto Health Care System, Palo Alto, California, USA. huzhwei@leland.stanford.edu
Shi
X Y
XY
Lin
R Z
RZ
Chen
J
J
Hoffman
B B
BB
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D008934:Mitogens / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects; D009638:Norepinephrine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D008934:Mitogens; D019000:Phosphotyrosine; D004247:DNA; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium; D009638:Norepinephrine