title: Angiotensin II stimulates vimentin phosphorylation via a Ca2+-dependent, protein kinase C-independent mechanism in cultured vascular smooth muscle cells.
abstract: Intermediate filaments have been proposed, via phosphorylation by protein kinase C, to be involved in sustained contraction of smooth muscle. We examined the effect of angiotensin II on the phosphorylation of the intermediate filament protein, vimentin, in cultured rat aortic vascular smooth muscle cells. Angiotensin II induced phosphorylation of a Triton X-100- and high salt-insoluble protein with a molecular weight of 58,000. This protein was identified as vimentin based on its specific interaction with anti-vimentin antibody as detected by immunoblot analysis. Angiotensin II-induced phosphorylation of vimentin was time- and dose-dependent. Phosphorylation was detectable at 15 s, peaked at 2 min after angiotensin II stimulation, and gradually declined to a new plateau which was sustained for at least 30 min. The threshold, half-maximal and maximal concentrations of angiotensin II that stimulated vimentin phosphorylation were 0.01, 0.1, and 10 nM, respectively. The Ca2+ ionophore, ionomycin, stimulated vimentin phosphorylation to the same extent as angiotensin II, whereas the protein kinase C-activating phorbol ester, phorbol 12-myristate 13-acetate, had only marginal effects on this reaction. Pretreatment of the cells with [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid attenuated angiotensin II- and ionomycin-induced vimentin phosphorylation to the same extent. Down-regulation of protein kinase C induced by prolonged treatment of the cells with phorbol 12,13-dibutyrate did not inhibit angiotensin II-induced vimentin phosphorylation. These results indicate that angiotensin II stimulates vimentin phosphorylation via a Ca2+-dependent, protein kinase C-independent mechanism in vascular smooth muscle cells and suggest that cytoskeletal proteins are major targets for angiotensin II-induced phosphorylation events.
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D001013:Aorta, Thoracic / Q000201:enzymology; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D007381:Intermediate Filament Proteins / Q000302:isolation & purification; D007382:Intermediate Filaments / Q000378:metabolism; D015759:Ionomycin; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009206:Myocardium / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014746:Vimentin / Q000378:metabolism*
chemical_list: D004987:Ethers; D007381:Intermediate Filament Proteins; D014746:Vimentin; D000804:Angiotensin II; D004533:Egtazic Acid; D015759:Ionomycin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium