Regulation of MAP kinase activity by growth stimuli in vascular smooth muscle.
Intracellular signaling pathways regulating vascular smooth muscle (VSM) cell growth and hypertrophy can be initiated by activation of receptor tyrosine kinases and/or protein kinase C (PKC). Mitogen-activated protein kinases (MAP kinases) are cytosolic serine/threonine kinases, proposed to act as a point of convergence for diverse growth factors utilizing these signaling pathways. The goals of this study were (1) to determine whether MAP kinase is expressed in cultured rat aortic VSM, (2) to assess the activation of MAP kinase by known proliferative and hypertrophic stimuli, and (3) to determine if stimulation of a PKC-dependent signaling pathway in these cells results in MAP kinase activation. MAP kinase activity was measured in cytosolic extracts of aortic VSM by quantifying myelin basic protein phosphorylation. Three peaks of activity were resolved chromatographically and identified as MAP kinase isoforms (MW 42, 44, and 46 kDa) by immunoblotting with antipeptide antibodies specific for MAP kinase. MAP kinase activity in quiescent growth-arrested cells (157 +/- 19 pmole 32P/min/mg) was markedly stimulated within 15 min by known mitogens (10% serum, 731 +/- 40 pmole 32P/min/mg; 40 ng/ml PDGF, 670 +/- 105 pmole 32P/min/mg; P < 0.01) and partially sustained for at least 90 min (serum, 606 +/- 34 pmole 32P/min/mg; PDGF, 323 +/- 59 pmole 32P/min/mg P < 0.05). Angiotensin II (AII, 0.1 microM) and a pharmacological PKC activator, phorbol 12,13-dibutyrate (PDB, 0.1 microM), are reported to be nonmitogenic hypertrophic stimuli in these cells. These stimuli transiently increased MAP kinase activity with a peak at 5 min (AII, 328 +/- 15 pmole 32P/min/mg; PDB, 592 +/- 41 pmole 32P/min/mg; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Langan
E M
EM
Department of Vascular Surgery, Geisinger Clinic, Danville, Pennsylvania 17822.
Youkey
J R
JR
Elmore
J R
JR
Franklin
D P
DP
Singer
H A
HA
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D006133:Growth Substances / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D006133:Growth Substances; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D000804:Angiotensin II; D015240:Phorbol 12,13-Dibutyrate; D017871:Calcium-Calmodulin-Dependent Protein Kinases