title: PKC-induced ERK1/2 interactions and downstream effectors in ovine cerebral arteries.
abstract: Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric tension and intracellular free calcium concentration ([Ca(2+)](i)) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 x 10(-6) M] in the absence or presence of ERK1/2 inhibition (U-0126, 10(-5) M). After PDBu +/- ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, caldesmon(Ser789), myosin light chain(20) (MLC(20)), and CPI-17. PDBu induced significant increase in tension in the absence of increased [Ca(2+)](i). PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated caldesmon(Ser789) and MLC(20) levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 x 10(-7) M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases MLC(20) phosphorylation in the thick filament pathway and increases Ca(2+) sensitivity. In addition, ERK1/2-dependent phosphorylation of caldesmon(Ser789) was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase.
authors.affiliation: Center for Perinatal Biology, Department of Physiology and Pharmacology, Loma Linda University, School of Medicine, Loma Linda, California 92350, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
authors.forename: Lubo
authors.initials: L
authors.lastname: Zhang
authors.forename: Lawrence D
authors.initials: LD
authors.lastname: Longo
mesh_terms: D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002536:Cerebral Arteries / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D004347:Drug Interactions; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012756:Sheep; D014661:Vasoconstriction; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D002070:Butadienes; D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D009570:Nitriles; D020033:Protein Isoforms; C113580:U 0126; D015240:Phorbol 12,13-Dibutyrate; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020742:rhoA GTP-Binding Protein