title: Endothelin-1--induced enhancement of coronary smooth muscle contraction via MAPK-dependent and MAPK-independent [Ca(2+)](i) sensitization pathways.
abstract: Endothelin-1 (ET-1) has been implicated in coronary vasospasm by enhancing coronary vasoconstriction to vasoactive eicosanoids, and a role for protein kinase C (PKC) activation has been suggested. However, the cellular mechanisms downstream from PKC activation are unclear. We investigated whether physiological concentrations of ET-1 enhance coronary smooth muscle contraction by activating a PKC-mediated signaling pathway involving tyrosine phosphorylation and activation of mitogen-activated protein kinase (MAPK). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca(2+)](i) was measured in fura-2 loaded cells, and tissue fractions were examined for reactivity with anti-phosphotyrosine (P-Tyr) and anti-MAPK antibodies using immunoprecipitation and immunoblot analysis. In Hanks' solution (1 mmol/L Ca(2+)), ET-1 (10 pmol/L) did not increase basal [Ca(2+)](i) (81 +/- 2 nmol/L) but caused cell contraction (10%) that was inhibited by calphostin C (10(-6) mol/L), inhibitor of PKC, tyrphostin (10(-6) mol/L), inhibitor of tyrosine kinase, and PD098059 (10(-6) mol/L), inhibitor of MAPK kinase. The vasoactive eicosanoid prostaglandin F(2alpha) (PGF(2alpha); 10(-7) mol/L) caused increases in cell contraction (11%) and [Ca(2+)](i) (122 +/- 9 nmol/L) that were inhibited by the Ca(2+) channel blocker verapamil (10(-6) mol/L) but not by calphostin C, tyrphostin, or PD098059. Pretreatment with ET-1 for 10 minutes enhanced cell contraction to PGF(2alpha) (33%) with no additional increase in [Ca(2+)](i) (124 +/- 10 nmol/L). Activation of PKC by phorbol 12-myristate 13-acetate (PMA; 10(-7) mol/L) caused cell contraction and enhanced PGF(2alpha) contraction (32%) with no additional increase in [Ca(2+)](i) (126 +/- 9 nmol/L). The ET-1-- and PMA-induced enhancement of PGF(2alpha) contraction was abolished by verapamil or calphostin C but not by tyrphostin or PD098059. ET-1 and PMA caused significant increases in tyrosine phosphorylation of MAPK that were inhibited by calphostin C, tyrphostin, and PD098059. PGF(2alpha) did not cause any additional increases in tyrosine phosphorylation of MAPK in tissues untreated or pretreated with ET-1 or PMA. Thus, physiological concentrations of ET-1 activate a Ca(2+)-independent PKC-mediated signaling pathway that involves tyrosine phosphorylation and activation of MAPK. The enhancement of PGF(2alpha)-induced coronary smooth muscle contraction by ET-1 involves additional activation of a Ca(2+)-sensitive PKC-mediated pathway but not tyrosine phosphorylation or activation of MAPK. The MAPK-dependent and MAPK-independent signaling pathways represent possible cellular mechanisms by which ET-1 could enhance coronary vasoconstriction to vasoactive eicosanoids in coronary vasospasm.
authors.affiliation: Department of Physiology and Biophysics and, Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, MS 39216, USA.
authors.forename: Ashley E
authors.initials: AE
authors.lastname: Cain
authors.forename: Dennis M
authors.initials: DM
authors.lastname: Tanner
authors.forename: Raouf A
authors.initials: RA
authors.lastname: Khalil
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003329:Coronary Vasospasm / Q000139:chemically induced; D015777:Eicosanoids / Q000009:adverse effects; D019332:Endothelin-1 / Q000494:pharmacology*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013552:Swine; D014661:Vasoconstriction / Q000187:drug effects
chemical_list: D015777:Eicosanoids; D019332:Endothelin-1; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium