Endothelin-1-induced enhancement of coronary smooth muscle contraction via MAPK-dependent and MAPK-independent [Ca²⁺]_i sensitization pathways

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, [Ca²⁺]_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 Ca²⁺), ET-1 (10 pmol/L) did not increase basal [Ca²⁺]_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_2α (PGF_2α; 10^-7 mol/L) caused increases in cell contraction (11%) and [Ca²⁺]_i (122±9 nmol/L) that were inhibited by the Ca²⁺ 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_2α (33%) with no additional increase in [Ca²⁺]_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_2α contraction (32%) with no additional increase in [Ca²⁺]_i (126±9 nmol/L). The ET-1- and PMA-induced enhancement of PGF_2α 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_2α 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 Ca²⁺-independent PKC-mediated signaling pathway that involves tyrosine phosphorylation and activation of MAPK. The enhancement of PGF_2α-induced coronary smooth muscle contraction by ET-1 involves additional activation of a Ca²⁺-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.