Endothelin-1 Enhances Eicosanoids-Induced Coronary Smooth Muscle Contractility and [Ca²⁺]_I by Activating a Protein Kinase C-Sensitive Ca²⁺ Entry Pathway

Abstract

P151

Endothelin-1 (ET-1) has been implicated in the pathogenesis of coronary vasospasm by enhancing the coronary vasoconstrictor response to vasoactive eicosanoids; however, the cellular mechanisms involved are unclear. We investigated whether physiological concentrations of ET-1 enhance prostaglandin F_2α (PGF_2α)-induced coronary smooth muscle contraction by increasing [Ca²⁺]_i and the Ca²⁺ mobilization mechanisms of coronary vasoconstriction. Single smooth muscle cells were freshly isolated from the left anterior descending coronary artery of castrated male pigs and labeled with the Ca²⁺ indicator fura-2 or with antibodies to specific protein kinase C (PKC) isoforms. In Hank’s solution (1 mM Ca²⁺), ET-1 (30 pM) caused a small increase in cell contraction (9%) and [Ca²⁺]_i (106±5 nM). PGF_2α (10^-7 M) increased cell contraction to 11% and [Ca²⁺]_i to 121±7 nM. In cells pretreated with ET-1 (30 pM) for 30 min, PGF_2α (10^-7 M) significantly increased cell contraction to 32% and [Ca²⁺]_i to 217±8 nM. Membrane depolarization by 24 mM KCl, which stimulates Ca²⁺ entry from the extracellular space, caused 12% cell contraction and increased [Ca²⁺]_i to 152±6 nM. In cells pretreated with ET-1, KCl significantly increased cell contraction to 34% and [Ca²⁺]_i to 306±8 nM. In Ca²⁺-free Hank’s, caffeine (10 mM), which stimulates Ca²⁺ release from intracellular stores, caused cell contraction (9%) and a transient increase in [Ca²⁺]_i (417±14 nM). ET-1 did not significantly affect the caffeine contraction or [Ca²⁺]_i. ET-1 caused significant translocation of α-PKC from the cytosol to the cell membrane, suggesting activation of the isoform. The PKC inhibitor Gö6976 (10^-6 M) completely inhibited the ET-1 induced α-PKC translocation and the enhancement of PGF_2α− and KCl-induced cell contraction and [Ca²⁺]_i. Thus, ET-1 enhances PGF_2α-induced coronary smooth muscle contractility and [Ca²⁺]_i by enhancing Ca²⁺ entry from the extracellular space, but not Ca²⁺ release from intracellular stores. The results suggest that the enhancement of PGF_2α-induced Ca²⁺ entry by ET-1 involves activation and translocation of α-PKC isoform.