Endothelin-1 Promotes Ca 2+ Antagonist-Insensitive Coronary Smooth Muscle Contraction Via Activation of ε-Protein Kinase C

Abstract

Certain forms of coronary artery disease do not respond to treatment with Ca 2+ channel blockers, and a role for endothelin-1 (ET-1) in Ca 2+ antagonist-insensitive forms of coronary vasospasm has been suggested; however, the signaling mechanisms involved are unclear. We tested the hypothesis that a component of ET-1–induced coronary smooth muscle contraction is Ca 2+ antagonist-insensitive and involves activation of protein kinase C (PKC). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca 2+ ] i was measured in fura-2 loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific PKC antibodies using Western blot analysis. In Hank’s solution (1 mmol/L Ca 2+ ), ET-1 (10 −7 mol/L) caused a transient increase in [Ca 2+ ] i (236±14 nmol/L) followed by a maintained increase in [Ca 2+ ] i (184±8 nmol/L) and 35% cell contraction. The Ca 2+ channel blockers verapamil and diltiazem (10 −6 mol/L) abolished the maintained ET-1–induced [Ca 2+ ] i , but only partially inhibited ET-1–induced cell contraction to 18%. The verapamil-insensitive component of ET-1 contraction was inhibited by the PKC inhibitors calphostin C and ε-PKC V1–2 . ET-1 caused translocation of Ca 2+ -dependent α-PKC and Ca 2+ -independent ε-PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1–induced translocation of α-PKC, but not that of ε-PKC. Phorbol 12-myristate 13-acetate (10 −6 mol/L), a direct activator of PKC, caused 22% cell contraction, with no increase in [Ca 2+ ] i , and translocation of ε-PKC that was inhibited by calphostin C, but not by verapamil. KCl (51 mmol/L), which stimulates Ca 2+ influx, caused 35% cell contraction and increase in [Ca 2+ ] i (291±11 nmol/L) that were inhibited by verapamil, but not by calphostin C, and did not cause translocation of α- or ε-PKC. In Ca 2+ -free (2 mmol/L EGTA) Hank’s solution, ET-1 caused 15% cell contraction, with no increase in [Ca 2+ ] i , and translocation of ε-PKC that were inhibited by ε-PKC V1–2 inhibitory peptide. Thus, a significant component of ET-1–induced contraction of coronary smooth muscle is Ca 2+ antagonist-insensitive and involves activation and translocation of Ca 2+ -independent ε-PKC, and may represent a signaling mechanism of Ca 2+ antagonist-resistant forms of coronary vasospasm.