Endothelin-1 enhances eicosanoids-induced coronary smooth muscle contraction by activating specific protein kinase C isoforms.

Endothelin-1 (ET-1), a potent vasoconstrictor, has been implicated in the pathogenesis of coronary vasospasm by enhancing coronary vasoconstriction to vasoactive eicosanoids; however, the cellular mechanisms involved are unclear. We investigated whether physiological concentrations of ET-1 enhance coronary smooth muscle contraction to vasoactive eicosanoids by activating specific protein kinase C (PKC) isoforms. Cell contraction was measured in single smooth muscle cells isolated from porcine coronary arteries, intracellular free Ca(2+) ([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 anti-PKC antibodies using Western blots. 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 it did cause cell contraction (9%) that was inhibited by GF109203X (10(-6) mol/L), an inhibitor of Ca(2+)-dependent and Ca(2+)-indpendent PKC isoforms. The vasoactive eicosanoid prostaglandin F(2alpha) (PGF(2alpha), 10(-7) mol/L) caused increases in cell contraction (11%) and [Ca(2+)](i) (108+/-7 nmol/L) that were inhibited by the Ca(2+) channel blocker diltiazem (10(-6) mol/L). Pretreatment with ET-1 (10 pmol/L) for 10 minutes enhanced cell contraction to PGF(2alpha) (35%) with no additional increase in [Ca(2+)](i) (112+/-8 nmol/L). Direct activation of PKC by phorbol 12,13-dibutyrate (PDBu, 10(-7) mol/L) caused cell contraction (10%) and enhanced PGF(2alpha) contraction (33%) with no additional increase in [Ca(2+)](i) (115+/-7 nmol/L). The ET-1-induced enhancement of PGF(2alpha) contraction was inhibited by Gö6976 (10(-6) mol/L), an inhibitor of Ca(2+)-dependent PKC isoforms. Both ET-1 and PDBu caused an increase in PKC activity in the particulate fraction and a decrease in the cytosolic fraction and increased the particulate/cytosolic PKC activity ratio. Western blots revealed the Ca(2+)-dependent alpha-PKC and the Ca(2+)-independent delta-, epsilon-, and zeta-PKC isoforms. In resting tissues, alpha- and epsilon-PKC were mainly cytosolic, delta-PKC was mainly in the particulate fraction, and zeta-PKC was equally distributed in the cytosolic and particulate fraction. ET-1 (10 pmol/L) alone or PDBu (10(-7) mol/L) alone caused translocation of epsilon-PKC from the cytosolic to the particulate fraction, localized delta-PKC more in the particulate fraction, but did not change the distribution of zeta-PKC. PGF(2alpha) (10(-7) mol/L) alone did not change PKC activity. In tissues pretreated with ET-1 or PDBu, PGF(2alpha) caused additional increases in alpha-PKC activity. Thus, the enhancement of PGF(2alpha)-induced coronary smooth muscle contraction by physiological concentrations of ET-1 involves activation and translocation of alpha-PKC in addition to delta- and epsilon-PKC isoforms, and this may represent one possible cellular mechanism by which ET-1 could enhance coronary vasoconstriction to vasoactive eicosanoids in coronary vasospasm.