Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoco nstriction are unclear, and a role for protein kinase C (PKC) has been sugg ested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activi ty of the specific PKC isoforms in coronary smooth muscle is unknown. In th e present study, Ca-45(2+) influx and isometric contraction were measured i n porcine coronary artery strips incubated at increasing extracellular calc ium concentrations ([Ca2+](e)) and stimulated with prostaglandin F-2 alpha (PGF(2 alpha)) or the stable thromboxane A(2) analog U46619, while in paral lel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysi s. At 0-300 mu M [Ca2+](e), both PGF(2 alpha) and U46619 (10(-5) M) signifi cantly increased PKC activity and contraction in the absence of a significa nt increase in Ca-45(2+) influx. At 600 mu M [Ca2+](e), PGF(2 alpha) and U4 6619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respecti vely, with a significant increase in Ca-45(2+) influx and contraction. The Ca-45(2+) influx - PKC activity contraction relationship showed a Ca-45(2+) -influx threshold of similar to 7 mu mol.kg(-1).min(-1) for maximal PKC act ivation by PGF(2 alpha) and U46619. Ca-45(2+) influx > 10 mu mol.kg(-1).min (-1) was associated with further increases in contraction despite a signifi cant decrease in PKC activity. Western blotting analysis revealed alpha-, d elta-, epsilon-, and zeta-PKC in porcine coronary artery. In unstimulated t issues, alpha- and epsilon-PKC were mostly distributed in the cytosolic fra ction. Significant eicosanoid-induced translocation of epsilon-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+](e), while t ranslocation of alpha-PKC was observed at 600 mu M [Ca2+](e). Thus, a signi ficant component of eicosanoid-induced coronary contraction is associated w ith significant PKC activity in the absence of significant increase in Ca2 entry and may involve activation and translocation of the Ca2+-independent epsilon-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal C a2+ entry and may involve activation and translocation of the Ca2+-dependen t alpha-PKC. The results also suggest that a smaller PKC activity at supram aximal Ca2+ entry may be sufficient during eicosanoid-induced contraction o f coronary smooth muscle.