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

Abstract

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2α (PGF2α) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 µM [Ca2+]e, both PGF2α and U46619 (10-5 M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 µM [Ca2+]e, PGF2α and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx - PKC activity - contraction relationship showed a 45Ca2+-influx threshold of ~7 µmol·kg-1·min-1 for maximal PKC activation by PGF2α and U46619. 45Ca2+ influx > 10 µmol·kg-1·min-1 was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed α-, δ-, ε-, and ζ-PKC in porcine coronary artery. In unstimulated tissues, α- and ε-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of ε-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of α-PKC was observed at 600 µM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent ε-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent α-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.Key words: protein kinase C, isoform, calcium influx, eicosanoids, vascular smooth muscle, contraction.