Oxidized-LDL enhances coronary vasoconstriction by increasing the activity of protein kinase C isoforms alpha and...

Oxidized low-density lipoprotein (ox-LDL) plays a critical role in the development of atherosclerotic coronary vasospasm; however, the cellular mechanisms involved are not fully understood. We tested the hypothesis that ox-LDL enhances coronary vasoconstriction by increasing the activity of specific protein kinase C (PKC) isoforms in coronary smooth muscle. Active stress was measured in de-endothelialized porcine coronary artery strips; cell contraction and [Ca(2+)](i) were monitored in single coronary smooth muscle cells loaded with fura-2; and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies with Western blots. Ox-LDL (100 microgram/mL) caused slow but significant increases in active stress to 1.3+/-0.4x10(3) N/m(2) and cell contraction (10%) that were completely inhibited by GF109203X (10(-6) mol/L), an inhibitor of Ca(2+)-dependent and -independent PKC isoforms, with no significant change in [Ca(2+)](i). 5-Hydroxytryptamine (5-HT; 10(-7) mol/L) and KCl (24 mmol/L) caused increases in cell contraction and [Ca(2+)](i) that were inhibited by the Ca(2+) channel blocker verapamil (10(-6) mol/L). Ox-LDL enhanced coronary contraction to 5-HT and KCl with no additional increases in [Ca(2+)](i). Direct activation of PKC by phorbol 12-myristate13-acetate (PMA; 10(-7) mol/L) caused a contraction similar in magnitude and time course to ox-LDL-induced contraction and enhanced 5-HT- and KCl-induced contraction with no additional increases in [Ca(2+)](i). The ox-LDL-induced enhancement of 5-HT and KCl contraction was inhibited by Gö6976 (10(-6) mol/L), an inhibitor of Ca(2+)-dependent PKC isoforms. Both ox-LDL and PMA caused an increase in PKC activity in the particulate fraction, a decrease in the cytosolic fraction, and an increase in the particulate/cytosolic PKC activity ratio. Western blots revealed the Ca(2+)-dependent PKC-alpha and the Ca(2+)-independent PKC-delta, -epsilon, and -zeta isoforms. In unstimulated tissues, PKC-alpha- and -epsilon were mainly cytosolic, PKC-delta was mainly in the particulate fraction, and PKC-zeta was equally distributed in the cytosolic and particulate fractions. Ox-LDL alone or PMA alone caused translocation of PKC-epsilon from the cytosolic to particulate fraction, whereas the distribution pattern of PKC-alpha, -delta, and -zeta remained unchanged. 5-HT (10(-7) mol/L) alone and KCl alone did not change PKC activity. In tissues pretreated with ox-LDL or PMA, 5-HT and KCl caused additional increases in PKC-alpha activity. Native LDL did not significantly affect coronary contraction, [Ca(2+)](i), or PKC activity. These results suggest that ox-LDL causes coronary contraction via activation of the Ca(2+)-independent PKC-epsilon and enhances the contraction to [Ca(2+)](i)-increasing agonists by activating the Ca(2+)-dependent PKC-alpha. Activation of PKC-alpha and -epsilon may represent a possible cellular mechanism by which ox-LDL could enhance coronary vasospasm.