Translocation and down-regulation of protein kinase C-alpha, -beta, and -gamma isoforms during ischemia-reperfusion in rat brain

We investigated the distribution of protein kinase C (PKC) isoforms in the subcellular fractions (P1, 1,000-g pellet; P2, 10,000-g pellet; P3, 100,000 -g pellet; S, 100,000-g supernatant) of rat forebrain after ischemia or rep erfusion by immunoblotting. PKC-delta and -epsilon isoforms were predominan t in the P2 (synaptosome-rich) fraction, whereas PKC-alpha, -beta, -gamma, -epsilon, and -zeta isoforms were rich in the S (cytosolic) fraction. With time of ischemia (5-30 min), PKC-alpha, -beta, and -gamma translocated to t he P2 and P3 fractions, whereas reperfusion for 60 min after 30 min of isch emia reduced PKC-beta activity greatly and PKC-alpha and -gamma activities to a lesser extent. There was no redistribution of PKC-delta, -epsilon, and -zeta after ischemia or reperfusion. A calpain inhibitor, acetylleucylleuc ylnorleucinal, inhibited the down-regulation of PKC-beta, through intraveno us injection. The PKC translocation to the P2 fraction was accompanied by t heir dephosphorylation, transition of PKC-alpha from dimer to trimer, and t he decrease in activity. These data show that PKC-alpha, -beta, and -gamma isoforms translocate chiefly to the synaptosome in ischemic brain in associ ation with the dephosphorylation, multimeric change, and inactivation, foll owed by the proteolysis of PKC-beta by calpain after postischemic reperfusi on.