TRANSLOCATION OF PKC, PROTEIN PHOSPHATASE INHIBITION AND PRECONDITIONING OF RABBIT CARDIOMYOCYTES

This study was designed to test the hypothesis that induction of the p reconditioned state results in a sustained translocation of protein ki nase C (PKC) which accounts for the memory associated with preconditio ning, Isolated rabbit cardiomyocytes were subjected to established pre conditioning protocols using either adenosine or transient ischemia. A t timed intervals during induction of preconditioning (PC), post-incub ation or final sustained ischemia, cells were harvested, subjected to digitonin lysis and separated into cytosolic and particulate fractions , Samples were evaluated by Western blot analysis with monoclonal anti bodies to alpha, epsilon, zeta and gamma PKC isozymes, and bands were quantified by densitometry. Internal controls for each experiment incl uded oxygenated cardiomyocytes and cells with PKC translocation evoked by treatment with phorbol 12-myristate 13-acetate (PMA). For control oxygenated cells. the particulate fraction contained about 30% of PKC epsilon, 5-10% of PKC alpha and 60-70% of PKC zeta, Preconditioning wi th adenosine (100 mu M) or 10 min ischemia had no significant effect o n these percentages, Furthermore, the relative amounts of the PKC isoz ymes associated with the particulate fraction of control and precondit ioned cells did not differ after a post-incubation in oxygenated buffe r or during a final ischemic incubation, PMA and ingenol completely tr anslocated the epsilon and alpha isoforms, while thymeleatoxin totally translocated PKC alpha but only partially (50%) translocated PKC epsi lon, The distribution of PKC zeta between fractions was not affected b y any drug, The protein phosphatase inhibitor calyculin A protected ce lls mimicking preconditioning. This protection was blocked by preincub ation with the selective PKC inhibitor calphostin C but was largely re tained if calphostin C was added only during the anal ischemic period. It is concluded that PKC activity is required for preconditioning, bu t a sustained translocation of PKC above basal levels is not necessary for protection of rabbit cardiomyocytes in vitro. (C) 1996 Academic P ress Limited.