title: Translocation of PKC, protein phosphatase inhibition and preconditioning of rabbit cardiomyocytes.
abstract: This study was designed to test the hypothesis that induction of the preconditioned state results in a sustained translocation of protein kinase C (PKC) which accounts for the memory associated with preconditioning. Isolated rabbit cardiomyocytes were subjected to established preconditioning protocols using either adenosine or transient ischemia. At timed intervals during induction of preconditioning (PC), post-incubation 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 antibodies to alpha, epsilon, zeta and gamma PKC isozymes, and bands were qualified by densitometry. Internal controls for each experiment included oxygenated cardiomyocytes and cell 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 with adenosine (100 microM) or 10 min ischemia had no significant effect on these percentages. Furthermore, the relative amounts of PKC isozymes associated with the particulate fraction of control and preconditioned cells did not differ after a postincubation in oxygenated buffer or during a final ischemic incubation. PMA and ingenol completely translocated the epsilon and alpha isoforms, while thymeleatoxin totally translocated PKC alpha but only partially (50%) translocated PKC epsilon. The distribution of PKC zeta between fractions was not affected by any drug. The protein phosphatase inhibitor calyculin A protected cells mimicking preconditioning. This protection was blocked by preincubation with the selective PKC inhibitor calphostin C but was largely retained if calphostin C was added only during the final ischemic period. It is concluded that PKC activity is required for preconditioning, but a sustained translocation of PKC above basal levels is not necessary for protection of rabbit cardiomyocytes in vitro.
authors.affiliation: Department of Veterans Affairs Medical Center, East Tennessee State University, James H. Quillen College of Medicine, Johnson City 37614, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Armstrong
authors.forename: D B
authors.initials: DB
authors.lastname: Hoover
authors.forename: M H
authors.initials: MH
authors.lastname: Delacey
authors.forename: C E
authors.initials: CE
authors.lastname: Ganote
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D006321:Heart / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism*; D008387:Marine Toxins; D017202:Myocardial Ischemia; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D010080:Oxazoles / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D007527:Isoenzymes; D008387:Marine Toxins; D010080:Oxazoles; C059041:calyculin A; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D000241:Adenosine; D013755:Tetradecanoylphorbol Acetate