NEUROPROTECTIVE EFFECTS OF PKC INHIBITION AGAINST CHEMICAL HYPOXIA

The effect of potassium cyanide-induced chemical hypoxia on protein ki nase C (PKC) translocation and cell injury was studied in differentiat ed PC12 cells. The cellular distribution of PKC in control cells and c ells exposed to 100 mu M and 1 mM KCN for 30 min. was visualized by us e of an anti-PKC antibody and confocal laser scanning microscope. In c ontrol differentiated PC12 cells, PKC was localized perinuclearly, whi le following 12-phorbol 13-myristate acetate (PMA) or KCN it was trans located to the plasma and organelle membranes. Western blot analysis w as used to quantify the translocation. Chemical hypoxia increased the membrane-bound PKC to 210% of control levels, while chelerythrine, a P KC inhibitor, and block of calcium influx into the cells (with calcium channel blocker and calcium-free medium) prevented this effect. Cyani de-induced PKC translocation persisted for at least 120 min. Cell inju ry was monitored by measuring lactate dehydrogenase (LDH) efflux from the cells 24 hr after addition of cyanide. PKC activation plays a role in hypoxic damage, since PKC down-regulation (by overnight exposure t o PMA) or inhibition (with chelerythrine or staurosporine) conferred p rotection against KCN-induced cytotoxicity. Ca2+ channel blocker nifed ipine also protected against chemical hypoxia. None of the pretreatmen ts rendered complete protection against cyanide-induced hypoxia, indic ating that PKC-independent mechanism(s) are also activated during chem ical hypoxia and contribute to cell injury.