Neuroprotective effects of PKC inhibition against chemical hypoxia.
The effect of potassium cyanide-induced chemical hypoxia on protein kinase C (PKC) translocation and cell injury was studied in differentiated PC12 cells. The cellular distribution of PKC in control cells and cells exposed to 100 microM and 1 mM KCN for 30 min. was visualized by use of an anti-PKC antibody and confocal laser scanning microscope. In control differentiated PC12 cells, PKC was localized perinuclearly, while following 12-phorbol 13-myristate acetate (PMA) or KCN it was translocated to the plasma and organelle membranes. Western blot analysis was used to quantify the translocation. Chemical hypoxia increased the membrane-bound PKC to 210% of control levels, while chelerythrine, a PKC inhibitor, and block of calcium influx into the cells (with calcium channel blocker and calcium-free medium) prevented this effect. Cyanide-induced PKC translocation persisted for at least 120 min. Cell injury 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 to PMA) or inhibition (with chelerythrine or staurosporine) conferred protection against KCN-induced cytotoxicity. Ca2+ channel blocker nifedipine also protected against chemical hypoxia. None of the pretreatments rendered complete protection against cyanide-induced hypoxia, indicating that PKC-independent mechanism(s) are also activated during chemical hypoxia and contribute to cell injury.
Pavlaković
G
G
Department of Pharmacology and Toxicology, Purdue University, West Lafayette, IN 47907-1334, USA.
Eyer
C L
CL
Isom
G E
GE
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009543:Nifedipine / Q000494:pharmacology; D016716:PC12 Cells / Q000378:metabolism; D010617:Phenanthridines / Q000494:pharmacology; D011190:Potassium Cyanide / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats
D000470:Alkaloids; D053119:Benzophenanthridines; D010617:Phenanthridines; C016299:chelerythrine; D011493:Protein Kinase C; D009543:Nifedipine; D011190:Potassium Cyanide