Pyruvate and oxaloacetate limit zinc-induced oxidative HT-22 neuronal cell injury.
During CNS ischaemia, accumulating evidence suggests that raised intracellular Zn(2+) levels may play a significant role in inducing neuronal cell death. Several mechanisms mediating Zn(2+)-induced cell death have been suggested, however the precise molecular mechanisms remain uncertain. Employing the HT-22 murine hippocampal neuronal cell line, we have evaluated possible mechanisms of cytotoxic extracellular Zn(2+) insults. Increased extracellular Zn(2+) levels was found to induce concentration-dependent cytotoxicity. When tested at 200muM, Zn(2+) increased intracellular Zn(2+) levels (determined via FluoZin-3 fluorescence) and rapidly induced cell death. However, neither L-type (nimodipine) nor T-type (mibefradil) voltage-activated Ca(2+) channel inhibitors limited Zn(2+)-induced cytotoxicity. Furthermore, and in contrast with staurosporine, Zn(2+) cytotoxic insults failed to induce significant caspase-3 activation and were insensitive to the poly-caspase inhibitor, zVAD-fmk. Antioxidant co-application (Trolox and N,N'-diphenyl-1,4-phenylenediamine (DPPD)) was neuroprotective versus 6h Zn(2+) insults. Additionally, despite inducing significant mitochondrial membrane potential loss, Zn(2+) failed to induce detectable increased superoxide production. However, both pyruvate and oxaloacetate were found to afford significant neuroprotection versus Zn(2+) cytotoxic insults, without significantly influencing intracellular Zn(2+) accumulation. We conclude that cultured HT-22 neurones are vulnerable to Zn(2+) cytotoxic insults via a non-caspase-3 mediated mechanism, which involves glycolytic inhibition.
Berry
Elizabeth V
EV
School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
Toms
Nick J
NJ
D000704:Analysis of Variance; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002461:Cell Line, Transformed; D003603:Cytotoxins / Q000378:metabolism / Q000633:toxicity*; D003952:Diagnostic Imaging / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005434:Flow Cytometry / Q000379:methods; D007306:Insecticides / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000473:pathology; D062907:Oxaloacetic Acid / Q000494:pharmacology*; D010617:Phenanthridines / Q000378:metabolism; D019289:Pyruvic Acid / Q000494:pharmacology*; D012402:Rotenone / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism / Q000633:toxicity*
D003603:Cytotoxins; D007306:Insecticides; D010617:Phenanthridines; D012402:Rotenone; D062907:Oxaloacetic Acid; C058428:hydroethidine; D019289:Pyruvic Acid; D053148:Caspase 3; D015032:Zinc