DYNAMICS OF INTRACELLULAR CALCIUM AND FREE RADICAL PRODUCTION DURING ISCHEMIC INJURY IN PYRAMIDAL NEURONS

P.L. Carlen, M.V. Frantseva and J.L. Perez Velazquez
Toronto Western Research Institute, and University of Toronto, Canada

Biochemical cascades initiated by oxidative stress and excitotoxic intracellular calcium rises are thought to converge on mitochondrial dysfunction, termed the mitochondrial permeability transition (MPT).  A gradual decrease of the mitochondrial potential, prevented by the MPT inhibitor cyclosporine A (CsA), was previously observed in our ischemia model. We investigated the contribution of mitochondrial dysfunction to FR overproduction in rat CA1 pyramidal neurons of organotypic slices subjected to a hypoxic-hypoglycemic insult. Ischemia-induced free radical generation (measured as changes in the fluorescence emission of dihydrorhodamine 123) was decreased by the mitochondrial complex I blocker, rotenone, indicating that mitochondria are the principal source of ischemic FR production. Measurements of mitochondrial calcium with the mitochondrial calcium probe dihydrorhod-2, revealed that FR production during and after the anoxic episode was preceded by the accumulation of mitochondrial calcium, suggesting that mitochondrial calcium overload is linked to mitochondrial FR generation during ischemia-reoxygenation injury.  CsA completely arrested both ischemic FR generation and mitochondrial calcium overload, and prevented neuronal depolarization and input resistance decrease during and after the ischemic episode, while having no effect on intracellular calcium levels, as measured by fluo-3 injected into individual pyramidal cells. A blocker of NAD+ hydrolysis, nicotinamide, reproduced CsA effects of blocking FR generation, mitochondrial calcium accumulation and cytoplasmic calcium increases.  Thus, hydrolysis of pyridine nucleotides seems to play an important role in hypoxia-hypoglycemia-induced mitochondrial calcium accumulation and generation of reactive oxygen species.

Supported by the Medical Research Council of Canada, the Ontario Neurotrauma Foundation, and the Bloorview Epilepsy Programme.

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