Quantitative evaluation of mitochondrial calcium content in rat cortical neurones following a glutamate stimulus
Author's present address M. Tassetto: Ecole Normale Supérieure, ACI Biologie Cellulaire des Homéoprotéines (UMR 8542), 46, rue d'Ulm, 75005 Paris, France.
Corresponding author I. J. Reynolds: Department of Pharmacology, W1351 Biomedical Science Tower, Pittsburgh, PA 15261, USA., Email: firstname.lastname@example.org
- 1Recent observations showed that a mitochondrial Ca2+ increase is necessary for an NMDA receptor stimulus to be toxic to cortical neurones. In an attempt to determine the magnitude of the Ca2+ fluxes involved in this phenomenon, we used carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial proton gradient uncoupler, to release mitochondrial free calcium ([Ca2+]m) during and following a glutamate stimulus, and magfura-2 to monitor cytoplasmic free calcium ([Ca2+]c).
- 2FCCP treatment of previously unstimulated neurones barely changed [Ca2+]c whereas when added after a glutamate stimulus it elevated [Ca2+]c to a much greater extent than did exposure to glutamate, suggesting a very large accumulation of Ca2+ in the mitochondria.
- 3Mitochondrial Ca2+ uptake was dependent on glutamate concentration, whereas the changes in the overall quantity of Ca2+ entering the cell, obtained by simultaneously treating neurones with glutamate and FCCP, showed a response that was essentially all-or-none.
- 4Mitochondrial Ca2+ uptake was also dependent on the nature and duration of a given stimulus as shown by comparing [Ca2+]m associated with depolarization and treatment with kainate, NMDA or glutamate. Large mitochondrial Ca2+ accumulation only occurred after a glutamate or NMDA stimulus.
- 5These studies provide a method of estimating the accumulation of Ca2+ in the mitochondria of neurones, and suggest that millimolar concentrations of Ca2+ may be reached following intense glutamate stimulation. It was shown that substantially more Ca2+ enters neurones following glutamate receptor activation than is reflected by [Ca2+]c increases.