Blockade of mitochondrial Ca2+ uptake by mitochondrial inhibitors amplifies the glutamate-induced calcium response in cultured cerebellar granule cells

The objective of this study was to evaluate the role of mitochondrial Ca2+ uptake (MCU) in modulation (shaping) of the glutamate (Glu)-induced changes in neuronal cytoplasmic Ca2+ ([Ca2+](i)), In order to block MCU, nerve cells mere treated with mitochondrial inhibitors (MI) inducing collapse of the mitochondrial potential (Delta Psi(m)). Measurements of changes in [Ca2+](i) acre performed using either the low-affinity (fura-2FF) or high-affinity (fura-2) Ca(2+)indicators, Loading of nerve cells with rhodamine 123 made it possible to monitor changes in Delta Psi m,. In the first series of experiments it was shown that blockade of MCU in fura-2FF-loaded cells with a cocktail of rotenone (2 mu M)+oligomycin (2.5 mu g/ml) greatly (2,53 +/- 0,4 times, n=61) increased the [Ca2+](i) response to a 1-min Glu (100 mu M) pulse. In fura-2-loaded cells, this increase was small (less than 1.3 times) or absent, In the second series of experiments, cocktails of rotenone+oligomycin or FCCP (1 mu M)+oligomycin were applied during a prolonged Glu application, This produced strong mitochondrial depolarisation and an additional [Ca2+](i) increase. In most cells the latter could be reversed or prevented by a removal of external Ca2+. The MI-induced additional [Ca2+](i) increase was especially pronounced in cells loaded with fura-2FF, In some neurones a removal of external Ca2+ did not produce a decrease in [Ca2+](i) during combined Glu+MI application, suggesting an impairment of [Ca2+](i) extrusion mechanisms of these cells, The conclusion is drawn that MCU makes a considerable contribution to regulation of [Ca2+](i) responses caused by Ca2+ influx via Glu-activated ionic channels. The reasons for a quantitative difference between [Ca2+](i) responses observed in fura-2- and fura-2FF-loaded neurones are discussed. (C) 1999 Federation of European Biochemical Societies.