Calcium sequestering ability of mitochondria modulates influx of calcium through glutamate receptor channel

The excitotoxicity of glutamate is believed to be mediated by sustained inc rease in the cytosolic Ca2+ concentration. Mitochondria play a vital role i n buffering the cytosolic calcium overload in stimulated neurons. Here we h ave studied the glutamate induced Ca2+ signals in cortical brain slices und er physiological conditions and the conditions that modify the mitochondria l functions. Exposure of slices to glutamate caused a rapid increase in [Ca 2+](i) followed by a slow and persistently rising phase. The rapid increase in [Ca2+](i) was mainly due to influx of Ca2+ through the N-methyl-D-aspar tate (NMDA) receptor channels. Glutamate stimulation in the absence of Ca2 in the extracellular medium elicited a small transient rise in [Ca2+](i) w hich can be attributed to the mobilization of Ca2+ from IF, sensitive endop lasmic reticulum pools conse quent to activation of metabotropic glutamate receptors. The glutamate induced Ca2+ influx was accompanied by depolarizat ion of the mitochondrial membrane, which was inhibited by ruthenium red, th e blocker of mitochondrial Ca2+ uniporter. These results imply that mitocho ndria sequester the Ca2+ loaded into the cytosol by glutamate stimulation. Persistent depolarization of mitochondrial membrane observed in presence of extracellular Ca2+ caused permeability transition and released the sequest ered Ca2+ which is manifested as slow rise in [Ca2+](i). Protonophore carbo nyl cyanide m-chlorophenyl-hydrazone (CCCP) depolarized the mitochondrial m embrane and enhanced the glutamate induced [Ca2+](i) response. Contrary to this, treatment of slices with mitochondrial inhibitor oligomycin or ruthen ium red markedly reduced the [Ca2+](i) response. Combined treatment with ol igomycin and rotenone further diminished the [Ca2+](i) response and also ab olished the CCCP mediated rise in [Ca2+](i). However, rotenone alone had no effect on glutamate induced [Ca2+](i) response. These changes in glutamate -induced [Ca2+](i) response could not be explained on the basis of deficien t mitochondrial Ca2+ sequestration or ATP dependent Ca2+ buffering. The mit ochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this wou ld have restrained the ATP dependent Ca2+ buffering processes leading to el evation of [Ca2+](i). In contrast our results showed repression of Ca2+ sig nal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions that hamper the Ca2+ sequestering a bility of mitochondria, the glutamate induced Ca2+ influx could be impeded. To validate this, influx of Mn2+ through ionotropic glutamate receptor cha nnel was monitored by measuring the quenching of Fura-2 fluorescence. Treat ment of slices with oligomycin and rotenone prior to glutamate exposure con spicuously reduced the rate of glutamate induced fluorescence quenching as compared to untreated slices. Thus our data establish that the functional s tatus of mitochondria can modify the activity of ionotropic glutamate recep tor and suggest that blockade of mitochondrial Ca2+ sequestration may desen sitize the NMDA receptor operated channel.