Pharmacological investigation of mitochondrial Ca2+transport in central neurons: studies with CGP-37157, an inhibitor of the mitochondrial Na+–Ca2+exchanger

Abstract

Mitochondria buffer large changes in [Ca2+]ifollowing an excitotoxic glutamate stimulus. Mitochondrial sequestration of [Ca2+]ican beneficially stimulate oxidative metabolism and ATP production. However, Ca2+overload may have deleterious effects on mitochondrial function and cell survival, particularly Ca2+–dependent production of reactive oxygen species (ROS) by the mitochondria. We recently demonstrated that the mitochondrial Na+–Ca2+exchanger in neurons is selectively inhibited by CGP-37157, a benzothiazepine analogue of diltiazem. In the present series of experiments we investigated the effects of CGP-37157 on mitochondrial functions regulated by Ca2+. Our data showed that 25μM CGP-37157 quenches DCF fluorescence similar to 100μM glutamate and this effect was enhanced when the two stimuli were applied together. CGP-37157 did not increase ROS generation and did not alter glutamate or 3mM hydrogen-peroxide-induced increases in ROS as measured by DHE fluorescence. CGP-37157 induces a slight decrease in intracellular pH, much less than that of glutamate. In addition, CGP-37157 does not enhance intracellular acidification induced by glutamate. Although it is possible that CGP-37157 can enhance mitochondrial respiration both by blocking Ca2+cycling and by elevating intramitochondrial Ca2+, we did not observe any changes in ATP levels or toxicity either in the presence or absence of glutamate. Finally, mitochondrial Ca2+uptake during an excitotoxic glutamate stimulus was only slightly enhanced by inhibition of mitochondrial Ca2+efflux. Thus, although CGP-37157 alters mitochondrial Ca2+efflux in neurons, the inhibition of Na+–Ca2+exchange does not profoundly alter glutamate-mediated changes in mitochondrial function or mitochondrial Ca2+content.