Pharmacological investigation of mitochondrial ca(2+) transport in central neurons: studies with CGP-37157, an inhibitor of the mitochondrial Na(+)-Ca(2+) exchanger.
Mitochondria buffer large changes in [Ca(2+)](i)following an excitotoxic glutamate stimulus. Mitochondrial sequestration of [Ca(2+)](i)can beneficially stimulate oxidative metabolism and ATP production. However, Ca(2+)overload may have deleterious effects on mitochondrial function and cell survival, particularly Ca(2+)-dependent production of reactive oxygen species (ROS) by the mitochondria. We recently demonstrated that the mitochondrial Na(+)-Ca(2+)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 Ca(2+). Our data showed that 25 microM CGP-37157 quenches DCF fluorescence similar to 100 microM 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 Ca(2+)cycling and by elevating intramitochondrial Ca(2+), we did not observe any changes in ATP levels or toxicity either in the presence or absence of glutamate. Finally, mitochondrial Ca(2+)uptake during an excitotoxic glutamate stimulus was only slightly enhanced by inhibition of mitochondrial Ca(2+)efflux. Thus, although CGP-37157 alters mitochondrial Ca(2+)efflux in neurons, the inhibition of Na(+)-Ca(2+)exchange does not profoundly alter glutamate-mediated changes in mitochondrial function or mitochondrial Ca(2+)content.
Scanlon
J M
JM
Department of Pharmacology, University of Pittsburgh, School of Medicine, Pittsburgh, USA.
Brocard
J B
JB
Stout
A K
AK
Reynolds
I J
IJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival; D002478:Cells, Cultured; D002998:Clonazepam / Q000031:analogs & derivatives* / Q000494:pharmacology; D005971:Glutamates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D016548:Prosencephalon / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors*; D013841:Thiazepines / Q000494:pharmacology*
D005971:Glutamates; D009498:Neurotoxins; D017382:Reactive Oxygen Species; D019831:Sodium-Calcium Exchanger; D013841:Thiazepines; D002998:Clonazepam; C079032:CGP 37157; D000255:Adenosine Triphosphate; D012964:Sodium; D002118:Calcium