Intracellular calcium concentrations during metabolic inhibition in the motoneuron cell line NSC-19.
Changes in the concentrations of intracellular free calcium ([Ca2+]i) and adenine nucleotides were determined in response to metabolic inhibitors in the motoneuron cell line NSC-19. The NADH dehydrogenase inhibitor amobarbital (Amytal) and the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) were used to alter energy metabolism. Exposure of cells to 5 mM Amytal did not significantly change ATP concentrations but produced transient elevations of [Ca2+]i of approximately 80 nM, which were reduced by 32% when cells were studied in Ca(2+)-free solutions. CCCP (10 microM) caused a transient reduction in ATP concentration of 33%. CCCP also produced sustained elevations of [Ca2+]i of about 280 nM, which were reduced by 47% when in Ca(2+)-free solutions. In spite of the sustained elevation of [Ca2+]i induced by CCCP, NSC-19 showed no reduction in cell viability after 48 h compared with controls. Ruthenium red, a blocker of Ca2+ uptake by mitochondria, had little effect on the CCCP-induced [Ca2+]i increment. KCl or glutamate did not produce significant changes in [Ca2+]i, indicating that these cells do not possess significant numbers of voltage-dependent Ca2+ channels or excitatory amino acid receptor-gated channels. [Ca2+]i values in these cells were modified by changes in extracellular Ca2+ concentrations. In Ca(2+)-containing solutions, inhibition of Na+/Ca2+ exchange by amiloride and bepridil led to increased [Ca2+]i, as did blockade of Ca2+ ATPase by vanadate, suggesting that membrane transporters are important in Ca2+ efflux in NSC-19. The present studies indicate that exposure of NSC-19 cells to Amytal and CCCP produces Ca2+ increments by release from internal stores, as well as by transmembrane influx. These results demonstrate that small increments in [Ca2+]i can be produced by metabolic inhibitors or other compounds and that such changes are not associated with immediate cell death. Changes in [Ca2+]i could potentially result in abnormal cell function secondary to altered action of Ca(2+)-dependent enzymes.
Hasham
M I
MI
Department of Medicine, University Hospital, University of British Columbia, Vancouver, BC, Canada.
Naumann
D
D
Kim
S U
SU
Cashman
N R
NR
Quamme
G A
GA
Krieger
C
C
D000227:Adenine Nucleotides / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000654:Amobarbital / Q000494:pharmacology; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004734:Energy Metabolism / Q000187:drug effects; D006822:Hybrid Cells; D051379:Mice; D008856:Microscopy, Fluorescence; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D014638:Vanadates / Q000494:pharmacology
D000227:Adenine Nucleotides; D000963:Antimetabolites; D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D014638:Vanadates; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D012964:Sodium; D000251:Adenosine Triphosphatases; D000654:Amobarbital; D002118:Calcium