title: Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons.
abstract: We sought to determine whether low-affinity, high-capacity mitochondrial Ca2+ uptake contributes to buffering physiological Ca2+ loads in sensory neurons. Intracellular free calcium concentration ([Ca2+]i) and intracellular free hydrogen ion concentration ([H+]i) were measured in single rat dorsal root ganglion (DRG) neurons grown in primary culture using indo-1 and carboxy-SNARF-based dual emission microfluorimetry. Field potential stimulation evoked action potential-mediated increases in [Ca2+]. Brief trains of action potentials elicited [Ca2+]i transients that recovered to basal levels by a single exponential process. Trains of > 25 action potentials elicited larger increases in [Ca2+]i, recovery from which consisted of three distinct phases. During a rapid initial phase [Ca2+]i decreased to a plateau level (450-550 nM). The plateau was followed by a slow return to basal [Ca2+]i [Ca2+]i transients elicited by 40-50 action potentials in the presence of the mitochondrial uncoupler carbonyl cyanide chlorophenyl hydrazone (CCCP), or the electron transport inhibitor antimycin A1, lacked the plateau, and the recovery to basal [Ca2+]i consisted of a single slow phase. Depolarization with 50 mM K+ produced a multiphasic [Ca2+]i transient and increased [H+]i from 74 +/- 3 to 107 +/- 8 nM. The rise in [H+]i was dependent upon extracellular Ca2+ and was inhibited by mitochondrial poisons. With mitochondrial Ca2+ buffering pharmacologically blocked, the recovery to basal [Ca2+]i was unaffected by removal of extracellular Na+. We conclude that large Ca2+ loads are initially buffered by fast mitochondrial sequestration that effectively uncouples electron transport from ATP synthesis, leading to an increase in [H+]i. Small Ca2+ loads are buffered by a nonmitochondrial, Na(+)-independent process.
authors.affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
authors.forename: J L
authors.initials: JL
authors.lastname: Werth
authors.forename: S A
authors.initials: SA
authors.lastname: Thayer
mesh_terms: D000200:Action Potentials; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002021:Buffers; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D002021:Buffers; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000968:Antimycin A; D002118:Calcium