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.

Calcium Homeostasis and Reactive Oxygen Species Production in Cells Transformed by Mitochondria from Individuals with Sporadic Alzheimer’s Disease

“…In fact, mitochondrial calcium rapidly and transiently increases after agonist-induced InsP 3 generation . The carbachol-induced peak calcium transient is comparable to the 450 -550 nM Ca 2ϩ range at which mitochondria are believed to play a buffering role in dorsal root ganglion neurons or chromaffin cells (Werth and Thayer, 1994;Herrington et al, 1996). Mitochondria can rapidly and significantly accumulate calcium from intra-or extracellular sources; they limit the exposure of a cell to high cytosolic calcium levels (Duchen et al, 1990;White and Reynolds, 1995).…”

Distinct Influx Pathways, Not Calcium Load, Determine Neuronal Vulnerability to Calcium Neurotoxicity

“…Given that most (>95%) Ca2~ions that enter a neuron under physiological conditions are buffered (Neher and Augustine, 1992;Zhou and Neher, 1993), one possibility is that the Ca2~buffering machinery of the cell is overwhelmed when a given Ca2~load is reached. For example, neuronalmitochondria are currently believed to buffer both physiological and pathological Ca2~loads (Werth and Thayer, 1994;White and Reynolds, 1995;Wang and Thayer, 1996). Exposure to EAAs produces mitochondrial dysfunction and the production of secondary excitotoxic products, such as reactive oxygen species (Dugan et al, 1995;Reynolds and Hastings, 1995;Schindler et al, 1996).…”

[Ca2+]iElevations Detected by BK Channels during Ca2+Influx and Muscarine-Mediated Release of Ca2+from Intracellular Stores in Rat Chromaffin Cells

“…The time course of this relaxation presumably reflects the nature of Ca 2ϩ sequestration and removal agents such as mitochondria, Na ϩ /Ca 2ϩ exchangers, and Ca 2ϩ /ATPases. Mitochondria have been shown to sequester Ca 2ϩ during Ca 2ϩ influx and gradually release it into the cytosol on termination of influx (Werth and Thayer, 1994). Furthermore, there is recent evidence that mitochondria are involved in shaping part of the [Ca 2ϩ ] i fall after very long influx steps in rat chromaffin cells Park et al, 1996).…”