Elevations in Sympathetic Neurons

We studied how mitochondrial Ca 2 1 transport influences [Ca 2 1 ] i dynamics in sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca 2 1 accumulation by SERCA pumps and depolarized to elevate [Ca 2 1 ] i ; the recovery that followed repolarization was then examined. The total Ca 2 1 flux responsible for the [Ca 2 1 ] i recovery was separated into mitochondrial and nonmitochondrial components based on sensitivity to the proton ionophore FCCP, a selective inhibitor of mitochondrial Ca 2 1 transport in these cells. The nonmitochondrial flux, representing net Ca 2 1 extrusion across the plasma membrane, has a simple dependence on [Ca 2 1 ] i , while the net mitochondrial flux (J mito ) is biphasic, indicative of Ca 2 1 accumulation during the initial phase of recovery when [Ca 2 1 ] i is high, and net Ca 2 1 release during later phases of recovery. During each phase, mitochondrial Ca 2 1 transport has distinct effects on recovery kinetics. J mito was separated into components representing mitochondrial Ca 2 1 uptake and release based on sensitivity to the specific mitochondrial Na 1 /Ca 2 1 exchange inhibitor, CGP 37157 (CGP). The CGP-resistant (uptake) component of J mito increases steeply with [Ca 2 1 ] i , as expected for transport by the mitochondrial uniporter. The CGP-sensitive (release) component is inhibited by lowering the intracellular Na 1 concentration and depends on both intraand extramitochondrial Ca 2 1 concentration, as expected for the Na 1 /Ca 2 1 exchanger. Above z 400 nM [Ca 2 1 ] i , net mitochondrial Ca 2 1 transport is dominated by uptake and is largely insensitive to CGP. When [Ca 2 1 ] i is z 200–300 nM, the net mitochondrial flux is small but represents the sum of much larger uptake and release fluxes that largely cancel. Thus, mitochondrial Ca 2 1 transport occurs in situ at much lower concentrations than previously thought, and may provide a mechanism for quantitative control of ATP production after brief or low frequency stimuli that raise [Ca 2 1 ] i to levels below z 500 nM. key words: mitochondria • calcium • calcium signaling • neurons • CGP 37157 I N T R O D U C T I O N There is a growing interest in mitochondrial Ca 2 1 transport. Ca 2 1 uptake and release by these organelles is thought to influence the dynamics of cytosolic free Ca 2 1 concentration ([Ca 2 1 ] i ) in a variety of cell types after stimuli that promote either Ca 2 1 entry from the extracellular medium or release from intracellular stores (for reviews see Miller, 1991, 1998; Babcock and Hille, 1998; Duchen, 1999). Modulation of [Ca 2 1 ] i dynamics by mitochondria may be a key factor in some forms of Ca 2 1 signaling (Hajnoczky et al., 1995; Budd and Nicholls, 1996; Hoth et al., 1997; Tang and Zucker, 1997; David et al., 1998). Moreover, changes in intramitochondrial free Ca 2 1 concentration ([Ca 2 1 ] m ) that occur as a result of stimulation are thought to regulate ATP synthesis in anticipation of cellular energy demands (McCormack and Denton, 1993; Robb-Gaspers et al., 1998). Mitochondrial Ca 2 1 transport has been studied extensively in isolated mitochondria. Ca 2 1 uptake is controlled by a Ca 2 1 -sensitive uniporter (EC 50 z 10–20 mM ; Gunter and Gunter, 1994) that permits Ca 2 1 to flow into the matrix down its steep electrochemical gradient. Ca 2 1 release from neuronal mitochondria is regulated primarily by a Na 1 /Ca 2 1 exchanger (Gunter and Pfeiffer, 1990) that is distinct from the plasma membrane exchanger found in many excitable cells (Crompton et al., 1978; Cox and Matlib, 1993). Overall, the magnitude and direction of net mitochondrial Ca 2 1 transport depends on the relative rates of Ca 2 1 uptake and release. When the extramitochondrial Ca 2 1 concentration is high, the rate of Ca 2 1 uptake via the uniporter greatly exceeds the maximal rate of Ca 2 1 release via the Na 1 /Ca 2 1 exchanger (Gunter and Pfeiffer, 1990), resulting in strong net mitochondrial Ca 2 1 accumulation. In contrast, at lower Ca 2 1 concentrations, where activity of the uniporter is far below its maximum, the net mitochondrial flux should depend on the relative rates of uptake and release. Despite the importance of mitochondrial Ca 2 1 uptake and release pathways in defining the rate of net mitochondrial Ca 2 1 transport, their individual contributions to [Ca 2 1 ] i dynamics in situ have not been determined, in part because they operate within an intracellular network of coupled transporters that makes contributions from individual transport systems difficult to resolve. Pharmacological agents have been useful in Address correspondence to David Friel, Ph.D, Department of Neuroscience, Case Western Reserve University, 10900 Euclid Ave. Cleveland, OH 44106. Fax: 216-368-4650; E-mail: [email protected] on M ay 7, 2017 D ow nladed fom Published February 28, 2000