SUMMARY1. We studied how in changes in cytosolic free Ca2+ concentration ([Ca2+]

This study describes a Ca2+ store in fura-2-loaded bullfrog sympathetic neurons that modulates [Ca2+]i responses elicited by either depolarization or Ca2+ release from a caffeine- and ryanodine-sensitive store. This store is insensitive to caffeine and ryanodine, but is sensitive to the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The FCCP-sensitive store slows both the rise in [Ca2+]i during stimulation (apparently by accumulating Ca2+ from the cytosol) and the recovery following stimulation (by releasing the accumulated Ca2+ into the cytosol). For a fixed level of depolarization, recovery is slowed to an extent that depends on stimulus duration. [Ca2+]i imaging shows that these effects are prominent in the soma but not in growth cones. Ca2+ uptake by the FCCP-sensitive store appears to be strongly [Ca2+]i dependent, since it becomes influential only when [Ca2+]i approaches approximately 500 nM. Therefore, this store may specifically influence [Ca2+]i during moderate and strong stimulation. The effect of the store on responses to depolarization can be accounted for by a simple three-compartment scheme consisting of the extracellular medium, the cytosol, and a single internal store with a [Ca2+]i-dependent uptake mechanism resembling the mitochondrial Ca2+ uniporter. The store's effect on responses to caffeine-induced Ca2+ release can be accounted for by including a second internal compartment to represent the caffeine-sensitive store. While the identity of the FCCP-sensitive store is unknown, its sensitivity to FCCP is consistent with a mitochondrial pool. It is suggested that by modulating the temporal properties of [Ca2+]i following stimulation, the FCCP-sensitive store may influence the degree of activation of intracellular [Ca2+]i-dependent processes.

Several lines of evidence suggest that neuronal mitochondria accumulate calcium when the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is elevated to levels approaching approximately 500 nM, but the spatial, temporal, and quantitative characteristics of net mitochondrial Ca uptake during stimulus-evoked [Ca(2+)](i) elevations are not well understood. Here, we report direct measurements of depolarization-induced changes in intramitochondrial total Ca concentration ([Ca](mito)) obtained by x-ray microanalysis of rapidly frozen neurons from frog sympathetic ganglia. Unstimulated control cells exhibited undetectably low [Ca](mito), but high K(+) depolarization (50 mM, 45 sec), which elevates [Ca(2+)](i) to approximately 600 nM, increased [Ca](mito) to 13.0 +/- 1.5 mmol/kg dry weight; this increase was abolished by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). The elevation of [Ca](mito) was a function of both depolarization strength and duration. After repolarization, [Ca](mito) recovered to prestimulation levels with a time course that paralleled the decline in [Ca(2+)](i). Depolarization-induced increases in [Ca](mito) were spatially heterogeneous. At the level of single mitochondria, [Ca](mito) elevations depended on proximity to the plasma membrane, consistent with predictions of a diffusion model that considers radial [Ca(2+)](i) gradients that exist early during depolarization. Within individual mitochondria, Ca was concentrated in small, discrete sites, possibly reflecting a high-capacity intramitochondrial Ca storage mechanism. These findings demonstrate that in situ Ca accumulation by mitochondria, now directly identified as the structural correlate of the "FCCP-sensitive store, " is robust, reversible, graded with stimulus strength and duration, and dependent on spatial location.

SUMMARY1. The whole-cell voltage-clamp technique was used to study the effects of extracellular ATP on smooth muscle cells isolated from the rat vas deferens.2. ATP (1-200 /,M) elicited an inward-rectifying current that was rapid in onset ( < 100 ms), reached a peak value that depended on [ATP], and desensitized in the continued presence of ATP (half-time 2 s).3. Cells recovered from desensitization when incubated in the absence of ATP (resensitization half-time -2 min).4. A comparison was made of the ability of ATP and several of its structural analogues to stimulate inward current at a negative holding potential. ATP was by far the most effective compound among the series ATP, ADP, AMP, adenosine, GTP, UTP and ITP. ADP elicited a current that was 20-25 % as large as that produced by ATP, while the other compounds were ineffective at a concentration which produced a maximal ATP response.5. AMP-CPP (a,,-methylene ATP), AMP-PCP (,f,y-methylene ATP), and AMP-PNP (fl,y-imido ATP), which are relatively resistant to hydrolysis, were similarly compared to ATP. While none of these analogues elicited a current resembling the ATP-induced current, AMP-CPP and AMP-PNP each produced a small, relatively sustained inward current; AMP-PCP had little or no effect.6. The ATP-sensitive conductance is cation selective, but does not appear to discriminate strongly between Na+, K+ and Mg2+.7. Analysis of the fluctuations which accompany the ATP-induced current suggests that ATP controls a population of channels with a unitary current > 0 5 pA at -130 mV.8. The ATP-evoked current discussed in this report may be responsible for the depolarizing effect of ATP previously described in multicellular preparations of the vas deferens.

We studied how mitochondrial Ca2+ transport influences [Ca2+]i dynamics in sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca2+ accumulation by SERCA pumps and depolarized to elevate [Ca2+]i; the recovery that followed repolarization was then examined. The total Ca2+ flux responsible for the [Ca2+]i recovery was separated into mitochondrial and nonmitochondrial components based on sensitivity to the proton ionophore FCCP, a selective inhibitor of mitochondrial Ca2+ transport in these cells. The nonmitochondrial flux, representing net Ca2+ extrusion across the plasma membrane, has a simple dependence on [Ca2+]i, while the net mitochondrial flux (Jmito) is biphasic, indicative of Ca2+ accumulation during the initial phase of recovery when [Ca2+]i is high, and net Ca2+ release during later phases of recovery. During each phase, mitochondrial Ca2+ transport has distinct effects on recovery kinetics. Jmito was separated into components representing mitochondrial Ca2+ uptake and release based on sensitivity to the specific mitochondrial Na+/Ca2+ exchange inhibitor, CGP 37157 (CGP). The CGP-resistant (uptake) component of Jmito increases steeply with [Ca2+]i, as expected for transport by the mitochondrial uniporter. The CGP-sensitive (release) component is inhibited by lowering the intracellular Na+ concentration and depends on both intra- and extramitochondrial Ca2+ concentration, as expected for the Na+/Ca2+ exchanger. Above ∼400 nM [Ca2+]i, net mitochondrial Ca2+ transport is dominated by uptake and is largely insensitive to CGP. When [Ca2+]i is ∼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 Ca2+ 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 [Ca2+]i to levels below ∼500 nM.