Mitochondrial Ca²⁺ uptake increasingly appears to be important in Ca²⁺ homeostasis of many cell types. Here, we examined the effect of a mitochondria Ca²⁺ uptake inhibitor on intracellular Ca²⁺ concentration ([Ca²⁺]_i) using rat arterial smooth muscle cells. Single cells were dissociated from femoral arteries of rats humanely killed according to a Schedule 1 protocol. [Ca²⁺]_i was measured using fura-2 from whole-cell voltage-clamped cells. Membrane potential was raised from -70 to -50 to -30 mV, yielding a concomitant increase in [Ca²⁺]_i. When a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP, 1 mM), was applied whilst membrane potential was maintained at -30 mV, an increase in [Ca²⁺]_i was noted in all cells tested. In some cells, the increase in [Ca²⁺]_i occurred in two phases. The initial, smaller increase in [Ca²⁺]_i was followed by a larger elevation in [Ca²⁺]_i. The average maximum increase in [Ca²⁺]_i caused by CCCP was 280 ± 180 nM (mean ± S.E.M., n = 6). Next, the effect of CCCP on Ca²⁺ transients caused by Ca²⁺ release was examined. Caffeine (20 mM) was applied repeatedly using a U-tube superfusion system (Evans & Kennedy, 1994) to cells voltage-clamped at -70 mV. When paired half-decay times (t_1/2, time required to reduced increase in [Ca²⁺]_i by 50 %) were measured in the control cells, no significant difference was noted (7.2 ± 2.0 s and 8.0 ± 1.7 s, n = 6, paired t test). In a separate set of experiments, caffeine transients were triggered, and upon the restoration of resting [Ca²⁺]_i, CCCP was applied. This treatment evoked a transient increase in [Ca²⁺]_i with the average peak increase of 500 ± 317 nM (n = 5). In the continued presence of CCCP, caffeine applications were repeated producing prolonged Ca²⁺ transients. t_1/2 after CCCP application was 33.2 ± 7.5 s, significantly different from that before CCCP (3.4 ± 0.2 s, paired t test, n = 5, P < 0.05). The effect of CCCP was partially reversible with the mean t_1/2 of 9 ± 1.2 s after wash-out of CCCP. CCCP is thought to inhibit mitochondria Ca²⁺ uptake by depolarization of the mitochondrial membrane. When mitochondria membrane potential was reported with Rhodamine 123, CCCP application increased fluorescence intensity, suggesting mitochondrial depolarization. Similar results were obtained with application of diazoxide. On the other hand, Rhodamine 123 signal was slightly reduced by application of oligomycin, suggesting mitochondria membrane hyperpolarization.

Our results suggest that mitochondrial Ca²⁺ uptake is important when [Ca²⁺]_i is raised by sustained membrane depolarization or Ca²⁺ release.

This work is supported by the British Heart Foundation. T.K. is a British Heart Foundation Intermediate Fellow. We would like to thank Dr Richard Evans (University of Leicester) for his help with the U-tube superfusion system.

All procedures accord with current UK legislation.