Mitochondrial regulation of the cytosolic Ca2+ concentration and the InsP3-sensitive Ca2+ store in guinea-pig colonic smooth muscle

1. Mitochondrial regulation of the cytosolic Ca2+ concentration ([Ca2+](c)) in guinea-pig single colonic myocytes has been examined, using whole-cell recording, flash photolysis of caged InsP(3) and microfluorimetry. 2. Depolarization increased [Ca2+](c) and triggered contraction. Resting [Ca2+](c) was virtually restored some 4 s after the end of depolarization, a time when the muscle had shortened to 50% of its fully relaxed length. The muscle then slowly relaxed (t(1/2) = 17 s). 3. The decline in the Ca2+ transient was monophasic but often undershot or overshot resting levels, depending on resting [Ca2+](c). The extent of the overshoot or undershoot increased when increasmg peak [Ca2+](c). 4. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 5 mu M), which dissipates the mitochondrial proton electrochemical gradient and therefore prevents mitochondrial Ca2+ accumulation, slowed Ca2+ removal at high (> 300 nM) but not at lower [Ca2+](c) and abolished [Ca2+](c) overshoots. Oligomycin B (5 mu M), which prevents mitchondrial ATP production, affected neither the rate of decline nor the magnitude of the overshoot. 5. During depolarization, the global rhod-2 signal (which represents the mitochondrial matrix Ca2+ concentration, [Ca2+](m)) rose slowly in a CCCP-sensitive manner during and for about 3 s after depolarization had ended. [Ca2+](m) then slowly decreased over tens of seconds. 6. Inhibition of sarcoplasmic reticulum Ca2+ uptake with thapsigargin (100 nM) reduced the undershoot and increased the overshoot. 7. Flash photolysis of caged InsP(3) (20 mu M) evoked reproducible increases in [Ca2+](c). CCCP (5 mu M) reduced the magnitude of the [Ca2+](c) transients evoked by flash photolysis of caged InsP(3). Oligomycin B (5 mu M) did not reduce the inhibition of the InsP(3)-induced Ca2+ transient by CCCP thus minimizing the possibility that CCCP lowered ATP levels by reversing the mitochondrial ATP synthase and so reducing SR Ca2+ refilling. 8. While CCCP reduced the magnitude of the InsP(3)-evoked Ca2+ signal, the internal Ca2+ store content, as assessed by the magnitude of ionomycin-evoked Ca2+ release, did not decrease significantly. 9. [Ca2+](c) decline in smooth muscle, follotving depolarization, mag involve mitochondrial Ca2+ uptake. Following InsP(3)-evoked Ca2+ release, mitochondrial uptake of Ca2+ may regulate the local [Ca2+](c) near the InsP(3) receptor so maintaining the sensitivity of the InsP(3) receptor to release Ca2+ from the SR.