oligomycin-b and rhod-2

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 InsP3 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 = 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 with increasing peak [Ca2+]c. 4. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 5 microM), 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 microM), 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 InsP3 (20 microM) evoked reproducible increases in [Ca2+]c. CCCP (5 microM) reduced the magnitude of the [Ca2+]c transients evoked by flash photolysis of caged InsP3. Oligomycin B (5 microM) did not reduce the inhibition of the InsP3-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 InsP3-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, following depolarization, may involve mitochondrial Ca2+ uptake. Following InsP3-evoked Ca2+ release, mitochondrial uptake of Ca2+ may regulate the local [Ca2+]c near the InsP3 receptor so maintaining the sensitivity of the InsP3 receptor to release Ca2+ from the SR.

Topics: Adenosine Triphosphatases; Animals; Antimetabolites; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Colon; Cytosol; Enzyme Inhibitors; Fluorescent Dyes; Guinea Pigs; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Ionophores; Male; Membrane Potentials; Mitochondria, Muscle; Muscle, Smooth; Oligomycins; Patch-Clamp Techniques; Thapsigargin