Control of InsP3-induced Ca2+ oscillations in permeabilized blowfly salivary gland cells: contribution of mitochondria.

Many agonists linked to the generation of inositol 1,4, 5-trisphosphate (InsP3) and release of Ca2+ from intracellular stores induce repetitive transients in cytosolic Ca2+ whose frequency increases over a certain range of agonist concentrations. In order to investigate the mechanisms underlying this frequency modulation, the fluorescent Ca2+ sensor mag-fura-2 was loaded into intracellular calcium stores and used to monitor InsP3-induced dynamics of the intraluminal calcium concentration ([Ca2+]L) in secretory cells of permeabilized blowfly Calliphora vicina salivary glands. In this preparation, increasing concentrations of InsP3 induced graded decreases in [Ca2+]L that were often superimposed with repetitive [Ca2+]L transients produced by sequential Ca2+ release and re-uptake. These [Ca2+]L oscillations developed at frequencies of 3-11 min-1 unrelated to the concentration of InsP3 present. In contrast, incremental concentrations of InsP3 applied in the presence of the oxidizable mitochondrial substrates citrate, succinate, or pyruvate-malate induced repetitive [Ca2+]L transients whose frequency increased with the concentration of InsP3. This InsP3 concentration-dependent modulation of oscillation frequency was abolished after dissipating the mitochondrial membrane potential (Delta psi m) by combined treatment with carbonyl cyanide p-trifluoromethoxyphenyl hydrazone + oligomycin or after application of Ruthenium Red, an inhibitor of mitochondrial Ca2+ uptake. Taken together, the data indicate that energized mitochondria exert negative control over the frequency of InsP3-induced Ca2+ oscillations. It is concluded that mitochondria play a crucial role in determining the duration of the interspike period and, therefore, for the encoding of amplitude-modulated, InsP3-liberating stimuli into the frequency of cytosolic Ca2+ oscillations.