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

1. Many agonists linked to the generation of inositol 1,4,5-trisphosphate (InsP(3)) and release of Ca2+ from intracellular stores induce repetitive transients in cytosolic Ca2+ whose frequency increases over a certain range of agonist concentrations. 2. 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 InsP(3)-induced dynamics of the intraluminal calcium concentration ([Ca2+](L)) in secretory cells of permeabilized blowfly Calliphora vicina salivary glands. 3. In this preparation, increasing concentrations of InsP(3) 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 InsP(3) present. 4. In contrast, incremental concentrations of InsP(3) applied in the presence of the oxidizable mitochondrial substrates citrate, succinate, or pyruvate-malate induced repetitive [Ca2+], transients whose frequency increased with the concentration of InsP(3). 5. This InsP(3) 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 InsP(3)-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, InsP(3)-liberating stimuli into the frequency of cytosolic Ca2+ oscillations.