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.
Zimmermann
B
B
Institut für Zoophysiologie und Zellbiologie, Universität Potsdam, D-14471 Potsdam, Germany.zimmerm@rz.uni-potsdam.de
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D002118:Calcium / Q000493:pharmacokinetics*; D015220:Calcium Channels / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004175:Diptera; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004928:Escin / Q000494:pharmacology; D005456:Fluorescent Dyes; D006493:Heparin / Q000494:pharmacology; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007476:Ionophores / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D009942:Organometallic Compounds; D010507:Periodicity; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D012117:Resins, Synthetic; D012430:Ruthenium Red / Q000494:pharmacology; D012469:Salivary Glands / Q000166:cytology / Q000378:metabolism; D013268:Stimulation, Chemical; D014475:Uncoupling Agents / Q000494:pharmacology
D000925:Anticoagulants; D015220:Calcium Channels; D002614:Chelating Agents; D004396:Coloring Agents; D005456:Fluorescent Dyes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007476:Ionophores; D009840:Oligomycins; D009942:Organometallic Compounds; D018160:Receptors, Cytoplasmic and Nuclear; D012117:Resins, Synthetic; D014475:Uncoupling Agents; C110932:tetramethyl rhodamine ethyl ester; D012430:Ruthenium Red; C024997:Chelex 100; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D004928:Escin; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D002118:Calcium