Calcium Accumulation by Organelles Within Myxicola Axoplasm

1. 45Ca2+ accumulation into inulin-inaccessible compartments within cytoplasm from the giant axon of Myxicola infundibulum was measured as a function of free calcium, pH, and time. Accumulation reached a maximum after 1 h and remained stable for at least 3 h. 2. At 0.5, 5, and 50 microM [Ca2+], in the presence of 1 mM ATP or 5 mM succinate, steady-state calcium uptake had a bell-shaped dependence on pH with a maximum near pH 7. Uptake was abolished by the proton uncoupling reagent carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 4 micrograms ml-1). 3. Uptake of the membrane permeant cation, [14C]-tetraphenylphosphonium (TPP+), also had a bell-shaped dependence on pH with a maximum pH approximately 7, suggesting a pH dependence of the electrical potential of a membrane enclosed cytoplasmic compartment. Cyanide (2 mM) inhibited TPP+ uptake. 4. Inositol 1,4,5-trisphosphate (IP3, 10 microM), reduced steady-state calcium accumulation by 20-22% at 0.5 microM free calcium, pH 7 (P < 0.01, n = 16) and at 5 microM free calcium, pH 8 (P < 0.0005, n = 35). No effects of IP3 were found at other pH or calcium concentrations. 5. Neither guanosine 5'-triphosphate (GTP) nor inositol 1,3,4,5-tetrakisphosphate (IP4) had an effect on calcium uptake (5 microM [Ca2+], pH 8). 6. At 0.5 microM free calcium; vanadate (10 microM) inhibited 20-30%, of the 45Ca2+ accumulation, thapsigargin (33 nM) inhibited 20-30%, and cyanide (2 mM) plus oligomycin B (2 micrograms ml-1), or valinomycin (1 microM), inhibited 70-80%. The fraction of uptake sensitive to thapsigargin fell as the free calcium increased; however, the sensitivity of uptake to cyanide plus oligomycin B was approximately 80% for 0.5, 5.0, and 50 microM [Ca2+]. 7. Thapsigargin had no additional inhibiting effect in the presence of cyanide plus oligomycin B. IP3 had no effect in the presence of cyanide plus oligomycin B or other mitochondrial inhibitors. 8. Results suggest the presence of both mitochondrial (70-80%) and non-mitochondrial (20-30%) calcium pools in this system (at 0.5-5.0 microM Ca2+). The apparent non-mitochondrial uptake (sensitive to thapsigargin, or IP3) is not detectable in the presence of mitochondrial inhibitors. We interpret these results as evidence of functional communication between mitochondrial and non-mitochondrial calcium stores.