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.