CALCIUM ACCUMULATION BY ORGANELLES WITHIN MYXICOLA AXOPLASM

1. Ca-45(2+) 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 muM [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 mug ml-1). 3. Uptake of the membrane permeant cation, [C-14]-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 mum), reduced steady-state calcium accumulation by 20-22 % at 0.5 mum free calcium, pH 7 (P < 0.01, n = 16) and at 5 muM 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 mum [Ca2+], pH 8). 6. At 0.5 muM free calcium; vanadate (10 muM) inhibited 20-30%, of the Ca-45(2+) accumulation, thapsigargin (33 nm) inhibited 20-30 %, and cyanide (2 mM) plus oligomycin B (2 mug ml-1), or valinomycin (1 mum), 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 - 80 % for 0-5, 5.0, and 50 muM [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 nonmitochondrial (20-30%) calcium pools in this system (at 0.5-5.0 mum 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.