Evidence for vacuolar-type proton pumps in nonmitochondrial and inositol 1,4,5-trisphosphate-sensitive calcium stores of insulin-secreting cells.
This study examines whether acidic, vacuolar-type, proton-pump-carrying organelles of insulin-secreting cells (clonal endocrine pancreatic cell line INS-1) function as rapidly exchanging, inositol 1,4,5-trisphosphate-sensitive calcium stores. Calcium uptake into calcium stores will be modulated by the proton concentration within the stores, since calcium pumps in general appear to mediate a countertransport of calcium with protons. We therefore tested for sensitivity of calcium sequestration by nonmitochondrial stores (inhibition of mitochondrial calcium uptake by 2 microM ruthenium red) in saponin-permeabilized cells to proton-conducting ionophores and proton pump inhibition, using this as a marker for involvement of acidic organelles. Calcium sequestration was partially inhibited by the protonophores nigericin (10-50 microM) and carbonylcyanide m-chlorophenylhydrazone (CCCP; 20-50 microM), as well as by inclusion of 30 mM NH4Cl. Bafilomycin A1, a potent and selective inhibitor of vacuolar-type proton pumps, alone (1 - 500 nM) had no effect on calcium sequestration. however, it induced an inhibitory effect in the presence of nigericin or CCCP, even at low concentrations (5 microM) of these ionophores, lacking itself an inhibitory action on calcium sequestration. Bafilomycin A1 then was already maximally active at a concentration as low as 10 nM. Corres ponding to inhibition of total nonmitochondrial calcium sequestration, filling of inositol 1,4,5-trisphosphate-sensitive stores was decreased or even abolished by the protonophores alone or the protonophores combined with bafilomycin A1. We conclude that vacuolar-type proton pumps are present in at least a part of nonmitochondrial and inositol 1,4,5-trisphosphate-sensitive calcium stores in INS-1 cells. This assigns these stores to organelles such as secretory granules, the trans Golgi network, or endosomes. Luminal acidity of these stores will stimulate calcium sequestration by providing more protons for countertransport of calcium by calcium pumps. High concentrations of protonophores may be required for inhibitory effects because otherwise the proton pumps may be able to compensate sufficiently for ionophore-mediated proton loss. The lack of effect of bafilomycin A1 without protonophores may be due to a sufficient luminal buffering capacity or to preceding inhibition of the pump by an inside-positive transmembrane potential.
Bode
H
H
Department of Pharmacology, Philipps-University, Karl-von-Frisch-Strasse, D-35033 Marburg, Germany.
Himmen
A
A
Göke
B
B
D000643:Ammonium Chloride / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007476:Ionophores / Q000494:pharmacology; D007515:Islets of Langerhans / Q000378:metabolism*; D018942:Macrolides; D008928:Mitochondria / Q000378:metabolism*; D009550:Nigericin / Q000494:pharmacology; D010539:Permeability; D017494:Proton Pumps / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D012430:Ruthenium Red / Q000494:pharmacology; D014617:Vacuoles / Q000378:metabolism
D000900:Anti-Bacterial Agents; D007328:Insulin; D007476:Ionophores; D018942:Macrolides; D017494:Proton Pumps; D000643:Ammonium Chloride; D012430:Ruthenium Red; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D015544:Inositol 1,4,5-Trisphosphate; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases; D009550:Nigericin; D002118:Calcium