In situ characterization of nonmitochondrial Ca2+ stores in individual pancreatic beta-cells.
Free Ca2+ was measured in intracellular stores of individual mouse pancreatic beta-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 micromol/l digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200-500 micromol/l. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 micromol/l ruthenium red, or 10 micromol/l antimycin A + 2 microg/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 micromol/l, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1-1 micromol/l ryanodine, or 1-5 micromol/l cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic beta-cell is mediated by IP3 receptors rather than ryanodine receptors.
Department of Medical Cell Biology, Uppsala University, Sweden.
A
A
Tengholm
C
C
Hagman
E
E
Gylfe
B
B
Hellman
D000818:Animals; D000001:Calcimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D004072:Digitonin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D016257:Fura-2 / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D007091:Image Processing, Computer-Assisted; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism; D009765:Obesity / Q000235:genetics; D009840:Oligomycins / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007476:Ionophores; D009840:Oligomycins; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D000001:Calcimycin; C033170:4-bromo-A-23187; D015544:Inositol 1,4,5-Trisphosphate; D004072:Digitonin; D002118:Calcium; D016257:Fura-2