IN-SITU CHARACTERIZATION OF NONMITOCHONDRIAL CA2-CELLS( STORES IN INDIVIDUAL PANCREATIC BETA)

Free Ca2+ was measured in intracellular stores of individual mouse pan creatic p-cells using dual-wavelength microfluorometry and the low-aff inity Ca2+ indicator furaptra. Controlled permeabilization of the plas ma membrane with 4 mu mol/l digitonin revealed that 22% of the furaptr a was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ mere simultaneously present, this cation rapid ly accumulated in the organelle pool, reaching an average concentratio n of 200-500 mu mol/l. Whereas agents affecting the mitochondrial func tion (5 mmol/l succinate, 2 mu mol/l ruthenium red, or 10 mu mol/l ant imycin A + 2 mu g/ml oligomycin) had Little effects, the Ca2+-ATPase i nhibitor thapsigargin released 92%, of the Ca2+ mobilizable with the i onophore Br-A23187. Digital imaging revealed regional differences in t he organelle Ca2+. The regions with the highest Ca2+ concentration wer e particularly responsive to inositol 1,4,5-trisphosphate (IP3). TP, m obilized Ca2+ in a dose-dependent way with half-maximal and maximal ef fects at about 1 and 5 mu mol/l, respectively. High concentrations of IF, released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, suc h as 10 mmol/l caffeine, 0.1-1 mu mol/l ryanodine, or 1-5 mu mol/l cyc lic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic p-cen is mediated by IP3 recepto rs rather than ryanodine receptors.