In Situ Characterization of Nonmitochondrial Ca²⁺ Stores in Individual Pancreatic β-Cells

Free Ca²⁺ was measured in intracellular stores of individual mouse pancreatic β-cells using dual-wavelength microfluorometry and the low-affinity Ca²⁺ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 umol/1 digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/1 Ca²⁺ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200–500 umol/1. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 umol/1 ruthenium red, or 10 umol/1 antimycin A + 2 ug/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 umol/1, 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 umol/1 ryanodine, or 1–5 umol/1 cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca²⁺ in the pancreatic β-cell is mediated by IP₃ receptors rather than ryanodine receptors.