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Abstract

Ca²⁺ uptake into isolated exocrine pancreatic cells with highly permeable plasma membrane was determined by measuring the decrease in free Ca²⁺ concentration of the surrounding incubation medium with a Ca²⁺-specific electrode. In the presence of Mg-ATP and respiratory substrates the free Ca²⁺ concentration of the incubation medium decreased rapidly after addition of leaky cells until a stable medium free Ca²⁺ concentration of 4.2 +/- 0.1 X 10^-7 mol/l was obtained. Changes in the medium free Ca²⁺ concentration at steady state by addition of Ca²⁺ or EGTA were buffered by cellular uptake or release, respectively, until the steady-state free Ca²⁺ concentration was reestablished. When nonmitochondrial Ca²⁺ uptake was determined in the presence of a combination of mitochondrial inhibitors (10^-5 mol/l antimycin, 5 X 10^-6 mol/l oligomycin, and 10^-2 mol/l azide), the rate of uptake was considerably reduced, while the steady-state concentration was unaltered. In contrast, mitochondrial uptake that could be observed in the presence of the ATPase inhibitor vanadate (2 X 10^-3 mol/l) proceeded at the same rate as the control, but the minimal medium free Ca²⁺ concentration reached was 2.4 +/- 0.1 X 10^-7 mol/l higher than the control. Addition of secretagogues at steady-state free Ca²⁺ concentration resulted in a Ca²⁺ release of 0.73 +/- 0.08 nmol/mg protein. The increase in medium free Ca²⁺ concentration was entirely transient and followed by reuptake to the prestimulation level. The data indicate that a cytosolic free Ca²⁺ concentration of 4 X 10^-7 mol/l can be regulated in pancreatic acinar cells by a nonmitochondrial Mg²⁺-dependent Ca²⁺ pool.