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Abstract

Regulation of steady-state free Ca²⁺ concentration at rest and at stimulation have been studied in isolated permeabilized pancreatic acinar cells by measuring the free Ca²⁺ concentration of the surrounding incubation medium with a Ca²⁺-specific electrode. Ca²⁺ transport mechanisms have been further characterized in subcellular membrane fractions by measuring ⁴⁵Ca²⁺ uptake into membrane vesicles and protein phosphorylation using polyacrylamide gel electrophoresis. (a) In permeabilized isolated acinar cells from exocrine glands, inositol-1,4,5-trisphosphate (IP₃) releases Ca²⁺ from endoplasmic reticulum. (b) Secretagogue-induced Ca²⁺ release from permeabilized cells is accompanied by increased production of IP₃. At rest, steady-state free Ca²⁺ concentration is regulated at 4 X 10 mol/L by the rough endoplasmic reticulum (RER). Ca²⁺ uptake into this pool is promoted by a (Ca²⁺ + Mg²⁺)-ATPase, and is dependent on cations and anions in the incubation medium in the order K⁺ greater than Na⁺ greater than Li⁺ greater than choline⁺ and Cl^- greater than Br^- greater than SO₄^2- = NO_3- greater than I^- greater than cyclamate- greater than SCN^-, respectively. Similarly, Ca²⁺-stimulated ³²P incorporation from [gamma ³²P]ATP into a 130 kD protein intermediate of (Ca²⁺ + Mg²⁺)-ATPase, as well as ³²P liberation, indicating (Ca²⁺ + Mg²⁺)-ATPase activity, are cation dependent. While ³²P incorporation is highest in the presence of choline, ³²P liberation is higher with K⁺, as compared with Na⁺ or choline, indicating that K⁺ ions facilitate dephosphorylation