Isolation of subcellular agonist-sensitive calcium stores from the pancreatic acinar cell

Abstract

The purpose of the present study was to develop a technique to identify, isolate and partially purify these membrane bound compartments for further characterizations of their Ca 2+ transport and storage mechanisms. We 45Ca 2+-loaded the agonist-sensitive Ca 2+ stores in rat pancreatic acini. The loading was accomplished by first depleting the stores with carbachol stimulation followed by the addition of 45Ca 2+ and atropine to the extracellular media. After homogenization of the 45Ca 2+-loaded acini, subcellular fractions were resolved on sucrose and Nycodenz gradients. 45Ca 2+ fluxes were minimized during these procedures by inclusion in the media of LaCI 3. Five subcellular fractions were identified that specifically accumulated 45Ca 2+ after carbachol stimulation. Electron microscopic observations of the fractions demonstrated that three of the fractions consisted of rough membrane vesicles; that one consisted of a mixture of rough and smooth membrane vesicles; and that one consisted of smooth membrane vesicles. All fractions were enriched in glucose-6-phosphatase. All 5 fractions demonstrated ATP dependent 45Ca 2+ uptake. By Western blot analysis, all fractions contained calnexin, p58, sarcoplasmic reticulum type Ca 2+-ATPase, and IP 3 receptor. These results demonstrated that the 45Ca 2+-loading technique can be used to isolate and characterize distinct compartments of the agonist-sensitive Ca 2+ store in the pancreatic acinar cell.