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

Pandol, S. J., Fitzsimmons, T., Schoeffield-Payne, M., Carlile, G. W. and Evans, William Howard 1995. Isolation of subcellular agonist-sensitive calcium stores from the pancreatic acinar cell. Cell Calcium 18 (5) , pp. 364-376. 10.1016/0143-4160(95)90052-7

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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 Ca2+ transport and storage mechanisms. We 45Ca2+-loaded the agonist-sensitive Ca2+ stores in rat pancreatic acini. The loading was accomplished by first depleting the stores with carbachol stimulation followed by the addition of 45Ca2+ and atropine to the extracellular media. After homogenization of the 45Ca2+-loaded acini, subcellular fractions were resolved on sucrose and Nycodenz gradients. 45Ca2+ fluxes were minimized during these procedures by inclusion in the media of LaCI3. Five subcellular fractions were identified that specifically accumulated 45Ca2+ 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 45Ca2+ uptake. By Western blot analysis, all fractions contained calnexin, p58, sarcoplasmic reticulum type Ca2+-ATPase, and IP3 receptor. These results demonstrated that the 45Ca2+-loading technique can be used to isolate and characterize distinct compartments of the agonist-sensitive Ca2+ store in the pancreatic acinar cell.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Publisher: Elsevier
ISSN: 0143-4160
Last Modified: 04 Jun 2017 07:26

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