Isolation of subcellular agonist-sensitive calcium stores from the pancreatic acinar cell.
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 45Ca(2+)-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 45Ca(2+)-loaded acini, subcellular fractions were resolved on sucrose and Nycodenz gradients. 45Ca2+ fluxes were minimized during these procedures by inclusion in the media of LaCl3. 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 Ca(2+)-ATPase, and IP3 receptor. These results demonstrated that the 45Ca(2+)-loading technique can be used to isolate and characterize distinct compartments of the agonist-sensitive Ca2+ store in the pancreatic acinar cell.
Pandol
S J
SJ
Department of Medicine, University of California, San Diego, USA.
Fitzsimmons
T
T
Schoeffield-Payne
M
M
Carlile
G W
GW
Evans
W H
WH
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D008854:Microscopy, Electron; D010179:Pancreas / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000378:metabolism
D002118:Calcium