Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

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Abstract
ATP-dependent Ca2+ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca2+-specific electrode and45Ca2+. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na+,K+(Mg2+)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared,45Ca2+ uptake correlated with the RNA content and not with the Na+,K+(Mg2+)ATPase activity. The characteristics of nonmitochondrial Ca2+ uptake into leaky isolated cells and45Ca2+ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg2+, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca2+ concentration of 2 and 2 μmol/liter, respectively. Calcium uptake decreased at higher free Ca2+ concentration.45Ca2+ uptake was dependent on monovalent cations (Rb+>K+>Na+>Li+>choline+) and different anions (Cl−>Br−>SO 4 2− >NO 3 − >I−>cyclamate−>SCN−) in both preparations. Twenty mmol/liter oxalate enhanced45Ca2+ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca2+ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca2+ concentration to 0.4 μmol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca2+ concentration in pancreatic acinar cells.