Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

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 and 45Ca2+. 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 and 45Ca2+ 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 mumol/liter, respectively. Calcium uptake decreased at higher free Ca2+ concentration. 45Ca2+ uptake was dependent on monovalent cations (Rb+ greater than K+ greater than Na+ greater than Li+ greater than choline+) and different anions (Cl- greater than Br- greater than SO4(2-) greater than NO3- greater than I- greater than cyclamate- greater than SCN-) in both preparations. Twenty mmol/liter oxalate enhanced 45Ca2+ 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 mumol/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.

Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

“…Free Ca 2 + of the medium (corresponding in penneabilized cens to the cytosol) inhibits IP3-induced Ca 2 + release in some cell types and not in others. lt is known from studies of penneabilized rat pancreatic acinar cells or pancreatic subcellular fractions, that the amount of Ca 2 + stored in the endoplasmic reticulum can be increased [21,22]. An important finding in this study is that the additional stored Ca 2 + is directly available for the IP3 induced Ca 2 + release, which suggests that the regulation of intracellular Ca 2 + by the IP3 sensitive Ca 2 + store depends not only on the presence of IP3 but also is a function of the cytosolic free Ca 2 + as weIl as the filling state of the corresponding compartments.…”

Subcellular distribution of Ca2+ pumping sites in human neutrophils.

“…In some studies positive correlations between Ca2`uptake or Ins 1,4,5-P3-induced Ca2' release and marker enzymes for the endoplasmic reticulum have been found (24)(25)(26). However, a recent study with human neutrophils suggested that granules actively pump Ca2' and thus may be the nonmitochondrial Ca2+ pool (27).…”

Immunolocalization of Rap1 in the Rat Parotid Gland: Detection on Secretory Granule Membranes

“…Endoplasmic reticular membranes (ER) were prepared from whole rat parotid glands as described previously by Watson et al (1992), according to the method of Bayerdorffer et al (1984) for rat pancreas.…”