Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells

Abstract

ATP-dependent 45Ca2+ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl2 and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na+,K+)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca2+ uptake was maximal with 10 and 2 mumol/liter free Ca2+, and half-maximal with 0.9 and 0.5 mumol/liter free Ca2+. It was maximal at 3 and 0.2 mmol/liter free Mg2+ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg2+ was replaced by Mn2+ or Zn2+ ATP-dependent Ca2+ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn2+ could replace Mg2+ for Ca2+ uptake by 20%. Other divalent cations such as Ba2+ and Sr2+ could not replace Mg2+ in Ca2+ uptake. Ca2+ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca2+ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca2+ uptake. The sequence was K+ greater than Rb+ greater than Na+ greater than Li+ greater than choline+ in plasma membranes and Rb+ greater than or equal to K+ greater than or equal to Na+ greater than Li+ greater than choline+ for rough endoplasmic reticulum. The anion sequence was Cl greater than or equal to Br greater than or equal to 1 greater than SCN greater than NO3 greater than isethionate greater than cyclamate greater than gluconate greater than SO2(4) greater than or equal to glutarate and Cl- greater than Br greater than gluconate greater than SO2(4) greater than NO3 greater than 1 greater than cyclamate greater than or equal to SCN, respectively. Ca2+ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K+ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca2+ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca2+ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca2+ from the cell that might be involved in the regulation of the cytosolic Ca2+ level.

Acetylcholine and cholecystokinin receptors functionally couple by different G‐proteins to phospholipase C in pancreatic acinar cells

“…Preparation of a fraction enriched in plasma membranes was performed as described recently, using a MgClz precipitation method [19].…”

Tmp21 and p24A, Two Type I Proteins Enriched in Pancreatic Microsomal Membranes, Are Members of a Protein Family Involved in Vesicular Trafficking

“…Plasma membranes were purified using the MgCl 2 precipitation method (Bayerdörffer et al, 1985). Microsomal membranes and the cytosol were prepared by differential centrifugation, as described previously .…”

Sr2+ can Pass through Ca2+ Entry Pathway Activated by Ca2+ Depletion, but can be Hardly Taken up by the Ca2+ Stores in the Rat Salivary Acinar Cells.

“…In some cases, IP3 and caffeine were added in the medium 40 min after addition of ATP. At indicated times aliquots were removed from the incubation medium by a rapid filtration technique (Bayerdorffer et al 1985). The radioactivity was counted in a liquid scintillation counter (LSC 1,000; Aloka, Tokyo).…”