Compartmentation of calcium in digitonin-disrupted guinea pig pancreatic acinar cells

Lucas, Miguel; Galván, Aurora; Solano, Patricia; Goberna, R.

doi:10.1016/0005-2736(83)90001-9

Cited by 17 publications

(11 citation statements)

References 14 publications

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“…Wakasugi et al [61] could distinguish mitochondrial and nonmitochondrial intracellular calcium pools, which was later confirmed by Lucas et al [33]. Electron-dense precipitates in the presence of ATP and oxalate were localized clearly inside the rough endoplasmic reticulum and the presence of calcium in these precipitates was identified by laser microprobe mass analysis [61].…”

Section: Discussionmentioning

confidence: 81%

“…Calcium uptake into sarcoplasmic reticulum [1] and endoplasmic reticulum of various tissues [11,16,20,22,26,33,35,39,44,46,62] can be stimulated by oxalate. This is a characteristic feature of the endoplasmic reticulum, in contrast to calcium uptake into plasma membrane vesicles [30,62], due to the permeability of the endoplasmic reticulum to oxalate.…”

Section: Calcium-precipitating Anionsmentioning

confidence: 99%

“…At least three structures are considered to influence the cytosolic calcium concentration: plasma membrane [30,43,55], mitochondria [5,6,10,12] and one or more not yet clearly identified nonmitochondrial structures [12,33,44,46]. Attempts to determine these nonmitochondrial structures in leaky acinar cells [33,57,61], as well as in isolated microsomal fractions of pancreatic acinar cells [34,44,46], suggest that the endoplasmic reticulum is involved in the regulation of the cytosolic free calcium concentration. The main evidence for this conclusion was based on the presence of calcium oxalate precipitates in the rough endoplasmic reticulum [46,61] and not on Ca 2+ uptake studies in purified membrane fractions of the endoplasmic reticulum.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

et al. 1984

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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.

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Section: Discussionmentioning

confidence: 81%

Section: Calcium-precipitating Anionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

et al. 1984

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“…It should be noted that incubation media with sucrose as a main osmotic component are usually used in experiments with isolated mitochondria of pancreas . However, sucrose‐based medium is only rarely used for incubation of permeabilized acinar pancreacytes . Usually, these cells are incubated in media with the main osmotic component—KCl .…”

Section: Introductionmentioning

confidence: 99%

An implication of novel methodology to study pancreatic acinar mitochondria under in situ conditions

Manko

Klevets

Manko

2012

Cell Biochemistry & Function

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Mitochondria maintain numerous energy-consuming processes in pancreatic acinar cells, yet characteristics of pancreatic mitochondrial oxidative phosphorylation in native conditions are poorly studied. Besides, it is not known which type of solution is most adequate to preserve functions of pancreatic mitochondria in situ. Here we propose a novel experimental protocol suitable for in situ analysis of pancreatic mitochondria metabolic states. Isolated rat pancreatic acini were permeabilized with low doses of digitonin. Different metabolic states of mitochondria were examined in KCl- and sucrose-based solutions using Clark oxygen electrode. Respiration of digitonin-treated, unlike of intact, acini was substantially intensified by succinate or mixture of pyruvate plus malate. Substrate-stimulated respiration rate did not depend on solution composition. In sucrose-based solution, oligomycin inhibited State 3 respiration at succinate oxidation by 65.4% and at pyruvate plus malate oxidation by 60.2%, whereas in KCl-based solution, by 32.0% and 36.1%, respectively. Apparent respiratory control indices were considerably higher in sucrose-based solution. Rotenone or thenoyltrifluoroacetone severely inhibited respiration, stimulated by pyruvate plus malate or succinate, respectively. This revealed low levels of non-mitochondrial oxygen consumption of permeabilized acinar cells. These results suggest a stronger coupling between respiration and oxidative phosphorylation in sucrose-based solution.

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“…Although we cannot ascribe a subcellular localization for this compartment, endoplasmic reticulum is often suggested as being a Ca' +-transporting subcellular organelle, based on Ca+ +-transport properties of microsomal preparations (e.g., Moore and Pastan, 1977;Lucas et al, 1983). Of interest in this regard is a recent study on pancreatic acinar cells (Lucas et al, 1983) in which it was demonstrated that a Ca++ pool exists that is ATPdependent but insensitive to inhibitors of mitochondrial Ca + + uptake. It has transport characteristics similar to those found in a microsomal fraction from these cells.…”

Section: Discussionmentioning

confidence: 97%

Intracellular calcium pools and their metabolic dependence in normal versus simian virus 40‐transformed human fibroblasts

Smith¹,

Tupper²

1984

Journal Cellular Physiology

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Previous studies indicate that although normal and Simian virus (SV40)-transformed WI38 human fibroblasts have similar levels of intracellular Ca++ on a per mg protein basis, their ability to maintain this intracellular Ca++ against a low concentration of extracellular Ca++ differs markedly. The transformed but not the normal cells rapidly lose Ca++ when exposed to low extracellular Ca++, suggesting Ca++ transport and/or sequestration differ in the two cell types. In this study we have extended our investigations of Ca++ metabolism in the two cell types. We observe that normal WI38 cells, when exposed to metabolic inhibitors to deplete intracellular ATP, undergo a twofold increase in intracellular Ca++ levels. Under similar conditions and over the same time course, no comparable change in Ca++ level is observed in the SV40-transformed cell, despite the extensive depletion of ATP. 45Ca++ desaturation curves indicate that the bulk of the net increase in cell Ca++ following ATP depletion of the normal WI38 cell comes to reside in a slowly exchanging Ca++ pool. The data also indicate that glycolysis, and not oxidative phosphorylation, drives the active extrusion of Ca++ from these cells, an observation consistent with previous studies on the Na+-K+ pump in other cell types. Finally, the data indicate that in these cells mitochondria do not appear to be the major subcellular organelle responsible for regulation of at least the two cellular Ca++ pools measurable using isotope desaturation analysis. This is based on the inability of the respiratory inhibitor rotenone to alter significantly the size of either of these Ca++ pools. These pools compose 80-90% of total cell Ca++ in both cell types.

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Compartmentation of calcium in digitonin-disrupted guinea pig pancreatic acinar cells

Cited by 17 publications

References 14 publications

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

An implication of novel methodology to study pancreatic acinar mitochondria under in situ conditions

Intracellular calcium pools and their metabolic dependence in normal versus simian virus 40‐transformed human fibroblasts

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