Endoplasmic Reticulum Calcium Pump Research Articles

Carbon tetrachloride-induced release of calcium from isolated hepatocytes

Studies have shown that CCl 4 administration to rats inhibits endoplasmic reticulum calcium pump activity and reduces the amount of calcium associated with subsequently isolated microsomal subcellular fractions. This report confirms that exposure of isolated hepatocytes to CCl 4 rapidly produces these effects in isolated parenchymal cells and demonstrates that when isolated hepatocytes are exposed to CCl 4, calcium is rapidly released from cells. This release can be detected with a calcium ion-selective electrode when cells are incubated in a medium with low extracellular calcium. Calcium released from an intracellular pool(s) may initiate hepatotoxic changes in liver.

Calcium transport systems in the LLC-PK 1 renal epithelial established cell line

ATP-dependent calcium uptake was measured in membrane vesicles prepared from the renal epithelial LLC-PK 1 established cell line. The relative contribution of the nonmitochondrial versus the mitochondrial calcium uptake is larger in LLC-PK 1 cell homogenates than in homogenates from renal cortex. Two types of calcium pump, characterized by the formation of calcium-dependent phosphointermediates of 135 kDa and 115 kDa, were found in membrane fractions from LLC-PK 1 cells. The 135 kDa calcium pump was also detected by 125I-labelled calmodulin overlay. Although the subcellular localization in LLC-PK 1 cell membranes could not be unambiguously determined, it is conceivable that the 135 kDa and the 115 kDa molecules represent the plasma membrane calcium pump and the endoplasmic reticulum calcium pump respectively, in agreement with what was found for renal cortex preparations. Extravesicular sodium partially inhibits ATP-driven calcium uptake in a plasma-membrane-enriched fraction of the LLC-PK 1 cells. The effect is potentiated by a vesicle inside-negative membrane potential. Although the effect is less pronounced than in renal cortex basal-lateral membranes, this observation suggests that an Na +-Ca 2+ exchange mechanism is also present in LLC-PK 1 cells. ATP-dependent calcium uptake in nonmitochondrial intracellular stores was investigated, using saponin-permeabilized cells. Permeabilized LLC-PK 1 cells lowered the free calcium concentration in the medium to less than 0.4 μM. More than 60% of the accumulated calcium can be released by addition of inositol 1,4,5-trisphosphate. Our data indicate that the LLC-PK 1 cell line can be successfully used as model system for the study of renal calcium handling.

Enhanced hepatotoxicity and inhibition of liver endoplasmic reticulum calcium pump by CCl 4 in rats fed a thiamine deficient diet

Male Sprague-Dawley rats were fed a thiamine deficient diet for three weeks, then treated with a range of CCl 4 doses (0.01–1-ml/kg). Rats fed the deficient diet grew more slowly (body weight 65 percent of control).and had elevated liver glutathione (GSH) (220 percent of control). CCl 4 hepatotoxicity, assessed by serum glutamicpyruvic transaminase (SGPT) activity and histological examination 24 hours after hepatotoxin, was augmented in the group fed the thiamine deficient diet. Likewise, CCl 4 inhibition of liver endoplasmic reticulum (ER) function (glucose-6-phosphatase (G6Pase) and calcium pump activities one hour after CCl 4) was enhanced in rats fed the deficient diet. These results suggest that thiamine deficiency enhances CCl 4 damage to membranes of the ER and enhances CCl 4 hepatotoxicity.

1,1-Dichloroethylene inhibition of liver microsomal calcium pump in vitro

Rat liver microsomes were preincubated with 1,1-dichloroethylene (1,1-DCE) and a NADPH-generating system for 20 min, then ER calcium pump activity determined. Calcium pump activity was inhibited as a function of 1,1-DCE concentration. 1,1-DCE did not inhibit in the absence of a NADPH-generating system. Calcium pump activity was inhibited to a significantly greater extent by 1,1-DCE in microsomes isolated from phenobarbital-pretreated rats than in microsomes from control rats. These studies suggest that the mixed function oxidase system generates a metabolite that attacks the endoplasmic reticulum (ER) calcium pump. The time course of pump inhibition by 1,1-DCE and CCl 4 were compared in vitro. 1,1-DCE produced inhibition of the calcium pump at a substantially slower rate. CCl 4 at 1 μliter/ml inhibited calcium pump activity within l min. 1,1-DCE did not produce significant inhibition within 1 min, but produced 58 ± 5% inhibition by 20 min. CCl 4 but not 1,1-DCE, produced lipid peroxidation as judged by malonic dialdehyde production. It is possible that lipid peroxidation contributes to the rapid course of inhibition produced by CCl 4. These studies suggest that metabolic activation, but not lipid peroxidation, is a prerequisite for inhibition of the liver ER calcium pump by chlorinated hydrocarbon hepatotoxins in vitro.

Destruction of liver microsomal calcium pump activity by carbon tetrachloride and bromotrichloromethane

Disturbed cellular calcium homeostatis has been observed during carbon tetrachloride (CCl 4) poisoning, with large alterations in calcium content occurring 8 hr after administration. Moore el at. [10] have shown that the hepatic smooth endoplasmic reticulum can sequester calcium and that this ability is decreased severely within 30 min after CCl 4 administration to rats. It was suggested that disturbed endoplasmic reticulum calcium pump activity may have a critical role in the expression of CCl 4 hepatotoxicity. We examined the effect of bromotrichloromethane (BrCCl 3) and CCl 4 metabolism on the calcium pump of Fe 2+-free rat liver microsomes. It was determined that severe deficits in calcium uptake can be correlated with minimal lipid peroxidation induced by these agents. At a given level of lipid peroxidation, calcium uptake was affected more severely than were the activities of the microsomal enzymes glucose-6-phosphatase and aminopyrine demethylase. Calcium uptake was increased 7-fold by the presence of 5 mM ATP in incubations prior to assay of calcium sequestration. Lipid peroxidation induced by BrCCl 3-NADPH was accompanied by leakage of calcium from calcium-loaded microsomes. These results strengthen the possibility that disturbances in intracellular calcium homeostasis may be a key event in liver injury induced by BrCCl 3 and CCl 4.