Role of store-operated Ca2+ channels in primary hepatocytes under conditions of calcium overload and ethanol-induced injury

Abstract

To investigate the role of store-operated calcium channels (SOCs) in primary hepatocytes under conditions of calcium overload and ethanol-induced injury. The in vitro model of chronic ethanol-induced hepatocyte injury was established using primary hepatocytes isolated from Sprague-Dawley rats. Ethanol-induced changes (24, 48 and 72 h; 50, 100, 200, 400 and 800 mmol/L) in expression of the SOCs proteins stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Oria1) were detected by qualitative PCR analysis (mRNA) and western blotting (protein). The possible role of these two SOCs proteins in the ethanol-induced extracellular calcium influx and related liver cell injury was determined by treating the cell system with various channel blockers (EGTA, La3+, and 2-APB). Cell viability was determined by MTT assay and cytosolic free calcium ion concentration was determined by flow cytometry. After 24 h of exposure to 0 (untreated) to 800 mM/L ethanol, the cell viability was reduced in a concentration-dependent manner. The 400 mmol/L concentration of ethanol decreased cell viability by 57.34% +/- 2.34%. and was chosen for use in subsequent experiments. Compared with the untreated control cells, the ethanol-treated cells showed significantly up-regulated mRNA and protein expression of both STIM1 and Orai1 at all times examined, suggesting that the ethanol-stimulated expression of STIM1 and Orai1 could persist for at least 72 h. The ethanol treatment induced increase in cytoplasmic calcium levels was significantly (and similarly) reduced by co-treatment with any of the three channel blockers. Chronic ethanol exposure can increase the expression of STIM1 and Orai1 in primary liver cells, suggesting that ethanol may increase extracellular calcium influx by up-regulating expression of these SOCs protein molecules, ultimately aggravating liver cell damage.