Role of store-operated Ca2+ channels in primary hepatocytes under conditions of calcium overload and ethanol-induced injury / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of store-operated calcium channels (SOCs) in primary hepatocytes under conditions of calcium overload and ethanol-induced injury.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Role of store-operated Ca2+ channels in ethanol-induced intracellular Ca2+ increase in HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs).</p><p><b>METHODS</b>HepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate (2-APB). Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2+ concentration ([Ca2+]i) were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) were evaluated by qPCR and western blotting.</p><p><b>RESULTS</b>The ethanol treatment produced dose-dependent reduction in cell viability (r = -0.985, P less than 0.01) and increases in leakage of ALT (F = 15.286, P less than 0.01) and AST (F = 39.674, P less than 0.01). Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2+]i level (1.25+/-0.36, 1.31+/-0.15, 1.41+/-0.18, 2.29+/-0.25, 2.58+/-0.19; F = 15.286, P less than 0.01). Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2+]i increase (2.32+/-0.08 reduced to 1.79+/-0.15 (t = 7.201, P less than 0.01) and 1.86+/-0.09 (t = 8.183, P less than 0.01) respectively). EGTA and 2-APB also increased the ethanol-treated cells' viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orai1, and the up-regulation effect lasted at least 72 h after treatment.</p><p><b>CONCLUSION</b>Ethanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.</p>