Homeostasis of Mitochondrial Calcium in Alcoholic Liver Diseases

Abstract

The production of reactive oxygen species (ROS) and oxidative stress are thought to be important pathological factors in onset and development of alcoholic liver diseases (ALD). Elevated level of ROS has previously been shown to alter calcium homeostasis by changing the activity of calcium-release and calcium-uptake mechanisms in the ER and mitochondria. In these studies, we investigated the effects of chronic ethanol consumption on calcium content and calcium handling in the mitochondria. Hepatocytes from ethanol-fed and their paired-fed littermates were cultured in the absence of alcohol for 1 to 18 hrs then loaded with fura-2/AM. Cells were washed into calcium free buffer then treated with the mitochondrial uncoupler, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), plus oligomycin, an inhibitor of F1FO-ATPase, to induce calcium release from mitochondrial matrix. The resultant increase in cytosolic calcium was measured with fura-2. The data show that FCCP-induces more calcium release in hepatocytes isolated from alcohol-fed rats compared to controls. The differences in FCCP-releasable calcium content was observed in both overnight-cultured cells and in freshly plated hepatocytes suggesting that chronic alcohol exposure causes a long lasting increase mitochondrial matrix calcium levels. The rates of mitochondrial calcium-uptake and efflux were determined in fresh-isolated and digitonin-permeabilized cells using extramitochondrial fura-6 FF free acid. The data indicate that the rates of mitochondrial calcium uptake were significantly faster in alcoholics compared to controls. The rates of mitochondrial calcium efflux were not significantly different when the matrix calcium content was normalized between control and alcoholics. These data indicate that long-term alcohol exposure induces adaptive changes in the mitochondrial calcium-uptake mechanisms that lead to chronically elevated levels of calcium in the mitochondrial matrix. This adaptive process is predicted to increase mitochondrial ROS production and thereby contribute to the pathogenesis of ALD.