THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, NO. zyxwvutsrqponmlk 17. Issue of September zyxwvutsrqpon 10, pp. 10141-10146, 1982 zyxwvutsrqpon Printed in zyxwvutsrqponmlkjihg U.S A. Chronic Ethanol Ingestion Increases Calcium Uptake and Resistance to Molecular Disordering by Ethanol in Liver Microsomes* zy (Received for publication, April 12, 1982) Biddanda C. Ponnappa, Alan J. Waring, Jan B. Hoek, Hagai Rottenberg, and Emanuel Rubin From the Department zyxwvuts of Pathology and Laboratory Medicine, Hahnemann Medical College, Philadelphia, Pennsylvania 19102 Calcium uptake and molecular ordering were studied in hepatic microsomes from rats treated chronically with ethanol and were compared with the effects of ethanol in zyxwvutsrqpo vitro. Calcium uptakewas increased by 30% in microsomes from ethanol-fed animals, the V,, being increased and the K,,, remaining unchanged. In Arrhen- ius plots of calcium uptake, the transition temperature in the 25-30 “C range was higher by 1.5 “C in micro- somes from ethanol-fed rats. Chronic ethanol adminis- tration resulted in more rigid microsomal membranes, as evidenced by EPR spectra of the spin probe 5-dox- ylstearic acid.In a plot of the order parameter, S, as a function of temperature, microsomes from ethanol-fed rats showed a discontinuity at a higher temperature than those from controls (29.9 versus 24.2 “C). Ethanol, in vitro, inhibited microsomal calcium uptake in both preparations. However, the degree of inhibition was greater in controls. The rate of calcium uptake by mi- crosomes from ethanol-treated animals in the presence of 100 zyxwvuts nm ethanol was the same as those from controls in the absence of ethanol. In uitro, increasing concen- trations of ethanol progressively decreased molecular ordering of control microsomes, whereas little change was noted in microsomes after chronic ethanol treat- ment. Studies from this (1-3) and other (4) laboratories have demonstrated that chronic ingestion of ethanol by rats is associated with structural and functional changes in mem- brane properties of different tissues. Isolated liver mitochon- dria from rats treated chronically with ethanol exhibit reduced respiration rates and ATPase activity (5-7). In addition, mi- tochondrial membranes from ethanol-treated animals are more resistant to the uncoupling effect of ethanol in vitro at physiological temperature (2). Subsequent studies (3) meas- uring the membrane order parameter by EPR using spin labels demonstrated that ethanol, in vitro, had a disordering effect on mitochondrial membranes from control rats, whereas mitochondria from rats fed ethanol chronically were highly resistant to this change. Interestingly, enzyme activities and order parameters in mitochondria from ethanol-fed rats in the presence of 100 mM ethanol were comparable to those of control mitochondria in the absence of ethanol (2, 3). These and other observations from our laboratory (1) suggest that the alterations in the activity of membrane-bound enzymes in * This work was supported inpart by United States Public Health Service Grant AA03442 from the National Institute of Alcoholism and Alcohol Abuse and Grants GM 28173 and AM31086 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. animals chronically exposed to ethanol reflect an adaptation to the presence of ethanol, mediated, at least partly, through a modification of the physical and chemical properties of the membranes. Chronic consumption of ethanol also exerts conspicuous effectson the membranes of the endoplasmic reticulum of the liver (5). Morphologically,an extensive proliferation of smooth endoplasmicreticulum is observed after chronic ethanol inges- tion, accompanied by an increase in microsomal protein, in microsomal drug-metabolizing activities, and in cytochrome P-450 concentration (8, 9). In the studies reported here, we have investigated the relationship between changes in structural and functional parameters of liver microsomal membranes induced by chronic ethanol ingestion. We focused onhepatic microsomal calcium uptake, a membrane-associated process which is not directly involved in ethanol metabolism but plays an impor- tant role in maintaining cellular calcium homeostasis (10). We examined the effects of ethanol, in vivo and in vitro, on microsomal calcium uptake and compared these effects with changes in the physical properties of the microsomal mem- branes, as detected by EPR. The results indicate that, similar to other systems, chronic ingestion of ethanol leads to adaptive changes in hepatic microsomal membranes, which counteract the acute effects of ethanol. MATERIALS AND METHODS Animals-Male Sprague-Dawley rats (Charles River Breeding Laboratories), initially weighing 90-130g, were fed a totally liquid diet (Bioserv, Inc.) in which ethanol provided 36% of total calories. Pair-fed littermate controls received the same diet except that car- bohydrates isocalorically replaced ethanol. Rats were maintained on this diet for 35-40 days, and ethanol consumption averaged 14-16 g/ kg of body weight/day. Preparation of the Microsomal Fraction-Rats werekilled by decapitation at 9-10 a.m. The livers were placed in 5 volumes of a medium containing SMB buffer ((0.25 M sucrose, 10 mM 4-mOrphO- linepropanesulfonic acid (pH 7.0), and 2 mM benzamidine), the last included as a protease inhibitor). Homogenization was carried out with 6 strokes of a motor-driven Potter-Elvehjem homogenizer at 1,OOO rpm. The homogenate was then sequentially centrifuged at 1,500 X g for 10 min and 15,000 X g for 15 min to remove the nuclear and mitochondrial fractions, respectively. The postmitochondrial su- pernatant was centrifuged at 125,000 X g for 45 min to obtain the microsomal pellet. This pellet was resuspended in SMB buffer at a concentration of approximately 10 mg of protein/ml. The operations were carried out at 0-4 “C. Measurement of Calcium Uptake Acti~ity-‘~Ca~+ uptake was measured at 37 “C according to a slightly modified procedure of Moore et al. (11). The incubation medium contained 100 mM KC1,20 mM imidazole (pH 6.8). 5 mM MgCL, 0.2 mM CaCI2, (0.5pCi of 45Ca), 0.36 IIIM ethylene glycol bis(P-aminoethyl ether)-N,N,N’,N’-tetraa- cetic acid, 5 mM ATP (adjusted to pH 6.8 with imidazole), 3 mM sodium azide, and 5 mM ammonium oxalate in a total volume of 1.0 ml. The free Ca2+ concentration, estimated as described by DeMeis and Hasselbach (12), was calculated to be 4.7 p ~. 45Ca2+ uptake was 10141