Alcohol-Induced Tolerance in Mitochondrial Membranes

centrations at equilibrium is essential for assessment of the uncertainty in binding data now available. PENTTI K. SIITERI Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco 94143 References 1. I. M. Klotz, Science 217, 1247 (1982); ibid. 220, 981 (1983). 2. P. J. Munson and D. Rodbard, ibid., p. 979. 3. G. Scatchard, Ann. N.Y. Acad. Sci. 51, 660 (1949). 4. J. F. Tait and S. Burstein, in The Hormones, G. Pincus, K. V. Thimann, E. B. Astwood, Eds. (Academic Press, New York, 1964). 5. G. L. Hammond, J. A. Nisker, L. A. Jones, P. K. Siiteri, J. Biol. Chem. 225, 5023 (1980). 29 June 1983; accepted 27 October 1983 Alcohol-Induced Tolerance in Mitochondrial Membranes other modulations of membrane structure and function. . . (6). Since Lieber and his colleagues (1) analyzed only total fatty acid composition of the mitochondrial membranes, and cardiolipin is a minor component, they could not detect this increased saturation of cardiolipin acyl chains. Nevertheless, their data regarding total fatty acid composition actually confirm our findings of a significant increase in stearic acid and a decrease in palmitic acid in both phosphatidylcholine and phosphatidylethanolamine. Our evidence for increased resistance to disordering by ethanol is based on studies with electron paramagnetic resonance (EPR) spin probes (2). The order parameter, measured by 5-doxyl stearic 13 JANUARY 1984 acid or 12-doxyl stearic acid, is significantly decreased by low concentrations of ethanol in liver mitochondria from normal rats but not in ethanol-fed rats. Similarly, the partition of doxyl-decane is greatly enhanced by ethanol in control rats but not ethanol-fed rats. Similar results were obtained earlier by several other groups in synaptosomal membranes and red blood cells (7) and more recently by us in liver microsomes (8). We, therefore, believe that this is a general phenomenon relevant to all membranes in all tissues (9). It is necessary to explain why Lieber and his colleagues could not confirm this observation in their studies. We have found that the resistance to disordering by ethanol is observed at high temperature (35°C) but not at low temperature (15°C) (2). Lieber and his colleagues measured 12-(9-anthroyloxy) stearic acid (12 AS) fluorescence anisotropy at 28°C, where the difference, if it exists, is expected to be small. The sensitivity of 12 AS anisotropy to small structural changes is at least one order of magnitude lower than that of the EPR technique, particularly in highly scattering membranes such as mitochondria. In fact, we suspect that scattering artifacts were not properly corrected for in their studies. Vanderkooi and Chance (10) studied fluorescence anisotropy of 12 AS in mitochondria at 200 to 45°C (Fig. 3 in 10); their measured polarization values ranged from 0.125 to 0.1. This value corresponds to an anisotropy range of 0.087 to 0.069. We measured the fluorescence anisotropy of 12 AS in mitochondria and obtained a value of 0.08 at 28°C. This value is in excellent agreement with those obtained by Vanderkooi and Chance (10), but is one-third those reported by Lieber and his colleagues (1). Nevertheless, because of the low values of fluorescence anisotropy and the large corrections for light scattering, the effect of low concentrations of ethanol on membrane fluidity cannot be easily detected. We suspect that Lieber and his colleagues, in fact, measured the Philadelphia, Pennsylvania 19102 References and Notes Gordon, J. Rochman, M. Arai, C. S. Lieber, Science 216, 1319 (1982). 2. H. Rottenberg, D. E. Robertson, E. Rubin, Lab. Invest. 42, 318 (1980); H. Rottenberg, A. J. Waring, E. Rubin, Science 213, 583 (1981); H. Rottenberg, T. Ohnishi, E. Rubin, Arch. Biochem. Biophys. 216, 51 (1982). 3. A. I. Cederbaum, C. S. Lieber, E. Rubin, Arch. Biochem. Biophys. 165, 560 (1974). 4. W. S. Thayer and E. Rubin, J. Biol. Chem. 256, 6090 (1981). 5. J. Burke and E. Rubin, Lab. Invest. 41, 393 (1979). 6. A. Waring, H. Rottenberg, T. Ohnishi, E. Rubin, Proc. Natl. Acad. Sci. U.S.A. 78, 2582 (1981). 7. J. H. Chin and D. B. Goldstein, Science 196, 684 (1977); D. A. Johnson, N. M. Lee, E. R. Cook, H. W. Loh, Mol. Pharmacol. 15, 739 (1979). 8. B. C. Ponnappa, A. J. Waring, J. B. Hoek, H. Rottenberg, E. Rubin, J. Biol. Chem. 257, 10141 (1982). 9. E. Rubin and H. Rottenberg, Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 2465 (1982). 10. J. M. Vanderkooi and B. Chance, FEBS Lett. 22, 23 (1972). 2 August 1982; accepted 12 November 1982 1. E. R. Gordon et al. (1) examined the basis of challenging theory proposed by Rottenberg and his colleagues (2) that the "chronic consumption of ethanol induces an adaptation of membrane composition causing increased membrane rigidity (decreased fluidity).... The increased rigidity impairs normal membrane function'. . . but in the presence of moderate concentrations of ethanol the membrane becomes sufficiently fluid to resemble normal membranes (depena dence)" (2, 3). To examine this theory, we designed a controlled nutritional experiment, which included a group of Chow-fed rats (for which respiratory functions have been well defined), ethanol-fed rats, and the pair-fed controls of the latter. Mitochondrial membranes from the Chow-fed animals contained a larger amount of saturated fatty acids than the mitochondrial preparations from the pair-fed controls and were more resistant to the fluidizing effects of ethanol, although respiratory functions in the membranes of the two groups were similar. In contrast, the 193 Downloaded from http://science.sciencemag.org/ on April 25, 2020 Lieber and his colleagues (1) state that they have found no evidence for a correlation between the structure of membrane phospholipids and function of mitochondrial membranes from ethanolfed rats, or for resistance to disordering by ethanol, which we reported earlier (2). However, extensive studies in our laboratory show that the decreased rate of respiration, first described by Cederbaum et al. (3) in intact mitochondria, can be explained as a direct consequence of decreased content and activity of individual protein components of the respiratory chain in mitochondrial inner membranes (4). What causes this decrease is not clear. It might be due to direct inhibition of mitochondrial protein synthesis (5) or to interference with membrane assembly. The latter may be influenced by the phospholipid composition of the membranes. Moreover, the respiratory activity indeed may be influenced by the phospholipid composition. We have shown that mitochondrial membranes from ethanol-fed rats display an increased saturation in the acyl chains of cardiolipin (6), an essential phospholipid component of the electron transport chain, which may contribute to the regulation of the respiration rate. Therefore, we suggested that "The phospholipid composition ... probably plays a role in considerable effect of alcohol on light scattering, which is caused by mitochondrial swelling. In summary, there is sufficient evidence from studies of rat liver mitochondria and other membrane systems to indicate that chronic alcoholism is associated with changes of membrane structure, composition, and function and that these changes lead to tolerance to the acute effects of ethanol. H. ROTTENBERG A. WARING E. RUBIN Department of Pathology and Laboratory Medicine, Hahnemann University School of Medicine, Alcohol-induced tolerance in mitochondrial membranes H Rottenberg, A Waring and E Rubin Science 223 (4632), 193. DOI: 10.1126/science.6691147 http://science.sciencemag.org/content/223/4632/193.1.citation PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions Use of this article is subject to the Terms of Service Science (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. The title Science is a registered trademark of AAAS. Copyright © 1984 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Downloaded from http://science.sciencemag.org/ on April 25, 2020 ARTICLE TOOLS