Abstract
The mechanisms underlying neuronal adaptation to ethanol are poorly understood but appear to involve alterations in cellular membrane structure and/or function. Using a two-dimensional gel analysis, we have recently identified Hsc70 as an ethanol-responsive gene (Miles, M.F. (1989) Neurology 39, (Suppl. 1), 425). Hsc70 is a constitutive member of the 70-kDa stress protein family which plays an important role in protein trafficking and coated vesicle processing. Thus, modulation of Hsc70 by ethanol could produce widespread changes in cellular membrane functioning. Here, we report a detailed study on the regulation of Hsc70 by ethanol in NG108-15 neuroblastoma x glioma cells. Northern and Western blot analyses showed that ethanol concentrations observed in actively drinking alcoholics caused an induction of Hsc70 mRNA and protein. Increases in Hsc70 mRNA were seen as early as 4 h after exposure to ethanol. In comparison with ethanol, propanol and butanol caused proportionally greater increases in Hsc70 mRNA. This is consistent with known anesthetic and intoxicating potencies of these aliphatic alcohols and suggested that lipophilicity, rather than an osmotic effect, was critical for ethanol induction of Hsc70. Induction of Hsc70 mRNA by ethanol resulted, at least in part, from increased Hsc70 gene transcription as determined by nuclear runoff studies. Stable transfection analysis revealed an ethanol-responsive cis-acting element in the proximal 2500 base pairs of the Hsc70 promoter. Regulation of Hsc70 by 50-200 mM ethanol appeared to be a specific change in expression of an ethanol-responsive gene rather than a typical stress protein response since no induction of the highly inducible stress protein, Hsp70, was seen at these ethanol concentrations. These results suggest that ethanol-induced changes in Hsc70 transcription may be important for neuronal adaptation to ethanol and the development of tolerance and dependence in alcoholics.
Highlights
From the $Ernest Gallo Clinic and Research Center and the §Departmenotf Neurology, University of California School of Medicine, Sun Francisco General Hospital, S a n Francisco, California 94110
We have conducteda detailed analysisof ethanolinduced changesin Hsc7O abundance.We show here that concentrations of ethanol observed in actively drinking alcoholicsproduced significant increases in Hsc7O mRNA and protein.,ourstudies provide thefirstdirect evidence for ethanol regulating gene expression at the level
chloramphenicol acetyltransferase (CAT) activity was expressed as percent of total chloramphenicol incorporated into product and under conditions used here was always less than 30%
Summary
From the $Ernest Gallo Clinic and Research Center and the §Departmenotf Neurology, University of California School of Medicine, Sun Francisco General Hospital, S a n Francisco, California 94110. Increases in Hsc7O mRNA were dependent calcium channels have alsobeen observed [11].It seen as early as 4h afterexposuretoethanol.In is generally unknown how ethanol produces changes in so comparison with ethanol, propanol and butanol causmedanymembraneproteins.Recently, several investigators proportionally greater increases in Hscm70RNA. This havereportedethanol-inducedchangesin specific mRNA is consistent with known anesthetic and intoxicating potencies of these aliphaticalcohols and suggested that lipophilicity, rather than aonsmotic effect,was critical for ethanol induction of Hsc7O. Ethanol Induces Hsc Gene Expression cations for the molecular mechanisms underlying cellular adaptation to chronic ethanol exposure
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