- Email: [email protected]
Effects of Acute and Chronic Ethanol Exposure on the Hepatic Gamma-Aminobutyric Acid Transport System in Rats
Alcohol, Vol. 19, No. 3, pp. 213–218, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/99/$–see front matter
PII S0741-8329(99)00049-X
Effects of Acute and Chronic Ethanol Exposure on the Hepatic Gamma-Aminobutyric Acid Transport System in Rats Y. GONG, L. CUI AND G. Y. MINUK Liver Diseases Unit, Departments of Medicine and Pharmacology, University of Manitoba, Winnipeg, Manitoba, Canada Received 10 February 1999; Accepted 11 June 1999 GONG, Y., L. CUI AND G. Y. MINUK. Effects of acute and chronic ethanol exposure on the hepatic gamma-aminobutyric acid transport system in rats. ALCOHOL 19(3) 213–218, 1999.—Ethanol-induced increases in gamma-aminobutyric (GABA)ergic activity contribute to the impairment in hepatic regeneration associated with alcohol-induced liver disease. To determine the mechanism(s) whereby ethanol increases GABAergic activity in the liver, we documented the effects of acute (5 g/kg 3 1) and chronic (36% of total calories over 6 weeks) ethanol exposure as well as exogenous GABA (500 mg/g body weight) administration on GABA transport protein (GABA-TP) mRNA expression in the livers of adult male Sprague– Dawley rats at various times (0–72 h) post 70% partial hepatectomy (PHx). We also documented the in vitro effects of ethanol (30–90 mM) on [3H]-GABA uptake in isolated rat hepatocytes. The results of the study revealed that compared to salineexposed controls, acute but not chronic ethanol exposure resulted in significant decreases in GABA-TP mRNA expression at 12, 24, and 48 h post PHx (saline exposed, 1.04 6 0.06, 1.19 6 0.21, and 1.15 6 0.05, vs. acute ethanol exposed, 0.80 6 0.16, 0.88 6 0.09, and 0.86 6 0.16 optical density units, p , 0.01, 0.05, and 0.05, respectively). An inhibitory effect was also observed following exogenous GABA administration (GABA-TP mRNA expression at 3 h was approximately 40% that of baseline, p , 0.05). [3H]-GABA uptake in isolated rat hepatocytes in vitro was unaffected by the presence of ethanol. In conclusion, the results of this study indicate that acute but not chronic ethanol exposure and exogenously administrated GABA inhibit hepatic GABA-TP mRNA expression following partial hepatectomy in the rat. These findings suggest that the increased GABAergic activity that occurs in the liver following acute ethanol exposure results from alterations in the hepatic GABA transport system at a transcriptional level. © 1999 Elsevier Inc. All rights reserved. Ethanol
GABA
Hepatic regeneration
GABA transport
ACUTE but perhaps not chronic ethanol exposure is a potent inhibitor of hepatic regenerative activity (12,31). How acute ethanol exposure inhibits hepatic regenerative activity remains unclear. Recently, we reported that at least some of this inhibitory effect could be prevented by the prior administration of gammaaminobutyric (GABAA) receptor antagonists suggesting that enhanced hepatic GABAergic activity may be involved (16). As in the brain, the liver contains both GABA receptor and transport systems (21,26). The latter is thought to be responsible for clearing GABA from the surface of hepatocytes and thereby regulating the amount of GABA available to bind to hepatic GABAA receptor sites (21). Thus, one would predict that interference with the GABA transport protein system (GABA-TP) would result in enhanced hepatic GABAA
Liver
receptor activity and impaired hepatic regeneration. In the present study, we tested this hypothesis by documenting the effects of acute and chronic ethanol exposure on hepatic GABA-TP expression in rats. METHOD
Effects of Acute Ethanol Exposure on GABA-TP mRNA Abundance To document the effects of acute ethanol exposure on GABA-TP, adult male Sprague–Dawley (SD) rats (200–250 g) were fed ethanol (5 g/kg) by gastric gavage 1 h prior to a 70% partial hepatectomy as described by Higgins and Anderson,
Requests for reprints should be addressed to Dr. G.Y. Minuk, Liver Diseases Unit, GF407, Health Sciences Centre, 820 Sherbrook Street, Winnipeg, Manitoba, Canada, R3A 1R9. Tel: (204) 787-4662; Fax: (204) 775-4255; E-mail: [email protected]
213
214
GONG, CUI AND MINUK
or sham surgery while under ether anesthesia (9). Groups of rats (n 5 4–6/group) were then sacrificed by cardiac puncture at times 6, 12, 24, and 48 h postsurgery. GABA-TP mRNA abundance was documented in the excised livers in the following manner: Total RNA was extracted from liver tissues by the lithium chloride/urea method (2). The RNA was resolved on 1% formaldehyde-agarose gels, and transferred onto GT nylon membranes (BioRad, Hercules, CA). The membranes were then hybridized with a radiolabeled mouse GABA transporter probe, GAT-3 (kindly provided by Dr. N. Nelson, Roche Institute of Molecular Biology) and 28S-rRNA cDNA probe respectively in a hybridization solution which consisted of 50% formamide, 0.2 M NaCl, 0.12 M Na2HPO4, and 7% SDS. Membranes were washed twice with 23 SSC/0.1% SDS at room temperature for 15 min and 0.23 SSC/0.1% SDS at 428C for 15 min. X-ray films exposed to the membranes were scanned, and the bands were quantitated by an NIH-IMAGE program (NIH, Bethesda, MD). All bands were standardized against the concurrently run 28S-rRNA. Effects of Chronic Ethanol Exposure on GABA-TP mRNA Abundance Chronic ethanol exposure was as described by Gill et al. (8). Briefly, rats received increasing concentrations of ethanol (3%, 5%, 7% to 9%, v/v) ad libitum for 16 days. Thereafter, all rats received 9% (v/v) ethanol for a total of 6 weeks. Isocaloric-fed rats served as controls. At 6 weeks, a 70% partial hepatectomy or sham surgery was performed. Thereafter, groups of rats (n 5 4–6/group) were sacrificed at 3, 6, 12, 24, and 48 h postsurgery. Hepatic GABA-TP mRNA abundance
was determined at each time interval in both ethanol-exposed and isocaloric-fed controls as described above. Effects of GABA on the GABA Transport System To determine whether the effects of ethanol on GABA-TP resulted from ethanol-induced increases in serum GABA concentrations, the following additional experiment was performed. Intraperitoneal injections of GABA (500 mg/g body weight) or equal volumes of saline were administered to adult SD rats (n 5 4/group). GABA-TP mRNA expression was then documented at times 0, 1, 3, 6, 12, and 24 h as described above. The dose of GABA had been predetermined to result in elevated serum GABA levels that approximate those reported in experimental animals and humans with advanced liver disease (20–50 mmol/liter) for approximately 4 h postinjection (10). Effects of Ethanol on GABA Transport by Isolated Hepatocytes Rat hepatocytes were isolated using a modification of the collagenase perfusion method described by Berry and Friend (4). Briefly, following the sacrifice of adult male SD rats by exsanguination under ether anesthesia, suspensions of liver cells were prepared by perfusing the livers in situ with calcium- and magnesium-free Hanks balanced salt solution (HBSS) followed by perfusion with complete HBSS containing collagenase (0.5% w/v). Following a series of filtrations and washes, 95% of isolated hepatocytes excluded trypan blue and contamination with sinusoidal cells as identified morphologically was less than 1%. Each assay was initiated by the addition of 1 ml of the suspen-
FIG. 1. Expression of GABA-TP mRNA in acute ethanol- (5 g/kg) or saline-exposed rats after sham or partial hepatectomy. The figure represents GABA-TP mRNA of one rat from four different groups as indicated, and at four different times after sham or partial hepatectomy (6, 12, 24, and 48 h). The same membranes were rehybridized with 28S-rRNA probe as a loading control.
ETHANOL AND GABA TRANSPORT
215
FIG. 2. Summary of the relative levels of GABA-TP expression in four different groups (see legend to Fig. 1), and four different times after sham or partial hepatectomy as indicated. The data represent the mean 6 SE (n 5 4–6/group). *p , 0.05; **p , 0.01.
sion of hepatocytes (standardized to 5 3 105 cells/ml) to 1 ml of buffer containing [3H]-GABA (final concentration 10 nM) and when indicated, unlabeled GABA (10 mM) or ethanol (30–90 mM). Incubation mixtures were shaken every 5 min. At designated time intervals, 250-ml samples were removed from the mixtures and placed in 400-ml microfuge tubes that already contained 100 ml of silicone oil and 50 ml of potassium hydroxide.
Following centrifugation through the microfuge tubes, specific uptake of [3H]-GABA was calculated by subtracting cell-associated [3H]-GABA in the presence of unlabeled GABA (nonspecific) from cell-associated [3H]-GABA in the absence of unlabeled GABA (total). Thus, specific 5 total 2 nonspecific. Ethical consent for this study was obtained from the Animal Care Committee at the University of Manitoba.
FIG. 3. Expression of GABA-TP mRNA in chronic ethanol-exposed rats and isocaloric-fed controls after partial hepatectomy. GABA-TP mRNA in two rats from ethanol or control groups, and at six different times as indicated are presented. The same membranes were rehybridized with 28S-rRNA probe as a loading control.
216
GONG, CUI AND MINUK
FIG. 4. Summary of the relative levels of GABA-TP expression in chronic ethanol-exposed rats and isocaloric-fed controls at 0, 3, 6, 12, 24, and 48 h after partial hepatectomy. The data represent the mean 6 SE (n 5 4–6/group). *p , 0.05.
Statistical Analyses
RESULTS
Analyses of variance followed by paired Student’s t-tests for parametric data and a Wilcoxon Rank Sum test for nonparametric data were performed where appropriate. p-Values ,0.05 were considered significant. The results provided represent the mean 6 standard error of the mean.
As demonstrated by the results in saline-treated sham and partial hepatectomized groups (Figs. 1 and 2), partial hepatectomy per se does not alter GABA-TP mRNA expression during the time intervals studied. The results of acute ethanol exposure on hepatic GABA-
FIG. 5. The time-dependent effect of GABA on rat liver GABA-TP mRNA expression. The figure represents GABA-TP mRNA expression in two rats after i.p. injection of GABA (500 mg/g body weight). The same membranes were rehybridized with 28SrRNA probe as a loading control.
ETHANOL AND GABA TRANSPORT
FIG. 6. Summary of the time-dependent effect of exogenous GABA administration on rat liver GABA-TP mRNA expression. The data represent the mean 6 SE (n 5 4/group). *p , 0.05.
TP mRNA expression following partial hepatectomy are also shown in Figures 1 and 2. Although ethanol exposure had no effect on sham-operated rats, in regenerating livers, ethanol exposure was associated with significant decreases in GABATP mRNA abundance at times 12, 24, and 48 h post partial hepatectomy (p , 0.01, 0.05, and 0.05 respectively). The results of chronic ethanol exposure on hepatic GABA-TP mRNA abundance are shown in Figures 3 and 4. Of interest, prior to partial hepatectomy (time 0) and for 3 h thereafter, chronic ethanol-exposed rats had significantly increased GABA-TP mRNA abundance when compared to isocaloric fed controls. However, by 6 h post partial hepatectomy this difference was no longer apparent. The effect of exogenous GABA administration on hepatic GABA transport mRNA abundance is shown in Figures 5 and 6. Within 1 h, GABA-TP mRNA abundance declined. By 3 h the decline was significant (approximately 40% of baseline). Thereafter, GABA-TP mRNA abundance increased such that by 12 h the levels approximated those documented prior to GABA administration. Ethanol exposure did not alter preformed GABA transport activity in that [3H]-GABA uptake by isolated hepatocytes in the presence of ethanol was similar to that documented in the absence of ethanol (data not shown). DISCUSSION
Previous data from our laboratory indicate that enhanced GABAergic activity interferes with hepatic regeneration. Specifically, we have demonstrated that exogenous GABA and/or GABAA receptor agonists decrease restituted liver mass, hepatic DNA and protein synthesis rates, ornithine decarboxylase activity, putrescine levels, peak insulin-like growth factor-1 (IGF-1), and IGF-binding protein mRNA expression and prevents hepatic depolarization following partial hepatectomy (15,17–19). Moreover, ciprofloxacin, a potent GABAA receptor antagonist, prevents or reverses many of these effects and appears to be of therapeutic benefit in animal models of liver disease where GABAergic activity is increased (11,16,32). That ethanol increases serum GABA concentrations and potentiates GABAA receptor activity in the brain, raises the possibility that ethanol’s inhibitory effects on hepatic regeneration are mediated by GABAergic mechanisms
217 (5). However, whether that is the case in the liver and the precise mechanism(s) involved, remain to be determined. The results of the present study suggest that acute ethanol exposure does indeed interfere with the GABAergic system in the liver by altering GABA-TP expression at a transcriptional level. One likely mechanism whereby acute ethanol exposure might interfere with GABA-TP mRNA expression in hepatocytes would be via ethanol-induced increases in intracellular GABA levels. Unfortunately, complex coelution profiles and rapid postmortem changes in GABA metabolic enzymes preclude accurate determinations of GABA concentrations in the liver [(13) and unpublished data]. Nonetheless, it is likely that intracellular GABA levels were increased in rats acutely exposed to ethanol for the following reasons. First, acute (but not chronic) ethanol exposure significantly increases diamine oxidase activity which is the principle enzyme responsible for GABA synthesis in the liver (28,29) while having no effect or an inhibitory effect on GABA transaminase, the enzyme responsible for GABA metabolism in the liver (30). Second, acute ethanol exposure results in increased serum GABA-like activity in experimental animals and humans (3,22). Given that ethanol did not interfere with hepatocyte uptake of GABA by preformed GABA-TP and the liver is the principal organ responsible for GABA clearance from the systemic circulation, one would predict that elevated serum GABA concentrations would have resulted in increased GABA uptake by hepatocytes and thereby increased liver tissue GABA levels (7). However, actual GABA determinations in the liver are required to confirm these assumptions. Controversy remains as to whether acute but not chronic ethanol exposure interferes with hepatic regenerative activity following partial hepatectomy (12,24). Certainly, significant differences have been reported in the synthesis and metabolism of specific hepatic growth promoters and inhibitors in rats exposed acutely versus chronically to ethanol (1). For example, whereas acute ethanol exposure decreases ornithine decarboxylase activity and thereby inhibits the synthesis of putrescine, a potent growth promoter, chronic ethanol exposure stimulates ornithine decarboxylase activity and enhances putrescine production (6,25). Similar discrepancies were identified in this study in that acute ethanol exposure decreased GABA-TP mRNA abundance following partial hepatectomy whereas, in chronic ethanol-exposed rats, GABA-TP mRNA abundance was increased prior to and to a lesser extent, following partial hepatectomy. Perhaps upregulation of the GABA-TP gene (which would be predicted to result in decreased GABA binding to GABAA receptor sites) contributes to the apparent increase in hepatic regenerative activity associated with chronic ethanol exposure reported by some groups (12,24). As is the case with determining GABA levels in the liver, assay difficulties precluded obtaining direct determinations of changes in GABA binding to GABAA receptor sites in the liver (14). Whereas membrane potential differences have served as a surrogate marker for GABAA receptor activity in the liver, ethanol appears to independently alter membrane potentials through changes in sodium/potassium ATPase activity and other non-GABA–related effects (16,23). Thus, the effects of ethanol on GABAA receptor activity could not be ascertained. Attempts to clone the GABAA receptor gene(s) in the liver are presently underway. If successful, more direct determinations of the effects of ethanol on hepatic GABAA receptor activity may be possible. In addition to the relevance of these findings to hepatic regeneration, the results of this study could also help to explain the mechanism whereby acute ethanol consumption results in elevated serum GABA concentrations (3,22). Such an effect
218
GONG, CUI AND MINUK
has been implicated in the pathogenesis of alcoholism, hepatic encephalopathy, and systemic hypotension (20,22,27). In conclusion, the results of this study indicate that acute but not chronic ethanol exposure and the exogenous administration of GABA are associated with downregulation of the GABA-TP gene in the liver.
ACKNOWLEDGEMENTS
The authors thank Ms. D. Byron for her prompt and accurate typing of the manuscript. This study was supported by a grant from the Medical Research Council of Canada. Dr. Gong is the recipient of a Canadian Liver Foundation Fellowship Award.
REFERENCES 1. Akerman, P.; Cote, P.; Yang, S.; McClain, C.; Nelson, S.; Bagby, G.; Diehl, A. M.: Chronic ethanol consumption alters liver cell sensitivity to TNF-a during liver regeneration (abstract). Hepatology 16:139A; 1992. 2. Auftray, C.; Rougean, F.: Purification of mouse immunoglobulin heavy-chain-messenger RNAs from total meyloma tumor RNA. Eur. J. Biochem. 107:303–304; 1980. 3. Bannister, P.; Levy, L.; Bolton, R.; Losowsky, M. S.: Acute ethanol ingestion raises plasma amino-butyric acid levels in healthy men. Alcohol Alcohol. 23:45–48; 1988. 4. Berry, M. N.; Friend, D. S.: High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study. J. Cell Biol. 43:506–520; 1969. 5. Charness, M. E.; Simon, R. P.; Greenberg, D. A.: Ethanol and the nervous system. N. Engl. J. Med. 321:442–454; 1989. 6. Diehl, A. M.; Chacon, M. A.; Wagner, P.: The effect of chronic ethanol feeding on ornithine decarboxylase activity and liver regeneration. Hepatology 8:237–242; 1988. 7. Ferenci, P.; Stehle, T.; Ebner, J.; Schmid, R.; Haussinger, D.: Uptake and catabolism of g-aminobutyric acid by the isolated perfused rat liver. Gastroenterology 95:402–407; 1988. 8. Gill, K.; Franc, C.; Amit, Z.: Voluntary ethanol consumption in rats: An examination of blood/brain ethanol levels and behaviour. Alcohol .Clin. Exp. Res. 10:457–462; 1986. 9. Higgins, G. M.; Anderson, R. M.: Experimental pathology of the liver: Restoration of liver of the white rat following partial surgical removal. Arch. Pathol. 12:186–202; 1931. 10. Jones, E. A.; Schafer, D. F.; Ferenci, P.; Pappas, S. C.: The GABA hypothesis of the pathogenesis of hepatic encephalopathy: Current status. Yale J. Biol. Med. 57:301–316; 1984. 11. Kaita, K. D. E.; Assy, N.; Gauthier, T.; Meyers, A. F. A.; Minuk, G. Y.: Beneficial effects of ciprofloxacin on survival and hepatic regenerative activity in a rat model of fulminant hepatic failure. Hepatology 27:533–536; 1998. 12. Leevy, C. M.: In vitro studies of hepatic DNA synthesis in percutaneous liver biopsy specimens. J. Lab. Clin. Med. 61:761–779; 1963. 13. Manyam, B. V.; Hare, T. A.: Cerebrospinal fluid GABA measurements: Basic and clinical considerations. Clin. Neuropharmacol. 6:25–36; 1983. 14. Minuk, G. Y.; Bear, C. E.; Sarjeant, E. J.: Sodium-independent, bicuculline-sensitive [3H]GABA binding to isolated rat hepatocytes. Am. J. Physiol. 252:642–647; 1987. 15. Minuk, G. Y.; Gauthier, T.; Ghahary, A.; Murphy, L. J.: The effect of gamma-aminobutyric acid (GABA) on serum and hepatic polyamine concentrations following partial hepatectomy in rats. Hepatology 14:685–689; 1991. 16. Minuk, G. Y.; Gauthier, T.; Zhang, X. K.; Wang, G. Q.; Pettigrew, N. M.; Burczynski, F. J.: Ciprofloxacin prevents the inhibitory effects of acute ethanol expsoure on hepatic regeneration in the rat. Hepatology 22:1797–1800; 1995.
17. Minuk, G. Y.; Gauthier, T.: The effect of gamma aminobutyric acid (GABA) on hepatic regenerative activity following partial hepatectomy in rats. Gastroenterology 104:217–221; 1993. 18. Minuk, G. Y.; Kaita, K.; Gauthier, T.; Dembinski, T.; Murphy, L. J.: Effect of exogenous g-aminobutyric acid (GABA) on hepatic insulin-like growth factor I (IGF-I) and IGF-I binding protein (IGFBP-I) mRNA abundance following partial hepatectomy in rats. Can. J. Physiol. Pharmacol. 73:1546–1551; 1995. 19. Minuk, G. Y.; Kren, B. T.; Xu, R.; Zhang, X. K.; Burczynski, F. J.; Mulrooney, N. P.; Fan, G.; Steer, C. J.: The effect of changes in hepatocyte membrane potential on immediate-early protooncogene expression following partial hepatectomy in rats. Hepatology 25:1123–1127; 1997. 20. Minuk, G. Y.; MacCannell, K. L.: Is the hypotension of cirrhosis a GABA-mediated process? Hepatology 8:73–77; 1998. 21. Minuk, G. Y.: Gamma-aminobutyric acid and the liver. Dig. Dis. Sci. 11:45–54; 1995. 22. Moss, H. B.; Yao, J. K.; Burns, M.; Maddock, J.; Tarter, R. E.: Plasma GABA-like activity in response to ethanol challenge in men at high risk for alcoholism. Biol. Psychiatry 23:617–625; 1990. 23. Moule, S. K.; McGiven, J. D.: Regulation of the plasma membrane potential in hepatocytes—Mechanism and physiological significance. Biochim. Biophys. Acta 1031:383–397; 1990. 24. Orrego, H.; Crossley, I. R.; Saldivia, V.; Medline, A.; Varghese, G.; Israel, Y.: Long-term ethanol administration and short- and long-term liver regeneration after partial hepatectomy. J. Lab. Clin. Med. 97:221–230; 1981. 25. Poso, A.; Poso, H.: Inhibition of ornithine decarboxylase in regenerating rat liver by acute ethanol treatment. Biochim. Biophys. Acta 606:338–346; 1980. 26. Roberts, E.: GABA: The road to neurotransmitter status. Benzodiazepine/GABA receptors and chloride channels: Structural and functional properties. New York: Alan R. Liss, Inc.; 1986:1–39. 27. Schafer, D. F.; Jones, E. A.: Hepatic encephalopathy and g-aminobutyric acid neurotransmitter system. Lancet I:18–19; 1992. 28. Sessa, A.; Desiderio, M. F.; Perin, A.: Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration. Biochim. Biophys. Acta 801:285–289; 1984. 29. Sessa, A.; Perin, A.: Diamine oxidase activity and related substrates in rat liver after chronic ethanol feeding. Agents Actions 37:107–110; 1992. 30. Sherif, F.; Wahlstrom, G.; Oreland, L.: Increase in brain GABAtransaminase activity after chronic ethanol treatment in rats. J. Neural Transm. Gen. Sect. 98:69–79; 1994. 31. Wands, J. R.; Carter, E. A.; Bucher, N. L. R.; Isselbacher, K. J.: Inhibition of hepatic regeneration in rats by acute and chronic ethanol intoxication. Gastroenterology 77:528–531; 1979. 32. Zhang, M.; Song, G.; Minuk, G. Y.: Effects of hepatic stimulator substance, herbal medicine, selenium/vitamin E, and ciprofloxacin on cirrhosis in the rat. Gastroenterology 110:1150–1155; 1996.
PII S0741-8329(99)00049-X
Effects of Acute and Chronic Ethanol Exposure on the Hepatic Gamma-Aminobutyric Acid Transport System in Rats Y. GONG, L. CUI AND G. Y. MINUK Liver Diseases Unit, Departments of Medicine and Pharmacology, University of Manitoba, Winnipeg, Manitoba, Canada Received 10 February 1999; Accepted 11 June 1999 GONG, Y., L. CUI AND G. Y. MINUK. Effects of acute and chronic ethanol exposure on the hepatic gamma-aminobutyric acid transport system in rats. ALCOHOL 19(3) 213–218, 1999.—Ethanol-induced increases in gamma-aminobutyric (GABA)ergic activity contribute to the impairment in hepatic regeneration associated with alcohol-induced liver disease. To determine the mechanism(s) whereby ethanol increases GABAergic activity in the liver, we documented the effects of acute (5 g/kg 3 1) and chronic (36% of total calories over 6 weeks) ethanol exposure as well as exogenous GABA (500 mg/g body weight) administration on GABA transport protein (GABA-TP) mRNA expression in the livers of adult male Sprague– Dawley rats at various times (0–72 h) post 70% partial hepatectomy (PHx). We also documented the in vitro effects of ethanol (30–90 mM) on [3H]-GABA uptake in isolated rat hepatocytes. The results of the study revealed that compared to salineexposed controls, acute but not chronic ethanol exposure resulted in significant decreases in GABA-TP mRNA expression at 12, 24, and 48 h post PHx (saline exposed, 1.04 6 0.06, 1.19 6 0.21, and 1.15 6 0.05, vs. acute ethanol exposed, 0.80 6 0.16, 0.88 6 0.09, and 0.86 6 0.16 optical density units, p , 0.01, 0.05, and 0.05, respectively). An inhibitory effect was also observed following exogenous GABA administration (GABA-TP mRNA expression at 3 h was approximately 40% that of baseline, p , 0.05). [3H]-GABA uptake in isolated rat hepatocytes in vitro was unaffected by the presence of ethanol. In conclusion, the results of this study indicate that acute but not chronic ethanol exposure and exogenously administrated GABA inhibit hepatic GABA-TP mRNA expression following partial hepatectomy in the rat. These findings suggest that the increased GABAergic activity that occurs in the liver following acute ethanol exposure results from alterations in the hepatic GABA transport system at a transcriptional level. © 1999 Elsevier Inc. All rights reserved. Ethanol
GABA
Hepatic regeneration
GABA transport
ACUTE but perhaps not chronic ethanol exposure is a potent inhibitor of hepatic regenerative activity (12,31). How acute ethanol exposure inhibits hepatic regenerative activity remains unclear. Recently, we reported that at least some of this inhibitory effect could be prevented by the prior administration of gammaaminobutyric (GABAA) receptor antagonists suggesting that enhanced hepatic GABAergic activity may be involved (16). As in the brain, the liver contains both GABA receptor and transport systems (21,26). The latter is thought to be responsible for clearing GABA from the surface of hepatocytes and thereby regulating the amount of GABA available to bind to hepatic GABAA receptor sites (21). Thus, one would predict that interference with the GABA transport protein system (GABA-TP) would result in enhanced hepatic GABAA
Liver
receptor activity and impaired hepatic regeneration. In the present study, we tested this hypothesis by documenting the effects of acute and chronic ethanol exposure on hepatic GABA-TP expression in rats. METHOD
Effects of Acute Ethanol Exposure on GABA-TP mRNA Abundance To document the effects of acute ethanol exposure on GABA-TP, adult male Sprague–Dawley (SD) rats (200–250 g) were fed ethanol (5 g/kg) by gastric gavage 1 h prior to a 70% partial hepatectomy as described by Higgins and Anderson,
Requests for reprints should be addressed to Dr. G.Y. Minuk, Liver Diseases Unit, GF407, Health Sciences Centre, 820 Sherbrook Street, Winnipeg, Manitoba, Canada, R3A 1R9. Tel: (204) 787-4662; Fax: (204) 775-4255; E-mail: [email protected]
213
214
GONG, CUI AND MINUK
or sham surgery while under ether anesthesia (9). Groups of rats (n 5 4–6/group) were then sacrificed by cardiac puncture at times 6, 12, 24, and 48 h postsurgery. GABA-TP mRNA abundance was documented in the excised livers in the following manner: Total RNA was extracted from liver tissues by the lithium chloride/urea method (2). The RNA was resolved on 1% formaldehyde-agarose gels, and transferred onto GT nylon membranes (BioRad, Hercules, CA). The membranes were then hybridized with a radiolabeled mouse GABA transporter probe, GAT-3 (kindly provided by Dr. N. Nelson, Roche Institute of Molecular Biology) and 28S-rRNA cDNA probe respectively in a hybridization solution which consisted of 50% formamide, 0.2 M NaCl, 0.12 M Na2HPO4, and 7% SDS. Membranes were washed twice with 23 SSC/0.1% SDS at room temperature for 15 min and 0.23 SSC/0.1% SDS at 428C for 15 min. X-ray films exposed to the membranes were scanned, and the bands were quantitated by an NIH-IMAGE program (NIH, Bethesda, MD). All bands were standardized against the concurrently run 28S-rRNA. Effects of Chronic Ethanol Exposure on GABA-TP mRNA Abundance Chronic ethanol exposure was as described by Gill et al. (8). Briefly, rats received increasing concentrations of ethanol (3%, 5%, 7% to 9%, v/v) ad libitum for 16 days. Thereafter, all rats received 9% (v/v) ethanol for a total of 6 weeks. Isocaloric-fed rats served as controls. At 6 weeks, a 70% partial hepatectomy or sham surgery was performed. Thereafter, groups of rats (n 5 4–6/group) were sacrificed at 3, 6, 12, 24, and 48 h postsurgery. Hepatic GABA-TP mRNA abundance
was determined at each time interval in both ethanol-exposed and isocaloric-fed controls as described above. Effects of GABA on the GABA Transport System To determine whether the effects of ethanol on GABA-TP resulted from ethanol-induced increases in serum GABA concentrations, the following additional experiment was performed. Intraperitoneal injections of GABA (500 mg/g body weight) or equal volumes of saline were administered to adult SD rats (n 5 4/group). GABA-TP mRNA expression was then documented at times 0, 1, 3, 6, 12, and 24 h as described above. The dose of GABA had been predetermined to result in elevated serum GABA levels that approximate those reported in experimental animals and humans with advanced liver disease (20–50 mmol/liter) for approximately 4 h postinjection (10). Effects of Ethanol on GABA Transport by Isolated Hepatocytes Rat hepatocytes were isolated using a modification of the collagenase perfusion method described by Berry and Friend (4). Briefly, following the sacrifice of adult male SD rats by exsanguination under ether anesthesia, suspensions of liver cells were prepared by perfusing the livers in situ with calcium- and magnesium-free Hanks balanced salt solution (HBSS) followed by perfusion with complete HBSS containing collagenase (0.5% w/v). Following a series of filtrations and washes, 95% of isolated hepatocytes excluded trypan blue and contamination with sinusoidal cells as identified morphologically was less than 1%. Each assay was initiated by the addition of 1 ml of the suspen-
FIG. 1. Expression of GABA-TP mRNA in acute ethanol- (5 g/kg) or saline-exposed rats after sham or partial hepatectomy. The figure represents GABA-TP mRNA of one rat from four different groups as indicated, and at four different times after sham or partial hepatectomy (6, 12, 24, and 48 h). The same membranes were rehybridized with 28S-rRNA probe as a loading control.
ETHANOL AND GABA TRANSPORT
215
FIG. 2. Summary of the relative levels of GABA-TP expression in four different groups (see legend to Fig. 1), and four different times after sham or partial hepatectomy as indicated. The data represent the mean 6 SE (n 5 4–6/group). *p , 0.05; **p , 0.01.
sion of hepatocytes (standardized to 5 3 105 cells/ml) to 1 ml of buffer containing [3H]-GABA (final concentration 10 nM) and when indicated, unlabeled GABA (10 mM) or ethanol (30–90 mM). Incubation mixtures were shaken every 5 min. At designated time intervals, 250-ml samples were removed from the mixtures and placed in 400-ml microfuge tubes that already contained 100 ml of silicone oil and 50 ml of potassium hydroxide.
Following centrifugation through the microfuge tubes, specific uptake of [3H]-GABA was calculated by subtracting cell-associated [3H]-GABA in the presence of unlabeled GABA (nonspecific) from cell-associated [3H]-GABA in the absence of unlabeled GABA (total). Thus, specific 5 total 2 nonspecific. Ethical consent for this study was obtained from the Animal Care Committee at the University of Manitoba.
FIG. 3. Expression of GABA-TP mRNA in chronic ethanol-exposed rats and isocaloric-fed controls after partial hepatectomy. GABA-TP mRNA in two rats from ethanol or control groups, and at six different times as indicated are presented. The same membranes were rehybridized with 28S-rRNA probe as a loading control.
216
GONG, CUI AND MINUK
FIG. 4. Summary of the relative levels of GABA-TP expression in chronic ethanol-exposed rats and isocaloric-fed controls at 0, 3, 6, 12, 24, and 48 h after partial hepatectomy. The data represent the mean 6 SE (n 5 4–6/group). *p , 0.05.
Statistical Analyses
RESULTS
Analyses of variance followed by paired Student’s t-tests for parametric data and a Wilcoxon Rank Sum test for nonparametric data were performed where appropriate. p-Values ,0.05 were considered significant. The results provided represent the mean 6 standard error of the mean.
As demonstrated by the results in saline-treated sham and partial hepatectomized groups (Figs. 1 and 2), partial hepatectomy per se does not alter GABA-TP mRNA expression during the time intervals studied. The results of acute ethanol exposure on hepatic GABA-
FIG. 5. The time-dependent effect of GABA on rat liver GABA-TP mRNA expression. The figure represents GABA-TP mRNA expression in two rats after i.p. injection of GABA (500 mg/g body weight). The same membranes were rehybridized with 28SrRNA probe as a loading control.
ETHANOL AND GABA TRANSPORT
FIG. 6. Summary of the time-dependent effect of exogenous GABA administration on rat liver GABA-TP mRNA expression. The data represent the mean 6 SE (n 5 4/group). *p , 0.05.
TP mRNA expression following partial hepatectomy are also shown in Figures 1 and 2. Although ethanol exposure had no effect on sham-operated rats, in regenerating livers, ethanol exposure was associated with significant decreases in GABATP mRNA abundance at times 12, 24, and 48 h post partial hepatectomy (p , 0.01, 0.05, and 0.05 respectively). The results of chronic ethanol exposure on hepatic GABA-TP mRNA abundance are shown in Figures 3 and 4. Of interest, prior to partial hepatectomy (time 0) and for 3 h thereafter, chronic ethanol-exposed rats had significantly increased GABA-TP mRNA abundance when compared to isocaloric fed controls. However, by 6 h post partial hepatectomy this difference was no longer apparent. The effect of exogenous GABA administration on hepatic GABA transport mRNA abundance is shown in Figures 5 and 6. Within 1 h, GABA-TP mRNA abundance declined. By 3 h the decline was significant (approximately 40% of baseline). Thereafter, GABA-TP mRNA abundance increased such that by 12 h the levels approximated those documented prior to GABA administration. Ethanol exposure did not alter preformed GABA transport activity in that [3H]-GABA uptake by isolated hepatocytes in the presence of ethanol was similar to that documented in the absence of ethanol (data not shown). DISCUSSION
Previous data from our laboratory indicate that enhanced GABAergic activity interferes with hepatic regeneration. Specifically, we have demonstrated that exogenous GABA and/or GABAA receptor agonists decrease restituted liver mass, hepatic DNA and protein synthesis rates, ornithine decarboxylase activity, putrescine levels, peak insulin-like growth factor-1 (IGF-1), and IGF-binding protein mRNA expression and prevents hepatic depolarization following partial hepatectomy (15,17–19). Moreover, ciprofloxacin, a potent GABAA receptor antagonist, prevents or reverses many of these effects and appears to be of therapeutic benefit in animal models of liver disease where GABAergic activity is increased (11,16,32). That ethanol increases serum GABA concentrations and potentiates GABAA receptor activity in the brain, raises the possibility that ethanol’s inhibitory effects on hepatic regeneration are mediated by GABAergic mechanisms
217 (5). However, whether that is the case in the liver and the precise mechanism(s) involved, remain to be determined. The results of the present study suggest that acute ethanol exposure does indeed interfere with the GABAergic system in the liver by altering GABA-TP expression at a transcriptional level. One likely mechanism whereby acute ethanol exposure might interfere with GABA-TP mRNA expression in hepatocytes would be via ethanol-induced increases in intracellular GABA levels. Unfortunately, complex coelution profiles and rapid postmortem changes in GABA metabolic enzymes preclude accurate determinations of GABA concentrations in the liver [(13) and unpublished data]. Nonetheless, it is likely that intracellular GABA levels were increased in rats acutely exposed to ethanol for the following reasons. First, acute (but not chronic) ethanol exposure significantly increases diamine oxidase activity which is the principle enzyme responsible for GABA synthesis in the liver (28,29) while having no effect or an inhibitory effect on GABA transaminase, the enzyme responsible for GABA metabolism in the liver (30). Second, acute ethanol exposure results in increased serum GABA-like activity in experimental animals and humans (3,22). Given that ethanol did not interfere with hepatocyte uptake of GABA by preformed GABA-TP and the liver is the principal organ responsible for GABA clearance from the systemic circulation, one would predict that elevated serum GABA concentrations would have resulted in increased GABA uptake by hepatocytes and thereby increased liver tissue GABA levels (7). However, actual GABA determinations in the liver are required to confirm these assumptions. Controversy remains as to whether acute but not chronic ethanol exposure interferes with hepatic regenerative activity following partial hepatectomy (12,24). Certainly, significant differences have been reported in the synthesis and metabolism of specific hepatic growth promoters and inhibitors in rats exposed acutely versus chronically to ethanol (1). For example, whereas acute ethanol exposure decreases ornithine decarboxylase activity and thereby inhibits the synthesis of putrescine, a potent growth promoter, chronic ethanol exposure stimulates ornithine decarboxylase activity and enhances putrescine production (6,25). Similar discrepancies were identified in this study in that acute ethanol exposure decreased GABA-TP mRNA abundance following partial hepatectomy whereas, in chronic ethanol-exposed rats, GABA-TP mRNA abundance was increased prior to and to a lesser extent, following partial hepatectomy. Perhaps upregulation of the GABA-TP gene (which would be predicted to result in decreased GABA binding to GABAA receptor sites) contributes to the apparent increase in hepatic regenerative activity associated with chronic ethanol exposure reported by some groups (12,24). As is the case with determining GABA levels in the liver, assay difficulties precluded obtaining direct determinations of changes in GABA binding to GABAA receptor sites in the liver (14). Whereas membrane potential differences have served as a surrogate marker for GABAA receptor activity in the liver, ethanol appears to independently alter membrane potentials through changes in sodium/potassium ATPase activity and other non-GABA–related effects (16,23). Thus, the effects of ethanol on GABAA receptor activity could not be ascertained. Attempts to clone the GABAA receptor gene(s) in the liver are presently underway. If successful, more direct determinations of the effects of ethanol on hepatic GABAA receptor activity may be possible. In addition to the relevance of these findings to hepatic regeneration, the results of this study could also help to explain the mechanism whereby acute ethanol consumption results in elevated serum GABA concentrations (3,22). Such an effect
218
GONG, CUI AND MINUK
has been implicated in the pathogenesis of alcoholism, hepatic encephalopathy, and systemic hypotension (20,22,27). In conclusion, the results of this study indicate that acute but not chronic ethanol exposure and the exogenous administration of GABA are associated with downregulation of the GABA-TP gene in the liver.
ACKNOWLEDGEMENTS
The authors thank Ms. D. Byron for her prompt and accurate typing of the manuscript. This study was supported by a grant from the Medical Research Council of Canada. Dr. Gong is the recipient of a Canadian Liver Foundation Fellowship Award.
REFERENCES 1. Akerman, P.; Cote, P.; Yang, S.; McClain, C.; Nelson, S.; Bagby, G.; Diehl, A. M.: Chronic ethanol consumption alters liver cell sensitivity to TNF-a during liver regeneration (abstract). Hepatology 16:139A; 1992. 2. Auftray, C.; Rougean, F.: Purification of mouse immunoglobulin heavy-chain-messenger RNAs from total meyloma tumor RNA. Eur. J. Biochem. 107:303–304; 1980. 3. Bannister, P.; Levy, L.; Bolton, R.; Losowsky, M. S.: Acute ethanol ingestion raises plasma amino-butyric acid levels in healthy men. Alcohol Alcohol. 23:45–48; 1988. 4. Berry, M. N.; Friend, D. S.: High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study. J. Cell Biol. 43:506–520; 1969. 5. Charness, M. E.; Simon, R. P.; Greenberg, D. A.: Ethanol and the nervous system. N. Engl. J. Med. 321:442–454; 1989. 6. Diehl, A. M.; Chacon, M. A.; Wagner, P.: The effect of chronic ethanol feeding on ornithine decarboxylase activity and liver regeneration. Hepatology 8:237–242; 1988. 7. Ferenci, P.; Stehle, T.; Ebner, J.; Schmid, R.; Haussinger, D.: Uptake and catabolism of g-aminobutyric acid by the isolated perfused rat liver. Gastroenterology 95:402–407; 1988. 8. Gill, K.; Franc, C.; Amit, Z.: Voluntary ethanol consumption in rats: An examination of blood/brain ethanol levels and behaviour. Alcohol .Clin. Exp. Res. 10:457–462; 1986. 9. Higgins, G. M.; Anderson, R. M.: Experimental pathology of the liver: Restoration of liver of the white rat following partial surgical removal. Arch. Pathol. 12:186–202; 1931. 10. Jones, E. A.; Schafer, D. F.; Ferenci, P.; Pappas, S. C.: The GABA hypothesis of the pathogenesis of hepatic encephalopathy: Current status. Yale J. Biol. Med. 57:301–316; 1984. 11. Kaita, K. D. E.; Assy, N.; Gauthier, T.; Meyers, A. F. A.; Minuk, G. Y.: Beneficial effects of ciprofloxacin on survival and hepatic regenerative activity in a rat model of fulminant hepatic failure. Hepatology 27:533–536; 1998. 12. Leevy, C. M.: In vitro studies of hepatic DNA synthesis in percutaneous liver biopsy specimens. J. Lab. Clin. Med. 61:761–779; 1963. 13. Manyam, B. V.; Hare, T. A.: Cerebrospinal fluid GABA measurements: Basic and clinical considerations. Clin. Neuropharmacol. 6:25–36; 1983. 14. Minuk, G. Y.; Bear, C. E.; Sarjeant, E. J.: Sodium-independent, bicuculline-sensitive [3H]GABA binding to isolated rat hepatocytes. Am. J. Physiol. 252:642–647; 1987. 15. Minuk, G. Y.; Gauthier, T.; Ghahary, A.; Murphy, L. J.: The effect of gamma-aminobutyric acid (GABA) on serum and hepatic polyamine concentrations following partial hepatectomy in rats. Hepatology 14:685–689; 1991. 16. Minuk, G. Y.; Gauthier, T.; Zhang, X. K.; Wang, G. Q.; Pettigrew, N. M.; Burczynski, F. J.: Ciprofloxacin prevents the inhibitory effects of acute ethanol expsoure on hepatic regeneration in the rat. Hepatology 22:1797–1800; 1995.
17. Minuk, G. Y.; Gauthier, T.: The effect of gamma aminobutyric acid (GABA) on hepatic regenerative activity following partial hepatectomy in rats. Gastroenterology 104:217–221; 1993. 18. Minuk, G. Y.; Kaita, K.; Gauthier, T.; Dembinski, T.; Murphy, L. J.: Effect of exogenous g-aminobutyric acid (GABA) on hepatic insulin-like growth factor I (IGF-I) and IGF-I binding protein (IGFBP-I) mRNA abundance following partial hepatectomy in rats. Can. J. Physiol. Pharmacol. 73:1546–1551; 1995. 19. Minuk, G. Y.; Kren, B. T.; Xu, R.; Zhang, X. K.; Burczynski, F. J.; Mulrooney, N. P.; Fan, G.; Steer, C. J.: The effect of changes in hepatocyte membrane potential on immediate-early protooncogene expression following partial hepatectomy in rats. Hepatology 25:1123–1127; 1997. 20. Minuk, G. Y.; MacCannell, K. L.: Is the hypotension of cirrhosis a GABA-mediated process? Hepatology 8:73–77; 1998. 21. Minuk, G. Y.: Gamma-aminobutyric acid and the liver. Dig. Dis. Sci. 11:45–54; 1995. 22. Moss, H. B.; Yao, J. K.; Burns, M.; Maddock, J.; Tarter, R. E.: Plasma GABA-like activity in response to ethanol challenge in men at high risk for alcoholism. Biol. Psychiatry 23:617–625; 1990. 23. Moule, S. K.; McGiven, J. D.: Regulation of the plasma membrane potential in hepatocytes—Mechanism and physiological significance. Biochim. Biophys. Acta 1031:383–397; 1990. 24. Orrego, H.; Crossley, I. R.; Saldivia, V.; Medline, A.; Varghese, G.; Israel, Y.: Long-term ethanol administration and short- and long-term liver regeneration after partial hepatectomy. J. Lab. Clin. Med. 97:221–230; 1981. 25. Poso, A.; Poso, H.: Inhibition of ornithine decarboxylase in regenerating rat liver by acute ethanol treatment. Biochim. Biophys. Acta 606:338–346; 1980. 26. Roberts, E.: GABA: The road to neurotransmitter status. Benzodiazepine/GABA receptors and chloride channels: Structural and functional properties. New York: Alan R. Liss, Inc.; 1986:1–39. 27. Schafer, D. F.; Jones, E. A.: Hepatic encephalopathy and g-aminobutyric acid neurotransmitter system. Lancet I:18–19; 1992. 28. Sessa, A.; Desiderio, M. F.; Perin, A.: Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration. Biochim. Biophys. Acta 801:285–289; 1984. 29. Sessa, A.; Perin, A.: Diamine oxidase activity and related substrates in rat liver after chronic ethanol feeding. Agents Actions 37:107–110; 1992. 30. Sherif, F.; Wahlstrom, G.; Oreland, L.: Increase in brain GABAtransaminase activity after chronic ethanol treatment in rats. J. Neural Transm. Gen. Sect. 98:69–79; 1994. 31. Wands, J. R.; Carter, E. A.; Bucher, N. L. R.; Isselbacher, K. J.: Inhibition of hepatic regeneration in rats by acute and chronic ethanol intoxication. Gastroenterology 77:528–531; 1979. 32. Zhang, M.; Song, G.; Minuk, G. Y.: Effects of hepatic stimulator substance, herbal medicine, selenium/vitamin E, and ciprofloxacin on cirrhosis in the rat. Gastroenterology 110:1150–1155; 1996.