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Chronic ethanol inhibits receptor-stimulated phosphoinositide hydrolysis in rat liver slices
Alcohol. Vol. 8. pp. 131-136. c Pergamon Press pie. 1991. Printed in the U.S.A.
0741-8329/91 $3.00 + .00
Chronic Ethanol Inhibits Receptor-Stimulated Phosphoinositide Hydrolysis in Rat Liver Slices I R U E B E N A. G O N Z A L E S *
A N D F U L T O N T. C R E W S t - "
*Department of Pharmacology, Universit3' of" Texas, Austin, TX 78712 attd ?Department of Pharmacology, Universita' of Florida, Gainesville, FL 32610 R e c e i v e d 6 F e b r u a r y 1990; A c c e p t e d 3 O c t o b e r 1990
GONZALES, R. A. AND F. T..CREWS. Chronic ethanol inhibits receptor.stimulated phosphoinositide hydrolysis in rat liver slices. ALCOHOL 8(2) 131-136, 1991.--The effects of chronic ethanol feeding on norepinephrine (NE)- and arginine-vasopressin (AVP)-stimulated phosphoinositide (PI) hydrolysis in rat liver slices was determined. The maximum NE-stimulated PI response was significantly reduced by 40% in liver slices from 8-month-old rats which had been treated for 5 months with a liquid diet containing ethanol compared to pair-fed controls. The maximum AVP-stimulated PI response was decreased by 39% in liver slices from the ethanol-fed rats compared to control. ECso values for NE- and AVP-stimulated PI hydrolysis in liver slices were not affected by the chronic ethanol treatment. Similar reductions in the maximal NE- and AVP-stimulated PI hydrolysis (28% and 27%, respectively) were found in 22-month-old rats which had been maintained on an ethanol containing diet for 5 months compared to pair-fed controis. The binding of [3H]prazosin and [~H]AVP to liver plasma membranes from 8-month-old ethanol-fed rats was not significantly different from binding to liver membranes from sucrose-fed controls. Our data suggest that chronic ethanol ingestion may lead to a reduction in Pl-linked signal transduction in liver. Ethanol
Liver
Phosphoinositide hydrolysis
Inositol phosphates
Arginine-vasopressin
a:Adrenergic receptors
cium concentrations (12). Stimulation of a : a d r e n e r g i c or vasopressin receptors in the liver results in the phospholipase-mediated hydrolysis of phosphoinositides and the generation of second messengers inositol 1,4,5-trisphosphate and diacylglycerol (I, 10). We have recently shown that c~:stimulated PI hydrolysis in brain slices is inhibited by ethanol in vitro which suggests that the membrane effects of ethanol may interfere with certain receptors which are linked to PI hydrolysis (6). Smith et al. (18) have previously reported that chronic ethanol in combination with a high fat diet causes an increase in cq-adrenergic-stimulated [32p] incorporation into phosphatidylinositol in hepatocytes. More recently, ethanol has been shown to alter calcium homeostasis in hepatocytes by transiently stimulating the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C (7,8). However, there is still little data available regarding the effects of chronic ethanol treatment on receptor-stimulated PI hydrolysis in liver. The present investigation was undertaken to further examine the effects of chronic treatment of rats with ethanol on stimulated PI hydrolysis in liver slices. Two different ages of rats were used to determine if aging alters the effects of the chronic ethanol treatment on PI hydrolysis in liver. Our results suggest that chronic ingestion of ethanol interferes with the transmission of a :adrenergic and vasopressin signals which are carried through the PI system in liver.
ONE of the most important pharmacological effects of ethanol ingestion is the disruption of liver function. Some of the effects of ethanol on 4iver function may be due to the metabolic consequences of the oxidation of ethanol (2). However, ethanol is also known to perturb the organization of cell membranes (4,5). Several investigators have reported that mitochondrial, microsomal, and plasma membranes from hepatocytes adapt to the presence of ethanol as measured by a resistance to the membrane disordering effects of ethanol (13, 14, 22). This membrane adaptation to chronic ethanol has been suggested to be involved in ethanol-induced changes in the oxidative phosphorylation capability of liver mitochondria (22). Other membrane-bound enzyme activities such as Na,K-ATPase may also be affected by chronic ethanol ingestion (24). These data suggest that the membrane disordering effects of ethanol lead to an adaptation of liver membranes with concomitant changes in the activity of membrane-dependent enzymes. Although it is clear that chronic ethanol ingestion results in changes in intracellular organelles in the liver, the demonstrated effects of ethanol on the liver plasma membrane suggest that the functions of the plasma membrane may be affected. Receptorstimulated phosphoinositide (PI) hydrolysis is known to be an important signal transduction system in liver for hormones such as catecholamines and 'vasopressin which regulate cytosolic ca]-
~This work was supported by grants from NIAAA (AA06069 and AA07297). -'Requests for reprints should be addressed to Dr. Fulton T. Crews. Box J-267 JHMHC, University of Florida Medical School, Gainesville, FL 32610.
131
132
GONZALES AND CREWS
METHOD
Materials Experimental subjects were male Fischer 344 rats (3 or 15 months old) obtained from the colony maintained by NIA by Harlan Sprague-Dawley (Indianapolis, IN). myo-[3H]lnositol ( 12-15 Ci/mmol), [3H]prazosin (85 Ci/mmol), and [3H]arginine-vasopressin (AVP, 64 Ci/mmol) were obtained from Amersham Corporation (Arlington Heights, IL). Norepinephrine (NE), AVP and other drugs were obtained from Sigma Chemical Company (St. Louis, MO). Prazosin was kindly provided by Pfizer, Inc. (Groton, CT). Solvents were reagent grade from Fisher Scientific Company (Orlando, FL).
PI Hydrolysis in Liver Slices The method of Berridge et al. (3) was modified for use with liver slices. Rats were decapitated and the liver was rapidly removed and washed in Krebs-Ringer bicarbonate buffer (KRB) (118 mM NaCI, 4.7 mM KCI, 0.75 mM CaCI 2, 1.18 mM KH 2PO.~, 1.18 mM MgSO.,, 24.8 mM NaHCO3, 10 mM glucose) which had been bubbled with O2/CO2 (95:5) to give a pH of 7.4. Thin slices (2-3 mm) were cut from several lobes, placed flat on a Teflon disk, and the tissue was further sliced on a McIlwain tissue chopper (0.35 mm) in two perpendicular directions. The liver slices were then transferred to a conical tube containing fresh warmed (37°C) KRB and washed four times in 30 min with constant bubbling with O2/CO 2. [3H]lnositol was added (0.3 I.tM final concentration), the tube was saturated with O2/CO 2, and the capped tube was incubated for 60 min with gentle shaking in a warm water bath. The labelled slices were washed twice with fresh KRB and distributed (50 p.l) into polypropylene tubes containing KRB (190 I.tl) which had 10 mM LiCI and 108 mM NaCI instead of the usual NaC1 concentration and were incubated for 10 min. Buffer or stimulants were added, and tubes were gassed with O2/CO2, capped and incubated for 60 rain at 37°C. Incubations were terminated by adding 1.0 ml of chloroform-methanol (1:2). Water and chloroform (0.35 ml) were added to the tubes, and the tissue was extracted for 10 min. After separation of phases by centrifugation, an aliquot of the upper phase (0.75 ml) was taken for analysis of [3H]inositol phosphates. The remaining upper phase was aspirated, and an aliquot of the lower phase was transferred to a scintillation vial. The chloroform was dried, and the radioactivity incorporated into phospholipids was determined with a liquid scintillation counter. The aqueous aliquot was diluted to 3 ml with water, and 1.0 ml of a slurry of Dowex- 1 (X8, formate form) was added. This was then poured into a polypropylene column fitted with a fritted disk, and the liquid was allowed to drain. The column was washed with 10 ml of water, and the total [3H]inositol phosphates were eluted directly into a vial with 5 ml of 1.0 M ammonium formate/0.1 M formic acid. Ten milliliters of Liquiscint (National Diagnostics, Manville, N J) were added, and the radioactivity eluted was determined with a liquid scintillation counter. PI hydrolysis was expressed as the percent of [3H]inositol phosphates released. This value was arrived at by taking the ratio of the [3H]inositol phosphates recovered (DPM from column) to the total [3H]inositol which had been incorporated into phospholipids (DPM from column + DPM in chloroform) and multiplying this by 100.
buffer A (250 mM sucrose, 10 mM Tris base, I mM EGTA, pH 7.5) and minced into small pieces. The tissue was homogenized in 10 volumes of modified buffer A with a Dounce homogenizer using 4 strokes of the loose pestle followed by 10 strokes with the tight-fitting pestle. The homogenate was further diluted with modified buffer A (7 volumes) and centrifuged at 1464×g for 10 min at 4°C. The pellet was resuspended with 17 volumes of modified buffer A and filtered through 2-ply nylon gauze. Percoll (5.6 ml) was added to the homogenate and mixed well. The Percoll-homogenate mixture was distributed into four 15-ml Corex tubes and centrifuged at 34,500 x g for 30 min at 4°C. Plasma membranes which banded near the top of the tube were collected and washed twice with modified buffer A. The final pellet was resuspended in incubation buffer (50 mM Tris, 10 mM MgCI 2, 1 mM EGTA, pH 7.4) and frozen in liquid nitrogen for up to two weeks before assaying for [3H]prazosin or [3H]arginine-vasopressin binding sites. Protein content of membranes was determined with the method of Lowry et al. ( 11 ).
[~H]Prazosin and [~H]AVP Binding to Liver Membranes For the binding assays. 30-40 p.g membrane protein was added to tubes containing 50 mM Tris, 10 mM MgCI 2 (pH 7.4). and the appropriate concentration of [3H]prazosin (0.5 nM) or [3H]AVP (I nM) and incubated for 20 min at 37°C or 30°C, respectively, to determine total binding. Nonspecific binding was determined by adding 10 ~M phentolamine or 10 p-M AVP. Nonspecific binding was subtracted from total to obtain specific binding. Scatchard analyses were performed at concentrations of 0.075-20 nM of each ligand. Bound ligand was separated from free by rapid filtration under vacuum through Whatman GF/C filters with a Brandel cell harvester. Filter bound radioactivity was determined with a liquid scintillation counter.
Chronic Ethanol Treaonent Rats were maintained on a nutritionally complete liquid diet containing either 35% of calories as ethanol (ethanol-fed) or pairfed with a diet containing isocaloric sucrose substituted for the ethanol (sucrose-fed) for 5 months following the procedure of Walker et al. (21). Chocolate-flavored Sustacal (Mead-Johnson) was mixed with an ethanol or sucrose containing solution to give 1.3 kcal/ml diet. Both diets were also supplemented with 0.3 g/ dl Vitamin Diet Fortification Mixture and 0.5 g/dl Salt Mixture XIV (both from ICN Nutritional Biochemicals). The ethanoltreated rats were originally matched by weight to controls, and the sucrose-fed animals were given the amount of diet which its ethanol-fed counterpart had consumed the previous day. The rats were maintained in a temperature controlled room on a standard 12 h/12 h light/dark cycle. The weight gain for the young sucrose- and ethanol-treated rats was 169 ± 11 g (n = 18) and 138 ± 18 g (n= 17), respectively, throughout the 5-month liquid diet period. Weight gain in the 22-month-old rats was 147--- 12 g (n= 6) and 129± 10 g (n=7), for the sucrose- and ethanol-treated rats, respectively. Ethanol-treated animals consumed an average of 11 g/kg of ethanol each day with blood levels ranging from approximately 50 mg/dl at 8 a.m. to 140 mg/dl at 8 p.m. Rats were killed on the morning after the last evening of ethanol feeding.
Preparation of Liver Plasma Membranes
Statistical Analysis
Liver plasma membranes were isolated according to the method of Prpic et al. (15). Pieces of rat liver were initially frozen over dry ice. The frozen pieces were then thawed in ice-cold modified
Significant differences between experimental groups and controis were determined using two-way analysis of variance. Comparison of individual means was carried out using Newman-Keuls
ETHANOL AND LIVER PI HYDROLYSIS
133
procedure. Differences were considered significant if p < 0 . 0 5 . RESULTS
Effects of Age and Chronic Ethanol Treatment of NE- and AVP-Stimulated PI Responses in Liver Slices To determine if aging or chronic ethanol treatment in vivo has any effects on stimulated PI hydrolysis in vitro, concentration-effect curves for NE- and AVP-stimulated PI hydrolysis were performed in liver slices from young and aged ethanol- and sucrosefed rats. Three- and 17-month-old rats were maintained on liquid diets containing either sucrose or ethanol for 5 months so that the animals were sacrificed when they were 8 (young) or 22 (aged) months old. Concentration-effect curves for NE- and AVP-stimulated PI hydrolysis in liver slices from young ethanol- and sucrose-fed rats are shown in Figs. 1 and 2, respectively. The maximal NE-stimulated PI response is 40% lower in the liver slices from the ethanol-fed rats compared to sucrose-treated controls (Fig. 1). A comparable 39% reduction in the maximal AVPstimulated PI response was observed in the liver slices from ethanol-fed rats compared to control (Fig. 2). The concentrationeffect curve for NE-stimulated PI hydrolysis was similar for control sucrose-fed young and aged rats (Figs. 1 and 31. A similar lack of effect of aging on the AVP-stimulated PI response was observed (Figs. 2 and 4). However, the chronic ethanol treatment caused a significant 23% reduction in the maximal NE-stimulated response in the aged animals (Fig. 3). The maximal AVP-stimulated PI response in liver slices was also reduced by 28% in the aged ethanol-fed rats compared to the aged sucrose-fed rats (Fig. 4). The ECso values for NE- or AVP-stimulated PI hydrolysis were not significantly different for the aged ethanol-fed rats compared to the aged sucrose-fed rats.
Effects of Chronic Ethanol Treaonent on [3H]Prazosin and [3H]A VP Binding to Liver Plasma Membranes To determine if the reduction of maximal NE- and AVP-stimulated PI responses in liver slices from the ethanol-fed rats was due to changes in eq-adrenergic or AVP binding sites, the binding of [3H]piazosin and [3H]AVP to liver plasma membranes was assayed. Scatchard analyses of saturation experiments with different concentrations of [3H]prazosin revealed that neither the Bm,,~ nor the Ka for [3H]prazosin to liver plasma membranes from ethanol-fed rats was significantly different from sucrose-fed rats (Table 1). Similar experiments comparing the binding of {3H]AVP to liver plasma membranes from ethanol to sucrose fed rats indicated that the Ka and Bmax values were not significantly different (Table 1). DISCUSSION
Liver cell functions such as glycogenolysis are controlled by a variety of circulating hormones which act through cell surface receptors. Activation of eq-adrenergic and vasopressin V, receptors initiates a cascade of reactions which control intracellular calcium concentrations via the release of inositol trisphosphate (IPO from the phosphodiesteratic breakdown of phosphatidylinositol 4,5-bisphosphate (10,12). Increases in cytosolic free calcium from the inosital trisphosphate mediated mobilization of calcium from endoplasmic reticulum modulate the activity of p h o s p h o r y l a s e a and s u b s e q u e n t cell r e s p o n s e s such as glycogenolysis in hepatocytes (9,19). We have investigated the effects of chronic in vivo ethanol ingestion on the accumulation of [3H]inositol phosphates in liver slices exposed to NE and AVP
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FIG. I. Effect of chronic ethanol in vivo on NE-stimulated PI hydrolysis in rat liver slices. Three-month-old rats were maintained on a liquid diet containing 35% calories as ethanol (ETOH) or pair-fed with an identical diet containing isocaloric sucrose substituted for the ethanol (sucrose) for 5 months. NE-stimulated PI hydrolysis was determined as described in the Method section. Each point represents the mean ___SEM of five experiments. The curve for ethanol-fed rats was significantly different from the control curve by factorial analysis of variance, F(1,64)=22.7, p<0.01. *Indicates p<0.05 compared to sucrose by Newman-Keuls test. to determine if ethanol alters Pl-linked signal transduction in the liver cell. Our results indicate that chronic ethanol treatment may interfere with ctradrenergic- and vasopressin-mediated PI hydrolysis in liver slices. Chronic treatment of rats with ethanol for 5 months on a liquid diet caused a decrease in the maximum PI hydrolysis response stimulated by NE or AVP compared to pair-fed controis. We did not detect any effects of the chronic ethanol treatment on the binding parameters for [3H]prazosin or [3H]AVP in liver plasma membranes suggesting that ethanol's effects are exerted distal to the receptor. The present observation of a reduction in the maximal ~t,- and AVP-stimulated PI response due to chronic ethanol treatment is opposite the findings of Smith et al. (18) who reported an increase in the stimulated synthesis of phosphatidylinositol in hepatocytes from ethanol-fed rats. This discrepancy may be due to differences in the chronic ethanol diet, tissue preparation, or the fact that we measured PI hydrolysis directly instead of the synthesis of phosphatidylinositol. It is also possible that our results may be due to small changes in the specific activity of the phosphoinositides which have been hydrolyzed due to receptor activation. However. we think that this is unlikely because we did not observe any consistent decrease in the basal levels of inositol phosphates in the liver slices from ethanol-treated rats. Aging of rats did not have any significant effects on NE- or AVP-stimulated PI hydrolysis in liver slices. Chronic ethanol in-' gestion had similar inhibitory effects on the PI responses of liver slices from 8-month-old and 22-month-old rats. These results suggest that the initial event in the PI-linked signal transduction pathway is not altered by aging. Furthermore, the response of the
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FIG. 2. Effect of chronic ethanol in vivo on AVP-stimulated PI hydrolysis in rat liver slices. Data were obtained as described in Fig. 1 except that AVP was the stimulant. Each point represents the mean_ SEM of five experiments. The curve for ethanol-fed rats was significantly different from the control curve by factorial analysis of variance, F(1,45)=56.0. p<0.01. *Indicates p<0.05 and **indicates p<0.01 compared to sucrose by Newman-Keuls test.
FIG. 3. Effect of chronic ethanol in vivo on NE-stimulated PI hydrolysis in liver slices from old rats. Data were obtained as described in Fig. 1 except that 18-month-old rats were maintained on the ethanol- or sucrosecontaining liquid diets (final age 22 months old). Each point represents the mean ---SEM of five experiments. The curve for ethanol-fed rats was significantly different from the control curve by factorial analysis of variance, F(1,49) = 13.2, p<0.01.
liver to chronic ethanol as measured by NE- and AVP-stimulated PI hydrolysis in liver slices is similar in 8-month-old rats compared to 22-month-old rats. Recent work has suggested that ctt-adrenergic and vasopressin receptors are coupled through a guanine nucleotide binding protein to phosphoinositide phosphodiesterase which is separate from those which couple receptors to adenylate cyclase (20). Ethanol may have effects on the coupling of a~-adrenergic or vasopressin receptors to the coupling protein and/or the interaction of the activated receptor complex with the phosphoinositide phosphodiesterase. Recently, Hoek et al. (7,8) have presented evidence that in vitro ethanol may transiently activate the PI phosphodiesterase in liver causing calcium mobilization and phosphorylase activation. Studies on isolated rat hepatocytes have indicated that in vitro ethanol increases the concentration of intracellular calcium in less than 1 minute. The response rapidly desensitizes within another minute apparently due to protein kinase C activation (8). It is possible that ethanol acts rapidly to cause a short transient activation of protein kinase C which subsequently leads to a prolonged and extended desensitization of hormone signals. Further experiments with broken membrane preparations are necessary to distinguish these possibilities. The reduction in maximal NE- and AVP-stimulated PI response in rat liver slices we observed after chronic ethanol treatment may be due, in part, to changes in the structure or composition of the liver plasma membrane. Previous studies have reported that liver plasma membranes from rats fed an ethanol-containing diet for 30 days or liver cells in culture grown in ethanol-containing media for 3 weeks exhibited lower diphenylhexatriene fluorescence polarization values (13, 17, 23). These results suggest that the liver plasma membrane becomes less rigid after chronic exposure to ethanol in contrast to liver mitochondrial membranes
which may become more rigid after exposure to chronic ethanol (16). However, we found that ethanol in vitro has little or no effect on the fluorescence polarization of diphenylhexatriene in membranes of rats which had received the ethanol-containing diet for 5 months compared to the sucrose controls (unpublished observations). Thus, it seems that factors other than bulk changes in membrane fluidity may be involved in the loss of maximal PI responses after chronic ethanol treatment. Changes in the lipid or protein composition of the plasma membrane may also be involved in the modulation of the PI response. Increases, decreases, and no change have been variously reported for the effects of chronic ethanol on the cholesterol/phospholipid ratio in liver plasma membranes in studies using different paradigms (13, 23, 24). Small changes in particular lipids or proteins (e.g., the coupling protein) may be responsible for the observed reduction in maximal P1 responses in the present study. The relationship of ethanol's inhibition of hormone-activated PI hydrolysis in liver slices after chronic treatment to subsequent hepatocyte cell responses such as calcium mobilization or phosphorylase activation is not known. Although the coupling of receptors to the phosphoinositide phospholipase C which mediates PI hydrolysis is 1:1, there appears to be a receptor reserve for eqand AVP-mediated calcium mobilization and phosphorylase activation (10,12). Approximately 20% of the maximal stimulated PI response is required for maximal increases in cytosolic calcium or phosphorylase activation. A reduction in the ability of hormones to stimulate the breakdown of PI may lead to a shift to the right of the concentration-effect curve for more distal events which are dependent upon the initial release of IP 3. However, chronic ethanol may have effects on calcium mobilization or phosphorylase activation in addition to inhibiting the initial event in the biochemical cascade. Changes in the membrane organization and
E T H A N O L A N D L I V E R PI H Y D R O L Y S I S
135
TABLE 1
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HG. 4. Effect of chronic ethanol in vivo on AVP-stimulated PI hydrolysis in liver slices from old rats. Data were obtained as described in Fig. 2 except that 22-month-old rats were subjects. Each point represents the mean ± SEM of five experiments. The curve for ethanol-fed rats was significantly different from the control curve by factorial analysis of variance, F( 1,561 = 37.1, p<0.01.
calcium uptake o f liver m i c r o s o m e s have been reported after chronic ethanol ingestion (141. Further e x p e r i m e n t s are required to determine if chronic ethanol ingestion alters these other cell responses. Furthermore, it should be noted that the liver slice
preparation used in this study contains cell types other than hepatocytes. It m a y be possible that s o m e of the c h a n g e s in the PI response we observed due to the chronic ethanol treatment were due to effects on a variety o f cells within the liver slice. In s u m m a r y , we have s h o w n that a 5 - m o n t h chronic ethanol treatment causes a 20--40% reduction in the m a x i m a l NE- or A V P - s t i m u l a t e d PI response in liver slices without affecting the binding o f [3H]prazosin or [3H]AVP to liver plasma m e m b r a n e s . This suppression of h o r m o n e - s t i m u l a t e d PI hydrolysis was observed in both y o u n g and old rats. T h e s e results s u g g e s t that chronic ethanol ingestion m a y interfere with H - l i n k e d h o r m o n e stimulated signal transduction in liver. Further e x p e r i m e n t s are necessary to understand the possible relationship between ethan o l ' s effects on PI hydrolysis and the chronic effects o f ethanol on liver function. ACKNOWLEDGEMENTS
The authors thank Dr. Don Walker and Dr. Bruce Hunter for maintaining the rats on the liquid diets.
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GONZALES AND CREWS
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0741-8329/91 $3.00 + .00
Chronic Ethanol Inhibits Receptor-Stimulated Phosphoinositide Hydrolysis in Rat Liver Slices I R U E B E N A. G O N Z A L E S *
A N D F U L T O N T. C R E W S t - "
*Department of Pharmacology, Universit3' of" Texas, Austin, TX 78712 attd ?Department of Pharmacology, Universita' of Florida, Gainesville, FL 32610 R e c e i v e d 6 F e b r u a r y 1990; A c c e p t e d 3 O c t o b e r 1990
GONZALES, R. A. AND F. T..CREWS. Chronic ethanol inhibits receptor.stimulated phosphoinositide hydrolysis in rat liver slices. ALCOHOL 8(2) 131-136, 1991.--The effects of chronic ethanol feeding on norepinephrine (NE)- and arginine-vasopressin (AVP)-stimulated phosphoinositide (PI) hydrolysis in rat liver slices was determined. The maximum NE-stimulated PI response was significantly reduced by 40% in liver slices from 8-month-old rats which had been treated for 5 months with a liquid diet containing ethanol compared to pair-fed controls. The maximum AVP-stimulated PI response was decreased by 39% in liver slices from the ethanol-fed rats compared to control. ECso values for NE- and AVP-stimulated PI hydrolysis in liver slices were not affected by the chronic ethanol treatment. Similar reductions in the maximal NE- and AVP-stimulated PI hydrolysis (28% and 27%, respectively) were found in 22-month-old rats which had been maintained on an ethanol containing diet for 5 months compared to pair-fed controis. The binding of [3H]prazosin and [~H]AVP to liver plasma membranes from 8-month-old ethanol-fed rats was not significantly different from binding to liver membranes from sucrose-fed controls. Our data suggest that chronic ethanol ingestion may lead to a reduction in Pl-linked signal transduction in liver. Ethanol
Liver
Phosphoinositide hydrolysis
Inositol phosphates
Arginine-vasopressin
a:Adrenergic receptors
cium concentrations (12). Stimulation of a : a d r e n e r g i c or vasopressin receptors in the liver results in the phospholipase-mediated hydrolysis of phosphoinositides and the generation of second messengers inositol 1,4,5-trisphosphate and diacylglycerol (I, 10). We have recently shown that c~:stimulated PI hydrolysis in brain slices is inhibited by ethanol in vitro which suggests that the membrane effects of ethanol may interfere with certain receptors which are linked to PI hydrolysis (6). Smith et al. (18) have previously reported that chronic ethanol in combination with a high fat diet causes an increase in cq-adrenergic-stimulated [32p] incorporation into phosphatidylinositol in hepatocytes. More recently, ethanol has been shown to alter calcium homeostasis in hepatocytes by transiently stimulating the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C (7,8). However, there is still little data available regarding the effects of chronic ethanol treatment on receptor-stimulated PI hydrolysis in liver. The present investigation was undertaken to further examine the effects of chronic treatment of rats with ethanol on stimulated PI hydrolysis in liver slices. Two different ages of rats were used to determine if aging alters the effects of the chronic ethanol treatment on PI hydrolysis in liver. Our results suggest that chronic ingestion of ethanol interferes with the transmission of a :adrenergic and vasopressin signals which are carried through the PI system in liver.
ONE of the most important pharmacological effects of ethanol ingestion is the disruption of liver function. Some of the effects of ethanol on 4iver function may be due to the metabolic consequences of the oxidation of ethanol (2). However, ethanol is also known to perturb the organization of cell membranes (4,5). Several investigators have reported that mitochondrial, microsomal, and plasma membranes from hepatocytes adapt to the presence of ethanol as measured by a resistance to the membrane disordering effects of ethanol (13, 14, 22). This membrane adaptation to chronic ethanol has been suggested to be involved in ethanol-induced changes in the oxidative phosphorylation capability of liver mitochondria (22). Other membrane-bound enzyme activities such as Na,K-ATPase may also be affected by chronic ethanol ingestion (24). These data suggest that the membrane disordering effects of ethanol lead to an adaptation of liver membranes with concomitant changes in the activity of membrane-dependent enzymes. Although it is clear that chronic ethanol ingestion results in changes in intracellular organelles in the liver, the demonstrated effects of ethanol on the liver plasma membrane suggest that the functions of the plasma membrane may be affected. Receptorstimulated phosphoinositide (PI) hydrolysis is known to be an important signal transduction system in liver for hormones such as catecholamines and 'vasopressin which regulate cytosolic ca]-
~This work was supported by grants from NIAAA (AA06069 and AA07297). -'Requests for reprints should be addressed to Dr. Fulton T. Crews. Box J-267 JHMHC, University of Florida Medical School, Gainesville, FL 32610.
131
132
GONZALES AND CREWS
METHOD
Materials Experimental subjects were male Fischer 344 rats (3 or 15 months old) obtained from the colony maintained by NIA by Harlan Sprague-Dawley (Indianapolis, IN). myo-[3H]lnositol ( 12-15 Ci/mmol), [3H]prazosin (85 Ci/mmol), and [3H]arginine-vasopressin (AVP, 64 Ci/mmol) were obtained from Amersham Corporation (Arlington Heights, IL). Norepinephrine (NE), AVP and other drugs were obtained from Sigma Chemical Company (St. Louis, MO). Prazosin was kindly provided by Pfizer, Inc. (Groton, CT). Solvents were reagent grade from Fisher Scientific Company (Orlando, FL).
PI Hydrolysis in Liver Slices The method of Berridge et al. (3) was modified for use with liver slices. Rats were decapitated and the liver was rapidly removed and washed in Krebs-Ringer bicarbonate buffer (KRB) (118 mM NaCI, 4.7 mM KCI, 0.75 mM CaCI 2, 1.18 mM KH 2PO.~, 1.18 mM MgSO.,, 24.8 mM NaHCO3, 10 mM glucose) which had been bubbled with O2/CO2 (95:5) to give a pH of 7.4. Thin slices (2-3 mm) were cut from several lobes, placed flat on a Teflon disk, and the tissue was further sliced on a McIlwain tissue chopper (0.35 mm) in two perpendicular directions. The liver slices were then transferred to a conical tube containing fresh warmed (37°C) KRB and washed four times in 30 min with constant bubbling with O2/CO 2. [3H]lnositol was added (0.3 I.tM final concentration), the tube was saturated with O2/CO 2, and the capped tube was incubated for 60 min with gentle shaking in a warm water bath. The labelled slices were washed twice with fresh KRB and distributed (50 p.l) into polypropylene tubes containing KRB (190 I.tl) which had 10 mM LiCI and 108 mM NaCI instead of the usual NaC1 concentration and were incubated for 10 min. Buffer or stimulants were added, and tubes were gassed with O2/CO2, capped and incubated for 60 rain at 37°C. Incubations were terminated by adding 1.0 ml of chloroform-methanol (1:2). Water and chloroform (0.35 ml) were added to the tubes, and the tissue was extracted for 10 min. After separation of phases by centrifugation, an aliquot of the upper phase (0.75 ml) was taken for analysis of [3H]inositol phosphates. The remaining upper phase was aspirated, and an aliquot of the lower phase was transferred to a scintillation vial. The chloroform was dried, and the radioactivity incorporated into phospholipids was determined with a liquid scintillation counter. The aqueous aliquot was diluted to 3 ml with water, and 1.0 ml of a slurry of Dowex- 1 (X8, formate form) was added. This was then poured into a polypropylene column fitted with a fritted disk, and the liquid was allowed to drain. The column was washed with 10 ml of water, and the total [3H]inositol phosphates were eluted directly into a vial with 5 ml of 1.0 M ammonium formate/0.1 M formic acid. Ten milliliters of Liquiscint (National Diagnostics, Manville, N J) were added, and the radioactivity eluted was determined with a liquid scintillation counter. PI hydrolysis was expressed as the percent of [3H]inositol phosphates released. This value was arrived at by taking the ratio of the [3H]inositol phosphates recovered (DPM from column) to the total [3H]inositol which had been incorporated into phospholipids (DPM from column + DPM in chloroform) and multiplying this by 100.
buffer A (250 mM sucrose, 10 mM Tris base, I mM EGTA, pH 7.5) and minced into small pieces. The tissue was homogenized in 10 volumes of modified buffer A with a Dounce homogenizer using 4 strokes of the loose pestle followed by 10 strokes with the tight-fitting pestle. The homogenate was further diluted with modified buffer A (7 volumes) and centrifuged at 1464×g for 10 min at 4°C. The pellet was resuspended with 17 volumes of modified buffer A and filtered through 2-ply nylon gauze. Percoll (5.6 ml) was added to the homogenate and mixed well. The Percoll-homogenate mixture was distributed into four 15-ml Corex tubes and centrifuged at 34,500 x g for 30 min at 4°C. Plasma membranes which banded near the top of the tube were collected and washed twice with modified buffer A. The final pellet was resuspended in incubation buffer (50 mM Tris, 10 mM MgCI 2, 1 mM EGTA, pH 7.4) and frozen in liquid nitrogen for up to two weeks before assaying for [3H]prazosin or [3H]arginine-vasopressin binding sites. Protein content of membranes was determined with the method of Lowry et al. ( 11 ).
[~H]Prazosin and [~H]AVP Binding to Liver Membranes For the binding assays. 30-40 p.g membrane protein was added to tubes containing 50 mM Tris, 10 mM MgCI 2 (pH 7.4). and the appropriate concentration of [3H]prazosin (0.5 nM) or [3H]AVP (I nM) and incubated for 20 min at 37°C or 30°C, respectively, to determine total binding. Nonspecific binding was determined by adding 10 ~M phentolamine or 10 p-M AVP. Nonspecific binding was subtracted from total to obtain specific binding. Scatchard analyses were performed at concentrations of 0.075-20 nM of each ligand. Bound ligand was separated from free by rapid filtration under vacuum through Whatman GF/C filters with a Brandel cell harvester. Filter bound radioactivity was determined with a liquid scintillation counter.
Chronic Ethanol Treaonent Rats were maintained on a nutritionally complete liquid diet containing either 35% of calories as ethanol (ethanol-fed) or pairfed with a diet containing isocaloric sucrose substituted for the ethanol (sucrose-fed) for 5 months following the procedure of Walker et al. (21). Chocolate-flavored Sustacal (Mead-Johnson) was mixed with an ethanol or sucrose containing solution to give 1.3 kcal/ml diet. Both diets were also supplemented with 0.3 g/ dl Vitamin Diet Fortification Mixture and 0.5 g/dl Salt Mixture XIV (both from ICN Nutritional Biochemicals). The ethanoltreated rats were originally matched by weight to controls, and the sucrose-fed animals were given the amount of diet which its ethanol-fed counterpart had consumed the previous day. The rats were maintained in a temperature controlled room on a standard 12 h/12 h light/dark cycle. The weight gain for the young sucrose- and ethanol-treated rats was 169 ± 11 g (n = 18) and 138 ± 18 g (n= 17), respectively, throughout the 5-month liquid diet period. Weight gain in the 22-month-old rats was 147--- 12 g (n= 6) and 129± 10 g (n=7), for the sucrose- and ethanol-treated rats, respectively. Ethanol-treated animals consumed an average of 11 g/kg of ethanol each day with blood levels ranging from approximately 50 mg/dl at 8 a.m. to 140 mg/dl at 8 p.m. Rats were killed on the morning after the last evening of ethanol feeding.
Preparation of Liver Plasma Membranes
Statistical Analysis
Liver plasma membranes were isolated according to the method of Prpic et al. (15). Pieces of rat liver were initially frozen over dry ice. The frozen pieces were then thawed in ice-cold modified
Significant differences between experimental groups and controis were determined using two-way analysis of variance. Comparison of individual means was carried out using Newman-Keuls
ETHANOL AND LIVER PI HYDROLYSIS
133
procedure. Differences were considered significant if p < 0 . 0 5 . RESULTS
Effects of Age and Chronic Ethanol Treatment of NE- and AVP-Stimulated PI Responses in Liver Slices To determine if aging or chronic ethanol treatment in vivo has any effects on stimulated PI hydrolysis in vitro, concentration-effect curves for NE- and AVP-stimulated PI hydrolysis were performed in liver slices from young and aged ethanol- and sucrosefed rats. Three- and 17-month-old rats were maintained on liquid diets containing either sucrose or ethanol for 5 months so that the animals were sacrificed when they were 8 (young) or 22 (aged) months old. Concentration-effect curves for NE- and AVP-stimulated PI hydrolysis in liver slices from young ethanol- and sucrose-fed rats are shown in Figs. 1 and 2, respectively. The maximal NE-stimulated PI response is 40% lower in the liver slices from the ethanol-fed rats compared to sucrose-treated controls (Fig. 1). A comparable 39% reduction in the maximal AVPstimulated PI response was observed in the liver slices from ethanol-fed rats compared to control (Fig. 2). The concentrationeffect curve for NE-stimulated PI hydrolysis was similar for control sucrose-fed young and aged rats (Figs. 1 and 31. A similar lack of effect of aging on the AVP-stimulated PI response was observed (Figs. 2 and 4). However, the chronic ethanol treatment caused a significant 23% reduction in the maximal NE-stimulated response in the aged animals (Fig. 3). The maximal AVP-stimulated PI response in liver slices was also reduced by 28% in the aged ethanol-fed rats compared to the aged sucrose-fed rats (Fig. 4). The ECso values for NE- or AVP-stimulated PI hydrolysis were not significantly different for the aged ethanol-fed rats compared to the aged sucrose-fed rats.
Effects of Chronic Ethanol Treaonent on [3H]Prazosin and [3H]A VP Binding to Liver Plasma Membranes To determine if the reduction of maximal NE- and AVP-stimulated PI responses in liver slices from the ethanol-fed rats was due to changes in eq-adrenergic or AVP binding sites, the binding of [3H]piazosin and [3H]AVP to liver plasma membranes was assayed. Scatchard analyses of saturation experiments with different concentrations of [3H]prazosin revealed that neither the Bm,,~ nor the Ka for [3H]prazosin to liver plasma membranes from ethanol-fed rats was significantly different from sucrose-fed rats (Table 1). Similar experiments comparing the binding of {3H]AVP to liver plasma membranes from ethanol to sucrose fed rats indicated that the Ka and Bmax values were not significantly different (Table 1). DISCUSSION
Liver cell functions such as glycogenolysis are controlled by a variety of circulating hormones which act through cell surface receptors. Activation of eq-adrenergic and vasopressin V, receptors initiates a cascade of reactions which control intracellular calcium concentrations via the release of inositol trisphosphate (IPO from the phosphodiesteratic breakdown of phosphatidylinositol 4,5-bisphosphate (10,12). Increases in cytosolic free calcium from the inosital trisphosphate mediated mobilization of calcium from endoplasmic reticulum modulate the activity of p h o s p h o r y l a s e a and s u b s e q u e n t cell r e s p o n s e s such as glycogenolysis in hepatocytes (9,19). We have investigated the effects of chronic in vivo ethanol ingestion on the accumulation of [3H]inositol phosphates in liver slices exposed to NE and AVP
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FIG. I. Effect of chronic ethanol in vivo on NE-stimulated PI hydrolysis in rat liver slices. Three-month-old rats were maintained on a liquid diet containing 35% calories as ethanol (ETOH) or pair-fed with an identical diet containing isocaloric sucrose substituted for the ethanol (sucrose) for 5 months. NE-stimulated PI hydrolysis was determined as described in the Method section. Each point represents the mean ___SEM of five experiments. The curve for ethanol-fed rats was significantly different from the control curve by factorial analysis of variance, F(1,64)=22.7, p<0.01. *Indicates p<0.05 compared to sucrose by Newman-Keuls test. to determine if ethanol alters Pl-linked signal transduction in the liver cell. Our results indicate that chronic ethanol treatment may interfere with ctradrenergic- and vasopressin-mediated PI hydrolysis in liver slices. Chronic treatment of rats with ethanol for 5 months on a liquid diet caused a decrease in the maximum PI hydrolysis response stimulated by NE or AVP compared to pair-fed controis. We did not detect any effects of the chronic ethanol treatment on the binding parameters for [3H]prazosin or [3H]AVP in liver plasma membranes suggesting that ethanol's effects are exerted distal to the receptor. The present observation of a reduction in the maximal ~t,- and AVP-stimulated PI response due to chronic ethanol treatment is opposite the findings of Smith et al. (18) who reported an increase in the stimulated synthesis of phosphatidylinositol in hepatocytes from ethanol-fed rats. This discrepancy may be due to differences in the chronic ethanol diet, tissue preparation, or the fact that we measured PI hydrolysis directly instead of the synthesis of phosphatidylinositol. It is also possible that our results may be due to small changes in the specific activity of the phosphoinositides which have been hydrolyzed due to receptor activation. However. we think that this is unlikely because we did not observe any consistent decrease in the basal levels of inositol phosphates in the liver slices from ethanol-treated rats. Aging of rats did not have any significant effects on NE- or AVP-stimulated PI hydrolysis in liver slices. Chronic ethanol in-' gestion had similar inhibitory effects on the PI responses of liver slices from 8-month-old and 22-month-old rats. These results suggest that the initial event in the PI-linked signal transduction pathway is not altered by aging. Furthermore, the response of the
134
GONZALES AND CREWS
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FIG. 2. Effect of chronic ethanol in vivo on AVP-stimulated PI hydrolysis in rat liver slices. Data were obtained as described in Fig. 1 except that AVP was the stimulant. Each point represents the mean_ SEM of five experiments. The curve for ethanol-fed rats was significantly different from the control curve by factorial analysis of variance, F(1,45)=56.0. p<0.01. *Indicates p<0.05 and **indicates p<0.01 compared to sucrose by Newman-Keuls test.
FIG. 3. Effect of chronic ethanol in vivo on NE-stimulated PI hydrolysis in liver slices from old rats. Data were obtained as described in Fig. 1 except that 18-month-old rats were maintained on the ethanol- or sucrosecontaining liquid diets (final age 22 months old). Each point represents the mean ---SEM of five experiments. The curve for ethanol-fed rats was significantly different from the control curve by factorial analysis of variance, F(1,49) = 13.2, p<0.01.
liver to chronic ethanol as measured by NE- and AVP-stimulated PI hydrolysis in liver slices is similar in 8-month-old rats compared to 22-month-old rats. Recent work has suggested that ctt-adrenergic and vasopressin receptors are coupled through a guanine nucleotide binding protein to phosphoinositide phosphodiesterase which is separate from those which couple receptors to adenylate cyclase (20). Ethanol may have effects on the coupling of a~-adrenergic or vasopressin receptors to the coupling protein and/or the interaction of the activated receptor complex with the phosphoinositide phosphodiesterase. Recently, Hoek et al. (7,8) have presented evidence that in vitro ethanol may transiently activate the PI phosphodiesterase in liver causing calcium mobilization and phosphorylase activation. Studies on isolated rat hepatocytes have indicated that in vitro ethanol increases the concentration of intracellular calcium in less than 1 minute. The response rapidly desensitizes within another minute apparently due to protein kinase C activation (8). It is possible that ethanol acts rapidly to cause a short transient activation of protein kinase C which subsequently leads to a prolonged and extended desensitization of hormone signals. Further experiments with broken membrane preparations are necessary to distinguish these possibilities. The reduction in maximal NE- and AVP-stimulated PI response in rat liver slices we observed after chronic ethanol treatment may be due, in part, to changes in the structure or composition of the liver plasma membrane. Previous studies have reported that liver plasma membranes from rats fed an ethanol-containing diet for 30 days or liver cells in culture grown in ethanol-containing media for 3 weeks exhibited lower diphenylhexatriene fluorescence polarization values (13, 17, 23). These results suggest that the liver plasma membrane becomes less rigid after chronic exposure to ethanol in contrast to liver mitochondrial membranes
which may become more rigid after exposure to chronic ethanol (16). However, we found that ethanol in vitro has little or no effect on the fluorescence polarization of diphenylhexatriene in membranes of rats which had received the ethanol-containing diet for 5 months compared to the sucrose controls (unpublished observations). Thus, it seems that factors other than bulk changes in membrane fluidity may be involved in the loss of maximal PI responses after chronic ethanol treatment. Changes in the lipid or protein composition of the plasma membrane may also be involved in the modulation of the PI response. Increases, decreases, and no change have been variously reported for the effects of chronic ethanol on the cholesterol/phospholipid ratio in liver plasma membranes in studies using different paradigms (13, 23, 24). Small changes in particular lipids or proteins (e.g., the coupling protein) may be responsible for the observed reduction in maximal P1 responses in the present study. The relationship of ethanol's inhibition of hormone-activated PI hydrolysis in liver slices after chronic treatment to subsequent hepatocyte cell responses such as calcium mobilization or phosphorylase activation is not known. Although the coupling of receptors to the phosphoinositide phospholipase C which mediates PI hydrolysis is 1:1, there appears to be a receptor reserve for eqand AVP-mediated calcium mobilization and phosphorylase activation (10,12). Approximately 20% of the maximal stimulated PI response is required for maximal increases in cytosolic calcium or phosphorylase activation. A reduction in the ability of hormones to stimulate the breakdown of PI may lead to a shift to the right of the concentration-effect curve for more distal events which are dependent upon the initial release of IP 3. However, chronic ethanol may have effects on calcium mobilization or phosphorylase activation in addition to inhibiting the initial event in the biochemical cascade. Changes in the membrane organization and
E T H A N O L A N D L I V E R PI H Y D R O L Y S I S
135
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HG. 4. Effect of chronic ethanol in vivo on AVP-stimulated PI hydrolysis in liver slices from old rats. Data were obtained as described in Fig. 2 except that 22-month-old rats were subjects. Each point represents the mean ± SEM of five experiments. The curve for ethanol-fed rats was significantly different from the control curve by factorial analysis of variance, F( 1,561 = 37.1, p<0.01.
calcium uptake o f liver m i c r o s o m e s have been reported after chronic ethanol ingestion (141. Further e x p e r i m e n t s are required to determine if chronic ethanol ingestion alters these other cell responses. Furthermore, it should be noted that the liver slice
preparation used in this study contains cell types other than hepatocytes. It m a y be possible that s o m e of the c h a n g e s in the PI response we observed due to the chronic ethanol treatment were due to effects on a variety o f cells within the liver slice. In s u m m a r y , we have s h o w n that a 5 - m o n t h chronic ethanol treatment causes a 20--40% reduction in the m a x i m a l NE- or A V P - s t i m u l a t e d PI response in liver slices without affecting the binding o f [3H]prazosin or [3H]AVP to liver plasma m e m b r a n e s . This suppression of h o r m o n e - s t i m u l a t e d PI hydrolysis was observed in both y o u n g and old rats. T h e s e results s u g g e s t that chronic ethanol ingestion m a y interfere with H - l i n k e d h o r m o n e stimulated signal transduction in liver. Further e x p e r i m e n t s are necessary to understand the possible relationship between ethan o l ' s effects on PI hydrolysis and the chronic effects o f ethanol on liver function. ACKNOWLEDGEMENTS
The authors thank Dr. Don Walker and Dr. Bruce Hunter for maintaining the rats on the liquid diets.
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pressin receptor-stimulated phosphatidylinositol synthesis in isolated rat hepatocytes. Biochem. Pharmacol. 32:3059-3063: 1983. 19. Thomas, A. P.; Marks. J. S.; Coll. K. E.; Williamson. J. R. Quantitation and early kinetics of inositol lipid changes induced by vasopressin in isolated and cultured hepatocytes. J. Biol. Chern. 258: 5716--5725; 1983. 20. Uhing, R. J.; Prpic. V.; Jiang, J.: Exton. J. H. Hormone-stimulated polyphosphoinositide breakdown in rat liver plasma membranes. J. Biol. Chem. 261:2140-2146; 1986. 21. Walker. D. W.; Barnes, D. E.; Zornetzer, S. F.: Hunter. B. E.: Kubanis, P. Neuronal loss in hippocampus induced by prolonged eth-
GONZALES AND CREWS
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