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Voluntary alcohol consumption and plasma beta-endorphin levels in alcohol-preferring rats chronically treated with naltrexone
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Physiology & Behavior 93 (2008) 1005 – 1010 www.elsevier.com/locate/phb
Voluntary alcohol consumption and plasma beta-endorphin levels in alcohol-preferring rats chronically treated with naltrexone Jadwiga Zalewska-Kaszubska a,⁎, Dorota Gorska a , Wanda Dyr b , Elzbieta Czarnecka a a
b
Department of Pharmacodynamics, Medical University of Lodz, Muszynskiego 1, PL 90-151 Lodz, Poland Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Warsaw, Poland Received 16 August 2007; received in revised form 18 December 2007; accepted 9 January 2008
Abstract It is well known that alcohol consumption leads to an increase in the levels of beta-endorphin (an endogenous opioid), which can contribute to the reinforcing effect of alcohol. Our previous studies have shown that repeated treatment with naltrexone, a nonselective opioid antagonist, results in increased plasma beta-endorphin levels. Ample studies in animals and humans have shown that naltrexone diminishes ethanol consumption. The aim of the present study in alcohol-preferring rats (Warsaw High Preferring; WHP) was to investigate the effect of 10 days of naltrexone treatment (2 mg/kg i.p.) on voluntary alcohol intake and on changes in plasma beta-endorphin levels while alcohol was available and 10 days after imposed abstinence. It was observed that voluntary alcohol intake induces an increase in plasma beta-endorphin levels in WHP rats. After a 10day period of alcohol withdrawal, the levels of this peptide were significantly reduced compared with the levels in rats with free access to ethanol and in control alcohol-naïve rats. In chronic naltrexone-treated rats with free access to alcohol, an increase in the levels of this peptide was also observed; however, voluntary alcohol intake was diminished. A similar increase in plasma beta-endorphin levels was observed in naltrexonetreated rats that did not have access to ethanol. As the endogenous opioid system has an important role in the development of a craving for alcohol, it is likely that chronic naltrexone treatment may have a beneficial effect leading to a compensatory increase in the beta-endorphin concentration and ameliorating its deficiency during ethanol withdrawal. Restoring the alcohol-induced deficiency of beta-endorphin may be an important factor in the prevention of craving and maintenance of abstinence. This finding supports the proposition that the endogenous opioid system plays an important role in developing a craving for alcohol. © 2008 Elsevier Inc. All rights reserved. Keywords: Alcohol-preferring rats; Beta-endorphin; Ethanol; Free-choice procedure; Naltrexone
1. Introduction Numerous human and animal studies have shown that opioid receptor antagonists significantly decrease alcohol consumption [1–7]. Naltrexone, a nonselective opioid antagonist, is one of the most effective drugs used clinically for the treatment of alcoholism. Naltrexone has been shown to reduce alcohol craving and relapse [8,9] probably by blocking the reward mechanisms in the central nervous system [10,11]. Several studies have shown that naltrexone also reduces the palatability of alcohol [3,4,12]. Naltrexone changes the taste of ethanol and, in this way, enhances
⁎ Corresponding author. E-mail address: [email protected] (J. Zalewska-Kaszubska). 0031-9384/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.01.007
the aversive response to ethanol [13]. However, changes in taste reactivity to ethanol are not the sole mechanism by which the opioid antagonist is responsible for reducing the intake of alcohol. Human and animal studies have demonstrated a relationship between the endogenous opioid system and alcohol drinking behaviour, supporting the hypothesis that this system has an important role in alcohol addiction [14,15]. It is well known that ethanol consumption increases the release of opioid peptides, especially beta-endorphin, which interacts with brain structures closely involved in the positive reinforcement system [15,16]. The release of beta-endorphin in the mesolimbic pathway may be associated with the activation of the central dopamine reward system [17–19]. In this way alcohol may have an indirect influence on the reward system. There is some indirect evidence that changes in pituitary beta-endorphin release may often reflect similar
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changes in central beta-endorphin release. Animal studies have shown that alcohol, which increases beta-endorphin release from the pituitary, also stimulates beta-endorphin release in distinct brain regions [20–24]. The concentrations of beta-endorphin in the cerebrospinal fluid as well as in the plasma of alcoholics are markedly decreased compared to those in normal individuals [25]. Our previous studies have shown that repeated naltrexone treatment in ethanol-naive Warsaw High Preferring (WHP) rats resulted in an increase in the plasma beta-endorphin concentrations [26], similar to the increase observed after a single administration of ethanol to untreated control rats. In light of these studies we have extended our research to examine the effects of naltrexone treatment on the beta-endorphin levels in rats with free access to ethanol and after enforced withdrawal. The aim of this study was also to investigate the effect of naltrexone treatment on voluntary ethanol consumption by alcohol-preferring rats. We used the WHP rat line as these rats have been characterized as consuming ethanol at the rate of 4–8 g/kg daily in a free-choice paradigm [27] and as having lower plasma beta-endorphin levels compared with their Warsaw Low Preferring (WLP) counterparts [28,29].
After the period when baseline ethanol consumption was established, the rats received intraperitoneally naltrexone (2 mg/kg; 2 ml/kg b.w., daily) or saline over the course of 10 days, according to the schedule in Table 1. The rats were given naltrexone or vehicle at 9–10 a.m., after the beginning of the daylight period.
2. Materials and methods
2.3. Materials
2.1. Animals
Sep-pak C 18 cartridges were obtained from Waters M.A., USA (Cat. No. WAT 020515); acetone (HPLC grade) and trifluoroacetic acid were from Baker. Aprotinin (Trascolan®) was purchased from Jelfa, Poland; naltrexone hydrochloride was from Sigma. Ether was purchased from POCh, Poland. The plasma beta-endorphin radioimmunoassay kit was obtained from Phoenix Pharmaceuticals, Inc., USA.
The experiments were carried out on 40 female adult rats weighing 220–280 g from the F36–37 generation of the WHP animal line that were kept under standard laboratory conditions. The animals were housed individually in stainless steel cages equipped with two graduated drinking tubes, containing tap water or 10% v/v alcohol. The alcohol solution was prepared from water and a stock solution of 95% reagent grade ethanol. Following the method of Rezvani et al. [30], the rats were given 3 days of free access to a solution of 10% (v/v) ethanol as a sole source of fluid. Food was available ad libitum. This procedure allowed them to become accustomed to drinking from the tubes and to experience the taste and pharmacological properties of alcohol. After the initial 3-day period the rats were given 24 hours of free-choice access to 10% ethanol and water during three consecutive weeks. Alcohol and water intake were recorded and the tubes were refilled daily. Consumption of ethanol and water was measured daily at the same time between 9.00 and 10.00 a.m., before naltrexone or saline administration. The position of the alcohol and water tubes was alternated daily to avoid the development of a position preference. Animals were weighed every 3 days. Prior to administration of naltrexone or saline, the average alcohol and water intakes were calculated for each rat over 5 consecutive days prior to the day of treatment. The data are expressed as the daily amount of ethanol ingested (g/kg), the total volume of fluid ingested daily (ml/kg) and preference. Alcohol consumption was determined by calculating grams of alcohol consumed per kilogram of body weight. The ethanol preference was calculated by the following formula: Ethanol preference ðkÞ ¼ intake of ethanol ðml=kg=dayÞ=total fluid intake ðml=kg=dayÞ 100:
2.2. Blood sampling procedure The rats were anaesthetized with ether 24 hours after the last administration of naltrexone or saline, and their blood samples were collected by heart puncture. Before blood collection the rats that previously had access to alcohol still had access to alcohol and water. Blood samples were collected in tubes containing EDTA (1.6 mg/ml) and gently rocked several times to prevent coagulation. Afterwards, the samples were transferred to centrifuge tubes containing aprotinin (500 KIU/ml) and gently rocked several times to inhibit proteinase activity. The samples were then cooled in an ice bath. The plasma was separated by centrifugation at 1600 × g for 15 minutes at 4 °C. The plasma was frozen and stored at − 20 °C until assessment.
2.4. Solid phase extraction of peptides from plasma Plasma beta-endorphin was determined after extraction by the acid-acetone method. The procedure for beta-endorphin extraction was based on the use of Sep-pak C 18 cartridges in accordance with the method of Angwin and Barchas [31] modified by Zalewska-Kaszubska and Obzejta [32]. Before loading on Sep-Pak C-18 cartridges, a 1-ml volume of plasma was acidified by mixing with the same volume of 1% trifluoroacetic acid (TFA) and centrifuged at 10 000 × g for Table 1 Experimental groups of rats Group
Number Initial procedure of rats
Treatment (10 days)
Control
8
Saline
Saline– 8 Ethanol Saline– 8 Water Naltrexone– 8 Ethanol Naltrexone– 8 Water
The control group was given water as its sole source of fluid The rats were given free access to 10% ethanol and water for 21 days
Saline during free ethanol access Saline during ethanol withdrawal Naltrexone (2 mg/kg) during free ethanol access Naltrexone (2 mg/kg) during ethanol withdrawal
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20 minutes at 4 °C. C-18 Sep-columns were activated by the passage of 2 ml of acetone and subsequently equilibrated twice with 2 ml of 1% TFA in distilled water. The supernatant of the acidified plasma solution was loaded onto the columns. The columns were washed twice with 2 ml of 1% TFA. Betaendorphins were eluted with 1.5 ml of 1% TFA/acetone (25:75) and dried under vacuum. Plasma levels of beta-endorphin were estimated by radioimmunoassay method. 2.5. Statistical analysis All data are expressed as mean ± SEM. Statistical analysis was performed using analysis of variance followed by post-hoc least significant differences (LSD). A normal distribution of data was tested using the Kolmogoroff–Smirnov test with Lillieforse correction. Differences were considered significant when p b 0.05. 3. Results Analysis of the amount of alcohol consumption in naltrexone-treated rats showed a significant decrease in comparison to the baseline. The one-way ANOVA demonstrated a significant main effect (F10,77 = 2.45, p b 0.05). There were no changes in ethanol intake during saline treatment (F10,77 = 0.61, p = 0.79). Baseline voluntary alcohol intake by WHP rats was 6.25 ± 0.24 g alcohol/kg bw/day before naltrexone treatment. As seen in Fig. 1, repeated single daily i.p. administration of naltrexone at a dose of 2 mg/kg for 10 consecutive days, but not the vehicle treatment, significantly reduced voluntary alcohol consumption without development of tolerance to the suppressant effect of the drug on alcohol intake. Two-way ANOVA revealed significant group effect (F1,140 = 91.38, p b 0.001) but no day effect (F9,140 = 1.01, p = 0.43) and two-way interaction effect for group × day treatment (F9,140 = 0.72, p = 0.68). Overall mean
Fig. 1. The effect of repeated treatment with naltrexone or saline on ethanol intake by WHP rats given a free choice between ethanol solution (10% v/v) and water. The rats were treated intraperitoneally with naltrexone (2 mg/kg; 2 ml/kg body weight/daily) or saline for 10 days. Rats were given free access to 10% v/v ethanol and water. The baseline is the mean of alcohol intake (g/kg/day) measured before naltrexone or saline treatment. Each point represents the mean ± SEM from 8 rats. ap b 0.05 in comparison to baseline.
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Table 2 Ethanol, water and total fluid intake (ml/kg/day) measured during the 5-day pretreatment period and the 10-day treatment with naltrexone or saline Group
Ethanol intake (ml/kg/day)
Water intake (ml/kg/day)
Total fluid (ml/kg/day)
Preference (%)
Pretreatment Saline Pretreatment Naltrexone
77.5 ± 2.8 76.7 ± 3.4 78.3 ± 3.0 57.7 ± 4.4 a
18.3 ± 2.2 17.7 ± 1.7 17.6 ± 1.9 18.6 ± 1,8
95.7 ± 2.9 94.4 ± 3.4 95.8 ± 2.6 76.3 ± 4.3 a
80.1 ± 2.3 81.1 ± 1.8 81.5 ± 2.0 74.6 ± 2.9 a
Data are expressed as the means ± SEM from 8 rats in each animal group over test sequences: baseline drinking period (5 days) and during naltrexone or saline injection (10 days). a p b 0.01 in comparison to pretreatment and saline treatment groups.
ethanol intake during the naltrexone administration period was 4.57 ± 0.38 g/kg/daily. As seen in Table 2, injection of 2 mg/kg naltrexone daily decreased the daily volume of alcohol ingested from 76.7 ± 3.4 ml/kg to 57.7 ± 4.4 ml/kg. The total fluid ingested also declined during the administration of naltrexone from 94.4 ± 3.4 ml/kg to 76.3 ± 4.3 ml/kg per day. Thus naltrexone administration suppressed the baseline volume of ethanol intake by 25% and the total fluid intake by 19% during the naltrexone treatment period. There was no effect of naltrexone treatment on water intake; however the total fluid intake decreased in the naltrexone treatment group (F3,236 = 52.09, p b 0,001) and the preference for ethanol changed from 81% to 75% (F2,236 = 34.59, p b 0.001). The mean amount of total fluid ingested fell by 18.1 ml/kg/day compared to saline treated rats. Naltrexone suppressed alcohol intake without altering water intake (Table 2).
Fig. 2. The effect of repeated treatment with naltrexone on beta-endorphin plasma levels in WHP rats. Rats were treated intraperitoneally with naltrexone (2 mg/kg; 2 ml/kg body weight, daily) or 0.9% NaCl for 10 days. Experimental groups: Control – group treated with 0.9% NaCl; Saline–Ethanol – group treated with 0.9% NaCl, with free access to ethanol and water; Saline–Water – alcohol withdrawal group treated with 0.9% NaCl; Naltrexone–Ethanol – group treated with naltrexone for 10 days with free access to ethanol; Naltrexone– Water – group treated with naltrexone for 10 days without access to ethanol. Except for the alcohol free control group, remaining groups were given free access to 10% v/v ethanol during the three weeks before the experiment. Values are expressed as mean ± SEM from 8 rats per group. ap b 0.05 in comparison to control group. b p b 0.05 in comparison to the groups: Saline–Ethanol, Naltrexone–Ethanol, Naltrexone–Water. cp b 0.05 in comparison to the ethanol withdrawal group (Saline–Water).
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The one-way ANOVA demonstrated a significant main effect for beta-endorphin concentration (F4,35 = 15.49, p b 0.05). As shown in Fig. 2, the basic plasma beta-endorphin level in WHP naïve rats was 368 ± 25 pg/ml. In the group of rats with free access to alcohol, the levels of this peptide increased to 556 ± 28 pg/ml. Similar levels of beta-endorphin were observed in rats treated with naltrexone both with and without concomitant ethanol access, 610 ± 49 pg/ml and 579 ± 62 pg/ml, respectively. A period of 10 days of enforced abstinence from ethanol consumption caused a significant decrease in plasma beta-endorphin levels (245 ± 17 pg/ml) in comparison both to rats with free access to ethanol and control rats. 4. Discussion Naltrexone at a dose of 2 mg/kg attenuated the consumption of alcohol in WHP rats. Rats treated with naltrexone drank significantly less ethanol than the saline-treated rats. In addition, the preference for ethanol in these rats was significantly decreased. Our study was performed with continuous access to ethanol; however, Kornet et al. [33] reported that, in rhesus monkeys, naltrexone is more effective after a period of imposed abstinence in comparison to a regimen with continuous supply of ethanol and water. Our results from the use of naltrexone at a dose of 2 mg/kg are in agreement with a few other studies in which the suppressant effect of naltrexone was also observed at similar doses, such as 1.8 mg/kg [34] or 2.5 mg/kg [35]. Davidson and Amit [35] have observed that naltrexone blocks acquisition of voluntary ethanol intake in rats at doses of 2.5 and 10 mg/kg, but no suppressant effect was observed at the intermediate doses of 5 and 7.5 mg/kg. They have surmised that the suppression of ethanol intake by the 2.5 mg/kg dose of naltrexone was mediated by different mechanisms to those operative at the higher 10 mg/kg dose of naltrexone. It was found that high doses of an opioid antagonist induce conditioned taste aversion (CTA) compared with low doses, which are not known to induce CTA [36]. However CTA is not the only mechanism underlying the suppressant effects of opioid antagonists on alcohol intake [37]. Kornet et al. [33] postulated that opioid modulation specifically interacts with positively reinforced behaviour and also suggested that alcohol abstinence results in altered opioid activity. This agrees with our observations that imposed abstinence led to a marked decrease in beta-endorphin concentration and administration of naltrexone could increase this level. It was reported that blockade of the opioid receptors reduces the opioid activity induced by ethanol [38]. We have previously suggested that the beta-endorphin endogenous opioid is an important factor in alcohol dependence [15,26,28,29]. In the present study we have observed that voluntary intake of ethanol led to a significant increase in the plasma beta-endorphin levels in WHP rats. The 10-day period of imposed abstinence from alcohol consumption decreased this peptide concentration both compared to the level observed in rats with free access to ethanol as well as in control alcohol-naïve rats in which the beta-endorphin level is genetically lower [28,29]. In human plasma, the beta-endorphin levels of subjects at high risk for excessive alcohol consumption also showed a lower basal activity in comparison to those at low risk [15,39]. We suppose
that a deficiency in beta-endorphin levels subsequently augmented by ethanol withdrawal is probably one of the main factors responsible for difficulties in maintaining abstinence. The use of alcohol as a form of “self-medication” is one of the methods for immediately increasing the beta-endorphin levels. In our previous studies we have observed an increase in the beta-endorphin level after a single injection of ethanol. The percentage of this increase was higher in WHP rats (about 120%) in comparison to WLP rats (about 30%) [26,29]. A greater increase in beta-endorphin release was also observed in humans with a high risk of alcoholism [40]. In our study, we have observed that repeated treatment with naltrexone leads to an increase in the beta-endorphin levels in the WHP rats both with and without concomitant free access to alcohol. The increase in this peptide concentration was similar in both naltrexone-treated groups of rats; however, rats in the freechoice paradigm group consumed significantly less ethanol in comparison to the pretreatment period. Our study confirmed also, at least in part, a study by Juarez and Eliana [41] who reported that alcohol consumption increased significantly when alcohol was offered immediately after naltrexone was interrupted. In our study we observed that treatment with naltrexone increased the betaendorphin concentrations to levels similar to those observed during voluntary alcohol intake. It is possible that interruption of naltrexone treatment leads to a decrease in beta-endorphin to the levels observed after ethanol withdrawal. During 10-day naltrexone administration we observed a decrease in voluntary alcohol consumption that persisted at a similar level during drug administration until the end of the experiment. Cowen et al. [42] suggested that, although naltrexone treatment may have a significant effect on reducing alcohol drinking in the short term, continuous long-term naltrexone treatment may not be effective in the treatment of alcoholism, possibly because of the induced increase in mu-opioid receptors. Some research has proven that long-lasting blockade of opioid receptors induces supersensitivity of the opioid receptors [43–45]. However, we have not observed the development of tolerance to naltrexone during 10 days of treatment. The amount of alcohol consumed during naltrexone treatment did not change significantly across the experiment. In summary, naltrexone was demonstrated to cause a significant decrease in ethanol consumption. The results obtained in this experiment may support conclusions from previous research that seemed to indicate that ethanol consumption is modulated, at least partially, by the endogenous opioid system [26,28,29]. Our findings revealed that the ability of naltrexone to decrease alcohol intake in alcohol-preferring rats may occur via an increase in betaendorphin levels. However, we have not excluded the contribution that naltrexone may also make in blocking the reinforcing aspects of the taste. It is possible that, when the opioid receptors are blocked via co-administration of naltrexone and alcohol, the effects after alcohol use may be more aversive. We suppose that naltrexone may be able to operate via at least in two different mechanisms. The main mechanism of naltrexone action is blockade of the opioid receptors, which results in a decrease in the positive reinforcement effect. The second mechanism of repeated naltrexone treatment produces an increase in betaendorphin concentrations. We suppose that increased pituitary beta-endorphin release may probably restore the disturbed balance
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Physiology & Behavior 93 (2008) 1005 – 1010 www.elsevier.com/locate/phb
Voluntary alcohol consumption and plasma beta-endorphin levels in alcohol-preferring rats chronically treated with naltrexone Jadwiga Zalewska-Kaszubska a,⁎, Dorota Gorska a , Wanda Dyr b , Elzbieta Czarnecka a a
b
Department of Pharmacodynamics, Medical University of Lodz, Muszynskiego 1, PL 90-151 Lodz, Poland Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Warsaw, Poland Received 16 August 2007; received in revised form 18 December 2007; accepted 9 January 2008
Abstract It is well known that alcohol consumption leads to an increase in the levels of beta-endorphin (an endogenous opioid), which can contribute to the reinforcing effect of alcohol. Our previous studies have shown that repeated treatment with naltrexone, a nonselective opioid antagonist, results in increased plasma beta-endorphin levels. Ample studies in animals and humans have shown that naltrexone diminishes ethanol consumption. The aim of the present study in alcohol-preferring rats (Warsaw High Preferring; WHP) was to investigate the effect of 10 days of naltrexone treatment (2 mg/kg i.p.) on voluntary alcohol intake and on changes in plasma beta-endorphin levels while alcohol was available and 10 days after imposed abstinence. It was observed that voluntary alcohol intake induces an increase in plasma beta-endorphin levels in WHP rats. After a 10day period of alcohol withdrawal, the levels of this peptide were significantly reduced compared with the levels in rats with free access to ethanol and in control alcohol-naïve rats. In chronic naltrexone-treated rats with free access to alcohol, an increase in the levels of this peptide was also observed; however, voluntary alcohol intake was diminished. A similar increase in plasma beta-endorphin levels was observed in naltrexonetreated rats that did not have access to ethanol. As the endogenous opioid system has an important role in the development of a craving for alcohol, it is likely that chronic naltrexone treatment may have a beneficial effect leading to a compensatory increase in the beta-endorphin concentration and ameliorating its deficiency during ethanol withdrawal. Restoring the alcohol-induced deficiency of beta-endorphin may be an important factor in the prevention of craving and maintenance of abstinence. This finding supports the proposition that the endogenous opioid system plays an important role in developing a craving for alcohol. © 2008 Elsevier Inc. All rights reserved. Keywords: Alcohol-preferring rats; Beta-endorphin; Ethanol; Free-choice procedure; Naltrexone
1. Introduction Numerous human and animal studies have shown that opioid receptor antagonists significantly decrease alcohol consumption [1–7]. Naltrexone, a nonselective opioid antagonist, is one of the most effective drugs used clinically for the treatment of alcoholism. Naltrexone has been shown to reduce alcohol craving and relapse [8,9] probably by blocking the reward mechanisms in the central nervous system [10,11]. Several studies have shown that naltrexone also reduces the palatability of alcohol [3,4,12]. Naltrexone changes the taste of ethanol and, in this way, enhances
⁎ Corresponding author. E-mail address: [email protected] (J. Zalewska-Kaszubska). 0031-9384/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.01.007
the aversive response to ethanol [13]. However, changes in taste reactivity to ethanol are not the sole mechanism by which the opioid antagonist is responsible for reducing the intake of alcohol. Human and animal studies have demonstrated a relationship between the endogenous opioid system and alcohol drinking behaviour, supporting the hypothesis that this system has an important role in alcohol addiction [14,15]. It is well known that ethanol consumption increases the release of opioid peptides, especially beta-endorphin, which interacts with brain structures closely involved in the positive reinforcement system [15,16]. The release of beta-endorphin in the mesolimbic pathway may be associated with the activation of the central dopamine reward system [17–19]. In this way alcohol may have an indirect influence on the reward system. There is some indirect evidence that changes in pituitary beta-endorphin release may often reflect similar
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changes in central beta-endorphin release. Animal studies have shown that alcohol, which increases beta-endorphin release from the pituitary, also stimulates beta-endorphin release in distinct brain regions [20–24]. The concentrations of beta-endorphin in the cerebrospinal fluid as well as in the plasma of alcoholics are markedly decreased compared to those in normal individuals [25]. Our previous studies have shown that repeated naltrexone treatment in ethanol-naive Warsaw High Preferring (WHP) rats resulted in an increase in the plasma beta-endorphin concentrations [26], similar to the increase observed after a single administration of ethanol to untreated control rats. In light of these studies we have extended our research to examine the effects of naltrexone treatment on the beta-endorphin levels in rats with free access to ethanol and after enforced withdrawal. The aim of this study was also to investigate the effect of naltrexone treatment on voluntary ethanol consumption by alcohol-preferring rats. We used the WHP rat line as these rats have been characterized as consuming ethanol at the rate of 4–8 g/kg daily in a free-choice paradigm [27] and as having lower plasma beta-endorphin levels compared with their Warsaw Low Preferring (WLP) counterparts [28,29].
After the period when baseline ethanol consumption was established, the rats received intraperitoneally naltrexone (2 mg/kg; 2 ml/kg b.w., daily) or saline over the course of 10 days, according to the schedule in Table 1. The rats were given naltrexone or vehicle at 9–10 a.m., after the beginning of the daylight period.
2. Materials and methods
2.3. Materials
2.1. Animals
Sep-pak C 18 cartridges were obtained from Waters M.A., USA (Cat. No. WAT 020515); acetone (HPLC grade) and trifluoroacetic acid were from Baker. Aprotinin (Trascolan®) was purchased from Jelfa, Poland; naltrexone hydrochloride was from Sigma. Ether was purchased from POCh, Poland. The plasma beta-endorphin radioimmunoassay kit was obtained from Phoenix Pharmaceuticals, Inc., USA.
The experiments were carried out on 40 female adult rats weighing 220–280 g from the F36–37 generation of the WHP animal line that were kept under standard laboratory conditions. The animals were housed individually in stainless steel cages equipped with two graduated drinking tubes, containing tap water or 10% v/v alcohol. The alcohol solution was prepared from water and a stock solution of 95% reagent grade ethanol. Following the method of Rezvani et al. [30], the rats were given 3 days of free access to a solution of 10% (v/v) ethanol as a sole source of fluid. Food was available ad libitum. This procedure allowed them to become accustomed to drinking from the tubes and to experience the taste and pharmacological properties of alcohol. After the initial 3-day period the rats were given 24 hours of free-choice access to 10% ethanol and water during three consecutive weeks. Alcohol and water intake were recorded and the tubes were refilled daily. Consumption of ethanol and water was measured daily at the same time between 9.00 and 10.00 a.m., before naltrexone or saline administration. The position of the alcohol and water tubes was alternated daily to avoid the development of a position preference. Animals were weighed every 3 days. Prior to administration of naltrexone or saline, the average alcohol and water intakes were calculated for each rat over 5 consecutive days prior to the day of treatment. The data are expressed as the daily amount of ethanol ingested (g/kg), the total volume of fluid ingested daily (ml/kg) and preference. Alcohol consumption was determined by calculating grams of alcohol consumed per kilogram of body weight. The ethanol preference was calculated by the following formula: Ethanol preference ðkÞ ¼ intake of ethanol ðml=kg=dayÞ=total fluid intake ðml=kg=dayÞ 100:
2.2. Blood sampling procedure The rats were anaesthetized with ether 24 hours after the last administration of naltrexone or saline, and their blood samples were collected by heart puncture. Before blood collection the rats that previously had access to alcohol still had access to alcohol and water. Blood samples were collected in tubes containing EDTA (1.6 mg/ml) and gently rocked several times to prevent coagulation. Afterwards, the samples were transferred to centrifuge tubes containing aprotinin (500 KIU/ml) and gently rocked several times to inhibit proteinase activity. The samples were then cooled in an ice bath. The plasma was separated by centrifugation at 1600 × g for 15 minutes at 4 °C. The plasma was frozen and stored at − 20 °C until assessment.
2.4. Solid phase extraction of peptides from plasma Plasma beta-endorphin was determined after extraction by the acid-acetone method. The procedure for beta-endorphin extraction was based on the use of Sep-pak C 18 cartridges in accordance with the method of Angwin and Barchas [31] modified by Zalewska-Kaszubska and Obzejta [32]. Before loading on Sep-Pak C-18 cartridges, a 1-ml volume of plasma was acidified by mixing with the same volume of 1% trifluoroacetic acid (TFA) and centrifuged at 10 000 × g for Table 1 Experimental groups of rats Group
Number Initial procedure of rats
Treatment (10 days)
Control
8
Saline
Saline– 8 Ethanol Saline– 8 Water Naltrexone– 8 Ethanol Naltrexone– 8 Water
The control group was given water as its sole source of fluid The rats were given free access to 10% ethanol and water for 21 days
Saline during free ethanol access Saline during ethanol withdrawal Naltrexone (2 mg/kg) during free ethanol access Naltrexone (2 mg/kg) during ethanol withdrawal
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20 minutes at 4 °C. C-18 Sep-columns were activated by the passage of 2 ml of acetone and subsequently equilibrated twice with 2 ml of 1% TFA in distilled water. The supernatant of the acidified plasma solution was loaded onto the columns. The columns were washed twice with 2 ml of 1% TFA. Betaendorphins were eluted with 1.5 ml of 1% TFA/acetone (25:75) and dried under vacuum. Plasma levels of beta-endorphin were estimated by radioimmunoassay method. 2.5. Statistical analysis All data are expressed as mean ± SEM. Statistical analysis was performed using analysis of variance followed by post-hoc least significant differences (LSD). A normal distribution of data was tested using the Kolmogoroff–Smirnov test with Lillieforse correction. Differences were considered significant when p b 0.05. 3. Results Analysis of the amount of alcohol consumption in naltrexone-treated rats showed a significant decrease in comparison to the baseline. The one-way ANOVA demonstrated a significant main effect (F10,77 = 2.45, p b 0.05). There were no changes in ethanol intake during saline treatment (F10,77 = 0.61, p = 0.79). Baseline voluntary alcohol intake by WHP rats was 6.25 ± 0.24 g alcohol/kg bw/day before naltrexone treatment. As seen in Fig. 1, repeated single daily i.p. administration of naltrexone at a dose of 2 mg/kg for 10 consecutive days, but not the vehicle treatment, significantly reduced voluntary alcohol consumption without development of tolerance to the suppressant effect of the drug on alcohol intake. Two-way ANOVA revealed significant group effect (F1,140 = 91.38, p b 0.001) but no day effect (F9,140 = 1.01, p = 0.43) and two-way interaction effect for group × day treatment (F9,140 = 0.72, p = 0.68). Overall mean
Fig. 1. The effect of repeated treatment with naltrexone or saline on ethanol intake by WHP rats given a free choice between ethanol solution (10% v/v) and water. The rats were treated intraperitoneally with naltrexone (2 mg/kg; 2 ml/kg body weight/daily) or saline for 10 days. Rats were given free access to 10% v/v ethanol and water. The baseline is the mean of alcohol intake (g/kg/day) measured before naltrexone or saline treatment. Each point represents the mean ± SEM from 8 rats. ap b 0.05 in comparison to baseline.
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Table 2 Ethanol, water and total fluid intake (ml/kg/day) measured during the 5-day pretreatment period and the 10-day treatment with naltrexone or saline Group
Ethanol intake (ml/kg/day)
Water intake (ml/kg/day)
Total fluid (ml/kg/day)
Preference (%)
Pretreatment Saline Pretreatment Naltrexone
77.5 ± 2.8 76.7 ± 3.4 78.3 ± 3.0 57.7 ± 4.4 a
18.3 ± 2.2 17.7 ± 1.7 17.6 ± 1.9 18.6 ± 1,8
95.7 ± 2.9 94.4 ± 3.4 95.8 ± 2.6 76.3 ± 4.3 a
80.1 ± 2.3 81.1 ± 1.8 81.5 ± 2.0 74.6 ± 2.9 a
Data are expressed as the means ± SEM from 8 rats in each animal group over test sequences: baseline drinking period (5 days) and during naltrexone or saline injection (10 days). a p b 0.01 in comparison to pretreatment and saline treatment groups.
ethanol intake during the naltrexone administration period was 4.57 ± 0.38 g/kg/daily. As seen in Table 2, injection of 2 mg/kg naltrexone daily decreased the daily volume of alcohol ingested from 76.7 ± 3.4 ml/kg to 57.7 ± 4.4 ml/kg. The total fluid ingested also declined during the administration of naltrexone from 94.4 ± 3.4 ml/kg to 76.3 ± 4.3 ml/kg per day. Thus naltrexone administration suppressed the baseline volume of ethanol intake by 25% and the total fluid intake by 19% during the naltrexone treatment period. There was no effect of naltrexone treatment on water intake; however the total fluid intake decreased in the naltrexone treatment group (F3,236 = 52.09, p b 0,001) and the preference for ethanol changed from 81% to 75% (F2,236 = 34.59, p b 0.001). The mean amount of total fluid ingested fell by 18.1 ml/kg/day compared to saline treated rats. Naltrexone suppressed alcohol intake without altering water intake (Table 2).
Fig. 2. The effect of repeated treatment with naltrexone on beta-endorphin plasma levels in WHP rats. Rats were treated intraperitoneally with naltrexone (2 mg/kg; 2 ml/kg body weight, daily) or 0.9% NaCl for 10 days. Experimental groups: Control – group treated with 0.9% NaCl; Saline–Ethanol – group treated with 0.9% NaCl, with free access to ethanol and water; Saline–Water – alcohol withdrawal group treated with 0.9% NaCl; Naltrexone–Ethanol – group treated with naltrexone for 10 days with free access to ethanol; Naltrexone– Water – group treated with naltrexone for 10 days without access to ethanol. Except for the alcohol free control group, remaining groups were given free access to 10% v/v ethanol during the three weeks before the experiment. Values are expressed as mean ± SEM from 8 rats per group. ap b 0.05 in comparison to control group. b p b 0.05 in comparison to the groups: Saline–Ethanol, Naltrexone–Ethanol, Naltrexone–Water. cp b 0.05 in comparison to the ethanol withdrawal group (Saline–Water).
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The one-way ANOVA demonstrated a significant main effect for beta-endorphin concentration (F4,35 = 15.49, p b 0.05). As shown in Fig. 2, the basic plasma beta-endorphin level in WHP naïve rats was 368 ± 25 pg/ml. In the group of rats with free access to alcohol, the levels of this peptide increased to 556 ± 28 pg/ml. Similar levels of beta-endorphin were observed in rats treated with naltrexone both with and without concomitant ethanol access, 610 ± 49 pg/ml and 579 ± 62 pg/ml, respectively. A period of 10 days of enforced abstinence from ethanol consumption caused a significant decrease in plasma beta-endorphin levels (245 ± 17 pg/ml) in comparison both to rats with free access to ethanol and control rats. 4. Discussion Naltrexone at a dose of 2 mg/kg attenuated the consumption of alcohol in WHP rats. Rats treated with naltrexone drank significantly less ethanol than the saline-treated rats. In addition, the preference for ethanol in these rats was significantly decreased. Our study was performed with continuous access to ethanol; however, Kornet et al. [33] reported that, in rhesus monkeys, naltrexone is more effective after a period of imposed abstinence in comparison to a regimen with continuous supply of ethanol and water. Our results from the use of naltrexone at a dose of 2 mg/kg are in agreement with a few other studies in which the suppressant effect of naltrexone was also observed at similar doses, such as 1.8 mg/kg [34] or 2.5 mg/kg [35]. Davidson and Amit [35] have observed that naltrexone blocks acquisition of voluntary ethanol intake in rats at doses of 2.5 and 10 mg/kg, but no suppressant effect was observed at the intermediate doses of 5 and 7.5 mg/kg. They have surmised that the suppression of ethanol intake by the 2.5 mg/kg dose of naltrexone was mediated by different mechanisms to those operative at the higher 10 mg/kg dose of naltrexone. It was found that high doses of an opioid antagonist induce conditioned taste aversion (CTA) compared with low doses, which are not known to induce CTA [36]. However CTA is not the only mechanism underlying the suppressant effects of opioid antagonists on alcohol intake [37]. Kornet et al. [33] postulated that opioid modulation specifically interacts with positively reinforced behaviour and also suggested that alcohol abstinence results in altered opioid activity. This agrees with our observations that imposed abstinence led to a marked decrease in beta-endorphin concentration and administration of naltrexone could increase this level. It was reported that blockade of the opioid receptors reduces the opioid activity induced by ethanol [38]. We have previously suggested that the beta-endorphin endogenous opioid is an important factor in alcohol dependence [15,26,28,29]. In the present study we have observed that voluntary intake of ethanol led to a significant increase in the plasma beta-endorphin levels in WHP rats. The 10-day period of imposed abstinence from alcohol consumption decreased this peptide concentration both compared to the level observed in rats with free access to ethanol as well as in control alcohol-naïve rats in which the beta-endorphin level is genetically lower [28,29]. In human plasma, the beta-endorphin levels of subjects at high risk for excessive alcohol consumption also showed a lower basal activity in comparison to those at low risk [15,39]. We suppose
that a deficiency in beta-endorphin levels subsequently augmented by ethanol withdrawal is probably one of the main factors responsible for difficulties in maintaining abstinence. The use of alcohol as a form of “self-medication” is one of the methods for immediately increasing the beta-endorphin levels. In our previous studies we have observed an increase in the beta-endorphin level after a single injection of ethanol. The percentage of this increase was higher in WHP rats (about 120%) in comparison to WLP rats (about 30%) [26,29]. A greater increase in beta-endorphin release was also observed in humans with a high risk of alcoholism [40]. In our study, we have observed that repeated treatment with naltrexone leads to an increase in the beta-endorphin levels in the WHP rats both with and without concomitant free access to alcohol. The increase in this peptide concentration was similar in both naltrexone-treated groups of rats; however, rats in the freechoice paradigm group consumed significantly less ethanol in comparison to the pretreatment period. Our study confirmed also, at least in part, a study by Juarez and Eliana [41] who reported that alcohol consumption increased significantly when alcohol was offered immediately after naltrexone was interrupted. In our study we observed that treatment with naltrexone increased the betaendorphin concentrations to levels similar to those observed during voluntary alcohol intake. It is possible that interruption of naltrexone treatment leads to a decrease in beta-endorphin to the levels observed after ethanol withdrawal. During 10-day naltrexone administration we observed a decrease in voluntary alcohol consumption that persisted at a similar level during drug administration until the end of the experiment. Cowen et al. [42] suggested that, although naltrexone treatment may have a significant effect on reducing alcohol drinking in the short term, continuous long-term naltrexone treatment may not be effective in the treatment of alcoholism, possibly because of the induced increase in mu-opioid receptors. Some research has proven that long-lasting blockade of opioid receptors induces supersensitivity of the opioid receptors [43–45]. However, we have not observed the development of tolerance to naltrexone during 10 days of treatment. The amount of alcohol consumed during naltrexone treatment did not change significantly across the experiment. In summary, naltrexone was demonstrated to cause a significant decrease in ethanol consumption. The results obtained in this experiment may support conclusions from previous research that seemed to indicate that ethanol consumption is modulated, at least partially, by the endogenous opioid system [26,28,29]. Our findings revealed that the ability of naltrexone to decrease alcohol intake in alcohol-preferring rats may occur via an increase in betaendorphin levels. However, we have not excluded the contribution that naltrexone may also make in blocking the reinforcing aspects of the taste. It is possible that, when the opioid receptors are blocked via co-administration of naltrexone and alcohol, the effects after alcohol use may be more aversive. We suppose that naltrexone may be able to operate via at least in two different mechanisms. The main mechanism of naltrexone action is blockade of the opioid receptors, which results in a decrease in the positive reinforcement effect. The second mechanism of repeated naltrexone treatment produces an increase in betaendorphin concentrations. We suppose that increased pituitary beta-endorphin release may probably restore the disturbed balance
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