- Email: [email protected]
Pharmacological characterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats
Peptides 23 (2002) 117–125
Pharmacological characterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats Roberto Ciccocioppoa,*, Carlo Polidoria, Lorena Antonellia, Severo Salvadorib, Remo Guerrinib, Maurizio Massia a
Department of Pharmacological Sciences and Experimental Medicine, University of Camerino, 62032 Camerino, Italy b Department of Pharmaceutical Sciences, University of Ferrara, 44100 Ferrara, Italy Received 21 May 2001; accepted 15 August 2001
Abstract Chronic intracerebroventricular (ICV) treatment with nociceptin/orphanin FQ (NC), the endogenous ligand for the opioid receptor-like 1 (ORL1) receptor, reduces ethanol intake in alcohol-preferring rats and abolishes the rewarding properties of ethanol in the place conditioning paradigm. To pharmacologically characterize the receptor involved, the present study evaluated the effect on ethanol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats of ICV injections for 8 days of NC or of the NC analogs NC(1–17)NH2, NC(1–13)NH2, NC(1–12)NH2 and [Nphe1]NC(1–13)NH2. In vitro studies indicate that NC, NC(1–17)NH2, NC(1–13)NH2 and NC(1–12)NH2 are agonists, while [Nphe1]NC(1–13)NH2 is a selective antagonist at the ORL1 receptor. Freely feeding and drinking rats were offered 10% ethanol 30 min/day at the beginning of the dark phase of the light cycle. NC significantly attenuated ethanol intake at 500 or 1000 ng/rat (210 or 420 pmol/rat). NC(1–17)NH2, markedly reduced ethanol intake, but its effect was statistically significant at 1000 (420 pmol/rat), not at 500 ng/rat (210 pmol/rat). After the end of treatment ethanol drinking promptly came back to baseline level. Ethanol consumption was also reduced by NC(1–13)NH2; however, its effect was less potent and pronounced. NC(1–12)NH2 did not modify ethanol intake at doses up to 4000 ng/rat (2339 pmol/rat). Water and food consumption were not modified. Treatment with [Nphe1]NC(1– 13)NH2, 66 or 99 g/rat, did not modify ethanol intake; however, [Nphe1]NC(1–13)NH2, 66 g/rat, given just before 1000 ng/rat of NC(1–17)NH2, abolished the effect of the agonist. The present results show that the 13 aminoacid N-terminal sequence of NC is essential for the effect on ethanol intake and indicate that [Nphe1]NC(1–13)NH2 acts as an antagonist to block the effect of NC. These findings provide further evidence that selective agonists at the ORL-1 receptor attenuate ethanol intake in alcohol-preferring rats and suggest that the NC/ORL1 system may represent an interesting target for treatment of alcohol abuse. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Nociceptin; Orphanin FQ; ORL1 receptors; Ethanol intake
1. Introduction Nociceptin/orphanin FQ (NC), the endogenous ligand of the opioid receptor-like1 (ORL1) receptor, is a 17 aminoacid neuropeptide, structurally related to the opioid peptide dynorphin A [29,30,42,43]. Despite its structural homology with opioid peptides, NC does not bind to , ␦ or opioid receptors, nor do opioid peptides activate the ORL1 receptor [41,42]. Activation of membrane ORL1 receptors by NC results in the same sequence of intracellular events induced by opioid receptors, namely negative coupling with adenylyl cyclase, activation of inwardly rectifying K⫹ channels * Corresponding author. Tel.: ⫹39-0737-403307; fax: ⫹39-0737630618. E-mail address: r.ciccocioppo@ca (R. Ciccocioppo).
and inhibition of Ca2⫹ current in a pertussis toxin sensitive manner [16,28,43,48]. However, these cellular responses to NC are insensitive to naloxone [11,17], confirming that the pharmacological actions of this peptide are not mediated by the classic opioid receptors. Detailed structure analysis revealed important differences between ORL1 and , ␦ or opioid receptors [36,41,42], suggesting that separate mutations might have led to a coordinated pharmacological separation of the NC system from the opioid system [27,42]. This view is supported by brain mapping studies showing that the neuroanatomical distribution of NC and its receptor differs from that of opioid peptides and opioid receptors [3,11,14,15,20,35,44,45,46]. NC has been reported to block opioid-induced supraspinal analgesia, and it has been proposed as a “functional antiopioid peptide” in the control of brain nociceptive pro-
0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 1 ) 0 0 5 8 7 - 3
118
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
cesses [31,32]. Thus, several studies have evaluated whether NC behaves as a functional antiopioid peptide also in relation to the motivational properties of opiates or the motivational properties of ethanol, that are largely dependent upon the endogenous opioid system [18,19]. These studies have shown that NC blocks the development of conditioned place preference induced by morphine or ethanol, suggesting that it reduces the rewarding properties of the two drugs [2,6,7, 9,26,33]. The effect may be mediated by interference of NC with activation of mesolimbic dopaminergic neurons of the VTA in response to administration of morphine [13,34] and possibly of ethanol. Moreover, studies of our group have shown that subchronic intracerebroventricular (ICV) treatment with NC reduces ethanol intake in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats [9]. The present study was aimed at the pharmacological characterization of the receptor involved in this effect of NC. The effect on ethanol drinking of ICV injections of the agonists NC, NC(1–17)NH2 and NC(1–13)NH2, of the low potency agonist NC(1–12)NH2, of the selective antagonist [Nphe1]NC(1–13)NH2 [for review 4,5] were tested in msP rats for 8 consecutive days.
2. Methods 2.1. Animals Male genetically selected alcohol-preferring rats were employed. They were bred in the Department of Pharmacological Sciences and Experimental Medicine of the University of Camerino (Marche, Italy) for 33 generations from Sardinian alcohol-preferring rats of the 13th generation, provided by the Department of Neurosciences of the University of Cagliari, Italy [1,10,24]. These animals are referred to as Marchigian Sardinian alcohol-preferring (msP) rats. At the time of the experiments their body weight ranged between 400 and 450 g. They were kept in a room with a reverse 12:12 h light/dark cycle (lights off at 10:00 a.m.), temperature of 20 –22°C and humidity of 45–55%. Rats were offered free access to tap water and food pellets (4RF18, Mucedola, Settimo Milanese, Italy). All the procedures were conducted in adherence to the European Community Council Directive for Care and Use of Laboratory Animals. 2.2. Surgery For intracranial (i.c.) surgery msP rats were anaesthetized by intraperitoneal (i.p.) injection of 100 –150 l/100 g body weight of a solution containing ketamine (86.2 mg/ml) and acepromazine(1.3 mg/ml). A guide cannula for ICV injections into the lateral cerebroventricle was stereotaxically implanted and cemented to the skull. The following coordinates, taken from the atlas of Paxinos and Watson
Table 1 Aminoacid sequence and molecular weight of NC and of the four analogs tested Peptide
Aminoacid sequence
MW
NC
H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-OH H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-NH2 H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-NH2 H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-NH2 H-Nphe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-NH2
2379
NC(1-17)NH2
NC(1-13)NH2 NC(1-12)NH2 [Nphe1]NC(1-13)NH2
2378
1952 1710 1952
[37] were used: antero-posterior ⫽ 1 mm behind the bregma, lateral ⫽ 2 mm from the sagittal suture, ventral ⫽ 2 mm from the surface of the skull. 2.3. Drug administration All the compounds tested are reported in Table 1. They were synthesized as salt derivatives at the Department of Pharmaceutical Sciences of the University of Ferrara, Italy. The compounds were dissolved in isotonic NaCl and ICV injected in a volume of 1 l/rat. NC was tested at doses of 250, 500 or 1000 ng/rat; NC(1–17)NH2, the amide derivative of NC, was tested at doses of 500 or 1000 ng/rat. The partial sequence NC(1–13)NH2 was tested at 500, 1000 or 2000 ng/rat, while NC(1–12)NH2 at 2000 or 4000 ng/rat. Lastly, doses of [Nphe1]NC(1–13)NH2 of 66 or 99 g/rat were used. The ICV injection was carried out by means of a stainless-steel injector 2.5 mm longer than the guide cannula, so that its tip protruded into the ventricle. To validate cannula placement, 1 l of black India ink was ICV injected just before the rat sacrifice and ink diffusion into the ventricles was evaluated. 2.4. Experimental procedure At the age of 3 months msP rats were selected for their preference for 10% ethanol (v/v), offering them free choice between water and 10% ethanol 24 h a day for 15 days. Water and 10% ethanol were offered in graduated drinking tubes equipped with metallic drinking spouts. The rats employed in the following experiments had a 24-h ethanol intake of about 6 g/kg with a percent ethanol preference [ml of ethanol solution/ml of total fluids (water ⫹ 10% ethanol) ingested in 24 h x 100] ranging from 80 to 90. For 3 weeks after surgery, rats had water and food available during the entire day, while 10% ethanol was offered for 30 min/day at the beginning of the dark phase(10:00 a.m.) of the reverse light/dark cycle. The restricted access to ethanol was
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
adopted in relation to the short half-life of NC and related peptides. All the experiments were carried out according to a between subject design, in which each group of rats received a single dose of a single compound. For 8 consecutive days, rats received a daily ICV injection of the peptide dose tested or of isotonic saline (controls), just before access to 10% ethanol. The 30-min ethanol intake was measured for additional 3 days after the end of the ICV treatment. The 30-min intake of 10% ethanol, food and water were measured and submitted to statistical analysis. Water and ethanol intakes were measured by reading the volume consumed from the graduated burettes. Food intake was measured by weighing the food containers and taking into account spillage. 2.5. Statistical analysis Data are reported as means ⫾ SEM. Statistical analysis of data were performed by means of split-plot analysis of variance (ANOVA) with between-groups comparisons for drug treatment and within-groups comparisons for day of treatment. Pairwise comparisons were carried out by means of the Dunnett’s test. Statistical significance was set at P ⬍ 0.05.
3. Results As shown in Fig. 1, upper panel, the 8-day ICV treatment with NC markedly reduced ethanol consumption. The analysis of variance revealed a statistically significant treatment effect [F(3,31) ⫽ 16.21, P ⬍ 0.01], but not significant day effect [F(7,217) ⫽ 1.21, P ⬎ 0.05] or treatment-day interaction. Post-hoc comparisons showed that the effect of NC was statistically significant at the doses of 500 or 1000 ng/rat (210 or 420 pmol/rat), but not at 250 ng/rat. After the end of NC treatment, msP rats promptly recovered their baseline ethanol intake. The analysis of variance revealed no significant difference in ethanol intake of the different experimental groups of rats either during the pre- or the post-treatment period. In the 8 days of NC treatment, food intake was copious during the 30 min of access to 10% ethanol, owing to the beginning of the nocturnal feeding, but was not significantly modified by NC treatment (Fig. 1, lower panel). As a consequence of the large food intake, the reduction in ethanol intake did not result in a significant modification of the overall caloric intake (calories from food ⫹ calories from ethanol). Water intake was very small; only a few animals took less than 0.5 ml of water. NC did not modify water intake (data not shown). The 8-day treatment with NC(1–17)NH2 induced a pronounced reduction of ethanol intake. The analysis of variance revealed a statistically significant treatment effect [F(2,21) ⫽ 6.10, P ⬍ 0.01], but a not significant day effect
119
[F(7,147) ⫽ 1.35, P ⬎ 0.05] or treatment-day interaction. Post-hoc comparisons showed that the effect was significant at the dose of 1000 ng/rat (420 pmol/rat), but not at 500 ng/rat (Fig. 2, upper panel). The analysis of variance revealed no significant difference in ethanol intake of the different experimental groups of rats either during the preor the post-treatment period. In the 8 days of treatment with NC(1–17)NH2, during the 30 min of access to ethanol, food intake ranged between 4.5 and 12.1 g/kg and was not significantly modified by NC(1– 17)NH2 treatment [F(2,21) ⫽ 0.99, P ⬎ 0.05]. The analysis of variance revealed a significant day effect [F(7,147) ⫽ 3.04, P ⬍ 0.01], but no significant treatment-day interaction (Fig. 2, lower panel). Water intake was very low and was not affected by NC(1–17)NH2 (data not shown). As shown in Fig. 3, upper panel, the 8-day ICV treatment with NC(1–13)NH2 induced a statistically significant reduction of ethanol intake [F(3,33) ⫽ 3.12, P ⬍ 0.05]. The analysis of variance revealed also a significant day effect [F(7,231) ⫽ 3.08, P ⬍ 0.01], but a not significant treatmentday interaction. Post-hoc comparisons showed that the effect was statistically significant only at the dose of 2000 ng/rat(1024 pmol/rat). The analysis of variance revealed no significant difference in ethanol intake of the different experimental groups of rats either during the pre- or the post-treatment period. Neither food intake nor water intake were modified by NC(1–13)NH2 (data not shown). NC(1–12)NH2 did not significantly affect ethanol intake [F(2,23) ⫽ 0.61, P ⬎ 0.05] at the doses of 2000 or 4000 ng/rat, that is 1169 or 2339 pmol/rat (Fig. 3, lower panel). The analysis of variance showed neither a significant day effect, nor a significant treatment-day interaction. Moreover, no significant difference in ethanol intake of the different experimental groups of rats was found either during the pre- or the post-treatment period. Neither food nor water intake were modified by NC(1– 12)NH2. As shown in Fig. 4, upper panel, the 8-day treatment with the selective ORL1 receptor antagonist [Nphe1]NC(1– 13)NH2 did not significantly modify ethanol intake following administration of 66 or 99 g/rat [F(2,21) ⫽ 0.89, P ⬎ 0.05], in the absence of significant day effect [F(7,147) ⫽ 1.82, P ⬎ 0.05] or of treatment-day interaction. The analysis of variance revealed no significant difference in ethanol intake of the different experimental groups of rats either during the pre- or the post-treatment period. The same doses of the ORL1 antagonist did not affect food or water intake. When [Nphe1]NC(1–13)NH2, 66 g/rat, was administered together with NC(1–17)NH2, 1000 ng/rat, the antagonist blocked the effect of NC(1–17)NH2 on ethanol intake. The overall analysis of variance showed a significant treatment effect [F(2,21) ⫽ 5.85, P ⬍ 0.01] and a significant day effect [F(7,147) ⫽ 3.22, P ⬍ 0.01], but a not significant treatment-day interaction. Ethanol intake was significantly
120
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
Fig. 1. Thirty-min ethanol intake (Upper Panel) and food intake (Lower Panel) in msP rats before (Pre), during (Treat) and after (Post) subchronic treatment with ICV injections of NC (250, 500 or 1000 ng/rat) or isotonic saline (Veh). Values represent the mean ⫾ SEM of 8 –9 subjects. Difference from controls: *P ⬍ 0.05; ** P ⬍ 0.01; where not indicated difference from controls was not statistically significant.
reduced by NC(1–17)NH2, [F(1,14) ⫽ 9.12, P ⬍ 0.01]; in rats pretreated with the antagonist the same dose of NC(1– 17)NH2 did not significantly modify ethanol intake in comparison to controls, which received two ICV injections of isotonic saline [F(1,14) ⫽ 2.68, P ⬎ 0.05] (Fig. 4B). The
analysis of variance revealed no significant difference in the intake of the different experimental groups of rats either during the pre- or the post-treatment period. Again, food and water intake were not significantly modified (data not shown).
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
121
Fig. 2. Thirty-min ethanol intake (Upper Panel) and food intake (Lower Panel) in msP rats before (Pre), during (Treat) and after (Post) subchronic treatment with ICV injections of NC(1–17)NH2 (500 or 1000 ng/rat) or isotonic saline (Veh). Values represent the mean ⫾ SEM of 8 subjects. Difference from controls: *P ⬍ 0.05; ** P ⬍ 0.01; where not indicated difference from controls was not statistically significant.
4. Discussion The results of the present study confirm that subchronic administration of NC reduces ethanol intake in alcoholpreferring rats, as shown by our previous study [9]. The effect is evoked at doses as low as 500, 1000 ng/rat, corresponding to 210 and 420 pmol/rat. The observed reduction in ethanol intake is reversible, as the intake promptly came
back to the baseline pretreatment levels after the end of treatment. The effect on ethanol intake of the heptadecapeptide amide, NC(1–17)NH2, was similar in intensity to that of NC, however, NC(1–17)NH2 was slightly less potent than NC, since it produced a statistically significant reduction of ethanol intake at 1000, but not 500 ng/rat. Because these peptides have almost identical molecular weight, NC(1–
122
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
Fig. 3. Thirty-min ethanol intake in msP rats before (Pre), during (Treat) and after (Post) subchronic treatment with ICV injections of NC(1–13)NH2 (500, 1000 or 2000 ng/rat) (Upper Panel) or of NC(1–12)NH2 (2000 or 4000 ng/rat) (Lower Panel). Values represent the mean ⫾ SEM of 8 –10 subjects for NC(1–13)NH2 and of 8 –9 subjects for NC(1–12)NH2. Difference from controls: *P ⬍ 0.05; ** P ⬍ 0.01; where not indicated difference from controls was not statistically significant.
17)NH2 was less potent also on a molar basis. The tridecapeptide, NC(1–13)NH2, induced a statistically significant, but less pronounced attenuation of ethanol intake only at 2000 ng/rat(1024 pmol/rat). Thus, NC(1–13)NH2 was markedly less potent than the parent compound. Lastly, the dodecapeptide NC(1–12)NH2 was ineffective at ICV doses up to 4000 ng/rat, that is up to 2339 pmol/rat. Thus, the tridecapeptide is apparently crucial for the effect of NC on ethanol consumption. This finding is similar to those re-
ported by our group for the pharmacological characterization of the NC receptor controlling food intake [38,39], as well as to those obtained in a variety of other in vitro and in vivo tests [see for review 4,5]. The results obtained with both NC and its partial sequences clearly show that changes in ethanol intake occurred in the absence of changes in other aspects of the ingestive behavior. Thus, the doses of NC and its partial sequences that reduced ethanol intake did not elicit the well
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
123
Fig. 4. Thirty-min ethanol intake in msP rats before (Pre), during (Treat) and after (Post) subchronic treatment with ICV injections of [Nphe1]NC(1–13)NH2 (66 or 99 g/rat) or isotonic saline (Veh) (Upper Panel) or of [Nphe1[NC(1–13)NH2 (66 g/rat) or isotonic saline ⫹ NC(1–17)NH2(1000 ng/rat) or isotonic saline (Lower Panel). Values represent the mean ⫾ SEM of 8 subjects. Difference from controls: *P ⬍ 0.05; ** P ⬍ 0.01; where not indicated difference from controls was not statistically significant.
known hyperphagic effect of NC [22,23,38,39,40,47], that appears to be mediated by the same ORL1 receptor. The threshold ICV dose for the hyperphagic effect of NC or NC(1–17)NH2 in msP rats is of about 3000 ng/rat. Also water intake was not significantly modified by NC or its partial sequences at the doses that reduced ethanol intake. Indeed, msP rats usually ingest very low amounts of water; moreover, having free access to water during the day, a lower intake of 10% ethanol in the 30-min test does not
necessarily require simultaneous increase in water intake for homeostatic reasons. Moreover, the effect on ethanol intake of these peptides was observed in conditions in which the rat gross behavior was not modified. Devine et al. [12] reported that hypolocomotion can be evoked by NC at doses of at least 1000 ng/rat, and that tolerance rapidly develops to this effect. As a matter of fact, in the present study slight hypolocomotion was observed in rats treated for the first time with 1000
124
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
ng/rat of NC or of NC(1–17)NH2, but it was not detected any more since the second day of treatment. Lastly, it is noteworthy that (with the exception of the anxiolytic and antistress effects of NC, which occurs at low doses similar to those required to reduce ethanol intake [8,21,25]) the other effects of NC, including the antianalgesic, the hyperphagic and the amnestic ones, are evoked in rats only at higher doses [5]. Taken together with the results of the present study, all these findings indicate that the effects of NC and its partial sequences on ethanol intake are highly selective. [Nphe1]NC(1–13)NH2 did not modify ethanol intake per se, but abolished the effect of NC(1–17)NH2 on ethanol intake. These findings confirm that [Nphe1]NC(1–13)NH2 is a pure antagonist, as previously reported for other tests [for review 4,5], and indicate that it may represent a valuable pharmacological tool for future studies aimed at evaluating the role of the NC/ORL1 receptor system in the control of ethanol intake. Moreover, the results obtained with this antagonist support the view that classic ORL1 receptors mediate the effect of NC and NC analogs on ethanol intake. Apparently, the results obtained with [Nphe1]NC(1– 13)NH2 suggest that ORL1 receptor signaling may have no tonic control over ethanol intake. However, taking into account that this antagonist has a rather low pA2 (about 6.3), other more potent antagonists should be tested to confirm this hypothesis. In conclusion, the present results confirm that agonists at the ORL1 receptor are able to reduce ethanol intake in alcohol-preferring rats, suggesting that the NC/ORL1 receptor system may represent an interesting target for treatment of alcohol abuse.
Acknowledgments The study was supported by Grants from the University of Camerino, Italy and from MURST, Italy (Cofin99).
References [1] Agabio R, Cortis G, Fadda F, Gessa GL, Lobina C, Reali R, Colombo G. Circadian drinking pattern of Sardinian alcohol-preferring rats. Alcohol Alcohol 1996;31:385– 8. [2] Angeletti S, Panocka I, Ciccocioppo R, Gini L, Massi M. Effect of nociceptin (NC) on morphine-induced conditioned place preference (CPP) in rats. Regul Pept 1999;80:122. [3] Anton B, Fein J, To T, Li X, Silberstein L, Evans CJ. Immunohistochemical localization of ORL-1 in the central nervous system of the rat. J Comp Neurol 1996;368:229 –51. [4] Calo’ G, Bigoni R, Rizzi A, Guerrini R, Salvadori S, Regoli D. Nociceptin/orphanin FQ receptor ligands. Peptides 2000;21:935– 47. [5] Calo’ G, Guerrini R, Rizzi A, Salvadori S, Regoli D. Pharmacology of nociceptin and its receptor: a novel therapeutic target. Br J Pharmacol 2000;129:1261– 83. [6] Ciccocioppo R, Angeletti S, Panocka I, Massi M. Nociceptin/orphanin FQ, and drugs of abuse. Peptides 2000;21:1071– 80.
[7] Ciccocioppo R, Angeletti S, Sanna PP, Weiss F, Massi M. Effect of nociceptin/orphanin FQ on the rewarding properties of morphine. Eur J Pharmacol 2000;404:153–59. [8] Ciccocioppo R, Martin-Fardon R, Weiss F, Massi M (2001) Nociceptin/orphanin FQ inhibits stress-, and CRF-induced anorexia in rats. NeuroReport 2001;12:1145–9. [9] Ciccocioppo R, Panocka I, Polidori C, Regoli D, Massi M. Effect of nociceptin on alcohol intake in alcohol-preferring rats. Psychopharmacology 1999;141:220 – 4. [10] Colombo G. Ethanol drinking behaviour in Sardinian alcohol-preferring rats. Alcohol Alcohol 1997;32:443–53. [11] Darland T, Heinricher MM, Grandy DK. Orphanin FQ/nociceptin: a role in pain, and analgesia, but so much more. Trends Neurosci 1998;21:215–21. [12] Devine DP, Taylor L, Reinscheid RK, Monsma FJ Jr, Civelli O, Akil H. Rats rapidly develop tolerance to the locomotor-inhibiting effects of the novel neuropeptide orphanin FQ. Neurochem Res 1996;21: 1387–96. [13] Di Giannuario A, Pieretti S. Nociceptin differentially affects morphine-induced dopamine release from the nucleus accumbens and nucleus caudate in rats. Peptides 2000;21:1125–30. [14] Florin S, Leroux-Nicollet I, Meunier JC, Costentin J. Autoradiographic localization of [3H]nociceptin binding sites from telencephalic to mesencephalic regions of the mouse brain. Neurosci Lett 1997;230:33– 6. [15] Florin S, Meunier JC, Costentin J. Autoradiographic localization of [3H]nociceptin binding sites in the rat brain. Brain Res 2000;880:11– 16. [16] Hawes BE, Graziano MP, Lambert DG. Cellular actions of nociceptin: transduction mechanisms. Peptides 2000;21:961–7. [17] Henderson G, McKnight AT. The orphan opioid receptor and its endogenous ligand nociceptin/orphanin FQ. Trends Pharmacol Sci 1997;18:293–300. [18] Herz A. Endogenous opioid systems and alcohol addiction. Psychopharmacology 1997;129:99 –111. [19] Herz A. Opioid reward mechanisms: a key role in drug abuse? Can J Physiol Pharmacol 1998;76:252– 8. [20] Ikeda K, Watanabe M, Ichikawa T, Kobayashi T, Yano R, Kumanishi T. Distribution of pre-pro-nociceptin/orphanin FQ mRNA and its receptor mRNA in developing and adult mouse central nervous systems. J Comp Neurol 1998;399:139 –51. [21] Jenck F, Moreau JL, Martin JR, Kilpatrick GJ, Reinscheid RK, Monsma FJ Jr, Nothacker HP, Civelli O. Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress. Proc Natl Acad Sci USA 1997;94:14854 –9. [22] Leventhal L, Mathis JP, Rossi GC, Pasternak GW, Bodnar RJ. Orphan opioid receptor antisense probes block orphanin FQ-induced hyperphagia. Eur J Pharmacol 1998;349:R1–3. [23] Leventhal L, Stevens LB, Rossi GC, Pasternak GW, Bodnar RJ. Antisense mapping of the MOR-1 opioid receptor clone: modulation of hyperphagia induced by DAMGO. J Pharmacol Exp Ther 1997; 282:1402–7. [24] Lobina C, Agabio R, Diaz G, Fa M, Fadda F, Gessa GL, Reali R, Colombo G. Constant absolute ethanol intake by Sardinian alcoholpreferring rats independent of ethanol concentrations. Alcohol Alcohol 1997;32:19 –22. [25] Martin-Fardon R, Ciccocioppo R, Massi M, Weiss F. Nociceptin prevents stress-induced ethanol- but not cocaine-seeking behavior in rats. NeuroReport 2000;9:1939 – 43. [26] Massi M, Panocka I, Angeletti S, Ciccocioppo R, Polidori C. Nociceptin (NC) attenuates ethanol intake, and ethanol-induced conditioned place preference (CPP) in alcohol-preferring rats. Brain Res 1999;848:A41–A42. [27] Meng F, Taylor LP, Hoversten MT, Ueda Y, Ardati A, Reinscheid RK, Monsma FJ, Watson SJ, Civelli O, Akil H. Moving from the orphanin FQ receptor to an opioid receptor using four point mutations. J Biol Chem 1996;271:32016 –20.
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125 [28] Meunier JC. Nociceptin/orphanin FQ, and the opioid receptor-like ORL1 receptor. Eur J Pharmacol 1997;340:1–15. [29] Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B, Mazarguil H, Vassart G, Parmentier M, Costentin J. Isolation and structure of the endogenous agonist of opioid receptor- like ORL1 receptor. Nature 1995;377:532–5. [30] Meunier JC, Mouledous L, Topham CM. The nociceptin/orphanin FQ-ORL1 receptor system. Peptides 2000;21:893–900. [31] Mogil JS, Grisel JE, Reinscheid RK, Civelli O, Belknap JK, Grandy DK. Orphanin FQ is a functional anti-opioid peptide. Neuroscience 1996;75:333–7. [32] Mogil JS, Grisel JE, Zhangs G, Belknap JK, Grandy DK. Functional antagonism of -, ␦- and -opioid antinociception by orphanin FQ. Neurosci Lett 1996;214:131– 4. [33] Murphy NP, Lee Y, Maidment NT. Orphanin FQ/nociceptin blocks acquisition of morphine place preference. Brain Res 1999;832:168–70. [34] Murphy NP, Maidment NT. Orphanin FQ/nociceptin modulation of mesolimbic dopamine transmission determined by microdialysis. J Neurochem 1999;73:179 – 86. [35] Neal CR, Jr., Mansour A, Reinscheid R, Nothacker HP, Civelli O, Watson SJ, Jr. Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J Comp Neurol 1999;406:503– 47. [36] Nothacker HP, Reinscheid RK, Mansour A, Henningsen RA, Ardati A, Monsma FJ, Jr., Watson SJ, Civelli O. Primary structure and tissue distribution of the orphanin FQ precursor. Proc Natl Acad Sci USA 1996;93:8677– 82. [37] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 2nd edition. North Ryde, N.S.W., Australia: Academic Press, 1986. [38] Polidori C, Calo’ G, Ciccocioppo R, Guerrini R, Regoli D, Massi M. Pharmacological characterization of the nociceptin receptor mediating hyperphagia: identification of a selective antagonist. Psychopharmacology 2009;148:430 –7.
125
[39] Polidori C, de Caro G, Massi M. The hyperphagic effect of nociceptin/orphanin FQ in rats. Peptides 2000;21:1051– 62. [40] Pomonis JD, Billington CJ, Levine AS. Orphanin FQ. agonist of orphan opioid receptor ORL1, stimulates feeding in rats. Neuroreport 1996;8:369 –71. [41] Reinscheid RK, Ardati A, Monsma FJ, Jr., Civelli O. Structureactivity relationship studies on the novel neuropeptide orphanin FQ. J Biol Chem 1996;271:14163– 8. [42] Reinscheid RK, Higelin J, Henningsen RA, Monsma FJ, Jr., Civelli O. Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities. J Biol Chem 1998;273:1490 –5. [43] Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ, Jr., Civelli O. Orphanin FQ: a neuropeptide that activates an opioid-like G proteincoupled receptor. Science 1995;270:792– 4. [44] Reinscheid RK, Nothacker HP, Civelli O. The orphanin FQ/nociceptin gene: structure, tissue distribution of expression and functional implications obtained from knockout mice. Peptides 2000;21:901– 6. [45] Schulz S, Schreff M, Nuss D, Gramsch C, Hollt V. Nociceptin/ orphanin FQ, and opioid peptides show overlapping distribution but not co-localization in pain-modulatory brain regions. NeuroReport 1996;7:3021–5. [46] Sim LJ, Childers SR. Anatomical distribution of mu, delta, and kappa opioid- and nociceptin/orphanin FQ-stimulated [35S]guanylyl-5⬘-O(␥-thio)-triphosphate binding in guinea pig brain. J Comp Neurol 1997;386:562–72. [47] Stratford TR, Holahan MR, Kelley AE. Injections of nociceptin into nucleus accumbens shell or ventromedial hypothalamic nucleus increase food intake. Neuroreport 1997;8:423– 6. [48] Vaughan CW, Christie MJ. Increase by the ORL1 receptor (opioid receptor-like1) ligand, nociceptin, of inwardly rectifying K⫹ conductance in dorsal raphe nucleus neurons. Br J Pharmacol 1996;117: 1609 –11.
Pharmacological characterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats Roberto Ciccocioppoa,*, Carlo Polidoria, Lorena Antonellia, Severo Salvadorib, Remo Guerrinib, Maurizio Massia a
Department of Pharmacological Sciences and Experimental Medicine, University of Camerino, 62032 Camerino, Italy b Department of Pharmaceutical Sciences, University of Ferrara, 44100 Ferrara, Italy Received 21 May 2001; accepted 15 August 2001
Abstract Chronic intracerebroventricular (ICV) treatment with nociceptin/orphanin FQ (NC), the endogenous ligand for the opioid receptor-like 1 (ORL1) receptor, reduces ethanol intake in alcohol-preferring rats and abolishes the rewarding properties of ethanol in the place conditioning paradigm. To pharmacologically characterize the receptor involved, the present study evaluated the effect on ethanol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats of ICV injections for 8 days of NC or of the NC analogs NC(1–17)NH2, NC(1–13)NH2, NC(1–12)NH2 and [Nphe1]NC(1–13)NH2. In vitro studies indicate that NC, NC(1–17)NH2, NC(1–13)NH2 and NC(1–12)NH2 are agonists, while [Nphe1]NC(1–13)NH2 is a selective antagonist at the ORL1 receptor. Freely feeding and drinking rats were offered 10% ethanol 30 min/day at the beginning of the dark phase of the light cycle. NC significantly attenuated ethanol intake at 500 or 1000 ng/rat (210 or 420 pmol/rat). NC(1–17)NH2, markedly reduced ethanol intake, but its effect was statistically significant at 1000 (420 pmol/rat), not at 500 ng/rat (210 pmol/rat). After the end of treatment ethanol drinking promptly came back to baseline level. Ethanol consumption was also reduced by NC(1–13)NH2; however, its effect was less potent and pronounced. NC(1–12)NH2 did not modify ethanol intake at doses up to 4000 ng/rat (2339 pmol/rat). Water and food consumption were not modified. Treatment with [Nphe1]NC(1– 13)NH2, 66 or 99 g/rat, did not modify ethanol intake; however, [Nphe1]NC(1–13)NH2, 66 g/rat, given just before 1000 ng/rat of NC(1–17)NH2, abolished the effect of the agonist. The present results show that the 13 aminoacid N-terminal sequence of NC is essential for the effect on ethanol intake and indicate that [Nphe1]NC(1–13)NH2 acts as an antagonist to block the effect of NC. These findings provide further evidence that selective agonists at the ORL-1 receptor attenuate ethanol intake in alcohol-preferring rats and suggest that the NC/ORL1 system may represent an interesting target for treatment of alcohol abuse. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Nociceptin; Orphanin FQ; ORL1 receptors; Ethanol intake
1. Introduction Nociceptin/orphanin FQ (NC), the endogenous ligand of the opioid receptor-like1 (ORL1) receptor, is a 17 aminoacid neuropeptide, structurally related to the opioid peptide dynorphin A [29,30,42,43]. Despite its structural homology with opioid peptides, NC does not bind to , ␦ or opioid receptors, nor do opioid peptides activate the ORL1 receptor [41,42]. Activation of membrane ORL1 receptors by NC results in the same sequence of intracellular events induced by opioid receptors, namely negative coupling with adenylyl cyclase, activation of inwardly rectifying K⫹ channels * Corresponding author. Tel.: ⫹39-0737-403307; fax: ⫹39-0737630618. E-mail address: r.ciccocioppo@ca (R. Ciccocioppo).
and inhibition of Ca2⫹ current in a pertussis toxin sensitive manner [16,28,43,48]. However, these cellular responses to NC are insensitive to naloxone [11,17], confirming that the pharmacological actions of this peptide are not mediated by the classic opioid receptors. Detailed structure analysis revealed important differences between ORL1 and , ␦ or opioid receptors [36,41,42], suggesting that separate mutations might have led to a coordinated pharmacological separation of the NC system from the opioid system [27,42]. This view is supported by brain mapping studies showing that the neuroanatomical distribution of NC and its receptor differs from that of opioid peptides and opioid receptors [3,11,14,15,20,35,44,45,46]. NC has been reported to block opioid-induced supraspinal analgesia, and it has been proposed as a “functional antiopioid peptide” in the control of brain nociceptive pro-
0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 1 ) 0 0 5 8 7 - 3
118
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
cesses [31,32]. Thus, several studies have evaluated whether NC behaves as a functional antiopioid peptide also in relation to the motivational properties of opiates or the motivational properties of ethanol, that are largely dependent upon the endogenous opioid system [18,19]. These studies have shown that NC blocks the development of conditioned place preference induced by morphine or ethanol, suggesting that it reduces the rewarding properties of the two drugs [2,6,7, 9,26,33]. The effect may be mediated by interference of NC with activation of mesolimbic dopaminergic neurons of the VTA in response to administration of morphine [13,34] and possibly of ethanol. Moreover, studies of our group have shown that subchronic intracerebroventricular (ICV) treatment with NC reduces ethanol intake in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats [9]. The present study was aimed at the pharmacological characterization of the receptor involved in this effect of NC. The effect on ethanol drinking of ICV injections of the agonists NC, NC(1–17)NH2 and NC(1–13)NH2, of the low potency agonist NC(1–12)NH2, of the selective antagonist [Nphe1]NC(1–13)NH2 [for review 4,5] were tested in msP rats for 8 consecutive days.
2. Methods 2.1. Animals Male genetically selected alcohol-preferring rats were employed. They were bred in the Department of Pharmacological Sciences and Experimental Medicine of the University of Camerino (Marche, Italy) for 33 generations from Sardinian alcohol-preferring rats of the 13th generation, provided by the Department of Neurosciences of the University of Cagliari, Italy [1,10,24]. These animals are referred to as Marchigian Sardinian alcohol-preferring (msP) rats. At the time of the experiments their body weight ranged between 400 and 450 g. They were kept in a room with a reverse 12:12 h light/dark cycle (lights off at 10:00 a.m.), temperature of 20 –22°C and humidity of 45–55%. Rats were offered free access to tap water and food pellets (4RF18, Mucedola, Settimo Milanese, Italy). All the procedures were conducted in adherence to the European Community Council Directive for Care and Use of Laboratory Animals. 2.2. Surgery For intracranial (i.c.) surgery msP rats were anaesthetized by intraperitoneal (i.p.) injection of 100 –150 l/100 g body weight of a solution containing ketamine (86.2 mg/ml) and acepromazine(1.3 mg/ml). A guide cannula for ICV injections into the lateral cerebroventricle was stereotaxically implanted and cemented to the skull. The following coordinates, taken from the atlas of Paxinos and Watson
Table 1 Aminoacid sequence and molecular weight of NC and of the four analogs tested Peptide
Aminoacid sequence
MW
NC
H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-OH H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-NH2 H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-NH2 H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-NH2 H-Nphe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-NH2
2379
NC(1-17)NH2
NC(1-13)NH2 NC(1-12)NH2 [Nphe1]NC(1-13)NH2
2378
1952 1710 1952
[37] were used: antero-posterior ⫽ 1 mm behind the bregma, lateral ⫽ 2 mm from the sagittal suture, ventral ⫽ 2 mm from the surface of the skull. 2.3. Drug administration All the compounds tested are reported in Table 1. They were synthesized as salt derivatives at the Department of Pharmaceutical Sciences of the University of Ferrara, Italy. The compounds were dissolved in isotonic NaCl and ICV injected in a volume of 1 l/rat. NC was tested at doses of 250, 500 or 1000 ng/rat; NC(1–17)NH2, the amide derivative of NC, was tested at doses of 500 or 1000 ng/rat. The partial sequence NC(1–13)NH2 was tested at 500, 1000 or 2000 ng/rat, while NC(1–12)NH2 at 2000 or 4000 ng/rat. Lastly, doses of [Nphe1]NC(1–13)NH2 of 66 or 99 g/rat were used. The ICV injection was carried out by means of a stainless-steel injector 2.5 mm longer than the guide cannula, so that its tip protruded into the ventricle. To validate cannula placement, 1 l of black India ink was ICV injected just before the rat sacrifice and ink diffusion into the ventricles was evaluated. 2.4. Experimental procedure At the age of 3 months msP rats were selected for their preference for 10% ethanol (v/v), offering them free choice between water and 10% ethanol 24 h a day for 15 days. Water and 10% ethanol were offered in graduated drinking tubes equipped with metallic drinking spouts. The rats employed in the following experiments had a 24-h ethanol intake of about 6 g/kg with a percent ethanol preference [ml of ethanol solution/ml of total fluids (water ⫹ 10% ethanol) ingested in 24 h x 100] ranging from 80 to 90. For 3 weeks after surgery, rats had water and food available during the entire day, while 10% ethanol was offered for 30 min/day at the beginning of the dark phase(10:00 a.m.) of the reverse light/dark cycle. The restricted access to ethanol was
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
adopted in relation to the short half-life of NC and related peptides. All the experiments were carried out according to a between subject design, in which each group of rats received a single dose of a single compound. For 8 consecutive days, rats received a daily ICV injection of the peptide dose tested or of isotonic saline (controls), just before access to 10% ethanol. The 30-min ethanol intake was measured for additional 3 days after the end of the ICV treatment. The 30-min intake of 10% ethanol, food and water were measured and submitted to statistical analysis. Water and ethanol intakes were measured by reading the volume consumed from the graduated burettes. Food intake was measured by weighing the food containers and taking into account spillage. 2.5. Statistical analysis Data are reported as means ⫾ SEM. Statistical analysis of data were performed by means of split-plot analysis of variance (ANOVA) with between-groups comparisons for drug treatment and within-groups comparisons for day of treatment. Pairwise comparisons were carried out by means of the Dunnett’s test. Statistical significance was set at P ⬍ 0.05.
3. Results As shown in Fig. 1, upper panel, the 8-day ICV treatment with NC markedly reduced ethanol consumption. The analysis of variance revealed a statistically significant treatment effect [F(3,31) ⫽ 16.21, P ⬍ 0.01], but not significant day effect [F(7,217) ⫽ 1.21, P ⬎ 0.05] or treatment-day interaction. Post-hoc comparisons showed that the effect of NC was statistically significant at the doses of 500 or 1000 ng/rat (210 or 420 pmol/rat), but not at 250 ng/rat. After the end of NC treatment, msP rats promptly recovered their baseline ethanol intake. The analysis of variance revealed no significant difference in ethanol intake of the different experimental groups of rats either during the pre- or the post-treatment period. In the 8 days of NC treatment, food intake was copious during the 30 min of access to 10% ethanol, owing to the beginning of the nocturnal feeding, but was not significantly modified by NC treatment (Fig. 1, lower panel). As a consequence of the large food intake, the reduction in ethanol intake did not result in a significant modification of the overall caloric intake (calories from food ⫹ calories from ethanol). Water intake was very small; only a few animals took less than 0.5 ml of water. NC did not modify water intake (data not shown). The 8-day treatment with NC(1–17)NH2 induced a pronounced reduction of ethanol intake. The analysis of variance revealed a statistically significant treatment effect [F(2,21) ⫽ 6.10, P ⬍ 0.01], but a not significant day effect
119
[F(7,147) ⫽ 1.35, P ⬎ 0.05] or treatment-day interaction. Post-hoc comparisons showed that the effect was significant at the dose of 1000 ng/rat (420 pmol/rat), but not at 500 ng/rat (Fig. 2, upper panel). The analysis of variance revealed no significant difference in ethanol intake of the different experimental groups of rats either during the preor the post-treatment period. In the 8 days of treatment with NC(1–17)NH2, during the 30 min of access to ethanol, food intake ranged between 4.5 and 12.1 g/kg and was not significantly modified by NC(1– 17)NH2 treatment [F(2,21) ⫽ 0.99, P ⬎ 0.05]. The analysis of variance revealed a significant day effect [F(7,147) ⫽ 3.04, P ⬍ 0.01], but no significant treatment-day interaction (Fig. 2, lower panel). Water intake was very low and was not affected by NC(1–17)NH2 (data not shown). As shown in Fig. 3, upper panel, the 8-day ICV treatment with NC(1–13)NH2 induced a statistically significant reduction of ethanol intake [F(3,33) ⫽ 3.12, P ⬍ 0.05]. The analysis of variance revealed also a significant day effect [F(7,231) ⫽ 3.08, P ⬍ 0.01], but a not significant treatmentday interaction. Post-hoc comparisons showed that the effect was statistically significant only at the dose of 2000 ng/rat(1024 pmol/rat). The analysis of variance revealed no significant difference in ethanol intake of the different experimental groups of rats either during the pre- or the post-treatment period. Neither food intake nor water intake were modified by NC(1–13)NH2 (data not shown). NC(1–12)NH2 did not significantly affect ethanol intake [F(2,23) ⫽ 0.61, P ⬎ 0.05] at the doses of 2000 or 4000 ng/rat, that is 1169 or 2339 pmol/rat (Fig. 3, lower panel). The analysis of variance showed neither a significant day effect, nor a significant treatment-day interaction. Moreover, no significant difference in ethanol intake of the different experimental groups of rats was found either during the pre- or the post-treatment period. Neither food nor water intake were modified by NC(1– 12)NH2. As shown in Fig. 4, upper panel, the 8-day treatment with the selective ORL1 receptor antagonist [Nphe1]NC(1– 13)NH2 did not significantly modify ethanol intake following administration of 66 or 99 g/rat [F(2,21) ⫽ 0.89, P ⬎ 0.05], in the absence of significant day effect [F(7,147) ⫽ 1.82, P ⬎ 0.05] or of treatment-day interaction. The analysis of variance revealed no significant difference in ethanol intake of the different experimental groups of rats either during the pre- or the post-treatment period. The same doses of the ORL1 antagonist did not affect food or water intake. When [Nphe1]NC(1–13)NH2, 66 g/rat, was administered together with NC(1–17)NH2, 1000 ng/rat, the antagonist blocked the effect of NC(1–17)NH2 on ethanol intake. The overall analysis of variance showed a significant treatment effect [F(2,21) ⫽ 5.85, P ⬍ 0.01] and a significant day effect [F(7,147) ⫽ 3.22, P ⬍ 0.01], but a not significant treatment-day interaction. Ethanol intake was significantly
120
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
Fig. 1. Thirty-min ethanol intake (Upper Panel) and food intake (Lower Panel) in msP rats before (Pre), during (Treat) and after (Post) subchronic treatment with ICV injections of NC (250, 500 or 1000 ng/rat) or isotonic saline (Veh). Values represent the mean ⫾ SEM of 8 –9 subjects. Difference from controls: *P ⬍ 0.05; ** P ⬍ 0.01; where not indicated difference from controls was not statistically significant.
reduced by NC(1–17)NH2, [F(1,14) ⫽ 9.12, P ⬍ 0.01]; in rats pretreated with the antagonist the same dose of NC(1– 17)NH2 did not significantly modify ethanol intake in comparison to controls, which received two ICV injections of isotonic saline [F(1,14) ⫽ 2.68, P ⬎ 0.05] (Fig. 4B). The
analysis of variance revealed no significant difference in the intake of the different experimental groups of rats either during the pre- or the post-treatment period. Again, food and water intake were not significantly modified (data not shown).
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
121
Fig. 2. Thirty-min ethanol intake (Upper Panel) and food intake (Lower Panel) in msP rats before (Pre), during (Treat) and after (Post) subchronic treatment with ICV injections of NC(1–17)NH2 (500 or 1000 ng/rat) or isotonic saline (Veh). Values represent the mean ⫾ SEM of 8 subjects. Difference from controls: *P ⬍ 0.05; ** P ⬍ 0.01; where not indicated difference from controls was not statistically significant.
4. Discussion The results of the present study confirm that subchronic administration of NC reduces ethanol intake in alcoholpreferring rats, as shown by our previous study [9]. The effect is evoked at doses as low as 500, 1000 ng/rat, corresponding to 210 and 420 pmol/rat. The observed reduction in ethanol intake is reversible, as the intake promptly came
back to the baseline pretreatment levels after the end of treatment. The effect on ethanol intake of the heptadecapeptide amide, NC(1–17)NH2, was similar in intensity to that of NC, however, NC(1–17)NH2 was slightly less potent than NC, since it produced a statistically significant reduction of ethanol intake at 1000, but not 500 ng/rat. Because these peptides have almost identical molecular weight, NC(1–
122
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
Fig. 3. Thirty-min ethanol intake in msP rats before (Pre), during (Treat) and after (Post) subchronic treatment with ICV injections of NC(1–13)NH2 (500, 1000 or 2000 ng/rat) (Upper Panel) or of NC(1–12)NH2 (2000 or 4000 ng/rat) (Lower Panel). Values represent the mean ⫾ SEM of 8 –10 subjects for NC(1–13)NH2 and of 8 –9 subjects for NC(1–12)NH2. Difference from controls: *P ⬍ 0.05; ** P ⬍ 0.01; where not indicated difference from controls was not statistically significant.
17)NH2 was less potent also on a molar basis. The tridecapeptide, NC(1–13)NH2, induced a statistically significant, but less pronounced attenuation of ethanol intake only at 2000 ng/rat(1024 pmol/rat). Thus, NC(1–13)NH2 was markedly less potent than the parent compound. Lastly, the dodecapeptide NC(1–12)NH2 was ineffective at ICV doses up to 4000 ng/rat, that is up to 2339 pmol/rat. Thus, the tridecapeptide is apparently crucial for the effect of NC on ethanol consumption. This finding is similar to those re-
ported by our group for the pharmacological characterization of the NC receptor controlling food intake [38,39], as well as to those obtained in a variety of other in vitro and in vivo tests [see for review 4,5]. The results obtained with both NC and its partial sequences clearly show that changes in ethanol intake occurred in the absence of changes in other aspects of the ingestive behavior. Thus, the doses of NC and its partial sequences that reduced ethanol intake did not elicit the well
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
123
Fig. 4. Thirty-min ethanol intake in msP rats before (Pre), during (Treat) and after (Post) subchronic treatment with ICV injections of [Nphe1]NC(1–13)NH2 (66 or 99 g/rat) or isotonic saline (Veh) (Upper Panel) or of [Nphe1[NC(1–13)NH2 (66 g/rat) or isotonic saline ⫹ NC(1–17)NH2(1000 ng/rat) or isotonic saline (Lower Panel). Values represent the mean ⫾ SEM of 8 subjects. Difference from controls: *P ⬍ 0.05; ** P ⬍ 0.01; where not indicated difference from controls was not statistically significant.
known hyperphagic effect of NC [22,23,38,39,40,47], that appears to be mediated by the same ORL1 receptor. The threshold ICV dose for the hyperphagic effect of NC or NC(1–17)NH2 in msP rats is of about 3000 ng/rat. Also water intake was not significantly modified by NC or its partial sequences at the doses that reduced ethanol intake. Indeed, msP rats usually ingest very low amounts of water; moreover, having free access to water during the day, a lower intake of 10% ethanol in the 30-min test does not
necessarily require simultaneous increase in water intake for homeostatic reasons. Moreover, the effect on ethanol intake of these peptides was observed in conditions in which the rat gross behavior was not modified. Devine et al. [12] reported that hypolocomotion can be evoked by NC at doses of at least 1000 ng/rat, and that tolerance rapidly develops to this effect. As a matter of fact, in the present study slight hypolocomotion was observed in rats treated for the first time with 1000
124
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125
ng/rat of NC or of NC(1–17)NH2, but it was not detected any more since the second day of treatment. Lastly, it is noteworthy that (with the exception of the anxiolytic and antistress effects of NC, which occurs at low doses similar to those required to reduce ethanol intake [8,21,25]) the other effects of NC, including the antianalgesic, the hyperphagic and the amnestic ones, are evoked in rats only at higher doses [5]. Taken together with the results of the present study, all these findings indicate that the effects of NC and its partial sequences on ethanol intake are highly selective. [Nphe1]NC(1–13)NH2 did not modify ethanol intake per se, but abolished the effect of NC(1–17)NH2 on ethanol intake. These findings confirm that [Nphe1]NC(1–13)NH2 is a pure antagonist, as previously reported for other tests [for review 4,5], and indicate that it may represent a valuable pharmacological tool for future studies aimed at evaluating the role of the NC/ORL1 receptor system in the control of ethanol intake. Moreover, the results obtained with this antagonist support the view that classic ORL1 receptors mediate the effect of NC and NC analogs on ethanol intake. Apparently, the results obtained with [Nphe1]NC(1– 13)NH2 suggest that ORL1 receptor signaling may have no tonic control over ethanol intake. However, taking into account that this antagonist has a rather low pA2 (about 6.3), other more potent antagonists should be tested to confirm this hypothesis. In conclusion, the present results confirm that agonists at the ORL1 receptor are able to reduce ethanol intake in alcohol-preferring rats, suggesting that the NC/ORL1 receptor system may represent an interesting target for treatment of alcohol abuse.
Acknowledgments The study was supported by Grants from the University of Camerino, Italy and from MURST, Italy (Cofin99).
References [1] Agabio R, Cortis G, Fadda F, Gessa GL, Lobina C, Reali R, Colombo G. Circadian drinking pattern of Sardinian alcohol-preferring rats. Alcohol Alcohol 1996;31:385– 8. [2] Angeletti S, Panocka I, Ciccocioppo R, Gini L, Massi M. Effect of nociceptin (NC) on morphine-induced conditioned place preference (CPP) in rats. Regul Pept 1999;80:122. [3] Anton B, Fein J, To T, Li X, Silberstein L, Evans CJ. Immunohistochemical localization of ORL-1 in the central nervous system of the rat. J Comp Neurol 1996;368:229 –51. [4] Calo’ G, Bigoni R, Rizzi A, Guerrini R, Salvadori S, Regoli D. Nociceptin/orphanin FQ receptor ligands. Peptides 2000;21:935– 47. [5] Calo’ G, Guerrini R, Rizzi A, Salvadori S, Regoli D. Pharmacology of nociceptin and its receptor: a novel therapeutic target. Br J Pharmacol 2000;129:1261– 83. [6] Ciccocioppo R, Angeletti S, Panocka I, Massi M. Nociceptin/orphanin FQ, and drugs of abuse. Peptides 2000;21:1071– 80.
[7] Ciccocioppo R, Angeletti S, Sanna PP, Weiss F, Massi M. Effect of nociceptin/orphanin FQ on the rewarding properties of morphine. Eur J Pharmacol 2000;404:153–59. [8] Ciccocioppo R, Martin-Fardon R, Weiss F, Massi M (2001) Nociceptin/orphanin FQ inhibits stress-, and CRF-induced anorexia in rats. NeuroReport 2001;12:1145–9. [9] Ciccocioppo R, Panocka I, Polidori C, Regoli D, Massi M. Effect of nociceptin on alcohol intake in alcohol-preferring rats. Psychopharmacology 1999;141:220 – 4. [10] Colombo G. Ethanol drinking behaviour in Sardinian alcohol-preferring rats. Alcohol Alcohol 1997;32:443–53. [11] Darland T, Heinricher MM, Grandy DK. Orphanin FQ/nociceptin: a role in pain, and analgesia, but so much more. Trends Neurosci 1998;21:215–21. [12] Devine DP, Taylor L, Reinscheid RK, Monsma FJ Jr, Civelli O, Akil H. Rats rapidly develop tolerance to the locomotor-inhibiting effects of the novel neuropeptide orphanin FQ. Neurochem Res 1996;21: 1387–96. [13] Di Giannuario A, Pieretti S. Nociceptin differentially affects morphine-induced dopamine release from the nucleus accumbens and nucleus caudate in rats. Peptides 2000;21:1125–30. [14] Florin S, Leroux-Nicollet I, Meunier JC, Costentin J. Autoradiographic localization of [3H]nociceptin binding sites from telencephalic to mesencephalic regions of the mouse brain. Neurosci Lett 1997;230:33– 6. [15] Florin S, Meunier JC, Costentin J. Autoradiographic localization of [3H]nociceptin binding sites in the rat brain. Brain Res 2000;880:11– 16. [16] Hawes BE, Graziano MP, Lambert DG. Cellular actions of nociceptin: transduction mechanisms. Peptides 2000;21:961–7. [17] Henderson G, McKnight AT. The orphan opioid receptor and its endogenous ligand nociceptin/orphanin FQ. Trends Pharmacol Sci 1997;18:293–300. [18] Herz A. Endogenous opioid systems and alcohol addiction. Psychopharmacology 1997;129:99 –111. [19] Herz A. Opioid reward mechanisms: a key role in drug abuse? Can J Physiol Pharmacol 1998;76:252– 8. [20] Ikeda K, Watanabe M, Ichikawa T, Kobayashi T, Yano R, Kumanishi T. Distribution of pre-pro-nociceptin/orphanin FQ mRNA and its receptor mRNA in developing and adult mouse central nervous systems. J Comp Neurol 1998;399:139 –51. [21] Jenck F, Moreau JL, Martin JR, Kilpatrick GJ, Reinscheid RK, Monsma FJ Jr, Nothacker HP, Civelli O. Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress. Proc Natl Acad Sci USA 1997;94:14854 –9. [22] Leventhal L, Mathis JP, Rossi GC, Pasternak GW, Bodnar RJ. Orphan opioid receptor antisense probes block orphanin FQ-induced hyperphagia. Eur J Pharmacol 1998;349:R1–3. [23] Leventhal L, Stevens LB, Rossi GC, Pasternak GW, Bodnar RJ. Antisense mapping of the MOR-1 opioid receptor clone: modulation of hyperphagia induced by DAMGO. J Pharmacol Exp Ther 1997; 282:1402–7. [24] Lobina C, Agabio R, Diaz G, Fa M, Fadda F, Gessa GL, Reali R, Colombo G. Constant absolute ethanol intake by Sardinian alcoholpreferring rats independent of ethanol concentrations. Alcohol Alcohol 1997;32:19 –22. [25] Martin-Fardon R, Ciccocioppo R, Massi M, Weiss F. Nociceptin prevents stress-induced ethanol- but not cocaine-seeking behavior in rats. NeuroReport 2000;9:1939 – 43. [26] Massi M, Panocka I, Angeletti S, Ciccocioppo R, Polidori C. Nociceptin (NC) attenuates ethanol intake, and ethanol-induced conditioned place preference (CPP) in alcohol-preferring rats. Brain Res 1999;848:A41–A42. [27] Meng F, Taylor LP, Hoversten MT, Ueda Y, Ardati A, Reinscheid RK, Monsma FJ, Watson SJ, Civelli O, Akil H. Moving from the orphanin FQ receptor to an opioid receptor using four point mutations. J Biol Chem 1996;271:32016 –20.
R. Ciccocioppo et al. / Peptides 23 (2002) 117–125 [28] Meunier JC. Nociceptin/orphanin FQ, and the opioid receptor-like ORL1 receptor. Eur J Pharmacol 1997;340:1–15. [29] Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B, Mazarguil H, Vassart G, Parmentier M, Costentin J. Isolation and structure of the endogenous agonist of opioid receptor- like ORL1 receptor. Nature 1995;377:532–5. [30] Meunier JC, Mouledous L, Topham CM. The nociceptin/orphanin FQ-ORL1 receptor system. Peptides 2000;21:893–900. [31] Mogil JS, Grisel JE, Reinscheid RK, Civelli O, Belknap JK, Grandy DK. Orphanin FQ is a functional anti-opioid peptide. Neuroscience 1996;75:333–7. [32] Mogil JS, Grisel JE, Zhangs G, Belknap JK, Grandy DK. Functional antagonism of -, ␦- and -opioid antinociception by orphanin FQ. Neurosci Lett 1996;214:131– 4. [33] Murphy NP, Lee Y, Maidment NT. Orphanin FQ/nociceptin blocks acquisition of morphine place preference. Brain Res 1999;832:168–70. [34] Murphy NP, Maidment NT. Orphanin FQ/nociceptin modulation of mesolimbic dopamine transmission determined by microdialysis. J Neurochem 1999;73:179 – 86. [35] Neal CR, Jr., Mansour A, Reinscheid R, Nothacker HP, Civelli O, Watson SJ, Jr. Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J Comp Neurol 1999;406:503– 47. [36] Nothacker HP, Reinscheid RK, Mansour A, Henningsen RA, Ardati A, Monsma FJ, Jr., Watson SJ, Civelli O. Primary structure and tissue distribution of the orphanin FQ precursor. Proc Natl Acad Sci USA 1996;93:8677– 82. [37] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 2nd edition. North Ryde, N.S.W., Australia: Academic Press, 1986. [38] Polidori C, Calo’ G, Ciccocioppo R, Guerrini R, Regoli D, Massi M. Pharmacological characterization of the nociceptin receptor mediating hyperphagia: identification of a selective antagonist. Psychopharmacology 2009;148:430 –7.
125
[39] Polidori C, de Caro G, Massi M. The hyperphagic effect of nociceptin/orphanin FQ in rats. Peptides 2000;21:1051– 62. [40] Pomonis JD, Billington CJ, Levine AS. Orphanin FQ. agonist of orphan opioid receptor ORL1, stimulates feeding in rats. Neuroreport 1996;8:369 –71. [41] Reinscheid RK, Ardati A, Monsma FJ, Jr., Civelli O. Structureactivity relationship studies on the novel neuropeptide orphanin FQ. J Biol Chem 1996;271:14163– 8. [42] Reinscheid RK, Higelin J, Henningsen RA, Monsma FJ, Jr., Civelli O. Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities. J Biol Chem 1998;273:1490 –5. [43] Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ, Jr., Civelli O. Orphanin FQ: a neuropeptide that activates an opioid-like G proteincoupled receptor. Science 1995;270:792– 4. [44] Reinscheid RK, Nothacker HP, Civelli O. The orphanin FQ/nociceptin gene: structure, tissue distribution of expression and functional implications obtained from knockout mice. Peptides 2000;21:901– 6. [45] Schulz S, Schreff M, Nuss D, Gramsch C, Hollt V. Nociceptin/ orphanin FQ, and opioid peptides show overlapping distribution but not co-localization in pain-modulatory brain regions. NeuroReport 1996;7:3021–5. [46] Sim LJ, Childers SR. Anatomical distribution of mu, delta, and kappa opioid- and nociceptin/orphanin FQ-stimulated [35S]guanylyl-5⬘-O(␥-thio)-triphosphate binding in guinea pig brain. J Comp Neurol 1997;386:562–72. [47] Stratford TR, Holahan MR, Kelley AE. Injections of nociceptin into nucleus accumbens shell or ventromedial hypothalamic nucleus increase food intake. Neuroreport 1997;8:423– 6. [48] Vaughan CW, Christie MJ. Increase by the ORL1 receptor (opioid receptor-like1) ligand, nociceptin, of inwardly rectifying K⫹ conductance in dorsal raphe nucleus neurons. Br J Pharmacol 1996;117: 1609 –11.