Mu-0Pio:Id Receptor Is Involved in P Endorphin- Induced Feeding in Goldfish

Home , Naloxonazine

Peptides, Vol. 17, No. 3, pp. 421-424, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781196 $15.00 + .@I PIISO196-9781(96)00006-X Mu-0pio:id Receptor Is Involved in P_Endorphin- Induced Feeding in Goldfish

NURIA DE PEDRO,’ MARiA VIRTUDES &PEDES, MA&A JESrjS DELGADO AND MERCEDES ALONSO-BEDATE

Departamento de Biologia Animal II (Fisiologfa Animal), Facultad de Ciencias Biolbgicas, Universidad Complutense, 28040 Madrid, Spain

Received 7 September 1995

DE PEDRO, N., M. V. Cl%PEDES, M. J. DELGADO AND M. ALONSO-BEDATE. Mu-opioid receptor is involved in p- endorphin-inducea'feeding in goldjsh. PEPTIDES 17( 3) 421-424, 1996.-The present study evaluated the central effects of selective opioid receptor subtype agonists and antagonists on food intake in satiated goldfish. Significant increases in feeding behavior occurred .m goldfish injected with P-endorphin, the kappa agonist, U-50488, the delta agonist, [D-Pen’,D-Pen5]enkephalin (DPEN), and the mu agonist, [o-Ala’,N-Me-Phe4,Gly5-ollenkephalin (DAMGO). On the other hand, the different receptor antagonists used: nor-binaltorphamine (nor-BNI) for kappa, 7-benzidilidenenaltrexone (BNTX) for delta,, naltriben for delta,, @imaltrexamine (P-FNA) for mu, and naloxonazine for mu,, by themselves, did not modify ingestion or slightly reduced it. The feeding stimulation by P-endorphin was antagonized by P-FNA and naloxonazine, but not by nor-BNI, BNTX, or naltriben. These data indicate that the mu-opioid receptor is involved in the modulation of the feeding behavior in goldfish.

P-Endorphin Food intake Goldfish Opioid receptors Opioid antagonists Mu receptor Delta receptor Kappa receptor

THE role of the endogenous opioid system in the regulation of kappa receptor subtypes (34) makes it difficult to identify the ingestive behavior is we:11 established (5,22,25). The general opioid receptor subtype involved in the feeding regulation theme in these studies shows that opioid agonists increase food (8,22,25,26). intake, whereas opioid receptor antagonists decrease consump- Selective centrally acting antagonists of opioid receptor sub- tion (8,22,25,26). types have been developed in the last decade, including nor-bin- Most of these reports about mediation of feeding by opioids altorphamine (nor-BNI), a reversible kappa and particularly have been performed in mammals, and some experimental ap- kappa, antagonist (32)) 7-benzidilidenenaltxone (BNTX), a proaches have also been performed in birds ( 12,15) and even in delta, antagonist (33)) naltriben, a delta* antagonist (37)) p-fun- invertebrate species, such as slugs (20,39). Recently, studies car- altrexamine (P-FNA), an irreversible mu antagonist ( 3 1) ,and ried out in our laboratory demonstrated for the first time that an naloxonazine, an irreversible mu, antagonist (30). The use of opioid (P-endorphin) is involved in the feeding central regula- these antagonists has facilitated the understanding of the role of tion in teleosts (14). Particularly, we reported that ICV admin- different opioid receptor subtypes in feeding behavior in mam- istration of P-endorphin stimulates food intake in goldfish at 2 h mals. postinjection ( 14)) in aga:ement with a number of previous stud- The present experiments were conducted to measure the ef- ies in rats, where this endogenous opioid increases consumption fects of different opioid agonists and antagonists on feeding in after central administration ( 19,22,24,25). goldfish, and mainly to identify the particular receptor sub- To date, opioid receptors have been classified into mu, delta, type(s) involved in @-endorphin-induced feeding in the same and kappa receptor subtypes (35,36,38). These subtypes have species. Thus, on one hand, the effects of ICV administration of been further divided into mu,, mul, delta,, delta,, kappa,, high-affinity, receptor-selective agonists and antagonists for mu, kappa,, and kappa, binding sites on the basis of competitive bind- delta, and kappa receptors on food intake were analyzed. On the ing and pharmacological studies in mammals ( 16,29). other hand, we evaluated the ability of these antagonists to block The involvement of endogenous opioid peptides on feeding the stimulatory effect of P-endorphin on feeding. has been demonstrated by the use of opioid receptors blockers. METHOD For example, the nonselective opioid antagonist naloxone usually attenuated food intake in a wide range of feeding situations and Animals species (14,15,17,22,25,;!6,40). Nevertheless, the failure of this Goldfish (Carassius auratus, 4.52 k 1.41 g b.wt.; Interzoo, general opioid antagonist to distinguish among mu, delta, and Madrid) were maintained under natural photoperiod in 100-l

’ Requests for reprints should be addressed to Nuria de Pedro.

421 422 DE PEDRO ET AL.

two ICV injections separated by 30 min. Treatment 1: two injections of 1 ~1 saline; treatment 2: 1 ~1 saline injection followed Osal t sal by 1 pg injection of delta agonist (DPEN); treatment 3: 1 ~1 Isal t U-50488 saline injection followed by P-endorphin ( 1 pg); treatment 4: 1 s 75. ~1 saline injection followed by 5 pg injection of delta, antagonist m qsal t D-E .z 50- (BNTX); treatment 5: BNTX (5 pg) injection followed by fl- endorphin ( 1 ,ug) injection; treatment 6: saline injection followed qsal t nor-BNI x 25. by 5 pg injection of delta, antagonist (naltriben) ; and treatment ILO qnnor-BNI t B-E 7: naltriben (5 pg) injection followed by P-endorphin ( 1 pg) O- injection. 1 2 3 4 5 Involvement of mu receptors in the feeding regulation. Fish were divided in seven groups (n = 6-71group) and ICV injected (two injections separated by 30 min). Treatment 1: two sequen- FIG. 1. Food intake (mg) after ICV administration of 1 ~1 saline alone (treatment 1); 1 pg U-50488 (treatment 2); 1 pg P-endorphin (treatment tial injections of 1 ~1 saline; treatment 2: 1 ~1 saline injection 3); 5 /lg nor-BNI (treatment 4); or both 5 pg nor-BNI + 1 pg ,&endorphin followed by 1 pg injection of mu agonist (DAMGO) ; treatment (treatment 5) at 2 h postinjection in goldfish (Carussius auratus), n = 3: 1 ~1 saline injection followed by p-endorphin ( 1 pg); treat- 7-g/group. Data are expressed as mean ? SEM. *p < 0.05, **p < 0.01 ment 4: 5 pg injection of mu (mu, + mu*) antagonist (fl-FNA) compared to control group (Duncan test). 22 h prior to 1 ~1 saline injection; treatment 5: P-FNA (5 pg) injection 22 h prior to the /?-endorphin ( 1 pg) injection; treatment 6: saline injection followed by the mu, antagonist naloxonazine aquaria, which received a constant flow of aerated and filtered (5 pg) injection; and treatment 7: naloxonazine (5 pg) injection water at 21 t 2°C. Fish were fed once daily with commercially followed by /?-endorphin (1 pg) injection. The 22-h interval prepared fish pellets (SeraBiogran) at ration of 1% b.wt. at guarantees an irreversible blockade of mu receptors (3,3 1). lOOO- 1100 h. Animals were acclimated to these conditions for at least 2 weeks prior to the experimental use. Statistical Analysis Fish were anesthetized in water containing tricaine methane- sulphonate (MS-222, l/10,000) and after loss of equilibrium Data were analyzed by an ANOVA test followed by Duncan’s they were weighed and ICV injected at 1000-1100 h. Fish re- multiple range test for multigroup comparisons. A probability covered equilibrium and normal activity in anesthetic-free water level of p < 0.05 was considered statistically significant. within l-2 min after the treatments. Daily food ration was available 1 h before the injections to be sure that fish were satiated at RESULTS the time of the feeding test. A detailed description of the feeding protocol used to evaluate food intake in goldfish has been re- Figure 1 shows the effects of agonist and antagonist for kappa ported elsewhere (13). Briefly, individual fish received pre- receptors on feeding in goldfish, F(4,31) = 8.1417,~ < 0.0001. weighed food in excess (7% b.wt.) and food intake (FI) was The ICV administration of both P-endorphin and U-50488 measured at 2 h after the last injection as FI = W, - (W f X F), (kappa agonist) significantly increased food intake (p < 0.01 where W i is the initial dry food weight, W f is the remaining dry and p < 0.05, respectively) in goldfish (Fig. 1) . The same figure food weight, and a correction factor, which represents the reduc- shows that the kappa antagonist, nor-BNI, by itself did not alter tion in food weight after remaining 2 h in the aquaria. feeding behavior in goldfish, and it was not able to counteract the @-endorphin stimulatory action on food consumption. Drugs and Surgery Figure 2 summarizes the role of delta receptors as mediators of /?-endorphin effect on feeding in goldfish, F( 6,42) = 5.12 11, Human P-endorphin, DAMGO, DPEN (Sigma); nor-BNI, p < 0.0005. As it can be observed, P-endorphin (p < 0.01) and BNTX, P-FNA, naltriben, U-50488 (RBI), and naloxonazine the delta agonist DPEN (p < 0.05) stimulated the food intake at (ICN) were dissolved in teleost saline (20 mg Na2C03 per 100 ml of 0.6% NaCl) alone or with methanol (10%) in the case of BNTX, P-FNA, naloxonazine, and naltriben. All drugs were ICV injected using a 0.3~mm Microlance needle connected to a 5~1 Hamilton microsyringe with a 18 Venocath cannula. The ICV injection procedure and the accuracy of injection placement into qsal t O-E the ventricular regions of the fish brain was previously estab- 2 75 lished (13). (u Hsal t BNTX .z 50 HBNTX t R-E Experimental Design u 0” 25 asal t naltriben Involvement of kappa receptors in the feeding regulation. lJ_ Fish were divided in five groups (n = 7-8/group), which re- qnaltriben t B-E ceived two ICV injections separated by 30 min. Treatment 1: two ‘1234567 injections of 1 ~1 saline; treatment 2: 1 pl saline injection fol- Treatments lowed by 1 pg injection of kappa agonist (U-50488) ; treatment FIG. 2. Food intake (mg) after ICV administration of 1 ~1 saline alone 3: 1 pl saline injection followed by P-endorphin ( 1 ,ug); treat- (treatment 1); 1 pg DPEN (treatment 2); 1 pg /?-endorphin (treatment ment 4: 1 ~1 saline injection followed by 5 pg injection of kappa 3); 5 pg BNTX (treatment 4); both 5 pg BNTX + 1 Kg fl-endorphin antagonist (nor-BNI) ; treatment 5: nor-BNI (5 kg) injection fol- (treatment 5); 5 fig naltriben (treatment 6); or both 5 pg naltiben + 1 lowed by P-endorphin ( 1 pg) injection. 1.18p-endorphin (treatment 7) at 2 h postinjection in goldfish (Curussius Involvement of delta receptors in the feeding regulation. Fish aurarus), n = 7-8/group. Data are expressed as mean 2 SEM. *p < were divided in seven groups (n = 7-8/group), which received 0.05, **p < 0.01 compared to control group (Duncan test). OPIOID ANTAGONISTS AND FEEDING 423

administration of nor-BNI (kappa antagonist), BNTX (delta, an- - 1251Zj Usal t sal tagonist), naltriben ( delta2 antagonist), P-FNA (mu antagonist), Isal t DAMGO or naloxonazine (mu, antagonist), by themselves, did not affect feeding behavior in goldfish. In fact, we found food intake values 75 qsal t D-E similar or slightly lower than that observed in the control groups. z i msal t fl-FNA These results are consistent with previous reports in mammals, E .- 50 where specific antagonists for the different opioid receptor Hfl-FNAt D-E -0 subtypes do not produce significantly modifications of food

8 25 Osal t naloxonazine intake or reduce ingestion, as it has been shown with the LL kappa antagonist nor-BNI (2,3,7-9,11,21,23,28), the delta, an- b Bnaloxonazinet O-E O 1 6 7 tagonist DALCE (4,7-9,21), the delta* antagonist naltrindol ,I (8,9,11,21,28), the mu antagonist P-FNA (3,7-9,21), and the Treatments mu, antagonist naloxonazine (7 -9,21). FIG. 3. Food intake (mg) after ICV administration of 1 ~1 saline alone The present study provides new evidence for a central role of (treatment 1); 1 pg DAMGO (treatment 2); 1 fig P-endorphin (treatment opioids on feeding regulation in poikilotherms, particularly in 3); 5 pg P-FNA (treatment 4); both 5 pg p-FNA + 1 pg P-endorphin teleost fish. Our results about the effect of opioid agonists and (treatment 5); 5 pg naloxonazine (treatment 6); or both 5 pg naloxona- antagonists on food intake in goldfish support the general agree- zine + 1 pg P-endorphin (treatment 7) at 2 h postinjection in goldfish ment in homeotherms that administration of opioid agonists in (Cur4ssius aurutus), n = &7/group. Data are expressed as mean t the central nervous system (CNS) stimulates food intake, SEM. **p < 0.01 compared. to control group (Duncan test). whereas opioid receptor antagonists reduce feeding or do not produce significant modifications in feeding (8,9,21,22,25, 2 h postinjection. After administration of either delta, (BNTX) 26,28). or delta2 (naltriben) antagonists no significant differences on Central injection of kappa (nor-BNI) and delta (BNTX, delta, naltriben, delta2) antagonists do not counteract p-endor- feeding were detected. These same antagonists immediately ad- ; ministered before /3-endorphin injection did not counteract the phin-induced feeding behavior in goldfish. In contrast, this food stimulatory action of the neuropeptide on feeding behavior in intake stimulation by the opioid P-endorphin is blocked by both goldfish. P-FNA (general mu antagonist) and naloxonazine (mu* antag- The effects of mu agonist (DAMGO) and antagonists (p- onist), suggesting that this stimulation of feeding is mediated via FNA, mu, + mu*; naloxonazine, mul) on food consumption in mu-opioidergic receptors in fish, particularly through the mu, goldfish are presented in Fig. 3, F( 6,40) = 17.7 169, p < 0.0001. subtype. However, the blockade by /3-FNA (mu1 + mu*) could Feeding was significantly increased by both P-endorphin (p < indicate an antagonism of either mu, or mu2, or even both re- 0.01) and DAMGO (p <: 0.05) compared to the control group ceptor subtypes. Thus, a possible role of the muz opioid receptor at 2 h postinjection. None of the antagonists tested significantly in the central regulation of feeding by P-endorphin in goldfish modified food intake by themselves. On the other hand, injection cannot be ruled out. Unfortunately, the lack of a high-affinity, of these antagonists, either P-FNA or naloxonazine, before fl- selective antagonist commercially available for mu2 receptors endorphin administration (treatment 7) totally blocked the /?- does not allow us to demonstrate the possible role of mu2 opioid endorphin-induced feeding increase. receptors as mediators of ,&endorphin-induced feeding in gold- There were no significant differences in food intake increases fish. after the treatment with any of the used agonists, F(2, 17) = On one hand, P-endorphin is an endogenous mu and delta 0.1968. receptor ligand ( 1). On the other hand, binding studies using selective radioligands have identified mu and kappa, receptors in DISCUSSION goldfish brain, but neither delta nor kappa1 sites can be detected ( 10). These studies, together with the present results on the in- The ICV administration of P-endorphin stimulated food in- ability of kappa and delta antagonists to counteract the feeding take in goldfish at 2 h postinjection, similar to results previously increase by /3-endorphin, lead us to discard kappa and delta re- obtained in our laboratory ( 14)) and in agreement with previous ceptors as mediators of this P-endorphin effect in goldfish. reports in mammals ( 19,22,24,25). In summary, the results presented in this article suggest that The opioid agonists used in the present study [kappa (U- P-endorphin-increased feeding in goldfish is mediated via mu 59488), delta (DPEN), .and mu (DAMGO)] increased feeding receptors, involving, at least, the mu, opioid receptor subtype. in satiated goldfish at 2 h postinjection, as it has been previously demonstrated in mammals (6,18,27). The magnitude of the stim- ACKNOWLEDGEMENTS ulatory effect observed was similar for the three agonists. Concerning the opioid antagonists, our results showed that, at This research was supported by DGICYT (PB 940236) and FPI grant the doses chosen and under our experimental conditions, the ICV from Ministry of Education and Science to N. de Pedro.

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