Neuropeptide Y receptor(s) mediating feeding in the rat: characterization with antagonists

Peptides 21 (2000) 29 –35

Neuropeptide Y receptor(s) mediating feeding in the rat: characterization with antagonists Carlo Polidoria,*, Roberto Ciccocioppoa, Domenico Regolib, Maurizio Massia a

Department of Pharmacological Sciences and Experimental Medicine, University of Camerino, 62032 Camerino, (MC) Italy b Department of Experimental and Clinical Medicine, University of Ferrara, 44100 Ferrara (FE), Italy Received 11 May 1999; accepted 27 September 1999

Abstract The present study evaluated the effect of the neuropeptide Y (NPY) Y1 receptor antagonists BIBO 3304 and SR 120562A and of the Y5 receptor antagonists JCF 104, JCF 109, and CGP 71683A on feeding induced either by NPY or food deprivation. In a preliminary experiment, NPY was injected into the third cerebroventricle (3V) at doses of 0.07, 0.15, 0.3, or 0.6 nmol/rat. The dose of 0.3 nmol/rat, which produced a cumulative 2-h food intake of 11.2 ⫾ 1.9 g/kg body weight, was chosen for the following experiments. The antagonists were injected in the 3V 1 min before NPY. The Y1 receptor antagonist BIBO 3304 significantly inhibited NPY-induced feeding at doses of 1 or 10 nmol/rat. The Y1 receptor antagonist SR 120562A, at the dose of 10 but not of 1 nmol/rat, significantly reduced the hyperphagic effect of NPY, 0.3 nmol/rat. The Y5 receptor antagonists JCF 104 and JCF 109 (1 or 10 nmol/rat) and CGP 71683A (10 or 100 nmol/rat) did not significantly modify the effect of NPY, 0.3 nmol/rat. However, JCF 104 (10 nmol/rat) and CGP 71683A (100 nmol/rat), but not JCF 109 (10 nmol/rat), significantly reduced food intake during the interval from 2 to 4 h after injection of a higher dose, 0.6 nmol/rat, of NPY. Feeding induced by 16 h of food deprivation was significantly reduced by the Y1 receptor antagonist BIBO 3304 (10 nmol/rat), but it was not significantly modified by the same dose of SR 120562A or JCF 104. These findings support the idea that the hyperphagic effect of NPY is mainly mediated by Y1 receptors. The results obtained with JCF 104 and CGP 71683A suggest that Y5 receptors may have a modulatory role in the maintenance of feeding induced by rather high doses of NPY after the main initial feeding response. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Neuropeptide Y; Neuropeptide Y antagonists; Food intake; BIBO 3304; JCF 104; JCF 109; SR 120562A; CGP 71683A; Nociceptin

1. Introduction Neuropeptide Y (NPY), a 36-amino acid neuropeptide isolated from porcine brain [32,33], is considered to be the most abundant and widely distributed neuropeptide present in the mammalian central and peripheral nervous system [2]. The hypothalamus and in particular the paraventricular nucleus (PVN) show high levels of NPY [1]; the hypothalamic levels of NPY have been reported to increase in fasting animals and to return to normal levels upon feeding [16,27]. Since the first reports of the hyperphagic effect of NPY [7,22], different investigators have striven to establish which NPY receptor subtype is involved in feeding behavior. * Corresponding author. Tel.: ⫹39-0737-403307; fax: ⫹39-0737630618. E-mail address: [email protected] (C. Polidori)

A decade ago, two NPY receptor subtypes, Y1 and Y2, were pharmacologically characterized [34]; later on, a Y3 receptor was proposed, but not cloned [35]. A fourth receptor (Y4) was cloned and characterized by its high affinity for the rat pancreatic polypeptide (PP) [3]. More recently, two additional NPY receptors have been cloned [11]; the first, described as Y5 receptor, has been pharmacologically characterized. The second, Y6, or PP2, has been cloned from the mouse, but it is inactive in man and absent in the rat [23]. Initially, it was proposed that the NPY Y1 receptor mediates the hyperphagic effect of NPY in rodents, because agonists selective for this receptor stimulate feeding after central administration [17]. The Y1 receptor antagonists 1229U91 and BIBO 3304 have been reported to inhibit NPY-induced food intake in freely feeding and drinking rats [19,36]. More recently, several authors have proposed that the hyperphagic effect of NPY may be mediated by Y5 recep-

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C. Polidori et al. / Peptides 21 (2000) 29 –35

tors [12–14]. Wyss et al. [37] observed that the orexigenic activity of a variety of NPY receptor agonists correlates with affinity for Y5, but not with affinity for Y1 receptors. Also, a study that used antisense oligonucleotides supports the involvement of Y5 receptors in NPY-induced feeding [28]. However, several results are inconsistent with this hypothesis: [D-Trp32]-NPY has been shown to be an agonist at Y5 receptors, but it has been reported to antagonize NPY-induced feeding, and the human PP weakly stimulates food intake, although it is a full agonist at the rat Y5 receptor in vitro [12,24]. Moreover, other findings suggest that NPY-induced feeding may involve either activation of more than one receptor subtype or of a novel ‘Y1-like’ receptor [24,30]. In this regard, the Y1 receptor antagonist 1229U91 has been reported to ‘hardly affect’ feeding induced by the Y5 receptor agonist bovine PP [18], and the Y1 agonist [Pro34]NPY has been reported to induce approximately 50% of the food intake observed after NPY injection [24]. The definitive assignment of a role of any NPY receptor in feeding behavior probably has been hampered by the lack of specific Y5 antagonists and by the limited number of Y1 receptor antagonists so far tested. Several selective Y1 or Y5 receptor antagonists are now available. CGP 71683A, a selective Y5 receptor antagonist, has been described by Criscione et al. [8]; two other Y5 receptor antagonists, indicated as JCF 104 [10] and JCF 109 [15], have been described in two patents by Synaptic Pharmaceutical Corp., Paramus, New Jersey, USA. Another Y1 antagonist has been reported by Serradeil–Le Gal et al. [29]. These compounds, in addition to the Y1 receptor antagonist BIBO 3304, have been used in the present study to evaluate the involvement of NPY receptor subtypes in feeding behavior in rats.

2. Methods 2.1. Animals Male Wistar rats weighing 300 –350 g were used. They were individually housed in a room with a 12:12 h light/ dark cycle, temperature of 20 –22°C and humidity of 45– 55%. They were offered free access to food pellets (4RF18, Mucedola, Settimo Milan, Italy) and tap water. 2.2. Drugs NPY was purchased from Bachem (Torrace, CA, USA); BIBO 3304, (R)-N-[[4-(aminocarbonyl)-phenyl]-methyl]N2-(diphenylacetyl)-argininamide trifluoroacetate, was a gift of Karl Thomae, Germany; CGP 71683A, [(4-{[(4amino-quinazolin-2-yl)amino]methyl}-cycloexyl)methyl] (naphthyl-sulphonyl)amine, was supplied by Dr K. G. Hofbauer, Novartis, Switzerland; JCF 104 and JCF 109 (two Synaptic products), were supplied by Dr J. L. Fauche`re,

Servier, France; SR 120562A was donated by Dr J. P. Maffran, Sanofi, France; nociceptin was obtained from Prof. S. Salvadori, Ferrara University, Ferrara, Italy. Nociceptin and NPY were dissolved in isotonic NaCl. The other compounds were dissolved in a solution prepared with equal parts of distilled water and dimethylsulfoxide (DMSO); in one experiment, CGP 71683A was dissolved in a vehicle containing 33% DMSO and 66% distilled water. 2.3. Surgery For intracranial surgery, rats were anesthetized by intramuscular (i.m.) injection of 100 to 150 ␮l/100 g body weight of a solution containing ketamine (86.2 mg/ml) and acepromazine (1.3 mg/ml). A guide cannula was stereotaxically implanted for injections into the third cerebroventricle (3V) and cemented to the skull. The following coordinates were used for the guide cannula: AP ⫽ 1 mm behind the bregma, L ⫽ 1 mm from the sagittal suture, V ⫽ 7.5 mm from the surface of the skull and with an angle of 10 degrees. Drug injections were made by means of a stainlesssteel injector 0.5 mm longer than the guide cannula, so that its tip protruded into the 3V. 2.4. Effect of NPY on food intake in freely feeding and drinking rats A group of freely feeding and drinking rats received a pulse intracerebroventricular (i.c.v.) injection (1 ␮l/rat) of either vehicle (isotonic saline) or NPY (0.07, 0.15, 0.3, or 0.6 nmol/rat). Their food and water intake were recorded at 30, 60, 120, and 240 min after the i.c.v. injection. This experiment was designed to define the hyperphagic doses of NPY to be used in the following experiments. 2.5. Effect of selective NPY receptor antagonists on NPYinduced food intake Freely feeding and drinking rats were used to test the effects of selective NPY receptor antagonists on food intake induced by ICV injection of either 0.3 nmol/rat or 0.6 nmol/rat of NPY. The following five NPY antagonists were tested against the hyperphagic effect of 0.3 nmol/rat of NPY: the doses of 1 or 10 nmol/rat were used with BIBO 3304, SR 120562A, JCF 104, and JCF 109, whereas the doses of 10 or 100 nmol/rat were used with CGP 71683A. The dose of 100 nmol/rat was used only for CGP 71683A, because the same dose of the other antagonists frequently induced alterations of the rats’ gross behavior. The i.c.v. injection of the antagonist (in 1 ␮l/rat) took place 1 min before NPY administration. Five groups of rats were used; according to a withinsubject design, each group received both vehicle (controls) and the different doses of each antagonist, at intervals of 3– 4 days. Food intake was recorded after 30, 60, 120, and 240 min.

C. Polidori et al. / Peptides 21 (2000) 29 –35

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Table 1 Cumulative food intake (g/kg) induced by ICV injections of NPY into the third ventricle of freely feeding and drinking rats Treatment (nmol/rat)

30 Min

60 Min

120 Min

240 Min

Vehicle NPY 0.07 NPY 0.15 NPY 0.3 NPY 0.6

0.0 ⫾ 0.0 1.6 ⫾ 0.6* 1.9 ⫾ 0.7* 4.9 ⫾ 1.3** 7.1 ⫾ 0.4**

0.0 ⫾ 0.0 2.1 ⫾ 0.8* 2.2 ⫾ 0.7* 8.1 ⫾ 1.6** 10.7 ⫾ 1.1**

0.9 ⫾ 0.6 4.2 ⫾ 1.2** 4.3 ⫾ 1.4** 11.2 ⫾ 1.9** 16.9 ⫾ 2.1**

4.8 ⫾ 1.3 6.5 ⫾ 1.4** 6.6 ⫾ 1.3** 13.9 ⫾ 1.8** 21.1 ⫾ 1.8**

Values are means ⫾ SEM of 14 subjects. Difference from controls: *P ⬍ 0.05; **P ⬍ 0.01; where not indicated, difference from controls was not statistically significant.

The highest doses of BIBO 3304, SR 120562A, JCF 104, JCF 109 (10 nmol/rat), and CGP 71683A (100 nmol/rat) were also tested (1 ␮l/rat) versus the effect of NPY, 0.6 nmol/rat. In one experiment, CGP 71683A (100 nmol/rat) was dissolved in 5 ␮l of a vehicle containing 33% DMSO and 66% water and infused into the 3V in 2 min, 1 h before injection of 0.6 nmol/rat of NPY. 2.6. Effect of selective NPY receptor antagonists on food intake induced by overnight food deprivation Three groups of overnight (6.00 p.m.–9.00 a.m.) food-, but not water-deprived rats were used. Before refeeding, the first group received i.c.v. injection of either vehicle or BIBO 3304 (1 or 10 nmol/rat), the second received either vehicle or SR 120562A (10 nmol/rat), and the third received vehicle or JCF 104 (10 nmol/rat). According to a within-subject design, each group received both vehicle and the dose of a single antagonist at interval of 4 –5 days. Food was presented 1 min later; food and water intake was measured 15, 30, 60, and 120 min afterward. The observation period was limited to 2 h, as feeding was almost complete in this time interval. 2.7. Effect of the selective NPY Y1 receptor antagonist BIBO 3304 on nociceptin-induced food intake Nociceptin is known to induce a potent hyperphagic effect in rats [25,31]. To evaluate the behavioral selectivity of the effect of BIBO 3304, the present experiment investigated the effect of BIBO 3304 on nociceptin-induced feeding. In a preliminary experiment, freely feeding and drinking rats were injected with nociceptin (0.8 or 1.7 nmol/rat) or vehicle (controls) into the 3V. The dose of 1.7 nmol/rat of nociceptin was then administered in the rats used as controls, after pretreatment either with BIBO 3304 (10 nmol/rat) or with vehicle, one min before nociceptin injection. Food and water intakes were recorded 30, 60, 120, and 240 min afterward. 2.8. Statistical analysis Data (g/kg) are reported as mean ⫾ SEM. Statistical analysis of data were performed by means of multifactorial

analysis of variance (ANOVA), with repeated measurements. Pairwise comparisons were made with the Dunnett’s test. Statistical significance was set at P ⬍ 0.05.

3. Results 3.1. Effect of NPY on food intake in freely feeding and drinking rats As shown in Table 1, the injection of NPY significantly increased food intake in freely feeding and drinking rats. The ANOVA revealed a statistically significant treatment effect (F(4,13) ⫽ 25.3; P ⬍ 0.001), time effect (F(3,39) ⫽ 69.7; P ⬍ 0.001), and treatment-time interaction (F(12,156) ⫽ 7.3; P ⬍ 0.001). The effect of NPY was statistically significant at 0.07 nmol/rat and higher doses. 3.2. Effect of selective NPY receptor antagonists on NPYinduced food intake BIBO 3304 markedly reduced the hyperphagic effect of NPY, 0.3 nmol/rat. The ANOVA revealed a statistically significant treatment effect both after 1 nmol/rat (F(1,6) ⫽ 9.6; P ⬍ 0.05) and 10 nmol/rat (F(1,8) ⫽ 26.9; P ⬍ 0.001; Fig. 1). BIBO 3304 (1 or 10 nmol/rat) slightly reduced food intake induced by NPY, 0.6 nmol/rat, but the effect was not statistically significant (Table 2). SR 120562A, 1 nmol/rat, did not significantly affect food intake induced by NPY (0.3 nmol/rat), whereas 10 nmol/rat significantly reduced it (F(1,5) ⫽ 30.6; P ⬍ 0.01; Fig. 2). SR 120562A (10 nmol/rat) did not significantly reduce food intake induced by NPY, 0.6 nmol/rat (F(1,5) ⫽ 0.1; P ⬎ 0.05; Table 2). As shown in Table 3, 10 nmol/rat JCF 104 did not reduce the orexigenic effect of NPY, 0.3 nmol/rat. The ANOVA revealed neither a significant treatment effect (F(1,4) ⫽ 1.4; P ⬎ 0.05), nor a significant treatment-time interaction. The same dose of JCF 104 did not significantly reduce food intake induced by NPY, 0.6 nmol/rat, in the first 2 h, but significantly reduced it at 4 h (Table 3). The ANOVA revealed a significant treatment effect (F(1,4) ⫽ 9.2; P ⬍ 0.05), time effect (F(1,3) ⫽ 14.8; P ⬍ 0.001), and treatmenttime interaction (F(3,12) ⫽ 9.3; P ⬍ 0.01).

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C. Polidori et al. / Peptides 21 (2000) 29 –35

Fig. 1. Effect of i.c.v. injections of BIBO 3304 (1 or 10 nmol/rat) on NPY (0.3 nmol/rat)-induced feeding in freely feeding and drinking rats. Values are mean ⫾ SEM from 7 (BIBO 3304, 1 nmol/rat) and 9 (BIBO 3304, 10 nmol/rat) subjects. Difference from controls: *P ⬍ 0.05; **P ⬍ 0.001; where not indicated difference from controls was not statistically significant.

JCF 109 at doses of 1 or 10 nmol/rat did not significantly reduce the hyperphagic effect of NPY 0.3 nmol/rat (F(1,5) ⫽ 1.0; P ⬎ 0.05) and (F(1,5) ⫽ 0.8; P ⬎ 0.05), respectively. The dose of 10 nmol/rat of JCF 109 did not reduce the hyperphagic effect of NPY, 0.6 nmol/rat (F(1,5) ⫽ 4.2; P ⬎ 0.05; Table 3). CGP 71683A, 10 to 100 nmol/rat, did not reduce the hyperphagic effect of NPY 0.3 nmol/rat (F(1,9) ⫽ 2.0; P ⬎ 0.05). However, 100 nmol/rat of CGP 71683A significantly inhibited the hyperphagic effect of NPY 0.6 nmol/rat (F(1,7) ⫽ 6.9; P ⬍ 0.05; Table 3); it did not modify NPY-induced Table 2 Effect of pretreatment with selective Y1 receptor antagonists, 10 nmol/ rat, on food intake induced in freely feeding and drinking rats by injection into the third ventricle on NPY, 0.6 nmol/rat Pretreatment Number 30 Min of rats

60 Min

120 Min

240 Min

Vehicle BIBO 3304

6 6

6.6 ⫾ 1.1 10.6 ⫾ 2.2 16.6 ⫾ 3.4 23.7 ⫾ 2.4 3.5 ⫾ 1.3 7.9 ⫾ 2.3 13.9 ⫾ 0.9 19.7 ⫾ 0.7

Vehicle SR120562A

6 6

8.6 ⫾ 1.4 12.2 ⫾ 1.5 18.6 ⫾ 2.1 22.6 ⫾ 2.3 5.4 ⫾ 1.1 10.2 ⫾ 1.8 16.2 ⫾ 1.0 21.6 ⫾ 1.7

Values are means ⫾ SEM. Difference from controls was not statistically significant.

Fig. 2. Effect of i.c.v. injections of SR 12562A (1 or 10 nmol/rat) on NPY (0.3 nmol/rat)-induced feeding in freely feeding and drinking rats. Values are mean ⫾ SEM from 6 subjects. Difference from controls, as in Fig. 1.

feeding in the first 2 h, but significantly reduced it at 4 h. Again, when 100 nmol/rat of CGP 71683A was infused in 5 ␮l of 33% DMSO and 66% water 1 h before NPY, 0.6 nmol/rat, feeding was not modified at 2 h, but was significantly reduced (P ⬍ 0.05) at 4 h (24.8 ⫾ 2.3 g/kg in controls versus 13.8 ⫾ 1.4 g/kg in CGP 71683A treated rats). 3.3. Effect of selective NPY receptor antagonists on food intake induced by overnight food deprivation BIBO 3304 reduced food intake induced by overnight food deprivation. The ANOVA revealed a statistically significant treatment effect at 10 nmol/rat (F(1,6) ⫽ 9.3; P ⬍ 0.05), but not at the dose of 1 nmol/rat (F(1,6) ⫽ 1.5; P ⬎ 0.05; Fig. 3). SR 120562A, 10 nmol/rat, only slightly reduced food intake in the first 60 min (from 9.3 ⫾ 0.7 to 7.3 ⫾ 1.5 g/kg). The ANOVA revealed no statistically significant effect. Food intake induced by overnight food deprivation in rats treated with the selective NPY Y5 receptor antagonist JCF 104, 10 nmol/rat, was essentially indistinguishable from that of controls (data not shown).

C. Polidori et al. / Peptides 21 (2000) 29 –35

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Table 3 Effect of pretreatment with selective Y5 receptor antagonists on food intake induced in freely feeding and drinking rats by injection into the third ventricle of NPY, 0.3 or 0.6 nmol/rat Pretreatment

Number of rats

30 Min

60 Min

120 Min

240 Min

NPY 0.3 nmol/rat 5 5 5

1.6 ⫾ 1.1 1.6 ⫾ 1.3 1.5 ⫾ 0.9

4.5 ⫾ 1.1 4.3 ⫾ 1.9 4.0 ⫾ 0.9

5.5 ⫾ 2.2 5.6 ⫾ 1.7 5.3 ⫾ 0.8

7.6 ⫾ 1.8 7.5 ⫾ 2.3 7.4 ⫾ 0.6

10 10 10

4.3 ⫾ 1.7 2.7 ⫾ 1.7 2.1 ⫾ 1.0

6.1 ⫾ 2.2 4.5 ⫾ 1.9 3.5 ⫾ 1.5

8.4 ⫾ 2.6 7.6 ⫾ 1.8 4.6 ⫾ 1.6

10.3 ⫾ 2.3 9.7 ⫾ 1.6 7.6 ⫾ 1.6

Vehicle JCF 109 (1) JCF 109 (10)

6 6 6

2.8 ⫾ 0.7 3.0 ⫾ 0.6 3.2 ⫾ 0.9

3.9 ⫾ 0.9 3.8 ⫾ 1.4 3.5 ⫾ 1.0

6.4 ⫾ 1.0 6.7 ⫾ 1.4 7.1 ⫾ 1.2

8.8 ⫾ 1.0 8.2 ⫾ 1.6 7.8 ⫾ 1.3

Vehicle JCF 104 (10)

5 5

7.8 ⫾ 0.8 8.7 ⫾ 2.2

12.7 ⫾ 2.6 13.4 ⫾ 3.3

23.2 ⫾ 5.8 15.0 ⫾ 3.4

27.7 ⫾ 5.3 15.0 ⫾ 3.5*

Vehicle CGP 71683A (100)

8 8

7.0 ⫾ 0.5 4.4 ⫾ 1.0

11.5 ⫾ 1.3 7.5 ⫾ 1.7

16.5 ⫾ 2.4 11.1 ⫾ 2.1

22.6 ⫾ 2.9 11.7 ⫾ 2.1*

Vehicle JCF 109 (10)

6 6

7.2 ⫾ 0.7 6.8 ⫾ 1.8

11.3 ⫾ 1.2 10.3 ⫾ 3.8

16.0 ⫾ 3.2 14.5 ⫾ 3.1

22.2 ⫾ 2.9 21.8 ⫾ 2.5

Vehicle JCF 104 (1) JCF 104 (10) Vehicle CGP 71683A (10) CGP 71683A (100)

NPY 0.6 nmol/rat

The dose of each antagonist (nmol/rat) is reported in parentheses. Values are means ⫾ SEM. Difference from controls: *P ⬍ 0.05; where not indicated, difference from controls was not statistically significant.

3.4. Effect of the selective NPY Y1 receptor antagonist BIBO 3304 on nociceptin-induced food intake Nociceptin (0.8 or 1.7 nmol/rat) injected into the 3V induced a significant increase in food intake. The ANOVA revealed a significant treatment (F(2,12) ⫽ 8.1; P ⬍ 0.01) and time effect (F(3,18) ⫽ 24.7; P ⬍ 0.001). In 4 h, controls took 1.4 ⫾ 0.8 g/kg, whereas rats treated with 0.8 and 1.7 nmol/rat of nociceptin took, respectively, 4.0 ⫾ 1.1 and 8.4 ⫾ 1.3 g/kg. BIBO 3304, 10 nmol/rat, did not significantly modify food intake induced by nociceptin, 1.7 nmol/rat (F(1,5) ⫽ 1.2; P ⬎ 0.05) during the 4-h period of observation (Fig. 4).

4. Discussion Several reports have shown that central injection of NPY induces a prompt and long-lasting increase in food intake in freely feeding and drinking rats [5,7,22,24]. Moreover, food deprivation has been reported to increase NPY-like immunoreactivity [6] and NPY release in the hypothalamus [4,27]. However, as stated in Section 1, the receptor subtype involved in the effect of NPY on feeding behavior is still a matter of debate. The results of the present study confirm previous findings of Wieland et al. [36] showing that the selective NPY Y1 receptor antagonist BIBO 3304 reduces feeding induced by either central injection of NPY or by overnight food deprivation. In the same report, BIBO 3304 was shown to

elicit a selective inhibition of NPY-induced feeding, as it did not block galanin- or noradrenaline-induced feeding. In accordance with these findings, in the present study, BIBO 3304 did not significantly inhibit the hyperphagic effect induced by central injection of nociceptin, 1.7 nmol/rat, which was of similar intensity as that of NPY (0.3 nmol/rat). In the present study, the Y1 receptor antagonist SR 120562A also significantly inhibited NPY-induced feeding, but it was less potent than BIBO 3304. It inhibited NPYinduced feeding at the dose of 10, but not of 1 nmol/rat; the dose of 10 nmol/rat did not significantly modify hyperphagia induced by overnight fasting. On the other hand, the dose of 100 nmol/rat of SR 120562A produced signs of toxicity (barrel rolling), and was not further used. These data concerning NPY Y1 receptor antagonists are in agreement with several reports suggesting that Y1 receptors mediate the hyperphagic effect NPY in rats [11,20,21]. However, it has been proposed that the Y5 receptor as well may be involved in feeding control [12,13]. This suggestion is supported by the finding that the orexigenic activity of a variety of NPY receptor agonists correlates with affinity for Y5, but not with affinity for Y1 receptors [37]. Moreover, a study that used rather high doses of antisense oligonucleotides for the Y5 receptor supports the involvement of this receptor in NPY-induced feeding [28]. Moreover, food intake is not stimulated by peptides, such as NPY(13–36) and rat PP, which exhibit low affinity for the Y5 receptor. Our data show that the selective Y5 receptor antagonists JCF 104, JCF 109, and CGP 71683A do not reduce feeding

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C. Polidori et al. / Peptides 21 (2000) 29 –35

Fig. 3. Effect of i.c.v. injections of BIBO 3304 (1 or 10 nmol/rat) on food intake induced by overnight food deprivation. Values are mean ⫾ SEM from seven subjects. Difference from controls, as in Fig. 1.

induced by NPY, 0.3 nmol/rat. However, JCF 104 and CGP 71683A significantly reduced the hyperphagic effect of NPY 0.6 nmol/rat, which occurred between the second and the fourth hour. This finding is apparently not related to problems

of solubility or of access of the antagonists to the brain parenchima; in fact, similar findings were obtained with compounds of different solubility (JCF 104 and CGP 71683A) and when administered in different vehicles and at different times before NPY injection. Thus, the results obtained with JCF 104 and CGP 71683A could induce to speculate that Y5 receptors are not critical receptors to confer hyperphagic activity to NPY, but they may be involved in the maintenance of the pronounced orexigenic effect induced by a rather high dose (0.6 nmol/rat) of NPY; at lower doses, only Y1 receptors may mediate the hyperphagic effect. In this regard, it is interesting to note that studies that have suggested the involvement of Y5 receptors in the hyperphagic effect of NPY have usually used rather high doses of NPY [12,24,37], such as 7 or even 10 nmol/rat. Moreover, in keeping with the present results, a recent study [9] reported that Y5 receptor antisense phosphothio-oligodeoxynucleotides do not reduce the 2-h food intake induced by 5 ␮g/rat of NPY or NPY(3–36) injected into the 3V, but markedly reduced the cumulative 10-h intake. In regard to feeding induced by overnight food deprivation, the Y1 receptor antagonist BIBO 3304, but not the Y5 receptor antagonist JCF 104, was able to inhibit feeding. These findings suggest that Y1, rather than Y5, receptors may be involved in deprivation-induced feeding. Accordingly, Xu et al. [38] have shown that hunger increases Y1 receptor gene expression, although no data have been so far reported for the Y5. In conclusion, the results obtained with NPY receptor antagonists suggest a major role of Y1 receptors in the hyperphagic effect of NPY and in feeding induced by food deprivation. They also suggest a modulatory role of Y5 receptors in the maintenance of feeding in response to high doses of NPY. However, testing of other antagonists for the Y5 receptors will be necessary to confirm this hypothesis. Finally, the present results indicate that the Y1 receptor antagonist BIBO 3304 inhibited NPY-induced feeding, but not the hyperphagic response to nociceptin. This finding not only provides evidence that the effect of BIBO 3304 is behaviorally selective, but it also indicates that the hyperphagic effect of nociceptin [25,31] is not mediated by NPY receptor mechanisms.

Acknowledgments The study was supported by grants from the University of Camerino, Italy. We thank Sheila Beatty for linguistic revision of this article.

References

Fig. 4. Effect of i.c.v. injections of BIBO 3304 (10 nmol/rat) on NC (1.7 nmol/rat)-induced feeding in freely feeding and drinking rats. Values are mean ⫾ SEM from six rats. Difference from controls, as in Fig. 1.

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