Evidence for an interaction between cannabinoidergic and dopaminergic systems with melanocortin MC3/ MC4 receptors in regulating food intake of neonatal chick

Central dopaminergic (DAergic) and adrenergic systems have a prominent role in appetite regulation; however, their interaction(s) have not been studied in neonatal layer chickens.Therefore, the current study aimed to determine the interaction of central DAergic and noradrenergic systems in food intake regulation in neonatal layer chickens. In the first experiment, chickens received the intracerebroventricular (ICV) injection of a control solution, prazosin (i.e., α1 adrenergic receptor antagonist; 10 nmol), dopamine (DA; 40 nmol), and prazosin plus DA. The second to fifth experiments were similar to the first experiment except that the birds were injected with yohimbine (i.e., α2 receptor antagonist; 13 nmol), metoprolol (i.e., β1 adrenergic receptor antagonist; 24 nmol), ICI 118,551 (i.e., β2 adrenergic receptor antagonist; 5 nmol), and SR59230R (i.e., β3 adrenergic receptor antagonist; 20 nmol) instead of prazosin. In the sixth experiment, the chickens received ICV injection with the control solution and noradrenaline (NA; 75, 150, and 300 nmol). In the seventh experiment, the birds were injected with the control solution, SCH23390 (i.e., D1 DAergic receptor antagonist; 5 nmol), NA (300 nmol), and SCH23390 plus NA In the eighth experiment, the control solution, AMI-193 (i.e., D2 DAergic receptor antagonist; 5 nmol), NA (300 nmol), and AMI-193 plus NA were injected. Then, cumulative food intake was recorded at 30, 60, and 120 min after the injection. According to the obtained results, the ICV injection of DA (40 nmol) significantly decreased food intake in comparison to that reported for the control group (p <0.05). The co-injection of yohimbine plus DA significantly amplified DA-induced hypophagia in the neonatal chickens (p <0.05). In addition, the co-administration of ICI 118,551 plus DA significantly inhibited the hypophagic effect of DA in the neonatal chickens (p <0.05). Furthermore, NA (75, 150, and 300 nmol) significantly reduced food intake in a dose-dependent manner (p <0.05). The co-injection of SCH23390 plus NA decreased the hypophagic effect of NA in the neonatal chickens, compared to that reported for the control group (p <0.05). The co-injection of AMI-193 plus NA diminished NA-induced hypophagia, compared to that reported for the control group (p <0.05). The aforementioned results suggested that there is an interconnection between central DAergic and noradrenergic systems through α2/β2 adrenergic and D1/D2 DAergic receptors in food intake regulation in neonatal chicks.

Background: Feeding behavior is regulated via a complex network which interacts through diverse signals from central and peripheral tissues. OBJECTIVES: The main purpose of the current study was to determine the role of central cannabinoidergic (CBergic) system on ghrelin-induced hypophagia in 3-h food deprived (FD3) neonatal chicken. METHODS: In experiment 1, chicks were ICV injected with control solution, ghrelin (0.6 nmol), SR141716A (selective CB1 receptors antagonist, 6.25 µg) and ghrelin + SR141716A. In experiment 2, chickens received ICV injection of (A) control solution, ghrelin (0.6 nmol), AM630 (selective CB2 receptors antagonist, 1.25 µg) and ghrelin + AM630. In experiment 3, chickens were ICV injected with control solution, 2-AG (selective CB1 receptors agonist, 2µg), GSK1614343 (selective ghrelin receptors antagonist, 6 nmol) and 2-AG + GSK1614343. In experiment 4, the birds received control solution, CB65 (selective CB2 receptors agonist, 6.25 µg), CB65 + GSK1614343. Then the cumulative food intake was measured until 120 min post injection. RESULTS: According to the results, ICV injection of the ghrelin, significantly decreased cumulative food intake (P<0.05). Co-injection of the ghrelin + SR141716A and/or ghrelin + AM630 significantly amplified ghrelin-induced hypophagia compared to control group (P<0.05). Hyperphagia observed by ICV injection of the 2-AG (2 µg) (P<0.05). Co-injection of the 2-AG + GSK1614343 increased food intake compared to control group (P<0.05). ICV injection of the CB65 (1.25 µg) significantly increased food intake (P<0.05). Also, co-injection of the CB65 + GSK1614343 significantly amplified cumulative food intake in FD3 neonatal layer-type chicken (P<0.05). CONCLUSIONS: These results suggested ghrelin-induced hypophagia mediates via CB1 and CB2 receptors in neonatal layer-type chicken

Central regulatory mechanisms for food intake regulation vary among animals. Evidence from animal studies suggests central opioids and dopamine have prominent role on appetite regulation but their interaction(s) have not been studied in layer-type chicken. Thus, in this study six experiments designed to investigate intracerebroventricular (ICV) administration of SCH23390 (D1 like receptors antagonist), Sulpride (D2 like receptors antagonist), DAMGO (μ-opioid receptors agonist), DPDPE (δ-opioid receptors agonist), U-50488H (κ-opioid receptors agonist) on feeding behavior in 3 h food deprived neonatal layer-type chickens. In experiment 1, chicks ICV injected with control solution, SCH23390 (2.5 nmol), DAMGO (125 pmol) and their combination (SCH23390 + DAMGO). In experiment 2: control solution, SCH23390 (2.5 nmol), DPDPE (δ-opioid receptors agonist, 40 pmol) and SCH23390 + DPDPE were applied to the birds. In experiment 3, injections were control solution, SCH23390 (2.5 nmol), U-50488H (30 nmol) and SCH23390 + U-50488H. In experiments 4–6 were similar to experiments 1–3 except Sulpride (2.5 nmol) applied instead of SCH23390. Then, cumulative food intake was recorded until 120 min after injection. According to the results, ICV injection of DAMGO (125 pmol) significantly decreased food intake but co-injection of DAMGO + SCH23390 diminished DAMGO-induced hypophagia (P 0.05). Furthermore, Sulpride had no role on DAMGO, DPDPE and U-50488H-induced food intake (P > 0.05). These results suggest there is an interaction between opioidergic and dopaminergic systems via μ and D1 receptors in appetite regulation in chicken.

Central regulatory mechanisms for neurotransmitters of food intake vary among animals. Endocannabinoids have crucial role on central food intake regulation in mammals but its role has not been studied in layer-type chicken. Thus, in this study 6 experiments designed to evaluate effects of intracerebroventricular (ICV) administration of 2-AG (2-Arachidonoylglycerol, selective CB1 receptors agonist), SR141716A (selective CB1 receptors antagonist), JWH015 (selective CB2 receptors agonist), AM630 (selective CB2 receptors antagonist) on feeding behavior in 3 h food deprived neonatal layer-type chickens. In experiment 1, birds ICV injected with control solution and 2-AG (0.25, 0.5 and 1 μg). In experiment 2: control solution, SR141716A (6.25, 12.5 and 25 μg) were ICV injected to birds. In experiment 3 animals received: control solution, SR141716A (6.25 μg), 2-AG (1 μg) and co-injection of SR141716A+2-AG. In experiment 4, chickens received control solution and JWH015 (6.25, 12.5 and 25 μg). In experiment 5, control solution and AM630 (1.25, 2.5 and 5 μg) were injected. In experiment 6, the birds received control solution, AM630 (1.25 μg), JWH015 (25 μg) and co-administration of AM630+JWH015. Then, cumulative food intake was recorded until 120 min after injection. According to the results, 2-AG dose dependently increased cumulative food intake while SR141716A reduced appetite compared to control group (P < 0.05). Injection of 2-AG (1 μg) amplified food intake and its effect minimized by SR141716A (6.25 μg) (P < 0.05). Also, ICV injection of JWH015 (25 μg) dose dependently increased food intake and co-injection of JWH015+AM630 decreased JWH015-induced food intake (P < 0.05). These results suggest CB1 and CB2 receptors have an important role on ingestive behavior in FD3 neonatal layer-type chicken.

The present study aimed to identify the role of dopaminergic and cannabinoidergic systems in the ghrelin-induced hypophagia among meat-type chickens. In the first experiment, intracerebroventricular (ICV) injection was applied to birds with control solution, D1 receptor antagonist (5 nmol), ghrelin (6 nmol), and D1 receptor antagonist plus ghrelin. The second to sixth experiments were similar to the first one, with the difference that D2 receptor antagonist (5 nmol), D3 receptor antagonist (6.4 nmol), D4 receptor antagonist (6 nmol), the precursor of dopamine (125 nmol), and 6-hydroxy dopamine (150 nmol) instead of D1 antagonist were injected into the broiler chickens. In experiment 7, control solution and different levels of ghrelin antagonists (5, 10, and 20 nmol) were injected. In experiment 8, the chickens were ICV injected with control solution, ghrelin antagonist (10 nmol), dopamine (40 nmol), and ghrelin antagonist plus dopamine. In experiments 9 and 10, CB1 and CB2 receptors antagonist (6.25µg and 5µg) were co-injected with ghrelin (6 nmol), respectively, measuring the food intake for 120 min after the injection. It was observed that ghrelin ICV injection considerably reduced food intake, whereas ghrelin antagonist increased food intake, depending on the dose (P<0.05). In addition, ghrelin-induced hypophagia was significantly attenuated by D1 receptor antagonist and 6-hydroxy dopamine (P<0.05), while the dopamine precursor considerably elevated the ghrelin-induced food intake (P<0.05). The dopamine-induced feeding behavior was diminished by the co-administration of [D-Lys-3]-GHRP-6 (10 nmol)+dopamine (40 nmol) (P<0.05). In addition, CB1 receptor antagonists enhanced the ghrelin influence on food intake (P<0.05). The results implied that the hypophagic impact of ghrelin was probably mediated by D1 and CB1 receptors within neonatal broilers.

ABSTRACTObjective: The present study was designed to examine the role of central γ-Aminobutyric acidA receptors and dopaminergic system on feeding behaviour in neonatal layer-type chicken.Methods: In this study, six experiments were designed, each with four treatment groups (n = 44 in each experiment). In experiment 1, four groups of 3-h food-deprived chicks received a dose of either the intracerebroventricular injection of (1) control solution, (2) Levo-dihydroxyphenylalanine as precursor of dopamine; 125 nmol, (3) Gaboxadol (γ-Aminobutyric acidA receptor agonist, 0.2 µg) and (4) Levo-dihydroxyphenylalanine (125 nmol) plus Gaboxadol (0.2 µg). Experiments 2–6 were similar to experiment 1, except that the chickens were intracerebroventricular-injected with 6-hydroxydopamine (is a neurotoxin; 2.5 nmol), SCH23390 (D1 receptor antagonist, 5 nmol), AMI-193 (D2 receptor antagonist, 5 nmol), NGB2904 (D3 receptor antagonist, 6.4 nmol) and L-741,742 (D4 receptor antagonist, 6 nmol) instead of levo-dihydroxyphenylalanine. Then, the cumulative food intake was measured until 120 min post-injection.Results: According to the results, intracerebroventricular injection of Gaboxadol (0.2 µg) significantly increased the food intake (P < 0.05). Co-injection of the 6-hydroxydopamine + Gaboxadol significantly amplified the food intake (P < 0.05). Intracerebroventricular injection of SCH23390 (5 nmol) + Gaboxadol (0.2 µg) significantly amplified the Gaboxadol-induced hyperphagia (P < 0.05). No significant effect was observed by co-injection of the D2–D4 receptor antagonists + Gaboxadol (P > 0.05).Conclusion: These results suggested the interconnection between central Dopaminergic and γ-Aminobutyric acidA on the feeding behaviour mediates via D1 and γ-Aminobutyric acidA receptors in 3-h food-deprived neonatal layer-type chicken.

Melanocortin 3 and 4 receptors (MC3R and MC4R) are known as the main receptors for melanocortin-induced hypophagia in mammalian and poultry. Also, central glutamatergic system has mediatory role on function of the melanocortin system in some brain areas. So, the aim of the current study was to determine the role of MC3/MC4 receptors agonist on food intake and its interaction with glutamatergic in 3-h food-deprived (FD3) neonatal broilers. In experiment 1, chickens were intracerebroventricular (ICV) injected with control solution, MTII (MC3/MC4 receptors agonist; 2.45, 4.8 and 9.8pmol). In experiment 2, control solution, SHU9119 (MC3/MC4 receptors antagonist; 0.5, 1 and 2nmol) were ICV injected. In experiment 3, birds ICV injected with control solution, SHU9119 (0.5nmol), MTII (9.8pmol) and co-injection of the SHU9119+MTII. Experiments 4-8 were similar to experiment 3, except birds injected with MK-801 (NMDA glutamate receptors antagonist, 15nmol), CNQX (AMPA glutamate receptors antagonist; 390nmol), AIDA (mGLUR1 glutamate receptors antagonist; 2nmol), LY341495 (mGLUR2 glutamate receptors antagonist; 150nmol) and UBP1112 (mGLUR3 glutamate receptors antagonist; 2nmol) instead of SHU9119. Then, cumulative food intake was recorded until 120min after injection. According to the results, dose dependent hypophagia observed after ICV injection of the MTII (p<0.05). ICV injection of SHU9119 significantly increased food intake in birds (p<0.05). Co-injection of SHU9119+MTII significantly inhibited MTII- induced hypophagia in neonatal chicks (p<0.05). In addition, hypophagia- induced by MTII was significantly attenuated with co-injection of MTII+MK-801(p<0.05). These results suggested MC3 and MC4 receptors have inhibitory role on food intake and this effect is probably mediated by NMDA glutamate receptors in neonatal chickens.

Recently, methylamine has been found as an endogenous amine, which is controlling food intake in mammals. However, there is no evidence about the effect of methylamine on feeding behavior in poultry. So, the present study was designed to evaluate the effect of intracerebroventricular (ICV) injection of methylamine and involvement of central methylamine/dopaminergic systems on feeding behavior in neonatal meat type chicks. In experiment 1, chicks were ICV injected with different doses of methylamine (0.48, 0.96, 1.44, 1.92 and 2.40 μmol). In experiment 2, chicks received a dose of either the control solution, 2.40 μmol methylamine, 125 nmol L-DOPA (dopamine precursor) or a combination of methylamine plus L-DOPA. Experiments 3-7 were similar to experiment 2 except that 150 nmol 6-OHDA (dopamine synthase inhibitor), 5 nmol SCH23390 (D1 receptor antagonist), 5 nmol AMI-193 (D2 receptor antagonist), 6.4 nmol NGB2904 (D3 receptor antagonist) and 6 nmol L-741, 742 (D4 receptor antagonist) were used instead of 125 nmol L-DOPA, respectively. Cumulative food intake was determined until 2 h post-injection. According to the results, methylamine significantly decreased food intake in a dose dependent manner (p < 0.05). The inhibitory effect of methylamine on food intake was significantly attenuated by 6-OHDA, SCH23390 and AMI-193 (P < 0.05), but NGB2904 and L-741, 742 had no effect on food intake induced by methylamine. In addition, hypophagic effect of methylamine significantly amplified by L-DOPA (P < 0.05). These results suggest that methylamine decrease food intake and there is an interaction between methylamine and dopaminergic system via D1 and D2 receptors in chickens.

The aim of the current study was to investigate the interaction of the nitric oxide and cannabinoidergic systems on feeding behaviour in neonatal chicken.A total of 6 experiments were designed to evaluate the interaction between cannabinoidergic and nitrergic systems on food intake in 3-h food-deprived (FD3) neonatal chickens. In Experiment 1, chickens received intracerebroventricular (ICV) injections of saline, 2-arachidonoylglycerol (2-AG) (a CB1 receptor agonist, 2 µg), l-arginine (nitric oxide precursor, 200 nmol) and co-administration of 2-AG + l-arginine. In Experiment 2, ICV injection of saline, 2-AG (2 µg), l-NAME (a nitric oxide synthesis inhibitor, 100 nmol) and their combination (2-AG + l-NAME) were applied to the birds. In Experiment 3, injections were saline, CB65 (a CB2 receptor agonist, 1.25 µg), l-arginine (200 nmol) and CB65 + l-arginine. In Experiment 4, birds received ICV injection of saline, CB65 (1.25 µg), l-NAME (100 nmol) and CB65 + l-NAME. In Experiment 5, chickens were ICV injected with saline, l-arginine (800 nmol), SR141716A (a selective CB1 receptor antagonist, 6.25 µg) and l-arginine + SR141716A. In Experiment 6, birds were injected with saline, l-arginine (800 nmol), AM630 (a selective CB2 receptor antagonist, 5 µg) and l-arginine + AM630. Cumulative food intake was recorded until 2-h post injection.ICV injection of CB1 and CB2 receptor agonists increased food intake. Co-injection of 2-AG + l-NAME increased the hyperphagic effects of CB1 receptors. CB2 receptor-induced food intake was not affected by co-administration of CB65 + l-NAME. l-Arginine decreased food intake and this effect was amplified by co-injection of l-arginine + SR141716A. However; CB2 receptor antagonists had no effect on l-arginine-induced hypophagia.The results suggest that there is an interaction between endogenous nitric oxide and the cannabinoidergic system on feeding behaviour which is mediated via CB1 receptors in the neonatal chicken.

An endogenous metal-ion site in the melanocortin MC1 and MC4 receptors was characterized mainly in transiently transfected COS-7 cells. ZnCl(2) alone stimulated signaling through the Gs pathway with a potency of 11 and 13 microm and an efficacy of 50 and 20% of that of alpha-melanocortin stimulating hormone (alpha-MSH) in the MC1 and MC4 receptors, respectively. In the presence of peptide agonist, Zn(II) acted as an enhancer on both receptors, because it shifted the dose-response curves to the left: most pronounced was a 6-fold increase in alpha-MSH potency on the MC1 receptor. The effect of the metal ion appeared to be additive, because the maximal cAMP response for alpha-MSH in the presence of Zn(II) was 60% above the maximal response for the peptide alone. The affinity of Zn(II) could be increased through binding of the metal ion in complex with small hydrophobic chelators. The binding affinities and profiles were similar for a number of the 2,2'-bipyridine and 1,10-phenanthroline analogs in complex with Zn(II) in the MC1 and MC4 receptors. However, the potencies and efficacies of the metal-ion complexes were very different in the two receptors, and close to full agonism was obtained in the MC1 receptor. Metal ion-chelator complexes having antagonistic properties were also found. An initial attempt to map the metal-ion binding site in the MC1 receptor indicated that Cys(271) in extracellular loop 3 and possibly Asp(119) at the extracellular end of TM-III, which are both conserved among all MC receptors, are parts of the site. It is concluded that the function of the MC1 and MC4 receptors can be positively modulated by metal ions acting both as partial agonists and as potentiators for other agonists, including the endogenous peptide ligand alpha-MSH at Zn(II) concentrations that could be physiological. Furthermore, the metal ion-chelator complexes may serve as leads in the development of novel melanocortin receptor modulators.

Novel anti-inflammatory and chondroprotective effects of the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and human melanocortin MC3 receptor agonist PG-990 on lipopolysaccharide activated chondrocytes

The aim of the current study was to determine possible interaction of central oxytocin and opioidergic system on food intake regulation in neonatal layer-type chicken. In experiment 1, FD3 chicken ICV injected with control solution, oxytocin (10 µg), β-FNA (µ receptor antagonist, 5 µg) and oxytocin (10 µg) + β-FNA were injected. Experiments 2–6 were similar to experiments 1, except chicken injected with nor-BNI (κ receptor antagonist, 5 µg), NTI (δ receptor antagonist, 5 µg), DAMGO (µ receptor agonist, 62.25 pmol), U-50488H (κ receptor agonist, 10 nmol), DPDPE (δ receptor agonist, 20 pmol) instead of β-FNA. In experiment 7, control solution, DAMGO (125 pmol), d(CH2)5Tyr(Me)-[Orn8]-vasotocin (oxytocin antagonist, 5 µg) and DAMGO + d(CH2)5Tyr(Me)-[Orn8]-vasotocin were ICV injected to FD3 chicken. Experiments 8 and 9 were similar to experiments 7, except chicken injected with U-50488H (30 nmol) and DPDPE (40 pmol) instead of DAMGO. Then, cumulative food intake was recorded at 30, 60 and 120 min after injection. According to the results, ICV injection of the oxytocin (10 µg) significantly decreased food intake compared to control group (P < 0.05). Co-injection of the oxytocin + β-FNA and oxytocin + U-50488H significantly decreased hypophagic effect of the oxytocin (P < 0.05). While, co-injection of the oxytocin + nor-BNI or oxytocin + DAMGO significantly amplified hypophagic effect of the oxytocin in chicken (P < 0.05). In addition, ICV injection of DAMGO (125 pmol) significantly decreased cumulative food intake compared to control group (P < 0.05). However, co-addministration of the DAMGO + (CH2)5Tyr(Me)-[Orn8]-vasotocin significantly decreased hypophagic effect of the DAMGO (P < 0.05) in chicken. These results suggested there are interconnection between oxytocin and opioidergic system on central food intake regulation, which mediates via µ and κ opioidergic receptors in neonatal layer-type chicken.

Characterization of aliphatic, cyclic, and aromatic N-terminally “capped” His- d-Phe-Arg-Trp-NH 2 tetrapeptides at the melanocortin receptors

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A(2A), cannabinoid CB(1), and dopamine D(2) receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A(2A), CB(1), and D(2)) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB(1) receptor with A(2A) and D(2) receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A(2A)-CB(1)-D(2) receptor heteromers. Analysis of MAPK signaling in striatal slices of CB(1) receptor KO mice and wild-type littermates supported the existence of A(1)-CB(1)-D(2) receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.

Fish melanocortin system