RESUMEN
Beta-melanocyte-stimulating hormone (ß-MSH), when centrally injected, induces anorexigenic effects in rodents and chickens but its mechanism remains unclear. Thus, the primary goal of this research was to elucidate the hypothalamic mechanism using chickens. Intracerebroventricular injection of 0.3, 1.0 and 3.0 nmol of ß-MSH decreased food intake for 540 min. Expression of hypothalamic mRNAs were affected by ß-MSH injection, including corticotrophin-releasing factor (CRF) and its receptor subtype 1 (CRFR1), mesotocin (MT) and its receptor (MTR), pro-opiomelanocortin, cocaine- and amphetamine-regulated transcript (CART), growth hormone secretagogue receptor (GHSR) and neuropeptide Y (NPY) receptor subtype 5 (NPYR5). Within the arcuate nucleus, expressions of NPY, agouti-related peptide, MT and MTR were increased by ß-MSH injection. ß-MSH-treated chicks had more CRF, CRFR1, CRF receptor subtype 2, GHSR, NPY receptor subtype 1 (NPYR1) and NPYR5 mRNA but lower levels of CART and ghrelin, in the paraventricular nucleus. Greater amounts of mRNA for MTR, GHSR, NPYR1 and NPYR5 and less CRF expression were observed in the ventromedial hypothalamus. In conclusion, central injection of ß-MSH potently reduced food intake and was associated with changes in mRNA expression of some anorexigenic factors in a hypothalamic nucleus-specific manner.
Asunto(s)
Hormona Liberadora de Corticotropina/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Oxitocina/análogos & derivados , Receptores de Hormona Liberadora de Corticotropina/metabolismo , beta-MSH/farmacología , Animales , Pollos , Hipotálamo/metabolismo , Inyecciones Intraventriculares , Oxitocina/metabolismoRESUMEN
Broiler chicks eat more food than layer chicks. However, the causes of the difference in food intake in the neonatal period between these strains are not clear. In this study, we examined the involvement of proopiomelanocortin (POMC)-derived melanocortin peptides α-, ß- and γ-melanocyte-stimulating hormones (MSHs) in the difference in food intake between broiler and layer chicks. First, we compared the hypothalamic mRNA levels of POMC between these strains and found that there was no significant difference in these levels between broiler and layer chicks. Next, we examined the effects of central administration of MSHs on food intake in these strains. Central administration of α-MSH significantly suppressed food intake in both strains. Central administration of ß-MSH significantly suppressed food intake in layer chicks, but not in broiler chicks, while central administration of γ-MSH did not influence food intake in either strain. It is therefore likely that the absence of the anorexigenic effect of ß-MSH might be related to the increased food intake in broiler chicks.
Asunto(s)
Apetito/efectos de los fármacos , Pollos/metabolismo , Ingestión de Energía/efectos de los fármacos , alfa-MSH/fisiología , beta-MSH/fisiología , gamma-MSH/fisiología , Animales , Expresión Génica , Hipotálamo/metabolismo , Masculino , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Receptor de Melanocortina Tipo 4/genética , Receptor de Melanocortina Tipo 4/metabolismo , alfa-MSH/farmacología , beta-MSH/farmacología , gamma-MSH/farmacologíaRESUMEN
The solution structures and inter-molecular interaction of the cyclic melanocortin antagonists SHU9119, JKC363, HS014, and HS024 with receptor molecules have been determined by NMR spectroscopy and molecular modeling. While SHU9119 is known as a nonselective antagonist, JKC363, HS014, and HS024 are selective for the melanocortin subtype-4 receptor (MC4R) involved in modulation of food intake. Data from NMR and molecular dynamics suggest that the conformation of the Trp9 sidechain in the three MC4R-selective antagonists is quite different from that of SHU9119. This result strongly supports the concept that the spatial orientation of the hydrophobic aromatic residue is more important for determining selectivity than the presence of a basic, "arginine-like" moiety responsible for biological activity. We propose that the conformation of hydrophobic residues of MCR antagonists is critical for receptor-specific selectivity.
Asunto(s)
Hormonas Estimuladoras de los Melanocitos/química , Péptidos Cíclicos/química , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Receptores de Melanocortina/antagonistas & inhibidores , Secuencia de Aminoácidos , Sitios de Unión , Interacciones Farmacológicas , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Hormonas Estimuladoras de los Melanocitos/síntesis química , Hormonas Estimuladoras de los Melanocitos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Receptor de Melanocortina Tipo 4/efectos de los fármacos , Receptores de Melanocortina/efectos de los fármacos , Sensibilidad y Especificidad , Soluciones/química , Relación Estructura-Actividad , beta-MSH/síntesis química , beta-MSH/química , beta-MSH/farmacologíaRESUMEN
The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.
Asunto(s)
Proteína Relacionada con Agouti/antagonistas & inhibidores , Proteína Relacionada con Agouti/fisiología , Polimorfismo Genético , Proopiomelanocortina/fisiología , Receptor de Melanocortina Tipo 4/genética , Proteína Relacionada con Agouti/biosíntesis , Proteína Relacionada con Agouti/metabolismo , Secuencia de Aminoácidos , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Obesidad/genética , Obesidad/metabolismo , Proopiomelanocortina/agonistas , Proopiomelanocortina/antagonistas & inhibidores , Proopiomelanocortina/biosíntesis , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Receptor de Melanocortina Tipo 4/agonistas , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Receptor de Melanocortina Tipo 4/biosíntesis , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , alfa-MSH/farmacología , alfa-MSH/fisiología , beta-MSH/metabolismo , beta-MSH/farmacología , gamma-MSH/metabolismo , gamma-MSH/farmacologíaRESUMEN
Research over the past decade has indicated that melanocortin peptides are potent inhibitors of inflammation and a promising source of new anti-inflammatory and cytoprotective therapies. The purpose of the present paper is to compare protective effects of alpha-, beta-, and gamma-melanocyte stimulating hormone on acetaminophen induced liver lesions in male CBA mice. Acetaminophen was applied intragastrically in a dose of 150 mg/kg, and tested substances were applied intraperitoneally 1 hour before acetaminophen. Mice were sacrificed after 24 hours and intensity of liver injury was estimated by measurement of plasma transaminase activity (AST and ALT) and histopathological grading of lesions. It was found that alpha-, beta-, and gamma-MSH decrease intensity of lesions by both criteria in a dose-dependent manner.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , alfa-MSH/farmacología , beta-MSH/farmacología , gamma-MSH/farmacología , Hormona Adrenocorticotrópica/química , Alanina Transaminasa/sangre , Secuencia de Aminoácidos , Animales , Aspartato Aminotransferasas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , alfa-MSH/química , beta-MSH/química , gamma-MSH/químicaRESUMEN
This review documents my research for the past 45 years in peptide chemistry. Initially, in order to study the structure-activity relationships of active center of alpha- and beta-melanocyte stimulating hormones (H-His-Phe-Arg-Trp-Gly-OH), we employed D-amino acids. That approach yielded first published report in 1965 of antagonists containing D-amino acids. Monkey beta-melanocyte stimulating hormone (beta-MSH), an 18 amino acid peptide stimulated pigment cells. We synthesized beta-MSH and fragments thereof, and studied in detail structure-activity relationships. A major and valuable result revealed that the C-terminal pentadecapeptide of beta-MSH exhibited higher MSH activity than the parent hormone providing a new question; namely, what was the role of the N-terminal tripeptide? In order to identify the novel enzyme, spleen fibrinolytic proteinase (SFP), I developed a specific chromogenic substrate, Suc-Ala-Tyr-Leu-Val-pNA, and a specific inhibitor, Suc-Tyr-D-Leu-D-Val-pNA, once again employing my D-amino acid strategy. SFP was purified by affinity chromatography using Suc-Tyr-D-Leu-D-Val-pNA as the bound ligand. The success of this approach provided me the incentive to develop a variety of potential drugs. Thus, I prepared a specific plasmin inhibitor (YO-2) and a plasma kallikrein inhibitor (PKSI-527). Next, my research developed novel opioid receptor specific opioid agonists and antagonists based on 2',6'-dimethyl-L-tyrosine (Dmt) dimers coupled with unique pyrazinone ring as a spacer. They exhibited potent oral antinociceptive activity acting through the mu-opioid receptor. Potent mu-receptor agonists (H-Dmt-Pro-Phe/Trp- Phe-NH(2)) were transformed into highly selective mu-receptor antagonists (N-allyl-Dmt-Pro-Phe/Trp-Phe-NH(2)), which reversed ethanol-induced increases in GABAergic neurotransmission, suggesting the possibility that they may emerge as candidates for the treatment of ethanol addiction.
Asunto(s)
Aminoácidos , Hormonas Estimuladoras de los Melanocitos , Péptidos/síntesis química , Receptores Opioides mu , Alcoholismo/tratamiento farmacológico , Descubrimiento de Drogas , Humanos , Hormonas Estimuladoras de los Melanocitos/síntesis química , Hormonas Estimuladoras de los Melanocitos/farmacología , Péptidos/química , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Relación Estructura-Actividad , beta-MSH/síntesis química , beta-MSH/farmacologíaRESUMEN
Proopiomelanocortin (POMC, a precursor of anorexigenic neuropeptides) neurons in hypothalamus suppresses food intake in both mammals and chickens. In mammals, several lines of evidence suggest that POMC-derived anorexigenic peptides upregulate mRNA levels of anorexigenic peptides such as corticotropin-releasing factor (CRF) and thyrotropin-releasing factor and downregulate mRNA levels of orexigenic peptides such as orexin and melanin-concentrating hormone. However, the POMC-induced anorexigenic pathway in chickens has not been well characterized. In the present study, we investigated how POMC neurons regulate mechanisms of food intake using an anorexigenic peptide, beta-melanocyte-stimulating hormone (beta-MSH), derived from the post-transcriptional cleavage of POMC. Central administration of beta-MSH in chicks significantly suppressed food intake, and importantly, this suppression was accompanied by a significant upregulation of CRF mRNA levels. Furthermore, the CRF type 2 receptor antagonist alpha-helical CRF significantly reversed the anorexigenic action of beta-MSH. These findings indicate that CRF and its receptor, CRF type 2 receptor, act as the major mediators in beta-MSH-induced anorexigenic action in chicks. beta-MSH significantly increased orexin mRNA levels and did not alter mRNA levels of thyrotropin-releasing factor and melanin-concentrating hormone in chicks, suggesting that the beta-MSH-induced anorexigenic pathway in chicks is different from that in mammals. Increases in orexin mRNA levels were accompanied by significant decreases in plasma glucose concentration, suggesting that orexin mRNA might be stimulated by beta-MSH-induced hypoglycemia. Thus, this study demonstrates the direct evidence that CRF is a critical downstream target in the beta-MSH-induced anorexigenic pathway in chicks.
Asunto(s)
Anorexia/etiología , Hormona Liberadora de Corticotropina/fisiología , Proopiomelanocortina/fisiología , beta-MSH/farmacología , Animales , Anorexia/inducido químicamente , Apetito , Pollos , Hormona Liberadora de Corticotropina/genética , Cartilla de ADN , Conducta Alimentaria/efectos de los fármacos , Conducta Alimentaria/fisiología , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiología , Hipotálamo/fisiopatología , Inyecciones Intraventriculares , Mamíferos , Neuronas/fisiología , Proopiomelanocortina/genética , beta-MSH/administración & dosificaciónRESUMEN
Recent studies have suggested that melanocortins contribute to the generation and/or maintenance of pathological pain. Experimental evidence indicates a primary role for melanocortin 4 (MC4) receptors in pathological pain. In a previous study, we described the presence of MC4 receptor transcripts in the dorsal root ganglia (DRG). This finding prompted us to investigate the peripheral antinociceptive effects of MC4 receptor antagonists. In addition, we assess the expression of MC4 receptors in the spinal cord and the DRG of rats subjected to neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. Injection of the MC4 receptor antagonists Asp3-Lys8- Ac-Nle-Asp-His-D-Nal(2')-Arg-Trp-Lys-NH(2) (SHU9119) and Mpr1-Cys8-Mpr-Glu-His-(D-Nal)-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH(2) (JKC-363) into the ipsilateral paw resulted in a significant and dose-dependent alleviation of mechanical allodynia (assayed by the von Frey test) and thermal hyperalgesia (assayed by the Hargreaves test). Compared to naive control animals, immunohistochemistry revealed a 40% and 22% increase in MC4 receptor-immunoreactivity (IR) in the dorsal horn of the spinal cord ipsilateral to the injury at 3 and 14 days after CCI, respectively. Similarly, in the ipsilateral L4-L5 DRG, a 21.1% enhancement in MC4 receptor-IR was seen 3 days after CCI, as well as a 40.5% increase 14 days after CCI. Together, painful neuropathy resulted in the up-regulation of MC4 receptors in the spinal and peripheral nociceptive pathways. This up-regulation of MC4 receptors promotes the pronociceptive action of their endogenous ligands. Therefore, a block of the MC4 receptors results in the antagonism of neuropathic pain and such treatment could be beneficial therapeutically for individuals with chronic neuropathic pain.
Asunto(s)
Hormonas Estimuladoras de los Melanocitos/farmacología , Neuralgia/tratamiento farmacológico , Péptidos Cíclicos/farmacología , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Analgésicos/administración & dosificación , Analgésicos/farmacología , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ganglios Espinales/metabolismo , Masculino , Hormonas Estimuladoras de los Melanocitos/administración & dosificación , Neuralgia/fisiopatología , Péptidos Cíclicos/administración & dosificación , Ratas , Ratas Wistar , Receptor de Melanocortina Tipo 4/genética , Médula Espinal/metabolismo , Factores de Tiempo , Regulación hacia Arriba , beta-MSH/administración & dosificación , beta-MSH/farmacologíaRESUMEN
The melanocortin system together with other appetite-related systems plays a significant role in appetite regulation. The appetite-related effects of one such melanocortin, beta-melanocyte-stimulating hormone (MSH), are well documented in rodents; however, its effects in the avian class are not thoroughly understood. Thus, we designed a study to determine the effects of i.c.v. beta-MSH injection on food and water intake, plasma corticosterone concentration, ingestive and non-ingestive behaviours, and hypothalamic neuronal activation using Cobb-500 chicks. Chicks responded to beta-MSH-treatment with a reduction in food and water intake; however when water intake was measured independently of food intake, it was not affected. beta-MSH-treated chicks also had increased plasma corticosterone concentrations and increased c-Fos reactivity in the periventricular, paraventricular and infundibular nuclei, and the ventromedial hypothalamus; however, the lateral hypothalamus was not affected. The effect on food intake is primary because behaviours that may be competitive with food intake were not increased in beta-MSH-treated chicks. Based on these results, we conclude that beta-MSH causes anorexigenic effects that are likely primarily mediated via stimulation of satiety-related hypothalamic nuclei in broiler-type chicks.
Asunto(s)
Animales Recién Nacidos , Apetito/efectos de los fármacos , Pollos , Hipotálamo/efectos de los fármacos , beta-MSH/farmacología , Animales , Corticosterona/sangre , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Privación de Alimentos/fisiología , Inmunohistoquímica , Inyecciones Intraventriculares , Concentración Osmolar , alfa-MSH/farmacología , beta-MSH/administración & dosificaciónRESUMEN
The recent emergence of obesity as a major health threat in the industrialized world has intensified the search for novel and effective pharmacologic treatment. The proopiomelanocortin (POMC)-melanocortin 4 receptor (MC4R) axis has been shown to regulate food intake and energy homeostasis and is considered among the most promising antiobesity targets. Our initial efforts in this area have focused on affinity and selectivity directed optimization of the native beta-MSH(5-22) sequence and resulted in the discovery of a potent MC4R agonist: Ac-Tyr-Arg-[Cys-Glu-His-D-Phe-Arg-Trp-Cys]-NH(2) (10). Subcutaneous administration of this peptide produced an excellent in vivo efficacy in reducing food intake and increasing fat metabolism. Additionally, suppression of food intake was observed in wild type but not in MC4R deficient mice, suggesting that the effects observed in the wild type mice were mediated through MC4R signaling. Subsequent optimization efforts led to the identification of a novel series of disulfide constrained hexapeptides as exemplified by Ac-[hCys-His-D-Phe-Arg-Trp-Cys]-NH(2) (100). These cyclic hexapeptides showed a further improved potency in binding MC4R and an enhanced selectivity over MC1R. At a dose of 0.07 mg/kg analog 102 reduced food intake by 38% and increased fat utilization by 58% in rats. These cyclic peptides provide novel and enhanced reagents for the elucidation of melanocortin receptors biology and may find applications in the treatment of obesity and related metabolic disorders.
Asunto(s)
Receptor de Melanocortina Tipo 4/agonistas , beta-MSH/química , beta-MSH/farmacología , Aminoácidos/química , Animales , Simulación por Computador , Disulfuros/química , Humanos , Receptor de Melanocortina Tipo 4/metabolismo , Relación Estructura-Actividad , beta-MSH/síntesis químicaRESUMEN
We previously identified melanocortin receptor 4 (MC4R) in a search for genes associated with hypoglossal nerve regeneration. As melanocortins promote nerve regeneration after axonal injury, we investigated whether MC4R functions as a key receptor for peripheral nerve regeneration. In situ hybridization revealed that MC4R mRNA is induced in mouse hypoglossal motor neurons after axonal injury, whereas mRNAs for MC1R, MC2R, MC3R, and MC5R are not expressed either before or after nerve injury. This result was confirmed by RT-PCR. The level of MC4R mRNA expression increased significantly from day 3 after axotomy, reached a peak on day 5, and decreased to the control level on day 14. Similar induction of MC4R was observed in axotomized mouse dorsal root ganglia (DRGs). MC4R mRNA expression was induced exclusively among the MCR family in the L4-6 DRG after sciatic nerve injury. We further examined whether alpha-melanocortin stimulating hormone (alpha-MSH) promotes neurite elongation via MC4R. In mouse DRG neuron culture, alpha-MSH significantly promoted neurite outgrowth at a concentration of 10(-8) mol/L. This neurite-elongation effect was entirely inhibited by the addition of a selective MC4R blocker, JKC-363. Therefore, it is concluded that alpha-MSH could stimulate neurite elongation via MC4R in DRG neurons. The present results suggest that induction of MC4R is crucial for motor and sensory neurons to regenerate after axonal injury.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Enfermedades del Nervio Hipogloso/patología , Neuronas Motoras/metabolismo , Neuronas Aferentes/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ganglios Espinales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedades del Nervio Hipogloso/metabolismo , Hibridación in Situ/métodos , Masculino , Hormonas Estimuladoras de los Melanocitos/farmacología , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Factor de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas Aferentes/citología , Neuronas Aferentes/efectos de los fármacos , Péptidos Cíclicos/farmacología , ARN Mensajero/metabolismo , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Receptor de Melanocortina Tipo 4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , beta-MSH/farmacologíaRESUMEN
The human skin holds the full machinery for pro-opiomelanocortin processing. The alpha-melanocyte-stimulating hormone (alpha-MSH)/melanocortin-1-receptor cascade has been implicated as a major player via the cAMP signal in the control of melanogenesis. Only very recently the beta-endorphin/mu-opiate receptor signal has been added to the list of regulators of melanocyte dendricity and melanin formation. In this context it was reported that (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)) can act as an allosteric inhibitor of tyrosinase, the key enzyme in melanogenesis, and this inhibition is reversible by both alpha- and beta-MSH. It was also shown earlier that 7BH(4), the isomer of 6BH(4), is twice as active in this inhibition reaction. However, as yet it is not known whether 7BH(4) is indeed present in loco in the melanosome. We here provide evidence that this isomer is present in this organelle in a concentration range up to 50 x 10(-6) M. Determination of beta-MSH in melanosomal extracts yielded 10 pg/mg protein. Moreover, we demonstrate reactivation of the 7BH(4)/tyrosinase inhibitor complex by beta-MSH, whereas alpha-MSH failed to do so. Furthermore, we show intra-melanosomal l-dopa formation from dopachrome by 7BH(4) in a concentration range up to 134 x 10(-6) M. Based on these results, we propose a new receptor-independent mechanism in the control of tyrosinase/melanogenesis by beta-MSH and the pterin 7BH(4).
Asunto(s)
Biopterinas/análogos & derivados , Células Epidérmicas , Melanocitos/metabolismo , Pigmentación de la Piel/efectos de los fármacos , beta-MSH/farmacología , Animales , Biopterinas/farmacología , Western Blotting/métodos , Línea Celular Tumoral , Células Cultivadas , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/farmacología , Humanos , Indolquinonas/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Levodopa/análisis , Levodopa/metabolismo , Melanocitos/efectos de los fármacos , Ratones , Microscopía Inmunoelectrónica/métodos , Monofenol Monooxigenasa/antagonistas & inhibidoresRESUMEN
Chronic use of morphine is accompanied by the development of morphine tolerance, which is one of the major problems associated with opiate treatment. Possible modulation of opioid effects by melanocortin receptor ligands has been recently demonstrated. Therefore, we investigated the influence of repeated intrathecal injection of a melanocortin receptor antagonist (SHU9119, JKC-363) on the development of morphine tolerance as measured by tail-flick test. It was also examined whether a single i.t. SHU9119 and JKC-363 administration could counteract the loss of analgesic potency of morphine in morphine tolerant rats. We examined also the influence of chronic morphine administration on mu-opioid receptor (MOR) and melanocortin 4 receptor (MC4-R) mRNAs in the rat spinal cord and dorsal root ganglia (DRG) during morphine tolerance. Morphine treatment (10mg/kg, i.p. twice daily) over 8 days induced tolerance as reflected by a significant reduction of withdrawal latency from 181 to 25% above baseline in the tail-flick test. Repeated co-administration of morphine and SHU9119 or JKC-363, significantly prevented the development of morphine tolerance. A single administration of an MC4-R antagonist restored morphine analgesic potency in morphine tolerant rats. Using RT-PCR we demonstrated no changes in the spinal cord but there was a decrease in MOR and increase in MC4-R gene expression in the DRG of rats tolerant to morphine. These results suggest that MC4-R may be involved in the mechanisms of opioid tolerance and antagonists of this receptor may be a possible new target in the search for strategies preventing the development of opioid tolerance.
Asunto(s)
Tolerancia a Medicamentos/fisiología , Hormonas Estimuladoras de los Melanocitos/farmacología , Morfina/efectos adversos , Péptidos Cíclicos/farmacología , Receptores de Melanocortina/antagonistas & inhibidores , Médula Espinal/efectos de los fármacos , Análisis de Varianza , Animales , Área Bajo la Curva , Conducta Animal , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Interacciones Farmacológicas , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/instrumentación , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Tiempo de Reacción/efectos de los fármacos , Receptor de Melanocortina Tipo 4/genética , Receptor de Melanocortina Tipo 4/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Médula Espinal/metabolismo , Factores de Tiempo , beta-MSH/farmacologíaRESUMEN
We report the isolation of a novel human circulating proopiomelanocortin-derived peptide, VA-beta-MSH, from hemofiltrate and its pharmacological characterization. Screening for lipolytic activity in differentiated 3T3-L1 adipocytes led to the isolation from a hemofiltrate peptide library by alternating reverse phase and cation exchange chromatography. In the course of this isolation, we also identified human beta-MSH-(1-22). We synthesized VA-beta-MSH by the N-(9-fluorenyl)-methoxycarbonyl (F-moc) solid phase method and used synthetic beta-MSH-(1-22) to confirm that both isolated peptides are lipolytically active in a dose-dependent manner in differentiated 3T3-L1 adipocytes in the nanomolar range. Using cAMP ELISA, we demonstrate that stimulation with both peptides caused a strong cAMP elevation in this cell system. Furthermore, we show that the selective inhibitors of cAMP-dependent protein kinase, 8-(4-Chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS); N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), significantly reduce VA-beta-MSH- and beta-MSH-(1-22)-mediated lipolysis. Although isolated after its lipolytic activity on 3T3-L1 cells, this newly identified circulating human melanocortin may serve other functions in human physiology. Moreover, the fact that these peptides have been identified after a functional assay, but have been overseen in large proteomic approaches, underscores the importance of such approaches in identifying previously undescribed circulating bioactive molecules.
Asunto(s)
Hemofiltración , Fallo Renal Crónico/metabolismo , beta-MSH/aislamiento & purificación , Células 3T3-L1 , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Humanos , Lipólisis , Ratones , Datos de Secuencia Molecular , Receptor de Melanocortina Tipo 2/análisis , Receptores de Corticotropina/análisis , Receptores de Melanocortina , beta-MSH/química , beta-MSH/farmacologíaRESUMEN
Melanocortin receptor 4 (MCR4) and CB(1) cannabinoid receptors independently modulate food intake. Although an interaction between the cannabinoid and melanocortin systems has been found in recovery from hemorrhagic shock, the interaction between these systems in modulating food intake has not yet been examined. The present study had two primary purposes: 1) to examine whether the cannabinoid and melanocortin systems act independently or synergistically in suppressing food intake; and 2) to determine the relative position of the CB(1) receptors in the chain of control of food intake in relation to the melanocortin system. Rats were habituated to the test environment and injection procedure and then received intracerebroventicular injections of various combinations of the MCR4 receptor antagonist JKC-363, the CB(1) receptor agonist Delta(9)-tetrahydrocannabinol, the MCR4 receptor agonist alpha-MSH, or the cannabinoid CB(1) receptor antagonist SR 141716. Food intake and locomotor activity were then recorded for 120 min. When administrated alone, SR 141716 and alpha-MSH dose-dependently attenuated baseline feeding, whereas sub-anorectic doses of SR 141716 and alpha-MSH synergistically attenuated baseline feeding when combined. Delta(9)-Tetrahydrocannabinol-induced feeding was not blocked by alpha-MSH, whereas SR 141716 dose-dependently attenuated JKC-363-induced feeding. Locomotor activity was not significantly affected by any drug treatment, suggesting that the observed effects on feeding were not due to a nonspecific reduction in motivated behavior. These findings revealed a synergistic interaction between the cannabinoid and melanocortin systems in feeding behavior. These results further suggested that CB(1) receptors are located downstream from melanocortin receptors and CB(1) receptor signaling is necessary to prevent the melanocortin system from altering food intake.
Asunto(s)
Ingestión de Alimentos/fisiología , Receptor Cannabinoide CB1/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , beta-MSH/análogos & derivados , Animales , Interacciones Farmacológicas , Masculino , Péptidos Cíclicos/farmacología , Piperidinas/farmacología , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor de Melanocortina Tipo 4/agonistas , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Rimonabant , alfa-MSH/farmacología , beta-MSH/farmacologíaRESUMEN
The pro-opiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) mediates many diverse physiological actions, including anti-inflammatory and immunomodulatory effects. However, little is known about the physiological roles of the other melanocortins, beta- and gamma-MSH. Here, we investigated the effects of melanocortin peptides in an in vivo neuroinflammation model. Six hours following intracisternal (i.c.) administration of 10 microg lipopolysaccharide (LPS) to mice a five-fold increase in the nitric oxide (NO) level was seen in the animals' brains, when detected by electron paramagnetic resonance (EPR). All tested melanocortins, alpha-, beta-, gamma1- and gamma2-MSH (0.001-10 nmol/mouse i.c.), dose dependently reduced the LPS induced increases in brain NO, with an order of effectiveness: beta-MSH > or = gamma1-MSH=gamma2-MSH>alpha-MSH. Our results suggest specialized functions of beta- and gamma-MSH melanocortins in inflammatory signal modulation in the brain.
Asunto(s)
Encéfalo/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Óxido Nítrico/metabolismo , beta-MSH/metabolismo , gamma-MSH/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Masculino , Ratones , Ratones Endogámicos ICR , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , alfa-MSH/metabolismo , alfa-MSH/farmacología , beta-MSH/farmacología , gamma-MSH/farmacologíaRESUMEN
Agouti and agouti-related protein (AgRP) are endogenous antagonists of the melanocortin receptors (MCxR). Previous data showed that recombinant full-length agouti and a synthetic fragment of AgRP, AgRP (83-132), are inverse agonists at the MC1R and MC4R, respectively. This study demonstrates the smaller analogs AgRP (87-120) and ASIP [90-132 (L89Y)], and short peptides Yc[CRFFNAFC]Y and Qc[CRFFRSAC]S are also MC4R inverse agonists. Furthermore, the relative affinity of the series of MC4R ligands for displacement of radiolabeled antagonist 125I-AgRP (86-132) versus radiolabeled agonist 125I-NDP-MSH did not correlate with ligand efficacy, which is more consistent with an induced-fit model than a simple two-state model of MC4R activation. These data shed new light on the determinants and mechanism of inverse agonism at the MC4R.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/fisiología , Proteínas/fisiología , beta-MSH/análogos & derivados , Proteína de Señalización Agouti , Proteína Relacionada con Agouti , Secuencias de Aminoácidos , Línea Celular , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Hipotálamo/patología , Cinética , Ligandos , Hormonas Estimuladoras de los Melanocitos/farmacología , Modelos Moleculares , Obesidad , Péptidos/química , Péptidos Cíclicos/farmacología , Pigmentación , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Melanocortina/antagonistas & inhibidores , Receptores de Melanocortina/química , Proteínas Recombinantes/química , alfa-MSH/metabolismo , beta-MSH/farmacologíaRESUMEN
We report cloning, pharmacological characterization, tissue distribution, detailed brain mapping, and role in control of food intake of melanocortin 4 receptor in goldfish (gMC4R). The gMC4R protein has 68% identity with the human ortholog and is conserved in important functional domains. Pharmacological profiling showed similar affinities and potency order to hMC4R for MSH peptides, whereas MTII and HS024 were identified as high-affinity agonist and antagonist analogs, respectively. The gMC4R-mRNA was found in brain and some peripheral tissues including the ovary, gill, and spleen. Detailed MC4R-mRNA mapping showed expression in main neuroendocrine and food intake-controlling areas. High expression levels were found in the telencephalon, preoptic area, ventral thalamus, tuberal hypothalamus, and hypothalamic inferior lobe. By RT-PCR, low levels were also detected in the cerebellum, medulla, and spinal cord. Intracerebroventricular MTII administration inhibited food intake in 24-h fasted animals in a dose-dependent manner, whereas HS024 stimulated food intake in fed animals, suggesting that melanocortins exert a tonic inhibitory effect on food intake, which is mediated through central MC4R signaling. The conserved central expression pattern and physiological role in regulation of food intake for the MC4R suggests that neuronal pathways of the melanocortin system may be important for regulation of energy homeostasis in most vertebrates.
Asunto(s)
Química Encefálica/fisiología , Ingestión de Alimentos/fisiología , Carpa Dorada/fisiología , Receptores de Corticotropina/genética , alfa-MSH/análogos & derivados , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Mapeo Encefálico , Clonación Molecular , Femenino , Expresión Génica/fisiología , Inyecciones Intraventriculares , Masculino , Datos de Secuencia Molecular , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , ARN Mensajero/análisis , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/metabolismo , Especificidad de la Especie , alfa-MSH/metabolismo , alfa-MSH/farmacología , beta-MSH/metabolismo , beta-MSH/farmacología , gamma-MSH/metabolismo , gamma-MSH/farmacologíaRESUMEN
Intracerebroventricular (ICV) injection of Agouti related protein (AgRP), an endogenous melanocortin 3 and 4 receptor (MC3/4-R) antagonist, produces a prolonged increase in food intake. To clarify the roles of the MC3-R and MC4-R in AgRP-induced hyperphagia, the feeding effect of AgRP (83-132) was compared with that of the selective MC4-R antagonist, JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH11-22). Single ICV administration of AgRP (83-132) increased food intake for 48 h whilst ICV JKC-363 increased food intake for 8h. An increase in body weight at 24 and 48 h was observed following AgRP (83-132) but not JKC-363 treatment. These data suggest that the sustained orexigenic action of AgRP (83-132) may not be through MC4-R antagonism.
Asunto(s)
Apetito/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Proteínas/farmacología , Receptores de Corticotropina/metabolismo , Proteína Relacionada con Agouti , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Conducta Alimentaria , Humanos , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Masculino , Péptidos/química , Péptidos Cíclicos/farmacología , Ratas , Ratas Wistar , Receptor de Melanocortina Tipo 3 , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/antagonistas & inhibidores , Factores de Tiempo , beta-MSH/análogos & derivados , beta-MSH/farmacologíaRESUMEN
Hypothalamic pro-opiomelanocortin neurones have an established role in the control of feeding. While pro-opiomelanocortin is the precursor for at least three melanocortin peptides, alpha-, beta- and gamma-melanocyte-stimulating hormone (MSH), it has been widely assumed that alpha-MSH is the predominant ligand involved. We compared the effects of centrally administered alpha-, beta- and gamma(2)-MSH on hypothalamic neuronal activation and on food intake in rats fasted for 48 h. Significant reductions in food intake were seen with alpha-MSH (first hour) and gamma(2)-MSH (second hour) but not with beta-MSH. The pattern of neuronal activation, assessed by the detection of early growth response factor-1 protein, showed considerable overlap; all three melanocortins activated cells in the arcuate, ventromedial, paraventricular, periventricular and supraoptic nuclei, as well as the preoptic area. alpha-MSH and beta-MSH produced activation in the dorsomedial nuclei while gamma(2)-MSH was only weakly active here. Retrograde labelling by systemic Fluorogold injection revealed that many cells activated by MSH compounds in the arcuate, paraventricular, periventricular and supraoptic nuclei (but not dorsomedial or ventromedial) project outside the blood-brain barrier and are therefore likely to include neuroendocrine cells. Desacetyl-alpha-MSH, which has previously been reported to lack effects on feeding, produced no discernible neuronal activation in the hypothalamus. Our finding that both the pattern of neuronal activation and the distribution of neuroendocrine cells activated in response to these closely related peptides show only partial overlap suggests that, in addition to common pathways, there may exist distinct hypothalamic circuits activated by different pro-opiomelanocortin products. The slower time course of gamma(2)-MSH- versus alpha-MSH-induced suppression of feeding provides further support for the notion that the biological responses to individual melanocortin peptides may involve distinct neuronal mechanisms.