RESUMO
Several neuropeptides with the C-terminal RFamide sequence have been identified in the hypothalamus of a variety of vertebrates. Among the RFamide peptide groups, however, only LPXRFamide peptides, including gonadotropin-inhibitory hormone, have been characterized in the avian brain. In the present study, we sought for the presence of other RFamide peptides in the avian hypothalamus. We identified a cDNA encoding an RFamide peptide orthologous to 26RFa (also referred to as QRFP) in the hypothalamus of the Japanese quail. The deduced quail 26RFa precursor consisted of 120-amino-acid residues, encoding one RFamide peptide with 27 amino acids. This RFamide peptide was flanked at the N terminus by a dibasic amino acid cleavage site and at the C terminus by a glycine amidation signal. Quantitative RT-PCR analysis demonstrated specific expression of quail 26RFa mRNA in the diencephalon including the hypothalamus. Furthermore, mass spectrometry analysis revealed the presence of a peptide exhibiting the mass of mature 26RFa, indicating that the peptide is actually produced from the precursor in the diencephalon. 26RFa-producing cell bodies were localized in the anterior hypothalamic nucleus in the brain. Synthetic 26RFa increased intracellular Ca(2+) concentration in HEK293T cells transfected with the chicken G protein-coupled receptor GPR103. Intracerebroventricular injection of 26RFa in broiler chicks stimulated feeding behavior. These data provide the first evidence for the occurrence of the peptide 26RFa in the avian hypothalamus and indicate that this peptide exerts orexigenic activity.
Assuntos
Proteínas Aviárias/genética , Coturnix/genética , Hormônios Hipotalâmicos/genética , Hipotálamo/metabolismo , Neuropeptídeos/genética , Receptores Acoplados a Proteínas G/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/metabolismo , Proteínas Aviárias/farmacologia , Cálcio/metabolismo , Linhagem Celular , Galinhas/genética , Galinhas/metabolismo , Galinhas/fisiologia , Coturnix/metabolismo , DNA Complementar/química , DNA Complementar/genética , Ingestão de Alimentos/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Hormônios Hipotalâmicos/metabolismo , Hormônios Hipotalâmicos/farmacologia , Injeções Intraventriculares , Masculino , Dados de Sequência Molecular , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Melanin-concentrating hormone (MCH) is a neuropeptide that exhibits potent orexigenic activity. In rodents, it exerts its actions by interacting with one receptor, MCH(1) receptor which is expressed in many parts of the central nervous system (CNS). To study the physiological implications of the MCH system, we need to be able to block it locally and acutely. This necessitates the use of MCH(1) receptor antagonists. While MCH(1) receptor antagonists have been previously reported, they are mainly not accessible to academic research. We apply here a strategy that leads to the isolation of a high affinity and selective MCH(1) receptor antagonist amenable to in vivo analyses without further chemical modifications. This antagonist, TPI 1361-17, was identified through the screening of multiple non-peptide positional scanning synthetic combinatorial libraries (PS-SCL) totaling more than eight hundred thousand compounds in conditions that allow for the identification of only high-affinity compounds. TPI 1361-17 exhibited an IC(50) value of 6.1 nM for inhibition of 1 nM MCH-induced Ca(2+) mobilization and completely displaced the binding of [(125)I] MCH to rat MCH(1) receptor. TPI 1361-17 was found specific, having no affinity for a variety of other G-protein coupled receptors and channels. TPI 1361-17 was found active in vivo since it blocked MCH-induced food intake by 75%. Our results indicate that TPI 1361-17 is a novel and selective MCH(1) receptor antagonist and is an effective tool to study the physiological functions of the MCH system. These results also illustrate the successful application of combinatorial library screening to identify specific surrogate antagonists in an academic setting.
Assuntos
Técnicas de Química Combinatória , Proteínas do Citoesqueleto/antagonistas & inibidores , Etilenotioureia/análogos & derivados , Guanidinas/farmacologia , Animais , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ingestão de Alimentos/efeitos dos fármacos , Etilenotioureia/química , Etilenotioureia/farmacologia , Guanidinas/química , Humanos , Masculino , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato , Paladar/efeitos dos fármacos , Tioureia/química , Tioureia/farmacologiaRESUMO
Melanin-concentrating hormone (MCH) is a neuropeptide that was originally isolated from salmon pituitary where it causes pigment aggregation. MCH is also abundantly present in mammalian neurons and expressed in the lateral hypothalamus and zona incerta, brain regions that are known to be at the center of feeding behavior. MCH binds to and activates two G protein-coupled receptors, MCH1R and MCH2R. Although MCH2R is non-functional in rodents, genetic and pharmacological studies have demonstrated that rodent MCH1R is involved in the regulation of feeding behavior and energy balance. Unexpectedly, some antagonists have provided evidence that MCH signaling participates in the regulation of other processes, such as emotion and stress. The discovery of MCH receptors has extensively promoted the progress of MCH studies and may represent an ideal example of how deorphanized receptors can open new directions toward more detailed physiological studies.