RESUMO
BACKGROUND AND PURPOSE: The neuropeptide 26RFa and its cognate receptor GPR103 are involved in the control of food intake and bone mineralization. Here, we have tested, experimentally, the predicted ligand-receptor interactions by site-directed mutagenesis of GPR103 and designed point-substituted 26RFa analogues. EXPERIMENTAL APPROACH: Using the X-ray structure of the ß2 -adrenoceptor, a 3-D molecular model of GPR103 has been built. The bioactive C-terminal octapeptide 26RFa(19-26) , KGGFSFRF-NH2 , was docked in this GPR103 model and the ligand-receptor complex was submitted to energy minimization. KEY RESULTS: In the most stable complex, the Phe-Arg-Phe-NH2 part was oriented inside the receptor cavity, whereas the N-terminal Lys residue remained outside. A strong intermolecular interaction was predicted between the Arg(25) residue of 26RFa and the Gln(125) residue located in the third transmembrane helix of GPR103. To confirm this interaction experimentally, we tested the ability of 26RFa and Arg-modified 26RFa analogues to activate the wild-type and the Q125A mutant receptors transiently expressed in CHO cells. 26RFa (10(-6) M) enhanced [Ca(2+) ]i in wild-type GPR103-transfected cells, but failed to increase [Ca(2+) ]i in Q125A mutant receptor-expressing cells. Moreover, asymmetric dimethylation of the side chain of arginine led to a 26RFa analogue, [ADMA(25) ]26RFa(20-26) , that was unable to activate the wild-type GPR103, but antagonized 26RFa-evoked [Ca(2+) ]i increase. CONCLUSION AND IMPLICATIONS: Altogether, these data provide strong evidence for a functional interaction between the Arg(25) residue of 26RFa and the Gln(125) residue of GPR103 upon ligand-receptor activation, which can be exploited for the rational design of potent GPR103 agonists and antagonists.
Assuntos
Modelos Moleculares , Neuropeptídeos/metabolismo , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligopeptídeos/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
The hypothalamus senses hormones and nutrients in order to regulate energy balance. In particular, detection of hypothalamic glucose levels has been shown to regulate both feeding behavior and peripheral glucose homeostasis, and impairment of this regulatory system is believed to be involved in the development of obesity and diabetes. Several data clearly demonstrate that glial cells are key elements in the perception of glucose, constituting with neurons a "glucose-sensing unit". Characterization of this interplay between glia and neurons represents an exciting challenge, and will undoubtedly contribute to identify new candidates for therapeutic intervention. The purpose of this review is to summarize the current data that stress the importance of glia in central glucose-sensing. The nature of the glia-to-neuron signaling is discussed, with a special focus on the endozepine ODN, a potent anorexigenic peptide that is highly expressed in hypothalamic glia.
Assuntos
Comunicação Celular/fisiologia , Glucose/metabolismo , Hipotálamo , Neuroglia , Neurônios , Transdução de Sinais/fisiologia , Animais , Humanos , Hipotálamo/citologia , Hipotálamo/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismoRESUMO
The present study aimed to investigate the distribution of the octadecaneuropeptide (ODN) in the goldfish brain and to look for a possible effect of ODN on somatolactin (SL) release from pituitary cells. A discrete population of ODN-immunoreactive neurones was localised in the lateral part of the nucleus lateralis tuberis. These neurones sent projections through the neurohypophyseal tract towards the neurohypophysis, and nerve fibres were seen in the close vicinity of SL-producing cells in the pars intermedia. Incubation of cultured goldfish pituitary cells with graded concentrations of ODN (10(-9) -10(-5 ) m) induced a dose-dependent stimulation of SL-ß, but not SL-α, release. ODN-evoked SL release was blocked by the metabotrophic endozepine receptor antagonist cyclo(1-8) [DLeu(5) ]OP but was not affected by the central-type benzodiazepine receptor antagonist flumazenil. ODN-induced SL release was suppressed by treatment with the phospholipase C (PLC) inhibitor U-73122 but not with the protein kinase A (PKA) inhibitor H-89. These results indicate that, in fish, ODN produced by hypothalamic neurones acts as a hypophysiotrophic neuropeptide stimulating SL release. The effect of ODN is mediated through a metabotrophic endozepine receptor positively coupled to the PLC/inositol 1,4,5-trisphosphate/protein kinase C-signalling pathway.
Assuntos
Proteínas de Peixes/metabolismo , Glicoproteínas/metabolismo , Neuropeptídeos/farmacologia , Hipófise/efeitos dos fármacos , Hormônios Hipofisários/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Carpa Dourada , Imuno-Histoquímica , Hipófise/citologia , Hipófise/metabolismo , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
Identification of novel molecules that can induce neuronal differentiation of embryonic stem (ES) cells is essential for deciphering the molecular mechanisms of early development and for exploring cell therapy approaches. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are known to be implicated early during ontogenesis in cell proliferation and neuronal differentiation. The aim of the present study was to determine the effects of VIP and PACAP on functional differentiation of ES cells. Quantitative-reverse transcription-polymerase chain reaction analysis showed an inversion of the expression pattern of PAC1 and VPAC1 receptors with time. ES cells expressed genes encoding extracellular signal-regulated kinase 1 and 2 and c-jun amino terminal kinase1. ES cells also expressed T-type α1I and α1G, L-type α1C and α1D, and N-type α1B calcium channel subunit mRNAs. Both peptides modified the shape of undifferentiated ES cells into bipolar cells expressing the neuronal marker neuron-specific enolase (NSE). Immunostaining indicated that PACAP intensified T-type α1I subunit immunoreactivity, whereas VIP increased L-types α1C and α1D, as well as N-type α1B subunit. Electrophysiological recording showed that VIP and PACAP enhanced transient calcium current. Moreover, VIP generated sustained calcium current. These findings demonstrate that PACAP and VIP induce morphological and functional differentiation of ES cells into a neuronal phenotype. Both peptides promote functional maturation of calcium channel subunits, suggesting that they can facilitate the genesis of cellular excitability.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Imuno-Histoquímica , Camundongos , Células-Tronco Neurais/citologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peptídeo Intestinal Vasoativo/genéticaRESUMO
The kisspeptin system has emerged as one of the main puberty gatekeepers among vertebrates. The European eel (Anguilla anguilla) is a remarkable model due to its phylogenetical position at the basis of teleosts, and its unique life cycle with a blockade of puberty before reproductive migration. We cloned the full-length coding sequence of a kisspeptin receptor (Kissr) in the eel. Comparison of Kissr sequences assigned the eel Kissr to a basal position in a clade including most of the known teleost Kissr, in agreement with the eel phylogenetical position. Eel Kissr tissue distribution was analyzed by quantitative real-time PCR. Eel Kissr was highly expressed in the brain, especially in the telencephalon and di-/mes-encephalon, while a very low or undetectable expression was observed in various peripheral organs. A high expression of Kissr was also found in the pituitary indicating a possible direct pituitary role of kisspeptin. Primary cultures of eel pituitary cells were performed to investigate the direct effects of kisspeptin on pituitary hormone expression. Human/lamprey kisspeptin exerted a time- and dose-dependent inhibitory effect on LHß expression. All other tested kisspeptins had a similar inhibitory effect on LHß expression. The inhibitory effect of kisspeptins was exerted specifically on LHß as no change was induced on the expression of other glycoprotein hormone subunits (GPα, FSHß and TSHß) nor of growth hormone. These data provide the first evidence for the existence, in the European eel, of a kisspeptin system, which may play a direct inhibitory role on pituitary LHß expression.
Assuntos
Kisspeptinas/farmacologia , Hormônio Luteinizante/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Anguilla , Animais , Sequência de Bases , Células Cultivadas , Feminino , Gonadotropinas/metabolismo , Dados de Sequência Molecular , Filogenia , Hipófise/citologia , Hipófise/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/classificação , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
In neurological insults, such as cerebral ischemia and traumatic brain injury, complex molecular mechanisms involving inflammation and apoptosis are known to cause severe neuronal cell loss, emphasizing the necessity of developing therapeutic strategies targeting simultaneously these two processes. Over the last decade, numerous in vitro and in vivo studies have demonstrated the unique therapeutical potential of pituitary adenylate cyclase-activating polypeptide (PACAP) for the treatment of neuronal disorders involving apoptotic cell death and neuroinflammation. The neuroprotective activity of PACAP is based on its capacity to reduce the production of deleterious cytokines from activated microglia, to stimulate the release of neuroprotective agents from astrocytes and to inhibit pro-apoptotic intracellular pathways. However, the use of PACAP as a clinically applicable drug is hindered by its peptidic nature. As most natural peptides, native PACAP shows poor metabolic stability, low bioavailability, inadequate distribution and rapid blood clearance. Moreover, injection of PACAP to human can induce peripheral adverse side effects. Therefore, targeted chemical modifications and/or conjugation of PACAP to different macromolecules are required to improve the pharmacokinetic and pharmacological properties of PACAP. This review presents the chemical, biochemical and pharmacological strategies that are currently under development to convert PACAP from a hypophysiotropic neurohormone into a clinically relevant neuroprotective drug.
Assuntos
Descoberta de Drogas/métodos , Fármacos Neuroprotetores/uso terapêutico , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/imunologia , Lesões Encefálicas/patologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/imunologia , Isquemia Encefálica/patologia , Sobrevivência Celular/efeitos dos fármacos , Citocinas/imunologia , Humanos , Fatores de Crescimento Neural/metabolismo , Fármacos Neuroprotetores/efeitos adversos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/efeitos adversos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologiaRESUMO
I.c.v. administration of the octadecaneuropeptide (ODN), a peptide derived from diazepam-binding inhibitor (DBI), induces anorexigenic and anxiogenic-like actions in rodents. We have recently shown that, in goldfish, i.c.v. injection of ODN also reduces food consumption via the metabotropic endozepine receptor. However, there is little information regarding the structure of DBI and the psychophysiological roles of endozepines in fish. Therefore, in the present study, we isolated and cloned a cDNA encoding goldfish DBI. The deduced sequence exhibits high similarity with non-mammalian DBIs, and we investigated the effect of homologous ODN on psychomotor activity in goldfish. i.c.v. injection of synthetic goldfish ODN at 10 pmol/g body weight (BW) stimulated locomotor activity. Since intact goldfish placed in a tank with both black and white background areas prefers the black compartment, we developed a method for measuring the time taken for fish to move from the black to the white area. I.c.v. administration of diazepam (35 and 350 pmol/g BW) decreased, whereas i.c.v. administration of ODN (10 pmol/g BW) or the central-type benzodiazepine receptor inverse agonist FG-7142 (9 pmol/g BW) increased the time taken to move from the black to the white background area. The anxiogenic-like effect of ODN was blocked by the central-type benzodiazepine receptor antagonist flumazenil (100 pmol/g BW), but was not affected by the metabotropic endozepine receptor antagonist cyclo1-8[d-Leu(5)]octapeptide (100 pmol/g BW). These data indicate that ODN can potently affect locomotor and psychomotor activities in goldfish and that this action is mediated via the central-type benzodiazepine receptor-signaling pathway.
Assuntos
Transtornos de Ansiedade/induzido quimicamente , Transtornos de Ansiedade/fisiopatologia , Inibidor da Ligação a Diazepam/fisiologia , Carpa Dourada/fisiologia , Atividade Motora/fisiologia , Neuropeptídeos/fisiologia , Fragmentos de Peptídeos/fisiologia , Animais , Comportamento Animal/fisiologia , Inibidor da Ligação a Diazepam/genética , Inibidor da Ligação a Diazepam/isolamento & purificação , Modelos Animais de Doenças , Feminino , Masculino , Neuropeptídeos/genética , Neuropeptídeos/isolamento & purificação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificaçãoRESUMO
Accumulating evidence indicate that the neuropeptide urotensin II and urotensin II receptors are expressed in subsets of mammal spinal motoneurons. In fact, a role for the peptide in the regulation of motoneuron function at neuromuscular junction has been suggested, while roles for urotensin II at central synapses in spinal cord have never been addressed. We found that urotensin II receptors were closely associated with cholinergic terminals apposed to a subset of motoneuron and non-motoneuron cell bodies in the ventral horn of the adult mouse cervical spinal cord; urotensin II receptor was also expressed on non-cholinergic nerve terminals. In particular, urotensin II receptor appeared associated with both large cholinergic C-boutons and standard cholinergic terminals contacting some motoneuron perikarya. Cholinergic nerve terminals from mouse cervical spinal cord were equipped with functional presynaptic urotensin II receptors linked to excitation of acetylcholine release. In fact, functional experiments conducted on cervical spinal synaptosomes demonstrated a urotensin II evoked calcium-dependent increase in [(3)H]acetylcholine release pharmacologically verified as consistent with activation of urotensin II receptors. In spinal cord these actions would facilitate cholinergic transmission. These data indicate that, in addition to its role at the neuromuscular junction, urotensin II may control motor function through the modulation of motoneuron activity within the spinal cord.
Assuntos
Acetilcolina/metabolismo , Vértebras Cervicais , Terminações Pré-Sinápticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Medula Espinal/metabolismo , Urotensinas/fisiologia , Animais , Masculino , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Junção Neuromuscular/citologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Medula Espinal/efeitos dos fármacos , Ureia/análogos & derivados , Ureia/farmacologia , Urotensinas/antagonistas & inibidoresRESUMO
Gonadotrophin-releasing hormone (GnRH) is the primary hypothalamic factor responsible for the control of gonadotrophin secretion in vertebrates. However, within the last decade, two other hypothalamic neuropeptides have been found to play key roles in the control of reproductive functions: gonadotrophin-inhibitory hormone (GnIH) and kisspeptin. In 2000, we discovered GnIH in the quail hypothalamus. GnIH inhibits gonadotrophin synthesis and release in birds through actions on GnRH neurones and gonadotrophs, mediated via GPR147. Subsequently, GnIH orthologues were identified in other vertebrate species from fish to humans. As in birds, mammalian and fish GnIH orthologues inhibit gonadotrophin release, indicating a conserved role for this neuropeptide in the control of the hypothalamic-pituitary-gonadal axis across species. Subsequent to the discovery of GnIH, kisspeptin, encoded by the KiSS-1 gene, was discovered in mammals. By contrast to GnIH, kisspeptin has a direct stimulatory effect on GnRH neurones via GPR54. GPR54 is also expressed in pituitary cells, but whether gonadotrophs are targets for kisspeptin remains unresolved. The KiSS-1 gene is also highly conserved and has been identified in mammals, amphibians and fish. We have recently found a second isoform of KiSS-1, designated KiSS-2, in several vertebrates, but not birds, rodents or primates. In this review, we highlight the discovery, mechanisms of action, and functional significance of these two chief regulators of the reproductive axis.
Assuntos
Glicoproteínas/metabolismo , Gonadotropinas/antagonistas & inibidores , Neuropeptídeos/metabolismo , Reprodução/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Evolução Biológica , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Gonadotropinas/genética , Gonadotropinas/metabolismo , Humanos , Dados de Sequência Molecular , Neurônios/metabolismo , Neuropeptídeos/classificação , Neuropeptídeos/genética , Filogenia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
In the central nervous system of mammals, the gene encoding diazepam-binding inhibitor (DBI) is exclusively expressed in glial cells. Previous studies have shown that central administration of a DBI processing product, the octadecaneuropeptide ODN, causes a marked inhibition of food consumption in rodents. Paradoxically, however, the effect of food restriction on DBI gene expression has never been investigated. Here, we show that in mice, acute fasting dramatically reduces DBI mRNA levels in the hypothalamus and the ependyma bordering the third and lateral ventricles. I.p. injection of insulin, but not of leptin, selectively stimulated DBI expression in the lateral ventricle area. These data support the notion that glial cells, through the production of endozepines, may relay peripheral signals to neurons involved in the central regulation of energy homeostasis.
Assuntos
Inibidor da Ligação a Diazepam/metabolismo , Jejum , Neuroglia/metabolismo , Neuropeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Regulação para Baixo , Epêndima/metabolismo , Hipotálamo/metabolismo , Injeções Intraperitoneais , Insulina/administração & dosagem , Ventrículos Laterais/metabolismo , Leptina/administração & dosagem , Masculino , Camundongos , Neuropeptídeos/genética , Fragmentos de Peptídeos/genética , Ligação Proteica , Terceiro Ventrículo/metabolismo , Transcrição GênicaRESUMO
Prolyl endopeptidase (PEP) is a serine protease that cleaves small peptides at the carboxyl side of L-proline. PEP has been reported to have important functions in the brain being implicated in learning and memory processes, psychological disorders and neurodegenerative diseases. Several PEP substrates have been shown to play a role during brain development and this observation led us to investigate the expression of PEP mRNA in the rat brain and spinal cord, from embryo to adult stages. In situ hybridization revealed that PEP mRNA is expressed early, from embryonic day 15, notably in germinative areas including the neocortical, hippocampal, pallidal, thalamic, anterior hypothalamic, tectal, cerebellar, pontine and medullary neuroepithelia. PEP mRNA was also found in the differentiating fields of the olfactory bulb, the orbital and cingulate cortex, the hippocampal formation, the cortical plate and the subventricular zone of the cortex. Quantitative RT-PCR analysis in various brain areas and the spinal cord showed that PEP mRNA levels are more abundant during the perinatal stages, coinciding with a period of neuronal migration and differentiation. From then on, PEP mRNA expression decreased, reaching its lowest levels at adulthood. Overall, the present data support the possibility that PEP exerts specific functions related to neurodevelopment besides those proposed to date.
Assuntos
Diferenciação Celular/genética , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/enzimologia , Neurogênese/genética , RNA Mensageiro/metabolismo , Serina Endopeptidases/genética , Animais , Padronização Corporal/genética , Mapeamento Encefálico , Movimento Celular/genética , Sistema Nervoso Central/crescimento & desenvolvimento , Feminino , Masculino , Neurônios/citologia , Neurônios/enzimologia , Organogênese/genética , Prolil Oligopeptidases , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Células-Tronco/enzimologiaRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide that was initially isolated from hypothalamus extracts on the basis of its ability to stimulate the production of cAMP in cultured pituitary cells. Recent studies have shown that PACAP exerts potent neuroprotective effects not only in vitro but also in in vivo models of Parkinson's disease, Huntington's disease, traumatic brain injury and stroke. The protective effects of PACAP are based on its capacity to prevent neuronal apoptosis by acting directly on neurons or indirectly through the release of neuroprotective factors by astrocytes. These biological activities are mainly mediated through activation of the PAC1 receptor which is currently considered as a potential target for the treatment of neurodegenerative diseases. However, the use of native PACAP, the endogenous ligand of PAC1, as an efficient neuroprotective drug is actually limited by its rapid degradation. Moreover, injection of PACAP to human induces peripheral side effects which are mainly mediated through VPAC1 and VPAC2 receptors. Strategies to overcome these compromising conditions include the development of metabolically stable analogs of PACAP acting as selective agonists of the PAC1 receptor. This review presents an overview of the structure-activity relationships of PACAP and summarizes the molecular and conformational requirements for activation of PAC1 receptor. The applicability of PACAP analogs as therapeutic agents for treatment of neurodegenerative diseases is also discussed.
Assuntos
Fármacos Neuroprotetores/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Fármacos Neuroprotetores/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Ratos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Relação Estrutura-AtividadeRESUMO
Hypothalamic magnocellular neurons express either one of the neurohypophysial hormones, vasopressin or oxytocin, along with different neuropeptides or neuromodulators. Axonal terminals of these neurons are generally accepted to release solely the two hormones but not others into the circulation. Here, we show that secretin, originally isolated from upper intestinal mucosal extract, is present throughout the hypothalamo-neurohypophysial axis and that it is released from the posterior pituitary under plasma hyperosmolality conditions. In the hypothalamus, it stimulates vasopressin expression and release. Considering these findings together with our previous findings that show a direct effect of secretin on renal water reabsorption, we propose here that secretin works at multiple levels in the hypothalamus, pituitary, and kidney to regulate water homeostasis. Findings presented here challenge previous understanding regarding the neurohypophysis and could provide new concepts in treating disorders related to osmoregulation.