RESUMO
Ghrelin is a peptide hormone involved in multiple functions, including growth hormone release stimulation, food intake regulation, and metabolic and cytoprotective effect. A novel family of peptides with internal cycles was designed as ghrelin analogs and the biological activity of two of them (A228 and A233) was experimentally studied in-depth. In this work, an in silico strategy was developed for describing and assessing the binding modes of A228 and A233 to GHS-R1a (ghrelin receptor) comparing it with ghrelin and GHRP-6 peptides. Several reported structures of different G protein coupled receptors were used as templates, to obtain a good quality model of GHS-R1a. The best model was selected by preliminary molecular docking with ghrelin and GHRP-6. Docking was used to estimate peptide orientations in the binding site of the best model, observing a superposition of its N-terminal and its first aromatic residue. To test the complex stability in time, the C-terminal fragments of each peptide were added and the complexes were inserted a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, performing a molecular dynamic simulation for 100 ns using the CHARMM36 force field. Despite of the structural differences, the studied peptides share a common binding mode; the N-terminal interacts with E124 and the aromatic residue close to it, with the aromatic cluster (F279, F309, and F312). A preliminary pharmacophore model, consisting in a positive charged amine and an aromatic ring at an approximate distance of 0.79 nm, can be proposed. The results here described could represent a step forward in the efficient search of new ghrelin analogs.
Assuntos
Simulação por Computador , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores de Grelina/agonistas , Sequência de Aminoácidos , Animais , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Receptores de Grelina/química , Receptores de Grelina/metabolismoRESUMO
Ghrelin is a recently discovered peptide, mainly produced in the stomach and involved in body's energy-maintenance processes. Ghrelin exerts its actions by activating the growth hormone secretagogue receptor (GHS-R). Recent analyses indicate that ghrelin targets the brain to regulate a wealth of functions, including behavioral responses that have been associated with stress and anxiety mechanisms. In this context, evidence shows the presence of GHS-R receptors in the dorsal raphe nucleus (DRN), the main source of serotonergic neurons that innervate encephalic structures involved in emotional control. Our study aims to evaluate the effects of the pharmacological manipulation of ghrelin receptors located in the DRN on the expression of the behavioral responses of Wistar rats. Such responses were assessed in the elevated T maze (ETM), an experimental model that allows the measurement, in the same animal, of two defensive tasks, inhibitory avoidance and escape. Our results showed that the intra-DRN infusion of ghrelin impaired the acquisition of inhibitory avoidance, an anxiolytic-like effect, and facilitated the expression of escape response in the ETM, indicating a panicogenic-like effect. The intra-DRN administration of the ghrelin receptor (GHS-R1a) antagonist PF-04628935 did not alter the behavioral tasks assessed in the ETM. Finally, our results revealed that intra-DRN infusions of PF-04628935 prior to the administration of ghrelin into this area neutralized the behavioral effects obtained in the ETM. Taken together, our data reveal the involvement of DRN GHS-R1a receptors in the regulation of defensive tasks that have been associated with generalized anxiety and panic disorders.
Assuntos
Aprendizagem da Esquiva/fisiologia , Núcleo Dorsal da Rafe/metabolismo , Reação de Fuga/fisiologia , Receptores de Grelina/metabolismo , Animais , Ansiolíticos/metabolismo , Ansiolíticos/farmacologia , Ansiedade/metabolismo , Transtornos de Ansiedade/metabolismo , Aprendizagem da Esquiva/efeitos dos fármacos , Núcleo Dorsal da Rafe/efeitos dos fármacos , Reação de Fuga/efeitos dos fármacos , Grelina/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Ratos , Ratos Wistar , Neurônios Serotoninérgicos/metabolismoRESUMO
Energy balance is regulated by ghrelin which is a neuroendocrine modulator. Ghrelin is expressed in reproductive organs. However, the role of ghrelin during in vitro maturation (IVM) and bovine preimplantational development is limited. The purpose of this study was to measure the expression of ghrelin (GHRL) and its receptor growth hormone secretagogue receptor 1A (GHS-R1A) mRNA, and determine cumulus oocyte complex (COC) viability after IVM with 0, 20, 40 and 60 pM of ghrelin. Also, pronuclear formation was recorded after in vitro fertilization (IVF). GHRL and GHS-R1A mRNA expression in oocyte and cumulus cells (CCs) was assessed using reverse transcription-polymerase chain reaction (PCR). Oocyte and CC viability were analyzed with the fluorescein diacetate fluorochrome-trypan blue technique. Pronuclear formation was determined 18 hours after IVF with Hoechst 33342. The results demonstrated that ghrelin mRNA is present in oocyte and CCs before and after 24 hours IVM with all treatments. Ghrelin receptor, GHS-R1A, was only detected in oocytes and CCs after 24 hours IVM with 20, 40 and 60 pM of ghrelin. Oocyte viability was not significantly different (P=0.77) among treatments. However, CC viability was significantly lower (P=0.04) when COCs were matured with ghrelin (77.65, 72.10, 66.32 and 46.86% for 0, 20, 40, and 60 pM of ghrelin, respectively). The chance of two pronuclei forming were higher (P=0.03) when ghrelin was not be added to the IVM medium. We found that ghrelin negatively impacts CC viability and pronuclear formation.
RESUMO
Prior evidence indicates that ghrelin is involved in the integration of cardiovascular functions and behavioral responses. Ghrelin actions are mediated by the growth hormone secretagogue receptor subtype 1a (GHS-R1a), which is expressed in peripheral tissues and central areas involved in the control of cardiovascular responses to stress. AIMS: In the present study, we assessed the role of ghrelin - GHS-R1a axis in the cardiovascular reactivity to acute emotional stress in rats. MAIN METHODS AND KEY FINDINGS: Ghrelin potentiated the tachycardia evoked by restraint and air jet stresses, which was reverted by GHS-R1a blockade. Evaluation of the autonomic balance revealed that the sympathetic branch modulates the ghrelin-evoked positive chronotropy. In isolated hearts, the perfusion with ghrelin potentiated the contractile responses caused by stimulation of the beta-adrenergic receptor, without altering the amplitude of the responses evoked by acetylcholine. Experiments in isolated cardiomyocytes revealed that ghrelin amplified the increases in calcium transient changes evoked by isoproterenol. SIGNIFICANCE: Taken together, our results indicate that the Ghrelin-GHS-R1a axis potentiates the magnitude of stress-evoked tachycardia by modulating the autonomic nervous system and peripheral mechanisms, strongly relying on the activation of cardiac calcium transient and beta-adrenergic receptors.