Systemic Ghrelin Treatment Induces Rapid, Transient, and Asymmetric Changes in the Metabolic Activity of the Mouse Brain.

INTRODUCTION: Ghrelin regulates a variety of functions by acting in the brain. The targets of ghrelin in the mouse brain have been mainly mapped using immunolabeling against c-Fos, a transcription factor used as a marker of cellular activation, but such analysis has several limitations. Here, we used positron emission tomography in mice to investigate the brain areas responsive to ghrelin. METHODS: We analyzed in male mice the brain areas responsive to systemically injected ghrelin using positron emission tomography imaging of 18F-fluoro-2-deoxyglucose (18F-FDG) uptake, an indicator of metabolic rate. Additionally, we studied if systemic administration of fluorescent ghrelin or native ghrelin displays symmetric accessibility or induction of c-Fos, respectively, in the brain of male mice. RESULTS: Ghrelin increased 18F-FDG uptake in few specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain at 0-10-min posttreatment. At the 10-20 and 20-30 min posttreatment, ghrelin induced mixed changes in 18F-FDG uptake in specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain, as well as in areas of the olfactory areas, hippocampal and retrohippocampal regions, hypothalamus, pons, medulla, and even the cerebellum. Ghrelin-induced changes in 18F-FDG uptake were transient and asymmetric. Systemically administrated fluorescent-ghrelin-labeled midline brain areas known to contain fenestrated capillaries and the hypothalamic arcuate nucleus, where a symmetric labeling was observed. Ghrelin treatment also induced a symmetric increased c-Fos labeling in the arcuate nucleus. DISCUSSION/CONCLUSION: Systemically injected ghrelin transiently and asymmetrically affects the metabolic activity of the brain of male mice in a wide range of areas, in a food intake-independent manner. The neurobiological bases of such asymmetry seem to be independent of the accessibility of ghrelin into the brain.

GHSR controls food deprivation-induced activation of CRF neurons of the hypothalamic paraventricular nucleus in a LEAP2-dependent manner.

OBJECTIVE: Prolonged fasting is a major challenge for living organisms. An appropriate metabolic response to food deprivation requires the activation of the corticotropin-releasing factor-producing neurons of the hypothalamic paraventricular nucleus (PVHCRF neurons), which are a part of the hypothalamic-pituitary-adrenal axis (HPA), as well as the growth hormone secretagogue receptor (GHSR) signaling, whose activity is up- or down-regulated, respectively, by the hormones ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2). Since ghrelin treatment potently up-regulates the HPA axis, we studied the role of GHSR in mediating food deprivation-induced activation of the PVHCRF neurons in mice. METHODS: We estimated the activation of the PVHCRF neurons, using immuno-staining against CRF and the marker of neuronal activation c-Fos in brain sections, and assessed plasma levels of corticosterone and glucose in different pharmacologically or genetically manipulated mouse models exposed, or not, to a 2-day food deprivation protocol. In particular, we investigated ad libitum fed or food-deprived male mice that: (1) lacked GHSR gene expression, (2) had genetic deletion of the ghrelin gene, (3) displayed neurotoxic ablation of the hypothalamic arcuate nucleus, (4) were centrally treated with an anti-ghrelin antibody to block central ghrelin action, (5) were centrally treated with a GHSR ligand that blocks ghrelin-evoked and constitutive GHSR activities, or (6) received a continuous systemic infusion of LEAP2(1-12). RESULTS: We found that food deprivation results in the activation of the PVHCRF neurons and in a rise of the ghrelin/LEAP2 molar ratio. Food deprivation-induced activation of PVHCRF neurons required the presence and the signaling of GHSR at hypothalamic level, but not of ghrelin. Finally, we found that preventing the food deprivation-induced fall of LEAP2 reverses the activation of the PVHCRF neurons in food-deprived mice, although it has no effect on body weight or blood glucose. CONCLUSION: Food deprivation-induced activation of the PVHCRF neurons involves ghrelin-independent actions of GHSR at hypothalamic level and requires a decrease of plasma LEAP2 levels. We propose that the up-regulation of the actions of GHSR associated to the fall of plasma LEAP2 level are physiologically relevant neuroendocrine signals during a prolonged fasting.

A Novel Truncated Liver Enriched Antimicrobial Peptide-2 Palmitoylated at its N-Terminal Antagonizes Effects of Ghrelin.

Ghrelin is secreted in the stomach during fasting and targets the growth hormone secretagogue receptor (GHSR1a) in the hypothalamus and brainstem to exert its orexigenic effect. Recently, liver enriched antimicrobial peptide-2 (LEAP2) was identified as an endogenous high-affinity GHSR1a antagonist. LEAP2 is a 40-amino acid peptide with two disulfide bridges and GHRS1a affinity in the N-terminal hydrophobic part. In this study, we tested modified truncated N-terminal peptide LEAP2 (1-14), along with its myristoylated, palmitoylated, and stearoylated analogs, to determine their affinity to and activation of GHSR1a and their anorexigenic effects after acute peripheral administration. The lipidized analogs bound GHSR1a with affinity similar to that of natural LEAP2, and lipidization significantly enhanced the affinity of LEAP2(1-14) to GHSR1a. According to the beta-lactamase reporter gene response, the natural GHSR1a agonist ghrelin activated the receptor with nanomolar EC50 LEAP2(1-14) analogs behaved as inverse agonists of GHSR1a and suppressed internal activity of the receptor with EC50 values in the 10-8 M range. LEAP2(1-14) analogs significantly lowered acute food intake in overnight fasted mice, and palmitoylated LEAP2(1-14) was the most potent. In free-fed mice, all LEAP2(1-14) analogs significantly decreased the orexigenic effect of the stable ghrelin analog [Dpr3]Ghrelin. Moreover, palmitoylated LEAP2(1-14) inhibited the growth hormone (GH) release induced by [Dpr3] Ghrelin and exhibited an increased stability in rat plasma compared with LEAP2(1-14). In conclusion, palmitoylated LEAP2(1-14) had the most pronounced affinity for GHSR1a, had an anorexigenic effect, exhibited stability in rat plasma, and attenuated [Dpr3]Ghrelin-induced GH release. Such properties render palmitoylated LEAP2(1-14) a promising substance for antiobesity treatment. SIGNIFICANCE STATEMENT: The agonist and antagonist of one receptor are rarely found in one organism. For ghrelin receptor (growth hormone secretagogue receptor, GHSR), endogenous agonist ghrelin and endogenous antagonist/inverse agonist liver enriched antimicrobial peptide-2 (LEAP2) co-exist and differently control GHSR signaling. As ghrelin has a unique role in food intake regulation, energy homeostasis, and cytoprotection, lipidized truncated LEAP2 analogs presented in this study could serve not only to reveal the relationship between ghrelin and LEAP2 but also for development of potential anti-obesity agents.

Effects of Motivational Downshifts on Specific Pavlovian-Instrumental Transfer in Rats.

BACKGROUND: Pavlovian stimuli predictive of appetitive outcomes can exert a powerful influence on the selection and initiation of action, a phenomenon termed outcome-selective Pavlovian-instrumental transfer (sPIT). Rodent studies suggest that sPIT is insensitive to motivational downshift induced by outcome devaluation, an effect that is, however, relatively underexplored. METHODS: Here we examined in detail the effects of distinct shifts in motivation from hunger to a state of relative satiety on sPIT in rats. RESULTS: A motivational downshift by outcome-specific devaluation immediately prior to testing markedly reduced overall lever responding and magazine entries but left intact the sPIT effect. A motivational downshift prior testing by (1) giving ad libitum rather than restricted access to maintenance diet in the home cage for 24 hours or by (2) a systemic blockade of hormone secretagogue receptor subtype 1A receptors to inhibit orexigenic actions of ghrelin both reduced overall lever responding and magazine entries. Moreover, these latter motivational downshifts reduced the sPIT effect; however, the sizes of the sPIT effects were still large. CONCLUSIONS: Collectively, our rodent findings indicate that major effects of various motivational downshifts are overall inhibition of lever pressing and magazine approach, possibly reflecting reduced general motivation. The observed effects of motivational downshifts on sPIT have implications with regard to the role of general motivating effects in sPIT and to the contribution of Pavlovian-instrumental interactions to excessive food seeking as well as obesity in humans.

Fluorescent P-Hydroxyphosphole for Peptide Labeling through P-N Bond Formation.

Synthesis of fluorescent P-hydroxybinaphtylphosphole-oxide or -sulfide was achieved by trapping a binaphtyl dianion with methyl dichlorophosphite or P-(N,N-diethylamino)dichlorophosphine, followed by oxidation or sulfuration of the P-center. After saponification or acid hydrolysis, the P-hydroxyphospholes were coupled to peptides using the coupling agent BOP, under the conditions required for the synthesis in solution or on a solid support. This new method was illustrated by the labeling of the JMV2959, a potent antagonist of the Growth Hormone Secretagogue Receptor type 1a (GHS-R1a). The labeled conjugates were used to characterize GHSR ligands by competition assays, based on Fluorescence Resonance Energy Transfer (FRET). Such P-hydroxyphosphole-oxide or -sulfide constitute a promising new class of compact fluorophores with large Stokes shift, for labeling biomolecules by grafting through the phosphorus atom.

Structure and dynamics of G protein-coupled receptor-bound ghrelin reveal the critical role of the octanoyl chain.

Ghrelin plays a central role in controlling major biological processes. As for other G protein-coupled receptor (GPCR) peptide agonists, the structure and dynamics of ghrelin bound to its receptor remain obscure. Using a combination of solution-state NMR and molecular modeling, we demonstrate that binding to the growth hormone secretagogue receptor is accompanied by a conformational change in ghrelin that structures its central region, involving the formation of a well-defined hydrophobic core. By comparing its acylated and nonacylated forms, we conclude that the ghrelin octanoyl chain is essential to form the hydrophobic core and promote access of ghrelin to the receptor ligand-binding pocket. The combination of coarse-grained molecular dynamics studies and NMR should prove useful in improving our mechanistic understanding of the complex conformational space explored by a natural peptide agonist when binding to its GPCR. Such information should also facilitate the design of new ghrelin receptor-selective drugs.

Biotinylated non-ionic amphipols for GPCR ligands screening.

We present herein the synthesis of biotin-functionalized polymers (BNAPols) that have been developed for the fixation of membrane proteins (MPs) onto surfaces. BNAPols were synthesized by free-radical polymerization of a tris(hydroxymethyl)acrylamidomethane (THAM)-derived amphiphilic monomer in the presence of a thiol-based transfer agent with an azido group. Then a Huisgen-cycloaddition reaction was performed with Biotin-(PEG)8-alkyne that resulted in formation of the biotinylated polymers. The designed structure of BNAPols was confirmed by NMR spectroscopy, and a HABA/avidin assay was used for estimating the percentage of biotin grafted on the polymer end chain. The colloidal characterization of these biotin-functionalized polymers was done using both dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. BNAPols were used to stabilize a model G protein-coupled receptor (GPCR), the human Growth Hormone Secretagogue Receptor (GHSR), out of its membrane environment. Subsequent immobilization of the BNAPols:GHSR complex onto a streptavidin-coated surface allowed screening of ligands based on their ability to bind the immobilized receptor. This opens the way to the use of biotinylated NAPols to immobilize functional, unmodified, membrane proteins, providing original sensor devices for multiple applications including innovative ligand screening assays.

GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation.

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

JMV5656, a short synthetic derivative of TLQP-21, alleviates acid-induced lung injury and fibrosis in mice.

TLQP-21, a peptide encoded by the prohormone VGF, is expressed in neuroendocrine cells and can modulate inflammatory processes. Since TLQP-21 can bind the complement 3a receptor 1 on macrophages, interest has risen in this peptide as a potential drug for the treatment of Acute Respiratory Distress Syndrome (ARDS), whose hospital mortality can reach 35-46%. Since no effective pharmacologic therapies are available, our aim was to exploit the potential of a short analog of TLQP-21(JMV5656) in order to modulate the inflammatory process in ARDS and the progression to pulmonary fibrosis in an experimental model of unilateral acid aspiration in mice. Mice were divided in 2 treatment groups. In the acute protocol, mice received intra-peritoneal injection of either vehicle or 0.6 mg/kg JMV5656 on experimental days 1 and 2, and ARDS was induced on day 3 under deep anesthesia by instillation of HCl (1.5 ml/kg of 0.1 M HCl in 0.9% NaCl) into the right lung; all measurements were performed 24 h later. In the subacute protocol, mice were treated as previously, but treatment with vehicle or JMV5656 was repeated also on day 4 and measurements were made 2 weeks later. Twenty-four hours after acid instillation, the total number of immune cell in the BAL rose sharply due primarily to an increase in the PMN population which increased from 1% up to 58% of total cell numbers. JMV5656 significantly reduced PMN recruitment into the alveolar space, but had no effects on cytokine levels in BAL. Two weeks after acid injury, static compliance of the right lung was significantly higher in the JMV5656-treated group compared to vehicle-treated group. Treatment with JMV5656 also blunted the acid-induced collagen deposition in the right lung. These results suggest that JMV5656 can ameliorate mechanical compliance, and reduce collagen deposition in acid-injured lungs in mice. This effect was likely due to the ability of JMV5656 to inhibit PMN recruitment in the injured lung.

Differential Effects of a Full and Biased Ghrelin Receptor Agonist in a Mouse Kindling Model.

The ghrelin system has received substantial recognition as a potential target for novel anti-seizure drugs. Ghrelin receptor (ghrelin-R) signaling is complex, involving Gαq/11, Gαi/o, Gα12/13, and ß-arrestin pathways. In this study, we aimed to deepen our understanding regarding signaling pathways downstream the ghrelin-R responsible for mediating anticonvulsive effects in a kindling model. Mice were administered the proconvulsive dopamine 1 receptor-agonist, SKF81297, to gradually induce a kindled state. Prior to every SKF81297 injection, mice were treated with a ghrelin-R full agonist (JMV-1843), a Gαq and Gα12 biased ligand unable to recruit ß-arrestin (YIL781), a ghrelin-R antagonist (JMV-2959), or saline. Mice treated with JMV-1843 had fewer and less severe seizures compared to saline-treated controls, while mice treated with YIL781 experienced longer and more severe seizures. JMV-2959 treatment did not lead to differences in seizure severity and number. Altogether, these results indicate that the Gαq or Gα12 signaling pathways are not responsible for mediating JMV-1843's anticonvulsive effects and suggest a possible involvement of ß-arrestin signaling in the anticonvulsive effects mediated by ghrelin-R modulation.

Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex.

How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.

Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles.

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.

Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling.

The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides ß-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger ß-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.

Anticonvulsant effect of a ghrelin receptor agonist in 6Hz corneally kindled mice.

Ghrelin has anticonvulsant and neuroprotective effects in models of chemoconvulsant-induced seizures and status epilepticus. In this study we investigated whether deletion of the ghrelin receptor could alter the kindling process in the 6 Hz corneal kindling model and whether ghrelin receptor ligands possess anticonvulsant effects in fully kindled mice. Ghrelin receptor wild-type and knockout mice were electrically stimulated at a subconvulsive current twice daily via corneal electrodes until they reached the fully kindled state. Mice lacking the ghrelin receptor showed similar seizure severity during kindling acquisition as well as in the maintenance phase when compared to their wild-type littermates. Subsequently we proceeded by investigating possible anticonvulsant effects of the ghrelin receptor ligands in the acute 6 Hz seizure model and the fully 6 Hz kindled mice. The ghrelin receptor agonist JMV-1843 decreased the seizure severity score both in acutely 6 Hz stimulated mice and in fully kindled ghrelin receptor wild-type mice, but not in fully kindled ghrelin receptor knockout mice. No effect on seizure severity was observed following the ghrelin receptor antagonist JMV-2959 in both models. This finding indicates that JMV-1843 exerts an anticonvulsant effect in kindled mice via the ghrelin receptor.

New ligands of the ghrelin receptor based on the 1,2,4-triazole scaffold by introduction of a second chiral center.

Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the 'C-terminal part' of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog.

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons.

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.

Antidiabetic potential of two medicinal plants used in Gabonese folk medicine.

BACKGROUND: Diabetes mellitus is a metabolic disorder which is rising globally in rich and developing countries. In the African region this rate is the highest, with 20 million diagnosed diabetics. Despite a noticeable progress in the treatment of diabetes mellitus by synthetic drugs, the search for new natural anti-diabetic agents is going on. Nauclea diderrichii (De Wild.) Merr. (ND) and Sarcocephalus pobeguinii Hua ex Pellegr. (SP) are used as traditional medicines in Gabon for the treatment of different diseases, especially in the case of diabetes. The aim of this study was to evaluate the antidiabetic potential of these two medicinal plants traditionally used in Gabon. METHODS: Pharmacological (inhibitory action on α and ß-glucosidases) and toxicological (effect on human T cell proliferation) studies were conducted on aqueous extracts of ND (leaves and bark) and SP (bark) collected in Gabon. All raw extracts were analyzed by HPTLC and their content in phenolic compounds was determined by using standard method. The most active extracts were submitted to preparative HPLC in order to evidence the most efficient subfractions by biological evaluation. RESULTS: The results showed that two extracts from ND were potent α-glucosidase inhibitors, the leaf extract being more active that the bark extract: the first one was more than 60 fold more active than Acarbose, which is an oral medication used to treat type 2 diabetes; the extract from SP bark was less efficient. The HPLC subfractions of the extracts of ND leaves and SP bark were tested in the same experimental conditions. In each case, the most active subfractions still show very potent inhibitory effect on α-glucosidase (80-90% inhibition at 0.1 mg/mL). The most efficient extract, from ND leaves, was also characterized by the highest percentage of phenolic compounds, which suggests a relationship between its inhibitory potential on α-glucosidase and its content in phenolic compounds. Conversely, only a moderate inhibitory activity of the three extracts was observed on ß-glucosidase. CONCLUSION: These results clearly indicated that active compounds present in N. diderrichii and S. pobeguinii leaves or/and bark were selective and highly potent inhibitors of α-glucosidase and validate their popular use for the treatment of diabetes.

Involvement of PKA and ERK pathways in ghrelin-induced long-lasting potentiation of excitatory synaptic transmission in the CA1 area of rat hippocampus.

Acute effects of ghrelin on excitatory synaptic transmission were evaluated on hippocampal CA1 synapses. Ghrelin triggered an enduring enhancement of synaptic transmission independently of NMDA receptor activation and probably via postsynaptic modifications. This ghrelin-mediated potentiation resulted from the activation of GHS-R1a receptors as it was mimicked by the selective agonist JMV1843 and blocked by the selective antagonist JMV2959. This potentiation also required the activation of PKA and ERK pathways to occur as it was inhibited by KT5720 and U0126, respectively. Moreover it most probably involved Ca(2+) influxes as both ghrelin and JMV1843 elicited intracellular Ca(2+) increases, which were dependent on the presence of extracellular Ca(2+) and mediated by L-type Ca(2+) channels opening. In addition, ghrelin potentiated AMPA receptor-mediated [Ca(2+) ]i increases while decreasing NMDA receptor-mediated ones. Thus the potentiation of synaptic transmission by GHS-R1a at hippocampal CA1 excitatory synapses probably results from postsynaptic mechanisms involving PKA and ERK activation, which are producing long-lasting enhancement of AMPA receptor-mediated responses.

New trisubstituted 1,2,4-triazoles as ghrelin receptor antagonists.

Ghrelin receptor ligands based on a trisubstituted 1,2,4-triazole scaffold were recently synthesized and evaluated for their in vitro affinity for the GHS-R1a receptor and their biological activity. In this study, replacement of the α-aminoisobutyryl (Aib) moiety (a common feature present in numerous growth hormone secretagogues described in the literature) by aromatic and heteroaromatic groups was explored. We found potent antagonists incorporating the picolinic moiety in place of the Aib moiety. In an attempt to increase affinity and activity of our lead compound 2, we explored the modulation of the pyridine ring. Herein we report the design and the structure-activity relationships study of these new ghrelin receptor ligands.

Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptor.

The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, µ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.