Signalling pathways activated by apelin receptor stimulation and responsible of the hypotensive effect of apelin.

L'apéline est un neuropeptide vasoactif qui joue un rôle crucial dans le maintien de l'équilibre hydrique et des fonctions cardiovasculaires. Des études réalisées au laboratoire sur les effets de l'apéline-17 (K17F) et du fragment d'apéline K16P, correspondant à K17F delétée de la phénylalanine (Phe) à son extrémité C-terminale, ont montré que la présence de cette Phe est nécessaire pour que l'apéline puisse d'une part, induire l'internalisation du récepteur de l'apéline, et d'autre part, provoquer une baisse de la pression artérielle. Par la suite, nous avons identifié dans les cellules CHO, exprimant de façon stable le récepteur murin de l'apéline que l'internalisation du récepteur de l'apéline induite par K17F avait pour conséquence d'induire l'activation d'une seconde voie de signalisation indépendante de la protéine Gi et dépendante de la beta-arrestine, correspondant à la voie des MAP kinases (Mitogen Activator Protein Kinase), qui pourrait être impliquée dans l'effet hypotenseur de l'apéline. Mes travaux ont ensuite consisté à caractériser dans un modèle physiologique, les artérioles afférentes juxtamédullaires de rein de rat (AAJM), si la voie de signalisation médiée par la beta-arrestine était impliquée dans l'effet vasodilatateur de K17F. Sachant que l'AngII induit une vasoconstriction en augmentant la mobilisation de calcium intracellulaire ([Ca2+]i), nous avons montré en mesurant les variations de diamètre artériolaire et de [Ca2+]i, que lorsque la voie Gi est bloquée par la toxine de pertussis (PTX), l'effet vasorelaxant induit par K17F n'est pas modifié. Ces données suggèrent que l'effet vasorelaxant de K17F sur les AAJM précontractées par l'AngII est protéine Gi-indépendant. En présence de PTX et de différents inhibiteurs d'internalisation, l'effet vasorelaxant induit par K17F sur les AAJM pré-contractées par l'AngII est aboli. De plus, en présence de PTX et de ces inhibiteurs, lorsque l'on applique K17F sur la phase plateau de la réponse calcique induite par l'AngII, aucune diminution significative de la mobilisation du [Ca2+]i est observée. Ceci est en accord avec notre hypothèse, à savoir que l'effet vasorelaxant de K17F est protéine Gi-indépendant et beta-arrestine-dépendant. L'ApélineR constitue une cible thérapeutique potentielle dans le traitement de l'insuffisance cardiaque et des rétentions hydriques. Sachant que la demi-vie de l'apéline dans la circulation sanguine est de l'ordre de la minute, un autre volet de mon travail de thèse a été de développer des analogues de K17F métaboliquement stables par deux stratégies différentes. Premièrement, nous avons substitué chacun des résidus de l'apéline par son énantiomère de la série D ou par un acide aminé synthétique. Deuxièmement, nous avons ajouté une chaîne fluoroalkyle à l'extrémité N-terminale de K17F. Ces deux stratégies nous ont permis d'obtenir plusieurs composés dont les plus actifs sont le P92 et le LIT01-196 qui conservent des propriétés pharmacologiques identiques à celles de K17F et qui présentent une demi-vie plasmatique largement supérieure à celle du peptide endogène. Ces deux analogues se sont révélés actifs in vivo avec une capacité à diminuer la pression artérielle et à réduire la sécrétion de vasopressine dans le sang conduisant à une augmentation de la diurèse aqueuse.Apeline is a vasoactive neuropeptide which plays a crucial role in maintaining water balance and cardiovascular functions. Laboratory studies on the effects of Aperlin-17 (K17F) and the K16P apelin fragment, corresponding to K17F deletion from phenylalanine (Phe) at its C-terminal part have shown the presence of this Phe is necessary for apelin to induce internalization of the Apelin receptor. Also cause a decrease in blood pressure. Subsequently, in the CHO cells expressing stably the murine receptor of the Apelin that the internalization of the Apelin receptor induced by K17F resulted in the activation of a second signaling pathway which is independent of the Gi protein, but dependent on beta-arrestin. This corresponds to the MAP kinase pathway (Mitogen Activator Protein Kinase), which could be involved in the hypotensive effect of the Apelin. My work consisted of characterizing a physiological model such as the rat kidney juxtamedullary afferent arterioles (JMAA), to study if the signaling pathway mediated by beta-arrestin was involved in the vasodilatory effect of K17F. Knowing that AngII induces vasoconstriction by increasing intracellular calcium mobilization ([Ca2+]i), we have showed by measuring variations in arteriolar diameter and [Ca2+]i, that when the Gi signaling pathway is blocked by pertussis toxin (PTX), the vasorelaxant effect induced by K17F is not modify. This data suggests that the vasorelaxing effect of K17F on AngII pre-contracted JMAAs is Gi-independent protein. In the presence of PTX and various internalization inhibitors the vasorelaxant effect induced by K17F on AngII-pre-contracted JMAAs is completely blocked. In addition, no significant decrease in [Ca2+]i mobilization is observed in the presence of PTX and these inhibitors, when K17F is applied to the plateau phase of the AngII-induced calcium response. This is in line with our hypothesis, that the vasorelaxing effect of K17F is Gi-independent protein and beta-arrestin-dependent. ApelineR is a potential therapeutic target for the treatment of heart failure and water retention. Knowing that the half-life of the aperitif in the bloodstream is approximatly one minute. Another aspect of my thesis was to develop metabolically stable K17F analogues by two different strategies. First, we have substituted each of the residues of the aperitif with its D-series enantiomer or a synthetic amino acid. Secondly, we added a fluoroalkyl chain to the N-terminal end of K17F. These two strategies have enabled us to obtain several compounds, the most active of which are P92 and LIT01-196. These retain pharmacological properties identical to those of K17F and have a plasma half-life significantly higher compared to the endogenous peptide. These two analogues have been shown to be active in vivo with the ability to reduce blood pressure and reduce vasopressin secretion in the blood leading to an increase in aqueous diuresis

Role of the Vasopressin/Apelin Balance and Potential Use of Metabolically Stable Apelin Analogs in Water Metabolism Disorders

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Role of the Vasopressin/Apelin Balance and Potential Use of Metabolically Stable Apelin Analogs in Water Metabolism Disorders

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New structural insights into the apelin receptor: identification of key residues for apelin binding

International audienceApelin is the endogenous ligand of the orphan 7-transmembrane domain GPCR APJ, now named the apelin receptor (ApelinR). Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To better understand the structural organization of the ApelinR, we built 3 homology 3-dimensional (3D) models of the human ApelinR using the validated cholecystokinin receptor-1 3D model or the X-ray structures of the β2-adrenergic and CXCR4 receptors as templates. Docking of the pyroglutamyl form of apelin 13 (pE13F) into these models revealed the conservation at the bottom of the binding site of a hydrophobic cavity in which the C-terminal Phe of pE13F was embedded. In contrast, at the top of the binding site, depending on the model, different interactions were visualized between acidic residues of the ApelinR and the basic residues of pE13F. Using site-directed mutagenesis, we showed that Asp 92, Glu 172, and Asp 282 of rat ApelinR are key residues in apelin binding by interacting with Lys 8, Arg 2, and Arg 4 of pE13F, respectively. These residues are only seen in the CXCR4-based ApelinR 3D model, further validating this model. These findings bring new insights into the structural organization of the ApelinR and the mode of apelin binding

Cellular localization of apelin and its receptor in the anterior pituitary: evidence for a direct stimulatory action of apelin on ACTH release.

International audienceApelin is a bioactive peptide recently identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ. The presence of apelin-immunoreactive nerve fibers, together with the detection of apelin receptor mRNA in the parvocellular part of the paraventricular nucleus and the stimulatory action of apelin on corticotropin-releasing hormone release, indicate that apelin modulates adrenocorticotropin (ACTH) release via an indirect action on the hypothalamus. However, a direct action of apelin in the anterior pituitary cannot be excluded. Here, we provided evidence for the existence of an apelinergic system within the adult male rat pituitary gland. Double immunofluorescence staining indicated that apelin is highly coexpressed in the anterior pituitary, mainly in corticotrophs (96.5 +/- 0.3%) and to a much lower extent in somatotropes (3.2 +/- 0.2%). Using in situ hybridization combined with immunohistochemistry, a high expression of apelin receptor mRNA was also found in corticotrophs, suggesting a local interaction between apelin and ACTH. In an ex vivo perifusion system of anterior pituitaries, apelin 17 (K17F, 10(-6) M) significantly increased basal ACTH release by 41%, whereas apelin 10 (R10F, 10(-6) M), an inactive apelin fragment, was ineffective. In addition, K17F but not R10F induced a dose-dependent increase in K(+)-evoked ACTH release, with maximal increase being observed for a 10(-6) M concentration. Taken together, these data outline the potential role of apelin as an autocrine/paracrine-acting peptide on ACTH release and provide morphological and neuroendocrine basis for further studies that explore the physiological role of apelin in the regulation of anterior pituitary functions

Functional dissociation between apelin receptor signaling and endocytosis: implications for the effects of apelin on arterial blood pressure

International audienceApelin is a peptide involved in the regulation of body fluid homeostasis and cardiovascular functions, that was recently isolated as the endogenous ligand for the human orphan APJ receptor, a G protein-coupled receptor which shares 31% amino-acid sequence identity with the angiotensin II type 1 receptor. The predominant molecular forms of apelin naturally occuring in vivo are apelin 36, apelin 17 (K17F) and the pyroglutamyl form of apelin 13 (pE13F). We investigated the structure-activity relationships of apelin at the rat apelin receptor, tagged at its C-terminal end with enhanced green fluorescent protein and stably expressed in CHO cells. We compared the abilities of N- and C-terminal deleted fragments of K17F (KFRRQRPRLSHKGPMPF) to bind with high affinity to the apelin receptor, to inhibit cAMP production and to induce apelin receptor internalization. The first five N-terminal and the last two C-terminal amino acids of K17F were not essential for apelin binding or cAMP response. In contrast, deletion of the arginine in position 6 drastically decreased binding and cAMP response. The full-length sequence of K17F was the most potent inducer of apelin receptor internalization because successive N-terminal amino-acid deletions progressively reduced internalization and the removal of a single amino acid, the phenylalanine in position 17 at the C-terminus of K17F abolished this process. Thus, K16P binds with high affinity to the apelin receptor and strongly inhibits cAMP production, but does not induce apelin receptor endocytosis. These data indicate that apelin receptor signaling (coupling to Gi) and endocytosis are functionally dissociated, possibly reflecting the existence of several conformational states of this receptor, stabilized by the binding of different apelin fragments to the receptor. We then investigated the consequences for biological activity of this functional dissociation by evaluating the effects of various apelin fragments, injected iv, on arterial blood pressure in normotensive Wistar Kyoto rats. We showed that apelin fragments, that did not induce receptor internalization in vitro but kept their ability to activate receptor coupling to Gi, did not decrease arterial blood pressure. Our data showed that hypotensive actions of apelin peptides correlate with the ability of those ligands to internalize. Thus, the depressor response of apelin may be controlled by apelin receptor endocytosis, which is probably required for initiation of a second wave of signal transduction. The development of biaised agonists of the apelin receptor capable of promoting only one specific signal transduction pathway may therefore offer new therapeutic avenues for the treatment of cardiovascular disorders

Optimization of photovoltaic solar power plant locations in northern Chile

The optimization of photovoltaic solar power plants location in Atacama Desert, Chile, is presented in this study. The study considers three objectives: (1) Find sites with the highest solar energy potential, (2) determine sites with the least impact on the environment, and (3) locate the areas which produce small social impact. To solve this task, multi-criteria decision analyses (MCDAs) such as analytical hierarchy process and ordered weighted averaging were applied in a GIS environment. In addition, survey results of social impacts were analyzed and included into the decision-making process, including landscape values. The most suitable sites for solar energy projects were found near roads and power lines throughout the study area. Large suitable areas were found also from central valley from Arica and Parinacota to the north edge of Atacama region. In Atacama region, most suitable sites were found in the Andes. On the contrary, Andes were also found to have high environmental values and scenically valuable landscapes. Moderate and low suitability were found on the coast, especially in Atacama region. Factors such as slope and distance to power lines and roads influenced largely the sensitivity analysis. Area of high suitability increased by 15% when distance to roads was excluded and 18% when distance to power lines or slope was removed. MCDA-GIS method was found to be useful and applicable to the optimization of solar power plant locations in northern Chile.peerReviewe

Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities

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Biased Signaling Favoring Gi over β-Arrestin Promoted by an Apelin Fragment Lacking the C-terminal Phenylalanine

International audienceApelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a Gi-independent but β-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes β-arrestin recruitment to the receptor, K16P had a much reduced ability to promote β-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further show that both β-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the β-arrestin-dependent ERK1/2 activation and not the Gi-dependent signaling may participate in K17F-induced BP decrease