RESUMEN
Exercise training leads to physiological cardiac hypertrophy and the protective axis of the renin-angiotensin system composed of angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor seems involved in this process. However, the role of the basal activity of the Mas receptor in exercise-induced physiological cardiac hypertrophy is still unclear. We evaluated the effects of the Mas receptor blockade on the left ventricular structure and function of rats submitted to running training. Rats were assigned to 4 groups: sedentary (S), sedentary + A-779 (Mas receptor antagonist, 120⯵g/kg/day, i.p.; SA), trained (60-minute treadmill running sessions, five days a week, 8 weeks; T), and trained + A-779 (TA). Systolic blood pressure was higher in sedentary and trained rats treated with A-779 at the end of the experimental period. The A-779 treatment prevented the left ventricular hypertrophy evoked by physical exercise and increased collagen deposition in sedentary and trained rats. Cardiomyocytes from the SA group presented increased length and thickness of the sarcomeres, elongated mitochondria, glycogen deposits, and enlarged cisterns of the sarcoplasmic reticulum. TA group presented a reduced sarcomere thickness and cytoplasm with a degenerative aspect. These findings show that the basal activity of the Mas receptor is essential for the proper turnover of the extracellular matrix in the myocardium and the maintenance of the sarcomeric structure of cardiomyocytes.
Asunto(s)
Cardiomegalia , Condicionamiento Físico Animal , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas , Ratas Wistar , Receptores Acoplados a Proteínas G , Animales , Ratas , Masculino , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Presión Sanguínea/efectos de los fármacos , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/patología , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/metabolismo , Angiotensina II/análogos & derivadosRESUMEN
Throughout evolution, the need for single-celled organisms to associate and form a single cluster of cells has had several evolutionary advantages. In complex, multicellular organisms, each tissue or organ has a specialty and function that make life together possible, and the organism as a whole needs to act in balance and adapt to changes in the environment. Sensory organs are essential for connecting external stimuli into a biological response, through the senses: sight, smell, taste, hearing, and touch. The G-protein-coupled receptors (GPCRs) are responsible for many of these senses and therefore play a key role in the perception of the cells' external environment, enabling interaction and coordinated development between each cell of a multicellular organism. The malaria-causing protozoan parasite, Plasmodium falciparum, has a complex life cycle that is extremely dependent on a finely regulated cellular signaling machinery. In this review, we summarize strong evidence and the main candidates of GPCRs in protozoan parasites. Interestingly, one of these GPCRs is a sensor for K+ shift in Plasmodium falciparum, PfSR25. Studying this family of proteins in P. falciparum could have a significant impact, both on understanding the history of the evolution of GPCRs and on finding new targets for antimalarials.
Asunto(s)
Señalización del Calcio/fisiología , Interacciones Huésped-Parásitos/fisiología , Malaria Falciparum/metabolismo , Percepción/fisiología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Terapia Molecular Dirigida/métodos , Percepción/efectos de los fármacos , Unión Proteica , Receptores Acoplados a Proteínas G/antagonistas & inhibidoresRESUMEN
GPR55 is a receptor expressed in several central nervous system areas, including the periaqueductal gray (PAG). Current knowledge of GPR55 physiology in PAG only covers pain integration, but it is involved in other actions such as anxiety, panic, motivated behaviors, and alcohol intake. In the present study, juvenile male Wistar rats were unexposed (alcohol-naïve group; A-naïve) or exposed to alcohol for 5 weeks (alcohol-pre-exposed group; A-pre-exposed). Posteriorly, animals received intra dorsal-PAG (D-PAG) injections of vehicle (10% DMSO), LPI (1 nmol/0.5 µl) and ML-193 (1 nmol/0.5 µl, a selective GPR55 antagonist). Finally, defensive burying behavior (DBB) paradigm and alcohol preference were evaluated. Compared to the A-naïve group, the A-pre-exposed vehicle group had higher (p < 0.05): (i) time of immobility; (ii) latency to and duration of burying; and (iii) alcohol consumption. In both groups (i.e., A-naïve and A-pre-exposed) treatment with LPI: (i) decreased duration of burying (p < 0.05); (ii) suppressed time of immobility; and (iii) increased alcohol intake (p < 0.05). On the other hand, treatment with ML-193: (i) decreased duration of immobility in A-pre-exposed (but not in A-naïve rats); (ii) promoted an aggressive response against the shock-probe in A-pre-exposed rats (p < 0.05); and (iii) increased alcohol intake (p < 0.05). Our results suggest that blockade of GPR55 in D-PAG is associated with anxiety-like behaviors, defensive aggressive behaviors, and higher alcohol intake, whereas LPI in D-PAG produced anxiolytic-like effects (probably GPR55-mediated), but not prevention of alcohol intake.
Asunto(s)
Agresión/efectos de los fármacos , Consumo de Bebidas Alcohólicas/fisiopatología , Ansiedad/inducido químicamente , Sustancia Gris Periacueductal/efectos de los fármacos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Agresión/fisiología , Animales , Ansiedad/fisiopatología , Conducta Animal , Lisofosfolípidos/administración & dosificación , Masculino , Modelos Animales , Sustancia Gris Periacueductal/metabolismo , Sustancia Gris Periacueductal/fisiopatología , Ratas , Receptores de Cannabinoides/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
We investigated the role of angiotensin II type 1 (AT1 receptor) and type 2 (AT2 receptor) and MAS receptors present in the medial amygdaloid nucleus (MeA) in behavioral changes in the forced swimming test (FST) evoked by acute restraint stress in male rats. For this, rats received bilateral microinjection of either the selective AT1 receptor antagonist losartan, the selective AT2 receptor antagonist PD123319, the selective MAS receptor antagonist A-779, or vehicle 10 min before a 60 min restraint session. Then, behavior in the FST was evaluated immediately after the restraint (15 min session) and 24 h later (5 min session). The behavior in the FST of a non-stressed group was also evaluated. We observed that acute restraint stress decreased immobility during both sessions of the FST in animals treated with vehicle in the MeA. The decreased immobility during the first session was inhibited by intra-MeA administration of PD123319, whereas the effect during the second session was not identified in animals treated with A-779 into the MeA. Microinjection of PD123319 into the MeA also affected the pattern of active behaviors (i.e., swimming and climbing) during the second session of the FST. Taken together, these results indicate an involvement of angiotensinergic neurotransmissions within the MeA in behavioral changes in the FST evoked by stress.
Asunto(s)
Angiotensinas/metabolismo , Conducta Animal , Complejo Nuclear Corticomedial/metabolismo , Actividad Motora , Sistema Renina-Angiotensina , Estrés Psicológico/metabolismo , Antagonistas de Receptores de Angiotensina/farmacología , Animales , Conducta Animal/efectos de los fármacos , Complejo Nuclear Corticomedial/efectos de los fármacos , Complejo Nuclear Corticomedial/fisiopatología , Modelos Animales de Enfermedad , Masculino , Actividad Motora/efectos de los fármacos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Ratas Wistar , Tiempo de Reacción , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Restricción Física , Transducción de Señal , Estrés Psicológico/etiología , Estrés Psicológico/fisiopatología , Estrés Psicológico/psicología , Natación , Factores de TiempoRESUMEN
Alamandine (Ala1-Arg2-Val3-Tyr4-Ile5-His6-Pro7), a heptapeptide hormone of the renin-angiotensin system (RAS), exerts its effects through the Mas-related G-protein coupled receptor of the type D, MrgD, which is expressed in different tissues, including the brain. In the present study, we tested the hypothesis that alamandine could attenuate the depression-like behavior observed in transgenic rats with low brain angiotensinogen, TGR (ASrAOGEN)680. Transgenic rats exhibited a significant increase in the immobility time in forced swim test, a phenotype reversed by intracerebroventricular infusion of alamandine. Pretreatment with D-Pro7-Ang-(1-7), a Mas/MrgD receptor antagonist, prevented the antidepressant-like effect induced by this peptide demonstrating, for the first time, that alamandine through MrgD receptor, can modulate depression-like behavior in TGR (ASrAOGEN)680. This result shows an action of alamandine which strengthens the importance of the counter-regulatory arms of the RAS in fight and treatment of neuropsychiatric diseases.
Asunto(s)
Angiotensinógeno/genética , Antidepresivos/farmacología , Encéfalo/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiología , Oligopéptidos/farmacología , Receptores Acoplados a Proteínas G/fisiología , Angiotensina I/farmacología , Angiotensinógeno/metabolismo , Animales , Encéfalo/metabolismo , Inyecciones Intraventriculares , Masculino , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oligopéptidos/administración & dosificación , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Ventricular hypertrophy is a risk factors for arrhythmias, ischemia and sudden death. It involves cellular modifications leading to a pathological remodeling and is associated with heart failure. The activation of the G protein-coupled estrogen receptor (GPER) mediates beneficial actions in the cardiovascular system. Our goal was to prevent and regress the hypertrophy by the activation of GPER in neonatal cardiac myocytes (NRCM) and SHR male rats. Aldosterone increased the neonatal cardiomyocytes cell surface area after 48 h of incubation. The aldo-induced hypertrophy was blocked by the mineralocorticoid receptor (MR) inhibitor Eplererone or the reduction of MR expression by siRNA. The activation of GPER by the agonist G-1 totally prevented the increase surface area by Ald. The transfection of neonatal rat cardiac myocytes with a siRNA against GPER or the incubation with GPER blockers G-15 and G-36 inhibited the protection of G-1. The significant increase of cell surface area after 48 h of incubation with Ald was totally regressed in 24 h by the presence of G-1, indicating that the activation of GPER not only prevent the hypertrophy but also regress the hypertrophy when it is already established. In the in vivo model, G-1 or Vehicle was constantly infused via the minipump to SHR. The reduction of the hypertrophy by G-1 was evident by the cross-sectional area, BNP and ANP markers and by echocardiography. In this studied we demonstrated that the activation of GPER prevented and regressed the hypertrophy induced by Ald in NRCM and regressed hypertrophy in SHR rats.
Asunto(s)
Cardiomegalia/prevención & control , Receptores Acoplados a Proteínas G/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Cardiomegalia/diagnóstico por imagen , Células Cultivadas , Ciclopentanos/farmacología , Ecocardiografía , Eplerenona/farmacología , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Quinolinas/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/fisiologíaRESUMEN
PURPOSE: Recent studies have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) is a key molecule in sustaining androgen-mediated prostate cancer cell survival. Thus, the aim of this study was to determine whether 20-HETE can affect the metastatic potential of androgen-insensitive prostate cancer cells, and the implication of the newly described 20-HETE receptor, GPR75, in mediating these effects. METHODS: The expression of GPR75, protein phosphorylation, actin polymerization and protein distribution were assessed by western blot and/or fluorescence microscopy. Additionally, in vitro assays including epithelial-mesenchymal transition (EMT), metalloproteinase-2 (MMP-2) activity, scratch wound healing, transwell invasion and soft agar colony formation were used to evaluate the effects of 20-HETE agonists/antagonists or GPR75 gene silencing on the aggressive features of PC-3 cells. RESULTS: 20-HETE (0.1â¯nM) promoted the acquisition of a mesenchymal phenotype by increasing EMT, the release of MMP-2, cell migration and invasion, actin stress fiber formation and anchorage-independent growth. Also, 20-HETE augmented the expression of HIC-5, the phosphorylation of EGFR, NF-κB, AKT and p-38 and the intracellular redistribution of p-AKT and PKCα. These effects were impaired by GPR75 antagonism and/or silencing. Accordingly, the inhibition of 20-HETE formation with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) elicited the opposite effects. CONCLUSIONS: The present results show for the first time the involvement of the 20-HETE-GPR75 receptor in the activation of intracellular signaling known to be stimulated in cell malignant transformations leading to the differentiation of PC-3 cells towards a more aggressive phenotype. Targeting the 20-HETE/GPR75 pathway is a promising and novel approach to interfere with prostate tumor cell malignant progression.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/metabolismo , Neoplasias de la Próstata/patología , Receptores Acoplados a Proteínas G/metabolismo , Amidinas/farmacología , Andrógenos/metabolismo , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Ácidos Hidroxieicosatetraenoicos/agonistas , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Células PC-3 , Neoplasias de la Próstata/tratamiento farmacológico , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacosRESUMEN
During the early preclinical phase, from hit identification and optimization to a lead compound, several medicinal chemistry strategies can be used to improve potency and/or selectivity. The conformational restriction is one of these approaches. It consists of introducing some specific structural constraints in a lead candidate to reduce the overall number of possible conformations in order to favor the adoption of a bioactive conformation and, as a consequence, molecular recognition by the target receptor. In this work, we focused on the application of the conformational restriction strategy in the last five years for the optimization of hits and/or leads of several important classes of therapeutic targets in the drug discovery field. Thus, we recognize the importance of several kinase inhibitors to the current landscape of drug development for cancer therapy and the use of G-protein Coupled Receptor (GPCR) modulators. Several other targets are also highlighted, such as the class of epigenetic drugs. Therefore, the possibility of exploiting conformational restriction as a tool to increase the potency and selectivity and promote changes in the intrinsic activity of some ligands intended to act on many different targets makes this strategy of structural modification valuable for the discovery of novel drug candidates.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Antineoplásicos/química , Química Farmacéutica , Descubrimiento de Drogas , Humanos , Estructura Molecular , Neoplasias/metabolismo , Neoplasias/patología , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Intracellular peptides are produced by proteasomes following degradation of nuclear, cytosolic, and mitochondrial proteins, and can be further processed by additional peptidases generating a larger pool of peptides within cells. Thousands of intracellular peptides have been sequenced in plants, yeast, zebrafish, rodents, and in human cells and tissues. Relative levels of intracellular peptides undergo changes in human diseases and also when cells are stimulated, corroborating their biological function. However, only a few intracellular peptides have been pharmacologically characterized and their biological significance and mechanism of action remains elusive. Here, some historical and general aspects on intracellular peptides' biology and pharmacology are presented. Hemopressin and Pep19 are examples of intracellular peptides pharmacologically characterized as inverse agonists to cannabinoid type 1 G-protein coupled receptors (CB1R), and hemopressin fragment NFKF is shown herein to attenuate the symptoms of pilocarpine-induced epileptic seizures. Intracellular peptides EL28 (derived from proteasome 26S protease regulatory subunit 4; Rpt2), PepH (derived from Histone H2B type 1-H), and Pep5 (derived from G1/S-specific cyclin D2) are examples of peptides that function intracellularly. Intracellular peptides are suggested as biological functional molecules, and are also promising prototypes for new drug development.
Asunto(s)
Descubrimiento de Drogas , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Oligopéptidos/farmacología , Animales , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Oligopéptidos/química , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Adenosine receptors (ARs) belong to family A of GPCRs that are involved in many diseases, including cerebral and cardiac ischemic diseases, immune and inflammatory disorders, etc. Thus, they represent important therapeutic targets to treat these conditions. Computational techniques such as molecular dynamics (MD) simulations permit researchers to obtain structural information about these proteins, and principal component analysis (PCA) allows for the identification of collective motions. There are available structures for the active form (3QAK) and the inactive form (3EML) of A2AR which permit us to gain insight about their activation/inactivation mechanism. In this work, we have proposed an inverse strategy using MD simulations where the active form was coupled to the antagonist caffeine and the inactive form was coupled to adenosine agonist. Moreover, we have included four reported thermostabilizing mutations in the inactive form to study A2AR structural differences under different conditions. Some observations stand out from the PCA studies. For instance, the apo structures showed remarkable similarities, and the principal components (PCs) were rearranged in a ligand-dependent manner. Additionally, the active conformation was less stable compared to the inactive one. Some PCs inverted their direction in the presence of a ligand, and comparison of the PCs between 3EML and 3EML_ADN showed that adenosine induced major changes in the structure of A2AR. Rearrangement of PCs precedes and drives conformational changes that occur after ligand binding. Knowledge about these conformational changes provides important insights about the activity of A2AR.
Asunto(s)
Simulación de Dinámica Molecular , Análisis de Componente Principal , Receptor de Adenosina A2A/química , Adenosina/agonistas , Adenosina/metabolismo , Humanos , Enlace de Hidrógeno , Ligandos , Conformación Molecular , Movimiento (Física) , Mutación , Conformación Proteica en Hélice alfa/efectos de los fármacos , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , EstereoisomerismoRESUMEN
The perivascular adipose tissue (PVAT) is located around the adventitia, composed primarily by adipocytes, stromal cells, leukocytes, fibroblasts and capillaries. It is well described that PVAT is an important modulator of the vascular tone being considered a biologically active tissue, releasing both vasoconstrictor and vasodilators factors. The literature shows that the anti-contractile effect induced by PVAT may be due to activation of the renin-angiotensin system (RAS). AIM: Investigate whether the renin-angiotensin system participates in the effect exerted by perivascular adipose tissue on the vascular tone. METHODS AND RESULTS: For this study we used thoracic aorta from Balb/c mice and performed vascular reactivity, nitric oxide and hydrogen peroxide quantification using selective probes and fluorescence microscopy, immunofluorescence to locate receptors and enzymes involved in this response. Our results demonstrated that perivascular adipose tissue induces an anti-contractile effect in endothelium-independent manner and involves Mas and AT2 receptors participation with subsequent PI3K/Akt pathway activation. This pathway culminated with nitric oxide and hydrogen peroxide production by neuronal nitric oxide synthase, being hydrogen peroxide most relevant for the anti-contractile effect of perivascular adipose tissue. CONCLUSION: For the first time in the literature, our results show the presence of Mas and AT2 receptors, as well as, nitric oxide synthase on perivascular adipose tissue. Furthermore, our results show the involvement of Mas and AT2 receptors and consequently nitric oxide synthase activation in the anti-contractile effect exerted by perivascular adipose tissue.
Asunto(s)
Tejido Adiposo/metabolismo , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico/metabolismo , Sistema Renina-Angiotensina/fisiología , Vasoconstricción/fisiología , Adventicia/anatomía & histología , Bloqueadores del Receptor Tipo 2 de Angiotensina II/farmacología , Animales , Aorta Torácica/metabolismo , Endotelio Vascular/metabolismo , Masculino , Ratones Endogámicos BALB C , Fenilefrina/farmacología , Receptor de Angiotensina Tipo 2/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatadores/metabolismoRESUMEN
BACKGROUND: Some reports have demonstrated the role of the G Protein-coupled Estrogen Receptor (GPER) in growth and proliferation of breast cancer cells. OBJECTIVE: In an effort to develop new therapeutic strategies against breast cancer, we employed an in silico study to explore the binding modes of tetrahydroquinoline 2 and 4 to be compared with the reported ligands G1 and G1PABA. METHODS: This study aimed to design and filter ligands by in silico studies determining their Lipinski's rule, toxicity and binding properties with GPER to achieve experimental assays as anti-proliferative compounds of breast cancer cell lines. RESULTS: In silico studies suggest as promissory two tetrahydroquinoline 2 and 4 which contain a carboxyl group instead of the acetyl group (as is needed for G1 synthesis), which add low (2) and high hindrance (4) chemical moieties to explore the polar, hydrophobic and hindrance effects. Docking and molecular dynamics simulations of the target compounds were performed with GPER to explore their binding mode and free energy values. In addition, the target small molecules were synthesized and assayed in vitro using breast cancer cells (MCF-7 and MDA-MB-231). Experimental assays showed that compound 2 decreased cell proliferation, showing IC50 values of 50µM and 25µM after 72h of treatment of MCF-7 and MDA-MB-231 cell lines, respectively. Importantly, compound 2 showed a similar inhibitory effect on proliferation as G1 compound in MDA-MB-231 cells, suggesting that both ligands reach the GPER-binding site in a similar way, as was demonstrated through in silico studies. CONCLUSION: A concentration-dependent inhibition of cell proliferation occurred with compound 2 in the two cell lines regardless of GPER.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Quinolinas/farmacología , Receptores de Estrógenos/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Modelos Moleculares , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-Actividad , Termodinámica , Células Tumorales CultivadasRESUMEN
Platelet activating factor is a lipid mediator of inflammation, and in recent decades, it has emerged as an important factor in tumor outcomes. Platelet activating factor acts by specific binding to its receptor, which is present in both tumor cells and cells that infiltrate tumors. Pro-tumorigenic effects of platelet activating factor receptor in tumors includes promotion of tumor cell proliferation, production of survival signals, migration of vascular cells and formation of new vessels and stimulation of dendritic cells and macrophages suppressor phenotype. In experimental models, blocking of platelet activating factor receptor reduced tumor growth and increased animal survival. During chemotherapy and radiotherapy, tumor cells that survive treatment undergo accelerated proliferation, a phenomenon known as tumor cell repopulation. Work from our group and others showed that these treatments induce overproduction of platelet activating factor-like molecules and increase expression of its receptor in tumor cells. In this scenario, antagonists of platelet activating factor markedly reduced tumor repopulation. Here, we note that combining chemo- and radiotherapy with platelet activating factor antagonists could be a promising strategy for cancer treatment.
Asunto(s)
Proliferación Celular , Neoplasias Experimentales/terapia , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Terapia Combinada/métodos , Neoplasias Experimentales/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/terapiaRESUMEN
Chemerin and its G protein-coupled receptor [chemerin receptor 23 (ChemR23)] have been associated with endothelial dysfunction, inflammation, and insulin resistance. However, the role of chemerin on insulin signaling in the vasculature is still unknown. We aimed to determine whether chemerin reduces vascular insulin signaling and whether there is interplay between chemerin/ChemR23, insulin resistance, and vascular complications associated with type 2 diabetes (T2D). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMCs) from C57BL/6J, nondiabetic lean db/m, and diabetic obese db/db mice as well as in human microvascular endothelial cells (HMECs). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, an effect prevented by CCX832 (ChemR23 antagonist) treatment. In VSMCs, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced Akt phosphorylation, glucose transporter 4 translocation to the membrane, and glucose uptake. In HMECs, chemerin decreased insulin-activated nitric oxide signaling. AMP-activated protein kinase phosphorylation was reduced by chemerin in both HMECs and VSMCs. CCX832 treatment of db/db mice decreased body weight, insulin, and glucose levels as well as vascular oxidative stress. CCX832 also partially restored vascular insulin responses in db/db and high-fat diet-fed mice. Our novel in vivo findings highlight chemerin/ChemR23 as a promising therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes. NEW & NOTEWORTHY Our novel findings show that the chemerin/chemerin receptor 23 axis plays a critical role in diabetes-associated vascular oxidative stress and altered insulin signaling. Targeting chemerin/chemerin receptor 23 may be an attractive strategy to improve insulin signaling and vascular function in obesity-associated diabetes.
Asunto(s)
Diabetes Mellitus/metabolismo , Arterias Mesentéricas/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal , Vasodilatación , Animales , Antioxidantes/farmacología , Células Cultivadas , Diabetes Mellitus/fisiopatología , Endotelio Vascular/metabolismo , Humanos , Insulina/metabolismo , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiopatología , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Compuestos Orgánicos/farmacología , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Vasodilatadores/farmacologíaRESUMEN
Chemerin, acting through its receptor ChemR23, is an adipokine associated with inflammatory response, glucose and lipid metabolism and vascular function. Although this adipokine has been associated with the development and progression of kidney disease, it is not clear whether the chemerin/ChemR23 system plays a role in renal function in the context of diabetes. Therefore, we sought to determine whether ChemR23 receptor blockade prevents the development and/or progression of diabetic nephropathy and questioned the role of oxidative stress and Nrf2 in this process. Renal redox state and function were assessed in non-diabetic lean db/m and diabetic obese db/db mice treated with vehicle or CCX832 (ChemR23 antagonist). Renal reactive oxygen species (ROS) production, which was increased in diabetic mice, was attenuated by CCX832. This was associated with an increase in Nox 4 expression. Augmented protein oxidation in db/db mice was not observed when mice were treated with CCX832. CCX832 also abrogated impaired Nrf2 nuclear activity and associated downregulation in antioxidants expression in kidneys from db/db mice. Our in vivo findings highlight the role of the redox signaling and Nrf2 system as renoprotective players during chemerin receptor blockade in diabetic mice. The chemerin/ChemR23 system may be an important target to limit renal dysfunction associated with obesity-related diabetes.
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Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/prevención & control , Riñón/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
We have synthesized a small series of five 3-[4-arylmethoxy)phenyl]propanoic acids employing an easy and short synthetic pathway. The compounds were tested in vitro against a set of four protein targets identified as key elements in diabetes: G protein-coupled receptor 40 (GPR40), aldose reductase (AKR1B1), peroxisome proliferator-activated receptor gama (PPARγ) and solute carrier family 2 (facilitated glucose transporter), member 4 (GLUT-4). Compound 1 displayed an EC50 value of 0.075 µM against GPR40 and was an AKR1B1 inhibitor, showing IC50 = 7.4 µM. Compounds 2 and 3 act as slightly AKR1B1 inhibitors, potent GPR40 agonists and showed an increase of 2 to 4-times in the mRNA expression of PPARγ, as well as the GLUT-4 levels. Docking studies were conducted in order to explain the polypharmacological mode of action and the interaction binding mode of the most active molecules on these targets, showing several coincidences with co-crystal ligands. Compounds 1-3 were tested in vivo at an explorative 100 mg/kg dose, being 2 and 3 orally actives, reducing glucose levels in a non-insulin-dependent diabetes mice model. Compounds 2 and 3 displayed robust in vitro potency and in vivo efficacy, and could be considered as promising multitarget antidiabetic candidates. This is the first report of a single molecule with these four polypharmacological target action.
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Diseño de Fármacos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Fenilpropionatos/química , Fenilpropionatos/farmacología , Aldehído Reductasa/antagonistas & inhibidores , Animales , Sitios de Unión , Línea Celular , Células Cultivadas , Técnicas de Química Sintética , Transportador de Glucosa de Tipo 4/agonistas , Transportador de Glucosa de Tipo 4/química , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Hipoglucemiantes/síntesis química , Ligandos , Ratones , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Terapia Molecular Dirigida , PPAR gamma/antagonistas & inhibidores , PPAR gamma/química , Fenilpropionatos/síntesis química , Unión Proteica , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/químicaRESUMEN
The N-terminal region of G protein-coupled receptors can be efficiently targeted for the generation of receptor-selective antibodies. These antibodies are useful for the biochemical characterization of the receptors. In this study, we developed a set of criteria to select the optimal epitope and applied them to generate antibodies to the N-terminal region of 34 different G protein-coupled receptors. The antibody characterization revealed that a subset of antibodies exhibited increased recognition of the receptor following agonist treatment and this increase could be blocked by treatment with the receptor antagonist. An analysis of the epitopes showed that those antibodies that exhibit increased recognition are on average twelve residues long, have an overall net negative charge and are enriched in aspartic and glutamic acids. These antibodies are useful since they facilitate studies examining dose dependent increases in recognition of receptors in heterologous cells as well as in native tissue. Another interesting use of these antibodies is that they facilitate measuring changes in receptor recognition in brain following peripheral drug administration; for example, systemic administration of cocaine, a blocker of dopamine transporter that increases local dopamine levels at the synapse, was found to lead to increases in antibody recognition of dopamine receptors in the brain. Taken together these studies, in addition to describing novel tools to study native receptors, provide a framework for the generation of antibodies to G protein-coupled receptors that can detect ligand-induced conformational changes.
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Anticuerpos/inmunología , Receptores Acoplados a Proteínas G/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/inmunología , Células HEK293 , Humanos , Masculino , Conformación Proteica , Conejos , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/químicaRESUMEN
Angiotensin-(1-7) (Ang [1-7]) and its receptor Mas are involved in a number of physiological processes, including control of arterial pressure and modulation of nervous system actions. However, the involvement of the Ang-(1-7)/Mas axis in peripheral nerve injury has not been investigated. Using a model of sciatic nerve injury in mice, we demonstrated opposing changes in Mas receptor expression at days 2 and 14 post-injury. Mas receptor expression was more intense 2days after the nerve lesion, compared with the intensity of the intact nerve. At this time point, the sciatic nerve functional index was -20. At day 14 after the lesion, the intensity of the immunostaining labeling in longitudinal sections of the nerve was reduced (â¼30%) and the functional index increased +36 (gait improvement). In the axotomized group treated with A779 (a Mas receptor antagonist), the functional recovery index decreased in relation to the untreated axotomized group. The Mas receptor inhibitor also altered the intensity of labeling of S-100, GAP43, and IBA-1 (morphological features compatible with delayed axon growth). This study demonstrated that Ang-(1-7)/Mas axis activity was differentially modulated in the acute and post-acute stages of nerve injury.
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Nervios Periféricos/efectos de los fármacos , Nervios Periféricos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neuropatía Ciática/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/farmacología , Animales , Proteína GAP-43/metabolismo , Masculino , Ratones , Fragmentos de Péptidos/farmacología , Nervios Periféricos/patología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Proteínas S100/metabolismoRESUMEN
Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gßγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Endotelio Vascular/metabolismo , Procesamiento Proteico-Postraduccional , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras del Transporte Vesicular/química , Animales , Células COS , Adhesión Celular , Células Cultivadas , Chlorocebus aethiops , Endotelio Vascular/citología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , Seudópodos/metabolismo , Interferencia de ARN , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rac/químicaRESUMEN
We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment in vitro. Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA-specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund's adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4+ T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response.