RESUMEN
Purines such as ATP are regulatory transmitters in motility of the gastrointestinal tract. The aims of this study were to propose functional roles of purinergic regulation of esophageal motility. An isolated segment of the rat esophagus was placed in an organ bath, and mechanical responses were recorded using a force transducer. Exogenous application of ATP (10-100 µM) evoked relaxation of the esophageal smooth muscle in a longitudinal direction under the condition of carbachol (1 µM) -induced precontraction. Pretreatment with a non-selective P2 receptor antagonist, suramin (500 µM), and a P2Y receptor antagonist, cibacron blue F3GA (200 µM), inhibited the ATP (100 µM) -induced relaxation, but a P2X receptor antagonist, pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (50 µM), did not affect it. A blocker of ATP-dependent potassium channels (KATP channels), glibenclamide (200 µM), inhibited the ATP-induced relaxation and application of an opener of KATP channels, nicorandil (50 µM), produced relaxation. The findings suggest that ATP is involved in inhibitory regulation of the longitudinal smooth muscle in the muscularis mucosae of the rat esophagus via activation of P2Y receptors and then opening of KATP channels.
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Adenosina Trifosfato , Esófago , Canales KATP , Músculo Liso , Receptores Purinérgicos P2Y , Animales , Ratas , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Músculo Liso/metabolismo , Masculino , Receptores Purinérgicos P2Y/metabolismo , Esófago/efectos de los fármacos , Esófago/fisiología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Canales KATP/metabolismo , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Ratas Wistar , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Motilidad Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/fisiología , Ratas Sprague-DawleyRESUMEN
As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly modulate important drug targets, such as G-protein coupled receptors (GPCRs). Here, we employed P2Y purinoceptor 14 (P2Y14), a drug target for inflammation and immune responses, to uncover the sugar nucleotide activation of GPCRs. Integrating molecular dynamics simulation with functional study, we identified the uridine diphosphate (UDP)-sugar-binding site on P2Y14, and revealed that a UDP-glucose might activate the receptor by bridging the transmembrane (TM) helices 2 and 7. Between TM2 and TM7 of P2Y14, a conserved salt bridging chain (K2.60-D2.64-K7.35-E7.36 [KDKE]) was identified to distinguish different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine. We identified the KDKE chain as a conserved functional motif of sugar binding for both P2Y14 and P2Y purinoceptor 12 (P2Y12), and then designed three sugar nucleotides as agonists of P2Y12. These results not only expand our understanding for activation of purinergic receptors but also provide insights for the carbohydrate drug development for GPCRs.
Sugars and other types of carbohydrates are biomolecules which play a range of key roles in the body. In particular, they are important messengers that help to coordinate immune responses. For example, a carbohydrate known as UDP-Glucose (a kind of UDP-sugar) can activate P2Y14, a receptor studded through the surface of many cells; this event then triggers a cascade of molecular events associated with asthma, kidney injury and lung inflammation. Yet it remains unclear how exactly UDP-Glucose recognizes P2Y14 and, more broadly, how carbohydrates interact with purinergic receptors, the class of proteins that P2Y14 belongs to. To examine this question, Zhao et al. combined functional experiments in the laboratory with molecular dynamics simulations, a computational approach. This work revealed that UDP-Glucose may activate P2Y14 by bridging its segments anchored within the cell membrane. A component of P2Y14, known as the KDKE chain, was found to have an important role in distinguishing between highly similar types of UDP-sugars. This allowed Zhao et al. to design three sugar molecules which could activate another purinergic receptor that also contained a KDKE chain. Purinergic receptors are promising therapeutic targets. A finer understanding of how they recognise the molecules that activate them is therefore important to be able to identify and design new drug compounds.
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Nucleótidos , Receptores Purinérgicos , Uridina Difosfato Glucosa , Azúcares , Receptores Purinérgicos P2YRESUMEN
The sex of an animal impacts glucose sensitivity, but little information is available regarding the mechanisms causing that difference, especially during acute inflammation. We examined sex-specific differences in the role of the P2Y2 receptor (P2Y2R) in glucose flux with and without LPS challenge. Male and female wild-type and P2Y2R knockout mice (P2Y2R-/-) were injected with LPS or saline and glucose tolerance tests (GTT) were performed. P2Y2R, insulin receptor, and GLUT4 transporter gene expression was also evaluated. Female mice had reduced fasting plasma glucose and females had reduced glucose excursion times compared to male mice during GTT. P2Y2R-/- males had significantly decreased glucose flux throughout the GTT as compared to all female mice. Acute inflammation reduced fasting plasma glucose and the GTT area under the curve in both sexes. While both wild-type and P2Y2R-/- male animals displayed reduced fasting glucose in LPS treatment, female mice did not have significant difference in glucose tolerance, suggesting that the effects of P2Y2R are specific to male mice, even under inflammatory conditions. Overall, we conclude that the role for the purinergic receptor, P2Y2R, in regulating glucose metabolism is minimal in females but plays a large role in male mice, particularly in the acute inflammatory state.
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Glucemia , Lipopolisacáridos , Femenino , Masculino , Animales , Ratones , Receptores Purinérgicos P2Y , Glucosa , Ratones Noqueados , InflamaciónRESUMEN
Our sense of hearing depends on the function of a specialised class of sensory cells, the hair cells, which are found in the organ of Corti of the mammalian cochlea. The unique physiological environment in which these cells operate is maintained by a syncitium of non-sensory supporting cells, which are crucial for regulating cochlear physiology and metabolic homeostasis. Despite their importance for cochlear function, the role of these supporting cells in age-related hearing loss, the most common sensory deficit in the elderly, is poorly understood. Here, we investigated the age-related changes in the expression and function of metabotropic purinergic receptors (P2Y1 , P2Y2 and P2Y4 ) in the supporting cells of the cochlear apical coil. Purinergic signalling in supporting cells is crucial during the development of the organ of Corti and purinergic receptors are known to undergo changes in expression during ageing in several tissues. Immunolabelling and Ca2+ imaging experiments revealed a downregulation of P2Y receptor expression and a decrease of purinergic-mediated calcium responses after early postnatal stages in the supporting cells. An upregulation of P2Y receptor expression was observed in the aged cochlea when compared to 1 month-old adults. The aged mice also had significantly larger calcium responses and displayed calcium oscillations during prolonged agonist applications. We conclude that supporting cells in the aged cochlea upregulate P2Y2 and P2Y4 receptors and display purinergic-induced Ca2+ responses that mimic those observed during pre-hearing stages of development, possibly aimed at limiting or preventing further damage to the sensory epithelium. KEY POINTS: Age-related hearing loss is associated with lower hearing sensitivity and decreased ability to understand speech. We investigated age-related changes in the expression and function of metabotropic purinergic (P2Y) receptors in cochlear non-sensory supporting cells of mice displaying early-onset (C57BL/6N) and late-onset (C3H/HeJ) hearing loss. The expression of P2Y1 , P2Y2 and P2Y4 receptors in the supporting cells decreased during cochlear maturation, but that of P2Y2 and P2Y4 was upregulated in the aged cochlea. P2Y2 and P2Y4 receptors were primarily responsible for the ATP-induced Ca2+ responses in the supporting cells. The degree of purinergic expression upregulation in aged supporting cells mirrored hearing loss progression in the different mouse strains. We propose that the upregulation of purinergic-mediated signalling in the aged cochlea is subsequent to age-related changes in the hair cells and may act as a protective mechanism to limit or to avoid further damage to the sensory epithelium.
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Calcio , Pérdida Auditiva , Humanos , Ratones , Animales , Anciano , Lactante , Calcio/metabolismo , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Receptores Purinérgicos/metabolismo , Receptores Purinérgicos P2Y , Receptores Purinérgicos P2Y2 , Receptores Purinérgicos P2Y1 , Adenosina Trifosfato/fisiología , Mamíferos/metabolismoAsunto(s)
Glándula Parótida , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Reordenamiento Génico , Pronóstico , Receptores de Citocinas/genética , Receptores Purinérgicos P2Y/genéticaRESUMEN
OBJECTIVES: CRLF2 alterations are associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This study aimed to explore the clinical, biological, and outcome features of pediatric BCP-ALL with CRLF2 abnormalities. METHODS: This study enrolled 630 childhood BCP-ALLs treated on CCLG-ALL 2008 or 2018 protocol. P2RY8-CRLF2 was determined by Sanger sequencing and CRLF2 expression was evaluated by qRT-PCR. The correlation between clinical, biological features and outcomes with P2RY8-CRLF2 or CRLF2 over-expression were analyzed. RESULTS: P2RY8-CRLF2 and CRLF2 over-expression were found in 3.33% and 5.71% respectively. P2RY8-CRLF2 was associated with male, higher frequency of CD7 expression, high WBC and MRD before consolidation. CRLF2 over-expression showed ETV6-RUNX1- , higher frequency of CD22, CD34, CD66c, CD86 expression, hyperdiploidy and high MRD at early treatment. The lower overall survival (OS) was found in patients with P2RY8-CRLF2 and confined only in IR group. Furthermore, adverse event-free survival and OS of P2RY8-CRLF2 were discovered comparing to those without known fusions or treated on CCLG-ALL 2008 protocol. However, P2RY8-CRLF2 was not confirmed as independent prognostic factors and no prognostic impact of CRLF2 over-expression was found. CONCLUSIONS: These findings indicate P2RY8-CRLF2 identifies a subset of patients with specific features and adverse outcomes that could be improved by risk-directed treatment.
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Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Niño , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Pronóstico , Supervivencia sin Progresión , Receptores Purinérgicos P2Y/genéticaRESUMEN
Although methylglyoxal (MGO), a highly reactive dicarbonyl compound, influences the functioning of the vasculature, modulating its effects on vascular reactivity to various substances remains unclear, especially purinoceptor ligands. Therefore, we sought to investigate the direct effects of MGO on relaxation induced by adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) in isolated rat carotid arteries. When carotid arteries were exposed to MGO (420 µM for 1 h), relaxation induced by acetylcholine or sodium nitroprusside was not affected by MGO. However, ATP- and UTP-induced relaxation was impaired by MGO compared with the control. In both ATP- and UTP-induced relaxation, endothelial denudation, incubation with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine or the selective P2Y purinoceptor 2 (P2Y2) receptor antagonist AR-C118925XX reduced relaxation in both the control and MGO groups, while the differences between the control and MGO groups were eliminated. The cyclooxygenase (COX) inhibitor indomethacin inhibited the differences in ATP/UTP-mediated relaxations between the control and MGO groups. Moreover, N-acetyl-L-cysteine (NAC), an antioxidant, could augment carotid arterial relaxation induced by ATP/UTP in the presence of MGO. MGO increased arachidonic acid-induced contraction, which was suppressed by NAC. Following both ATP/UTP stimulation, MGO increased the release of prostanoids. These results suggest that MGO impaired ATP- and UTP-induced relaxation in carotid arteries, which was caused by suppressed P2Y2 receptor-mediated signaling and reductions in endothelial NO. Moreover, MGO partially contributed to COX-derived vasoconstrictor prostanoids through increased oxidative stress.
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Acetilcolina , Piruvaldehído , Acetilcolina/farmacología , Acetilcisteína , Adenosina , Adenosina Trifosfato/farmacología , Animales , Antioxidantes/farmacología , Ácido Araquidónico , Arginina , Arterias Carótidas , Inhibidores de la Ciclooxigenasa , Indometacina/farmacología , Óxido de Magnesio , Óxido Nítrico , Polifosfatos , Prostaglandina-Endoperóxido Sintasas , Prostaglandinas , Piruvaldehído/farmacología , Ratas , Receptores Purinérgicos P2Y , Sodio , Uridina , Uridina Trifosfato/farmacología , VasoconstrictoresRESUMEN
The endothelium plays a key role in blood vessel health. At the interface of the blood, it releases several mediators that regulate local processes that protect against the development of cardiovascular disease. In this interplay, there is increasing evidence for a role of extracellular nucleotides and endothelial purinergic P2Y receptors (P2Y-R) in vascular protection. Recent advances have revealed that endothelial P2Y1-R and P2Y2-R mediate nitric oxide-dependent vasorelaxation as well as endothelial cell proliferation and migration, which are processes involved in the regeneration of damaged endothelium. However, endothelial P2Y2-R, and possibly P2Y1-R, have also been reported to promote vascular inflammation and atheroma development in mouse models, with endothelial P2Y2-R also being described as promoting vascular remodeling and neointimal hyperplasia. Interestingly, at the interface with lipid metabolism, P2Y12-R has been found to trigger HDL transcytosis through endothelial cells, a process known to be protective against lipid deposition in the vascular wall. Better characterization of the role of purinergic P2Y-R and downstream signaling pathways in determination of the endothelial cell phenotype in healthy and pathological environments has clinical potential for the prevention and treatment of cardiovascular diseases.
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Enfermedades Cardiovasculares , Sistema Cardiovascular , Animales , Enfermedades Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Células Endoteliales/metabolismo , Endotelio/metabolismo , Ratones , Nucleótidos , Receptores Purinérgicos P2Y , Vasodilatación/fisiologíaRESUMEN
Fibrosis contributes to ~45% of deaths in western countries. In chronic liver disease, fibrosis is a major factor determining outcomes, but efficient antifibrotic therapies are lacking. Although platelet-derived growth factor and transforming growth factor-ß constitute key fibrogenic mediators, they do not account for the well-established link between cell death and fibrosis in the liver. Here, we hypothesized that damage-associated molecular patterns (DAMPs) may link epithelial cell death to fibrogenesis in the injured liver. DAMP receptor screening identified purinergic receptor P2Y14 among several candidates as highly enriched in hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Conversely, P2Y14 ligands uridine 5'-diphosphate (UDP)-glucose and UDP-galactose were enriched in hepatocytes and were released upon different modes of cell death. Accordingly, ligand-receptor interaction analysis that combined proteomic and single-cell RNA sequencing data revealed P2Y14 ligands and P2Y14 receptor as a link between dying cells and HSCs, respectively. Treatment with P2Y14 ligands or coculture with dying hepatocytes promoted HSC activation in a P2Y14-dependent manner. P2Y14 ligands activated extracellular signal-regulated kinase (ERK) and Yes-associated protein (YAP) signaling in HSCs, resulting in ERK-dependent HSC activation. Global and HSC-selective P2Y14 deficiency attenuated liver fibrosis in multiple mouse models of liver injury. Functional expression of P2Y14 was confirmed in healthy and diseased human liver and human HSCs. In conclusion, P2Y14 ligands and their receptor constitute a profibrogenic DAMP pathway that directly links cell death to fibrogenesis.
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Células Estrelladas Hepáticas , Hepatocitos , Receptores Purinérgicos P2Y , Receptores Purinérgicos P2 , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/metabolismo , Humanos , Ligandos , Hígado/metabolismo , Cirrosis Hepática/patología , Ratones , Proteómica , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Análisis de la Célula Individual , Uridina Difosfato/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Neurofibromatosis type 1 (NF1) is characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. We identified the G-protein-coupled receptor (GPCR) P2ry14 in human neurofibromas, neurofibroma-derived SC precursors (SCPs), mature SCs, and mouse SCPs. Mouse Nf1-/- SCP self-renewal was reduced by genetic or pharmacological inhibition of P2ry14. In a mouse model of NF1, genetic deletion of P2ry14 rescued low cAMP signaling, increased mouse survival, delayed neurofibroma initiation, and improved SC Remak bundles. P2ry14 signals via Gi to increase intracellular cAMP, implicating P2ry14 as a key upstream regulator of cAMP. We found that elevation of cAMP by either blocking the degradation of cAMP or by using a P2ry14 inhibitor diminished NF1-/- SCP self-renewal in vitro and neurofibroma SC proliferation in in vivo. These studies identify P2ry14 as a critical regulator of SCP self-renewal, SC proliferation, and neurofibroma initiation.
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AMP Cíclico/metabolismo , Neurofibroma , Neurofibromatosis 1 , Receptores Purinérgicos P2Y/metabolismo , Animales , Autorrenovación de las Células , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Ratones , Neurofibroma/genética , Neurofibroma/metabolismo , Neurofibroma/patología , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Células de Schwann/metabolismoRESUMEN
Nucleotides are released from all cells through regulated pathways or as a result of plasma membrane damage or cell death. Outside the cell, nucleotides act as signalling molecules triggering multiple responses via specific plasma membrane receptors of the P2 family. In the nervous system, purinergic signalling has a key function in neurotransmission. Outside the nervous system, purinergic signalling is one of the major modulators of basal tissue homeostasis, while its dysregulation contributes to the pathogenesis of various disease, including inflammation and cancer. Pre-clinical and clinical evidence shows that selective P2 agonists or antagonists are effective treatments for many pathologies, thus highlighting the relevance of extracellular nucleotides and P2 receptors as therapeutic targets.
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Nucleótidos/metabolismo , Transducción de Señal , Animales , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Membrana Celular/metabolismo , Humanos , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Neoplasias/metabolismo , Neoplasias/patología , Nucleótidos/análisis , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismoRESUMEN
CD39/NTPDase1 has emerged as an important molecule that contributes to maintain inflammatory and coagulatory homeostasis. Various studies have hypothesized the possible role of CD39 in COVID-19 pathophysiology since no confirmatory data shed light in this regard. Therefore, we aimed to quantify CD39 expression on COVID-19 patients exploring its association with severity clinical parameters and ICU admission, while unraveling the role of purinergic signaling on thromboinflammation in COVID-19 patients. We selected a prospective cohort of patients hospitalized due to severe COVID-19 pneumonia (n=75), a historical cohort of Influenza A pneumonia patients (n=18) and sex/age-matched healthy controls (n=30). CD39 was overexpressed in COVID-19 patients' plasma and immune cell subsets and related to hypoxemia. Plasma soluble form of CD39 (sCD39) was related to length of hospital stay and independently associated with intensive care unit admission (adjusted odds ratio 1.04, 95%CI 1.0-1.08, p=0.038), with a net reclassification index of 0.229 (0.118-0.287; p=0.036). COVID-19 patients showed extracellular accumulation of adenosine nucleotides (ATP and ADP), resulting in systemic inflammation and pro-coagulant state, as a consequence of purinergic pathway dysregulation. Interestingly, we found that COVID-19 plasma caused platelet activation, which was successfully blocked by the P2Y12 receptor inhibitor, ticagrelor. Therefore, sCD39 is suggested as a promising biomarker for COVID-19 severity. As a conclusion, our study indicates that CD39 overexpression in COVID-19 patients could be indicating purinergic signaling dysregulation, which might be at the basis of COVID-19 thromboinflammation disorder.
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Apirasa/sangre , Apirasa/metabolismo , COVID-19/patología , Receptores Purinérgicos P2Y/metabolismo , Tromboinflamación/patología , Adenosina Difosfato/análisis , Adenosina Trifosfato/análisis , Biomarcadores/sangre , Plaquetas/inmunología , Hipoxia de la Célula/fisiología , Cuidados Críticos/estadística & datos numéricos , Femenino , Humanos , Virus de la Influenza A/inmunología , Gripe Humana/patología , Tiempo de Internación , Masculino , Persona de Mediana Edad , Activación Plaquetaria/inmunología , Pronóstico , Estudios Prospectivos , Antagonistas del Receptor Purinérgico P2Y/farmacología , SARS-CoV-2/inmunología , Índice de Severidad de la Enfermedad , Transducción de Señal/inmunología , Tromboinflamación/inmunología , Ticagrelor/farmacologíaRESUMEN
B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a "de novo" variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis.
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Síndrome Antifosfolípido/inmunología , Tolerancia Inmunológica/inmunología , Lupus Eritematoso Sistémico/inmunología , Mutación Missense/inmunología , Receptores Purinérgicos P2Y/inmunología , Animales , Síndrome Antifosfolípido/genética , Síndrome Antifosfolípido/metabolismo , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Tolerancia Inmunológica/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Nefritis Lúpica/genética , Nefritis Lúpica/inmunología , Nefritis Lúpica/metabolismo , Masculino , Ratones Endogámicos C57BL , Mutación Missense/genética , Linaje , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Receptores Purinérgicos P2Y/genética , Receptores Purinérgicos P2Y/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
In this issue of JEM, He et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20211004) associate novel P2RY8 genetic variants to lupus, expanding the field of monogenic autoimmunity. The authors demonstrate that P2RY8 prevents the expansion of DNA-reactive B cells by restraining B cell mobility and activation within the germinal center.
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Centro Germinal , Lupus Eritematoso Sistémico , Linfocitos B , ADN , Humanos , Lupus Eritematoso Sistémico/genética , Núcleo Familiar , Receptores Purinérgicos P2YRESUMEN
The P2Y14 nucleotide receptor, a subtype of P2Y receptors, is implicated in many human inflammatory diseases. Based on the identification of favorable residues of two screening hits in the almost symmetrical P2Y14 binding domain, we describe the structural optimization of previously identified virtual screening hits 6 and 7 that result in the development of P2Y14R antagonists with a novel 2-phenyl-benzoxazole acetamide chemical scaffold. Notably, compound 52 showed potent P2Y14R antagonistic activity (IC50 = 2 nM), and a stronger inhibitory effect on MSU-induced inflammatory in vitro, better than a previously described P2Y14R antagonist PPTN. In vivo evaluation demonstrated that compound 52 also had satisfactory inhibitory activity on the inflammatory response of gout flares in mice. Moreover, P2Y14R antagonist 52 decreased paw swelling and inflammatory cell infiltration through cAMP/NLRP3/GSDMD signaling pathways in MSU-induced acute gouty arthritis mice. The discussions on the binding mechanism that employ MM/GBSA free energy calculations/decompositions also provide some useful clues for further structural designing of compound 52. Taken together, 2-phenyl-benzoxazole acetamide derivative 52 with potent P2Y14R antagonistic activity and in vivo potency could be a promising strategy for gout therapy and deserves further optimization.
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Acetamidas/farmacología , Benzoxazoles/farmacología , Descubrimiento de Drogas , Gota/tratamiento farmacológico , Antagonistas del Receptor Purinérgico P2/farmacología , Receptores Purinérgicos P2Y/metabolismo , Acetamidas/síntesis química , Acetamidas/química , Animales , Benzoxazoles/síntesis química , Benzoxazoles/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Gota/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Estructura Molecular , Antagonistas del Receptor Purinérgico P2/síntesis química , Antagonistas del Receptor Purinérgico P2/química , Relación Estructura-ActividadRESUMEN
Purinergic P2Y receptors, by binding adenosine triphosphate (ATP), are known for enhancing glucose-stimulated insulin secretion (GSIS) in pancreatic ß cells. However, the impact of these receptors in the actin dynamics and insulin granule exocytosis in these cells is not established, neither in normal nor in glucotoxic environment. In this study, we investigate the involvement of P2Y receptors on the behavior of insulin granules and the subcortical actin network dynamics in INS-1 832/13 ß cells exposed to normal or glucotoxic environment and their role in GSIS. Our results show that the activation of P2Y purinergic receptors by ATP or its agonist increase the insulin granules exocytosis and the reorganization of the subcortical actin network and participate in the potentiation of GSIS. In addition, their activation in INS-1832/13 ß-cells, with impaired insulin secretion following exposure to elevated glucose levels, restores GSIS competence through the distal steps of insulin exocytosis. These results are confirmed ex vivo by perifusion experiments on islets from type 2 diabetic (T2D) Goto-Kakizaki (GK) rats. Indeed, the P2Y receptor agonist restores the altered GSIS, which is normally lost in this T2D animal model. Moreover, we observed an improvement of the glucose tolerance, following the acute intraperitoneal injection of the P2Y agonist concomitantly with glucose, in diabetic GK rats. All these data provide new insights into the unprecedented therapeutic role of P2Y purinergic receptors in the pathophysiology of T2D.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Islotes Pancreáticos , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Diabetes Mellitus Tipo 2/metabolismo , Exocitosis , Glucosa/metabolismo , Glucosa/toxicidad , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ratas , Receptores Purinérgicos P2Y/metabolismoRESUMEN
Gout flares require monosodium urate (MSU) to activate the NLRP3 inflammasome and secrete sufficient IL-1ß. However, MSU alone is not sufficient to cause a flare. This is supported by the evidence that most patients with hyperuricemia do not develop gout throughout their lives. Recent studies have shown that, besides MSU, various purine metabolites, including adenosine triphosphate, adenosine diphosphate, and adenosine bind to different purine receptors for regulating IL-1ß secretion implicated in the pathogenesis of gout flares. Purine metabolites such as adenosine triphosphate mainly activate the NLRP3 inflammasome through P2X ion channel receptors, which stimulates IL-1ß secretion and induces gout flares, while some purine metabolites such as adenosine diphosphate and adenosine mainly act on the G protein-coupled receptors exerting pro-inflammatory or anti-inflammatory effects to regulate the onset and resolution of a gout flare. Given that the purine signaling pathway exerts different regulatory effects on inflammation and that, during the inflammatory process of a gout flare, an altered expression of purine metabolites and their receptors was observed in response to the changes in the internal environment. Thus, the purine signaling pathway is involved in regulating gout flare and resolution. This study was conducted to review and elucidate the role of various purine metabolites and purinergic receptors during the process.
Asunto(s)
Gota/etiología , Receptores Purinérgicos/fisiología , Adenosina Trifosfato/metabolismo , Antígenos CD/fisiología , Apirasa/fisiología , Gota/fisiopatología , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Receptores Purinérgicos/clasificación , Receptores Purinérgicos P2Y/fisiología , Transducción de Señal/fisiologíaRESUMEN
G-protein-coupled receptors (GPCRs), especially chemokine receptors, play a central role in the regulation of T cell migration. Various GPCRs are upregulated in activated CD4 T cells, including P2Y10, a putative lysophospholipid receptor that is officially still considered an orphan GPCR, i.e., a receptor with unknown endogenous ligand. Here we show that in mice lacking P2Y10 in the CD4 T cell compartment, the severity of experimental autoimmune encephalomyelitis and cutaneous contact hypersensitivity is reduced. P2Y10-deficient CD4 T cells show normal activation, proliferation and differentiation, but reduced chemokine-induced migration, polarization, and RhoA activation upon in vitro stimulation. Mechanistically, CD4 T cells release the putative P2Y10 ligands lysophosphatidylserine and ATP upon chemokine exposure, and these mediators induce P2Y10-dependent RhoA activation in an autocrine/paracrine fashion. ATP degradation impairs RhoA activation and migration in control CD4 T cells, but not in P2Y10-deficient CD4 T cells. Importantly, the P2Y10 pathway appears to be conserved in human T cells. Taken together, P2Y10 mediates RhoA activation in CD4 T cells in response to auto-/paracrine-acting mediators such as LysoPS and ATP, thereby facilitating chemokine-induced migration and, consecutively, T cell-mediated diseases.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Receptores Purinérgicos P2Y/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Anciano , Animales , Comunicación Autocrina/inmunología , Linfocitos T CD4-Positivos/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Quimiocinas/metabolismo , Quimiotaxis de Leucocito/inmunología , Encefalomielitis Autoinmune Experimental/sangre , Femenino , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Lisofosfolípidos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Comunicación Paracrina/inmunología , Cultivo Primario de Células , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
This review article presents a collection of tool compounds that selectively block and are recommended for studying P2Y and P2X receptor subtypes, investigating their roles in physiology and validating them as future drug targets. Moreover, drug candidates and approved drugs for P2 receptors will be discussed.
Asunto(s)
Adenosina Trifosfato/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Animales , HumanosRESUMEN
Extracellular nucleosides and nucleotides activate a group of G protein-coupled receptors (GPCRs) known as purinergic receptors, comprising adenosine and P2Y receptors. Furthermore, purinergic P2X ion channels are activated by ATP. These receptors are expressed in liver resident cells and play a critical role in maintaining liver function. In the normal physiology, these receptors regulate hepatic metabolic processes such as insulin responsiveness, glycogen and lipid metabolism, and bile secretion. In disease states, ATP and other nucleotides serve as danger signals and modulate purinergic responses in the cells. Recent studies have demonstrated that purinergic receptors play a significant role in the development of metabolic syndrome associated non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fibrosis, hepatocellular carcinoma (HCC) and liver inflammation. In this concise review, we dissect the role of purinergic signaling in different liver resident cells involved in maintaining healthy liver function and in the development of the above-mentioned liver pathologies. Moreover, we discuss potential therapeutic strategies for liver diseases by targeting adenosine, P2Y and P2X receptors.