RESUMO
Neurodegenerative diseases are associated with chronic neuroinflammation in the brain, which can result in microglial phagocytosis of live synapses and neurons that may contribute to cognitive deficits and neuronal loss. The microglial P2Y6 receptor (P2Y6R) is a G-protein coupled receptor, which stimulates microglial phagocytosis when activated by extracellular uridine diphosphate, released by stressed neurons. Knockout or inhibition of P2Y6R can prevent neuronal loss in mouse models of Alzheimer's disease (AD), Parkinson's disease, epilepsy, neuroinflammation and aging, and prevent cognitive deficits in models of AD, epilepsy and aging. This review summarises the known roles of P2Y6R in the physiology and pathology of the brain, and its potential as a therapeutic target to prevent neurodegeneration and other brain pathologies.
Assuntos
Microglia , Doenças Neurodegenerativas , Receptores Purinérgicos P2 , Humanos , Animais , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Microglia/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/genética , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/efeitos dos fármacosRESUMO
The P2Y6 receptor (P2Y6R), a Gq-coupled receptor, is a potential drug discovery target for various inflammatory and degenerative conditions. Antagonists have been shown to attenuate colitis, acute lung injury, etc. In the search for competitive antagonists, we have investigated the SAR of 3-nitro-2-(trifluoromethyl)-2H-chromene derivatives, although high affinity is lacking. We now reveal that long-chain amino-functionalized congeners display greatly enhanced affinity in the antagonism of UDP-induced Ca2+ mobilization in human (h) P2Y6R-transfected 1321N1 astrocytoma cells. A 6-(Boc-amino-n-heptylethynyl) analogue 30 (MRS4940) had an IC50 of 162 nM, which was a 123-fold greater affinity than the corresponding unprotected primary alkylamine, 107-fold greater than the corresponding pivaloyl derivative 30, and 132-fold selective compared to the P2Y14R. However, similar Boc-amino chains attached at the 8-position produced weak µM affinity. Thus, the P2Y6R affinity depended on the chain length, attachment point, and terminal functionality. Off-target activities, at 45 sites, were tested for acylamino derivatives 20, 24, 26, 30, 31, and 37, which showed multiple interactions, particularly at the biogenic amine receptors. The more potent analogues may be suitable for evaluation in inflammation and cancer models, which will be performed in future studies.
Assuntos
Receptores Purinérgicos P2 , Humanos , Receptores Purinérgicos P2/metabolismo , Linhagem Celular Tumoral , Benzopiranos/farmacologia , Benzopiranos/química , Antagonistas do Receptor Purinérgico P2/farmacologia , Antagonistas do Receptor Purinérgico P2/química , Relação Estrutura-Atividade , Cálcio/metabolismoRESUMO
Ectonucleotidases play an important role in regulating the level of extracellular nucleotides and nucleosides and are an important part of the regulation of the effects of adenosine and ATP on adenosine and P2 receptors, respectively. We have previously established the ambiguous effect of P2 receptor agonists on the contractile activity of smooth muscle tissue in rats with the valproate model of autism. In this work, HPLC was used to evaluate the activity of ectonucleotidases in the smooth muscle tissues of the internal organs of rats with a valproate model of autism. The activity of ectonucleotidases was significantly higher in the smooth muscle tissues of the duodenum, vas deferens, and bladder, but lower in the ileum and uterus. The results obtained make it possible to compare the activity of ectonucleotidases identified here with changes in P2 receptor-mediated contractility of smooth muscle tissues revealed in our previous experiments.
Assuntos
Transtorno Autístico , Contração Muscular , Músculo Liso , Bexiga Urinária , Ácido Valproico , Ducto Deferente , Animais , Ratos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Ácido Valproico/farmacologia , Transtorno Autístico/metabolismo , Transtorno Autístico/induzido quimicamente , Transtorno Autístico/tratamento farmacológico , Masculino , Feminino , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/enzimologia , Contração Muscular/efeitos dos fármacos , Útero/efeitos dos fármacos , Útero/metabolismo , Íleo/efeitos dos fármacos , Íleo/metabolismo , Íleo/enzimologia , Modelos Animais de Doenças , Ratos Wistar , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfatases/metabolismoRESUMO
Despite the success of anti-programmed cell death-1 (anti-PD-1) immunotherapy, many cancer patients remain unresponsive, and reliable predictive biomarkers are lacking. Here, we show that aberrant expression of the pyrimidinergic receptor P2RY6 is frequent in human cancers and causes immune evasion. In mouse syngeneic and human xenograft tumor models, ectopic expression of P2RY6 shapes an immunosuppressive tumor microenvironment (TME) to enhance tumor growth and resistance to immunotherapy, whereas deletion of P2RY6 from tumors with high P2RY6 expression inflames the TME to inhibit tumor growth. As a G protein-coupled receptor, P2RY6 activates Gq/phospholipase C-ß signaling and stimulates the synthesis of prostaglandin E2, which is a key mediator of immunosuppression in the TME. In contrast to the essential role of P2RY6 in tumors, global deletion of P2ry6 from mice does not compromise viability. Our study thus nominates P2RY6 as a precision immunotherapy target for patients with high tumor-intrinsic P2RY6 expression.
Assuntos
Dinoprostona , Microambiente Tumoral , Animais , Dinoprostona/metabolismo , Humanos , Camundongos , Microambiente Tumoral/imunologia , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Terapia de Imunossupressão , Receptores Purinérgicos P2/metabolismo , Tolerância Imunológica , Transdução de SinaisRESUMO
The P2Y14 receptor has been proven to be a potential target for IBD. Herein, we designed and synthesized a series of 4-amide-thiophene-2-carboxyl derivatives as novel potent P2Y14 receptor antagonists based on the scaffold hopping strategy. The optimized compound 39 (5-((5-fluoropyridin-2-yl)oxy)-4-(4-methylbenzamido)thiophene-2-carboxylic acid) exhibited subnanomolar antagonistic activity (IC50: 0.40 nM). Moreover, compound 39 demonstrated notably improved solubility, liver microsomal stability, and oral bioavailability. Fluorescent ligand binding assay confirmed that 39 has the binding ability to the P2Y14 receptor, and molecular dynamics (MD) simulations revealed the formation of a unique intramolecular hydrogen bond (IMHB) in the binding conformation. In the experimental colitis mouse model, compound 39 showed a remarkable anti-IBD effect even at low doses. Compound 39, with a potent anti-IBD effect and favorable druggability, can be a promising candidate for further research. In addition, this work lays a strong foundation for the development of P2Y14 receptor antagonists and the therapeutic strategy for IBD.
Assuntos
Doenças Inflamatórias Intestinais , Receptores Purinérgicos P2 , Tiofenos , Animais , Tiofenos/farmacologia , Tiofenos/síntese química , Tiofenos/química , Tiofenos/uso terapêutico , Humanos , Camundongos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Receptores Purinérgicos P2/metabolismo , Relação Estrutura-Atividade , Antagonistas do Receptor Purinérgico P2/farmacologia , Antagonistas do Receptor Purinérgico P2/química , Antagonistas do Receptor Purinérgico P2/síntese química , Antagonistas do Receptor Purinérgico P2/uso terapêutico , Masculino , Descoberta de Drogas , Amidas/química , Amidas/farmacologia , Amidas/síntese química , Amidas/uso terapêutico , Microssomos Hepáticos/metabolismo , Simulação de Dinâmica Molecular , Colite/tratamento farmacológicoRESUMO
P2Y14 receptor (P2Y14R) is activated by uridine 5'-diphosphate-glucose, which is involved in many human inflammatory diseases. Based on the molecular docking analysis of currently reported P2Y14R antagonists and the crystallographic overlap study between the reported P2Y14R antagonist compounds 6 and 9, a series of N-substituted-acetamide derivatives were designed, synthesized, and identified as novel and potent P2Y14R antagonists. The most potent antagonist, compound I-17 (N-(1H-benzo[d]imidazol-6-yl)-2-(4-bromophenoxy)acetamide, IC50 = 0.6 nM) without zwitterionic character, showed strong binding ability to P2Y14R, high selectivity, moderate oral bioactivity, and improved pharmacokinetic profiles. In vitro and in vivo evaluation demonstrated that compound I-17 had satisfactory inhibitory activity on the inflammatory response of monosodium urate (MSU)-induced acute gouty arthritis. I-17 decreased inflammatory factor release and cell pyroptosis through the NOD-like receptor family pyrin domain-containing 3 (NLRP3)/gasdermin D (GSDMD) signaling pathway. Thus, compound I-17, with potent P2Y14R antagonistic activity, in vitro and in vivo efficacy, and favorable bioavailability (F = 75%), could be a promising lead compound for acute gouty arthritis.
Assuntos
Acetamidas , Simulação de Acoplamento Molecular , Receptores Purinérgicos P2 , Acetamidas/farmacologia , Acetamidas/química , Acetamidas/síntese química , Acetamidas/farmacocinética , Humanos , Animais , Receptores Purinérgicos P2/metabolismo , Camundongos , Masculino , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Relação Estrutura-Atividade , Antagonistas do Receptor Purinérgico P2/farmacologia , Antagonistas do Receptor Purinérgico P2/química , Antagonistas do Receptor Purinérgico P2/síntese química , Descoberta de Drogas , Ratos , Cristalografia por Raios X , Ratos Sprague-Dawley , Estrutura MolecularRESUMO
INTRODUCTION: The P2Y14 receptor (P2Y14R), a member of the G protein-coupled receptor family, is activated by extracellular nucleotides. Due to its involvement in inflammatory, immunological and other associated processes, P2Y14R has emerged as a promising therapeutic target. Despite lacking a determined three-dimensional crystal structure, the homology modeling technique based on closely related P2Y receptors' crystallography has been extensively utilized for developing active compounds targeting P2Y14R. Recent discoveries have unveiled numerous highly effective and subtype-specific P2Y14R inhibitors. This study presents an overview of the latest advancements in P2Y14R inhibitors. AREAS COVERED: This review presents an overview of the advancements in P2Y14R inhibitor research over the past five years, encompassing new patents, journal articles, and highlighting the therapeutic prospects inherent in these compounds. EXPERT OPINION: The recent revelation of the vast potential of P2Y14R inhibitors has led to the development of novel compounds that exhibit promising capabilities for the treatment of sterile inflammation of the kidney, potentially diabetes, and asthma. Despite being a relatively nascent class of compounds, certain members have already exhibited their capacity to surmount specific challenges posed by conventional P2Y14R inhibitors. Targeting P2Y14R through small molecules may present a promising therapeutic strategy for effectively managing diverse inflammatory diseases.
Assuntos
Desenvolvimento de Medicamentos , Inflamação , Patentes como Assunto , Animais , Humanos , Asma/tratamento farmacológico , Asma/fisiopatologia , Diabetes Mellitus/tratamento farmacológico , Desenho de Fármacos , Inflamação/tratamento farmacológico , Inflamação/fisiopatologia , Receptores Purinérgicos P2RESUMO
Many individuals with cancer are resistant to immunotherapies. Here, we identify the gene encoding the pyrimidine salvage pathway enzyme cytidine deaminase (CDA) among the top upregulated metabolic genes in several immunotherapy-resistant tumors. We show that CDA in cancer cells contributes to the uridine diphosphate (UDP) pool. Extracellular UDP hijacks immunosuppressive tumor-associated macrophages (TAMs) through its receptor P2Y6. Pharmacologic or genetic inhibition of CDA in cancer cells (or P2Y6 in TAMs) disrupts TAM-mediated immunosuppression, promoting cytotoxic T cell entry and susceptibility to anti-programmed cell death protein 1 (anti-PD-1) treatment in resistant pancreatic ductal adenocarcinoma (PDAC) and melanoma models. Conversely, CDA overexpression in CDA-depleted PDACs or anti-PD-1-responsive colorectal tumors or systemic UDP administration (re)establishes resistance. In individuals with PDAC, high CDA levels in cancer cells correlate with increased TAMs, lower cytotoxic T cells and possibly anti-PD-1 resistance. In a pan-cancer single-cell atlas, CDAhigh cancer cells match with T cell cytotoxicity dysfunction and P2RY6high TAMs. Overall, we suggest CDA and P2Y6 as potential targets for cancer immunotherapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Imunoterapia , Difosfato de Uridina , Humanos , Difosfato de Uridina/metabolismo , Imunoterapia/métodos , Resistencia a Medicamentos Antineoplásicos/imunologia , Animais , Camundongos , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/tratamento farmacológico , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Linhagem Celular Tumoral , Receptores Purinérgicos P2/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Microambiente Tumoral/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/tratamento farmacológico , Nucleotídeos/metabolismo , Tolerância Imunológica , Receptor de Morte Celular Programada 1RESUMO
BACKGROUND: Gouty arthritis (GA), a common inflammatory condition triggered by monosodium urate crystal accumulation, often necessitates safer treatment alternatives due to the limitations of current therapies. Astilbin, a flavonoid from Smilax glabra Roxb, has demonstrated potential in traditional Chinese medicine for its anti-inflammatory properties. However, the anti-GA effect and its underlying mechanism have not been fully elucidated. PURPOSE: This study aimed to investigate the therapeutic potential of astilbin in GA, focusing on its effects on neutrophil extracellular traps (NETs), as well as the potential molecular target of GA both in vitro and in vivo. STUDY DESIGN: Firstly, astilbin inhibited the citrullinated histone H3 (Cit h3) protein levels and reduced the NETs formation in neutrophils stimulated by monosodium urate (MSU). Secondly, we wondered the effect of astilbin on migration of neutrophils and dimethyl-sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells under the stimulation of MSU. Then, the effect of astilbin on suppressing NETs through purinergic P2Y6 receptor (P2Y6R) and Interlukin-8 (IL-8)/ CXC chemokine receptor 2 (CXCR2) pathway was investigated. Also, the relationship between P2Y6R and IL-8/CXCR2 was explored in dHL-60 cells under stimulation of MSU. Finally, we testified the effect of astilbin on reducing NETs in GA through suppressing P2Y6R and then down-regulating IL-8/CXCR2 pathway. METHODS: MSU was used to induce NETs in neutrophils and dHL-60 cells. Real-time formation of NETs and migration of neutrophils were monitored by cell living imaging with or without MSU. Then, the effect of astilbin on NETs formation, P2Y6R and IL-8/CXCR2 pathway were detected by immunofluorescence (IF) and western blotting. P2Y6R knockdown dHL-60 cells were established by small interfering RNA to investigate the association between P2Y6R and IL-8/CXCR2 pathway. Also, plasmid of P2Y6R was used to overexpress P2Y6R in dHL-60 cells, which was employed to explore the role of P2Y6R in astilbin inhibiting NETs. Within the conditions of knockdown and overexpression of P2Y6R, migration and NETs formation were assessed by transmigration assay and IF staining, respectively. In vivo, MSU-induced GA mice model was established to assess the effect of astilbin on inflammation by haematoxylin-eosin and ELISA. Additionally, the effects of astilbin on neutrophils infiltration, NETs, P2Y6R and IL-8/CXCR2 pathway were analyzed by IF, ELISA, immunohistochemistry (IHC) and western blotting. RESULTS: Under MSU stimulation, astilbin significantly suppressed the level of Cit h3 and NETs formation including the fluorescent expressions of Cit h3, neutrophils elastase, myeloperoxidase, and intra/extracellular DNA. Also, results showed that MSU caused NETs release in neutrophils as well as a trend towards recruitment of dHL-60 cells to MSU. Astilbin could markedly decrease expressions of P2Y6R and IL-8/CXCR2 pathway which were upregulated by MSU. By silencing P2Y6R, the expression of IL-8/CXCR2 pathway and migration of dHL-60 cells were inhibited, leading to the suppression of NETs. These findings indicated the upstream role of P2Y6R in the IL-8/CXCR2 pathway. Moreover, overexpression of P2Y6R was evidently inhibited by astilbin, causing a downregulation in IL-8/CXCR2 pathway, migration of dHL-60 cells and NETs formation. These results emphasized that astilbin inhibited the IL-8/CXCR2 pathway primarily through P2Y6R. In vivo, astilbin administration led to marked reductions in ankle swelling, inflammatory infiltration as well as neutrophils infiltration. Expressions of P2Y6R and IL-8/CXCR2 pathway were evidently decreased by astilbin and P2Y6R inhibitor MRS2578 either alone or in combination. Also, astilbin and MRS2578 showed notable effect on reducing MSU-induced NETs formation and IL-8/CXCR2 pathway whether used alone or in combination, parallelly demonstrating that astilbin decreased NETs formation mainly through P2Y6R. CONCLUSION: This study revealed that astilbin suppressed NETs formation via downregulating P2Y6R and subsequently the IL-8/CXCR2 pathway, which evidently mitigated GA induced by MSU. It also highlighted the potential of astilbin as a promising natural therapeutic for GA.
Assuntos
Artrite Gotosa , Armadilhas Extracelulares , Flavonóis , Interleucina-8 , Neutrófilos , Receptores Purinérgicos P2 , Armadilhas Extracelulares/efeitos dos fármacos , Humanos , Interleucina-8/metabolismo , Receptores Purinérgicos P2/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Artrite Gotosa/tratamento farmacológico , Células HL-60 , Flavonóis/farmacologia , Animais , Ácido Úrico/farmacologia , Receptores de Interleucina-8B/metabolismo , Masculino , Histonas/metabolismo , Anti-Inflamatórios/farmacologia , CamundongosRESUMO
Purinergic receptor P2Y11, a G protein-coupled receptor that is stimulated by extracellular ATP, has been demonstrated to be related to the chemotaxis of granulocytes, apoptosis of neutrophils, and secretion of cytokines in vitro. P2Y11 mutations were associated with narcolepsy. However, little is known about the roles of P2RY11 in the occurrence of narcolepsy and inflammatory response in vivo. In this study, we generated a zebrafish P2Y11 mutant using CRISPR/Cas9 genome editing and demonstrated that the P2Y11 mutant replicated the narcolepsy-like features including reduced HCRT expression and excessive daytime sleepiness, suggesting that P2Y11 is essential for HCRT expression. Furthermore, we accessed the cytokine expression in the mutant and revealed that the P2RY11 mutation disrupted the systemic inflammatory balance by reducing il4, il10 and tgfb, and increasing il6, tnfa, and il1b. In addition, the P2RY11-deficient larvae with caudal fin injuries exhibited significantly slower migration and less recruitment of neutrophils and macrophages at damaged site, and lower expression of anti-inflammatory cytokines during tissue damage. All these findings highlight the vital roles of P2RY11 in maintaining HCRT production and secreting anti-inflammatory cytokines in the native environment, and suggested that P2RY11-deficient zebrafish can serve as a reliable and unique model to further explore narcolepsy and inflammatory-related diseases with impaired neutrophil and macrophage responses.
Assuntos
Citocinas , Inflamação , Macrófagos , Neutrófilos , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Neutrófilos/metabolismo , Neutrófilos/imunologia , Macrófagos/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Citocinas/metabolismo , Mutação/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/deficiênciaRESUMO
Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.
Assuntos
Trifosfato de Adenosina , Adenilil Ciclases , Relaxamento Muscular , Músculo Liso , Testosterona , Traqueia , Uridina Trifosfato , Animais , Uridina Trifosfato/farmacologia , Uridina Trifosfato/metabolismo , Cobaias , Relaxamento Muscular/efeitos dos fármacos , Masculino , Trifosfato de Adenosina/metabolismo , Traqueia/metabolismo , Traqueia/efeitos dos fármacos , Testosterona/farmacologia , Testosterona/metabolismo , Adenilil Ciclases/metabolismo , Músculo Liso/metabolismo , Músculo Liso/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismoRESUMO
Microglial calcium signaling is rare in a baseline state but strongly engaged during early epilepsy development. The mechanism(s) governing microglial calcium signaling are not known. By developing an in vivo uridine diphosphate (UDP) fluorescent sensor, GRABUDP1.0, we discovered that UDP release is a conserved response to seizures and excitotoxicity across brain regions. UDP can signal through the microglial-enriched P2Y6 receptor to increase calcium activity during epileptogenesis. P2Y6 calcium activity is associated with lysosome biogenesis and enhanced production of NF-κB-related cytokines. In the hippocampus, knockout of the P2Y6 receptor prevents microglia from fully engulfing neurons. Attenuating microglial calcium signaling through calcium extruder ("CalEx") expression recapitulates multiple features of P2Y6 knockout, including reduced lysosome biogenesis and phagocytic interactions. Ultimately, P2Y6 knockout mice retain more CA3 neurons and better cognitive task performance during epileptogenesis. Our results demonstrate that P2Y6 signaling impacts multiple aspects of myeloid cell immune function during epileptogenesis.
Assuntos
Sinalização do Cálcio , Epilepsia , Camundongos Knockout , Microglia , Fagocitose , Receptores Purinérgicos P2 , Animais , Microglia/metabolismo , Microglia/imunologia , Camundongos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/genética , Sinalização do Cálcio/fisiologia , Epilepsia/metabolismo , Epilepsia/imunologia , Epilepsia/genética , Difosfato de Uridina/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Hipocampo/metabolismo , Neuroimunomodulação/fisiologiaRESUMO
ATP plays a crucial role as an energy currency in the body's various physiological functions, including the regulation of the sleep-wake cycle. Evidence from genetics and pharmacology demonstrates a strong association between ATP metabolism and sleep. With the advent of new technologies such as optogenetics, genetically encoded biosensors, and novel ATP detection methods, the dynamic changes in ATP levels between different sleep states have been further uncovered. The classic mechanism for regulating sleep by ATP involves its conversion to adenosine, which increases sleep pressure when accumulated extracellularly. However, emerging evidence suggests that ATP can directly bind to P2 receptors and influence sleep-wake regulation through both adenosine-dependent and independent pathways. The outcome depends on the brain region where ATP acts and the expression type of P2 receptors. This review summarizes the experimental evidence on the relationship between ATP levels and changes in sleep states and outlines the mechanisms by which ATP is involved in regulating the sleep-wake cycle through both adenosine-dependent and independent pathways. Hopefully, this review will provide a comprehensive understanding of the current research basis and progress in this field and promote further investigations into the specific mechanisms of ATP in regulating sleep.
Assuntos
Trifosfato de Adenosina , Adenosina , Sono , Vigília , Humanos , Adenosina/metabolismo , Adenosina/fisiologia , Sono/fisiologia , Trifosfato de Adenosina/metabolismo , Vigília/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/fisiologiaRESUMO
Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.
Assuntos
Trifosfato de Adenosina , Cálcio , Senescência Celular , Fibroblastos , Receptores Purinérgicos P2 , Humanos , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Pulmão/metabolismo , Pulmão/citologia , Mitocôndrias/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Linhagem Celular , Proliferação de CélulasRESUMO
Excessive lipolysis in white adipose tissue (WAT) leads to insulin resistance (IR) and ectopic fat accumulation in insulin-sensitive tissues. However, the impact of Gi-coupled receptors in restraining adipocyte lipolysis through inhibition of cAMP production remained poorly elucidated. Given that the Gi-coupled P2Y13 receptor (P2Y13-R) is a purinergic receptor expressed in WAT, we investigated its role in adipocyte lipolysis and its effect on IR and metabolic dysfunction-associated steatotic liver disease (MASLD). In humans, mRNA expression of P2Y13-R in WAT was negatively correlated to adipocyte lipolysis. In mice, adipocytes lacking P2Y13-R displayed higher intracellular cAMP levels, indicating impaired Gi signaling. Consistently, the absence of P2Y13-R was linked to increased lipolysis in adipocytes and WAT explants via hormone-sensitive lipase activation. Metabolic studies indicated that mice lacking P2Y13-R showed a greater susceptibility to diet-induced IR, systemic inflammation, and MASLD compared with their wild-type counterparts. Assays conducted on precision-cut liver slices exposed to WAT conditioned medium and on liver-specific P2Y13-R-knockdown mice suggested that P2Y13-R activity in WAT protects from hepatic steatosis, independently of liver P2Y13-R expression. In conclusion, our findings support the idea that targeting adipose P2Y13-R activity may represent a pharmacological strategy to prevent obesity-associated disorders, including type 2 diabetes and MASLD.
Assuntos
Adipócitos , Tecido Adiposo Branco , Fígado Gorduroso , Resistência à Insulina , Lipólise , Receptores Purinérgicos P2 , Animais , Feminino , Humanos , Masculino , Camundongos , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Tecido Adiposo Branco/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/deficiênciaRESUMO
The purinergic receptor P2Y6 receptor (P2Y6R) is a member of the G protein-coupled receptors (GPCR) family. P2Y6R is widely expressed in various cell types and plays a critical role in physiological processes, where it is activated by extracellular uridine diphosphate (UDP) and mobilizes Ca2+ via the Gαq/11 protein pathway. We have recently discovered the pathophysiological role of P2Y6R in cardiovascular and inflammatory diseases, including inflammatory bowel disease and non-alcoholic fatty liver disease. Furthermore, we uncovered the redox-dependent internalization of P2Y6R. In this review, we provide a comprehensive overview of the pathophysiological activity of P2Y6R in cardiovascular and inflammatory diseases. Additionally, we discuss the concept of atypical internalization control of GPCRs, which may be applied in the prevention and treatment of intestinal inflammation and cardiovascular remodeling.
Assuntos
Sistema Cardiovascular , Doenças Inflamatórias Intestinais , Hepatopatia Gordurosa não Alcoólica , Receptores Purinérgicos P2 , HumanosAssuntos
Aterosclerose , Cálcio , Receptores Purinérgicos P2 , Humanos , Cálcio/metabolismo , Células Espumosas/metabolismo , Calreticulina/metabolismo , Macrófagos/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Receptores Depuradores/metabolismo , Fosfolipases/metabolismoRESUMO
During the establishment of neuronal circuits, axons and dendrites grow and branch to establish specific synaptic connections. This complex process is highly regulated by positive and negative extracellular cues guiding the axons and dendrites. Our group was pioneer in describing that one of these signals are the extracellular purines. We found that extracellular ATP, through its selective ionotropic P2X7 receptor (P2X7R), negatively regulates axonal growth and branching. Here, we evaluate if other purinergic compounds, such as the diadenosine pentaphosphate (Ap5A), may module the dynamics of dendritic or axonal growth and branching in cultured hippocampal neurons. Our results show that Ap5A negatively modulates the dendrite's growth and number by inducing transient intracellular calcium increases in the dendrites' growth cone. Interestingly, phenol red, commonly used as a pH indicator in culture media, also blocks the P2X1 receptors, avoided the negative modulation of Ap5A on dendrites. Subsequent pharmacological studies using a battery of selective P2X1R antagonists confirmed the involvement of this subunit. In agreement with pharmacological studies, P2X1R overexpression caused a similar reduction in dendritic length and number as that induced by Ap5A. This effect was reverted when neurons were co-transfected with the vector expressing the interference RNA for P2X1R. Despite small hairpin RNAs reverting the reduction in the number of dendrites caused by Ap5A, it did not avoid the dendritic length decrease induced by the polyphosphate, suggesting, therefore, the involvement of a heteromeric P2X receptor. Our results are indicating that Ap5A exerts a negative influence on dendritic growth.
Assuntos
Trifosfato de Adenosina , Fosfatos de Dinucleosídeos , Receptores Purinérgicos P2 , Trifosfato de Adenosina/farmacologia , Receptores Purinérgicos P2/metabolismo , Neurônios/metabolismo , Dendritos/metabolismo , Hipocampo/metabolismoRESUMO
BACKGROUND AND AIMS: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. METHODS: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists. RESULTS: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cß/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified. CONCLUSIONS: Macrophage P2Y6R regulates phospholipase Cß/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate.