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1.
J Neuroinflammation ; 16(1): 71, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947729

RESUMO

BACKGROUND: HIV-associated neurocognitive disorders (HANDs) afflict more than half of HIV-1-positive individuals. The transactivator of transcription (Tat) produced by HIV virus elicits inflammatory process and is a major neurotoxic mediator that induce neuron damage during HAND pathogenesis. Activated astrocytes are important cells involved in neuroinflammation and neuronal damage. Purinergic receptors expressed in astrocytes participate in a positive feedback loop in virus-induced neurotoxicity. Here, we investigated that whether P2Y4R, a P2Y receptor subtype, that expressed in astrocyte participates in Tat-induced neuronal death in vitro and in vivo. METHODS: Soluble Tat protein was performed to determine the expression of P2Y4R and proinflammatory cytokines in astrocytes using siRNA technique via real-time PCR, Western blot, and immunofluorescence assays. Cytometric bead array was used to measure proinflammatory cytokine release. The TUNEL staining and MTT cell viability assay were analyzed for HT22 cell apoptosis and viability, and the ApopTag® peroxidase in situ apoptosis detection kit and cresyl violet staining for apoptosis and death of hippocampal neuron in vivo. RESULTS: We found that Tat challenge increased the expression of P2Y4R in astrocytes. P2Y4R signaling in astrocytes was involved in Tat-induced inflammatory cytokine production via PI3K/Akt- and ERK1/2-dependent pathways. Knockdown of P2Y4R expression significantly reduced inflammatory cytokine production and relieved Tat-mediated neuronal apoptosis in vitro. Furthermore, in vivo challenged with Tat, P2Y4R knockdown mice showed decreased inflammation and neuronal damage, especially in hippocampal CA1 region. CONCLUSIONS: Our data provide novel insights into astrocyte-mediated neuron damage during HIV-1 infection and suggest a potential therapeutic target for HANDs.


Assuntos
Astrócitos/efeitos dos fármacos , Citocinas/metabolismo , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/patologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/patologia , Proteína Oncogênica v-akt , Fosfatidilinositol 3-Quinases , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/genética , Transdução de Sinais/genética , Transdução Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
2.
Int J Mol Sci ; 20(6)2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30909368

RESUMO

BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.


Assuntos
Trifosfato de Adenosina/metabolismo , Apoptose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hipóxia/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina/metabolismo , Apoptose/genética , Biomarcadores , Espaço Extracelular/metabolismo , Expressão Gênica , Humanos , Hipóxia/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P2/genética , Transdução de Sinais , Estresse Fisiológico/genética
3.
Front Neural Circuits ; 13: 14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30894803

RESUMO

Little is known about the molecular and cellular mechanisms involved in the formation of the cranial peripheral sensory system in vertebrates. To identify genes involved in the formation of these circuits, we performed a forward genetic screen utilizing a transgenic zebrafish line (p2rx3.2:gfp sl1) that expresses green fluorescent protein (gfp) in sensory neurons of the Vth, VIIth, IXth and Xth cranial ganglia. Here, we describe a novel zebrafish mutant in which a missense mutation in the adam19b gene selectively affects the epibranchial sensory circuits.


Assuntos
Proteínas ADAM/metabolismo , Orientação de Axônios/fisiologia , Rombencéfalo/citologia , Rombencéfalo/fisiologia , Células Receptoras Sensoriais/metabolismo , Proteínas ADAM/genética , Animais , Animais Geneticamente Modificados , Orientação de Axônios/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Análise Mutacional de DNA , Embrião não Mamífero , Gânglios dos Invertebrados/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva , Mutação/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Arch Cardiovasc Dis ; 112(2): 124-134, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30600215

RESUMO

BACKGROUND: The P2Y13 purinergic receptor regulates hepatic high-density lipoprotein uptake and biliary sterol secretion; it acts downstream of the membrane ecto-F1-adenosine triphosphatase, which generates extracellular adenosine diphosphate that selectively activates P2Y13, resulting in high-density lipoprotein endocytosis. Previous studies have shown that the serum concentration of the F1-adenosine triphosphatase inhibitor inhibitory factor 1 is negatively associated with cardiovascular risk. AIM: To evaluate whether p2y13 genetic variants affect cardiovascular risk. METHODS: Direct sequencing of the p2y13 coding and flanking regions was performed in a subcohort of 168 men aged 45-74 years with stable coronary artery disease and 173 control subjects from the GENES study. The two most frequent mutations, rs3732757 and rs1466684, were genotyped in 767 patients with coronary artery disease and 789 control subjects, and their association with cardiovascular risk markers was analysed. RESULTS: Carriers of the rs3732757 261T and rs1466684 557G alleles represented 9% and 27.5% of the entire population, respectively. The allele frequencies were identical in patients with coronary artery disease and control subjects. The presence of 261T was associated with higher concentrations of plasma lipoprotein A-I and inhibitory factor 1, increased fat mass and a lower heart rate. Moreover, the proportion of patients with coronary artery disease with a pejorative systolic ankle-brachial index was lower in carriers of the 261T allele. In both populations, the 557G allele was associated with increased concentrations of lipoprotein(a), and an allele dose effect was observed. CONCLUSIONS: Two frequent p2y13 variants are associated with specific bioclinical markers of cardiovascular risk. Although neither one of these variants appears to be related to the development of atherosclerotic disease, they may modulate the risk of additional cardiovascular complications.


Assuntos
Adiposidade/genética , Doença da Artéria Coronariana/genética , Frequência Cardíaca/genética , Lipoproteína(a)/sangue , Polimorfismo de Nucleotídeo Único , Proteínas/análise , Receptores Purinérgicos P2/genética , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/fisiopatologia , França , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Fatores de Risco
5.
Int J Mol Sci ; 19(7)2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30029501

RESUMO

Uridine 5'-diphosphate (UDP)-activated purinergic receptor P2Y6 is a member of a G-protein-coupled purinergic receptor family that plays an important role in mammalian innate immunity. However, the role of the P2Y6 receptor (P2Y6R) in fish immunity has not been investigated. In this report, we characterized a P2Y6R gene from Japanese flounder (Paralichthys olivaceus) and examined its role in fish innate immunity. Sequence analysis reveals that the Japanese flounder P2Y6R protein is conserved and possesses four potential glycosylation sites. Quantitative real-time RT-PCR analysis shows that P2Y6R is broadly distributed in all examined Japanese flounder tissues with dominant expression in the liver. In addition, P2Y6R gene expression was up-regulated in head kidney macrophages (HKMs) upon lipopolysaccharides (LPS) and poly(I:C) stimulations but down-regulated by LPS challenge in peripheral blood leukocytes (PBLs). Furthermore, pharmacological inhibition of the endogenous P2Y6 receptor activity by the potently selective P2Y6R antagonist, MRS 2578, greatly up-regulated pro-inflammatory cytokine interleukin (IL)-1ß, IL-6 and TNF-α gene expression in PBL cells treated with UDP. Moreover, LPS- and poly(I:C)-induced gene expression of IL-1ß and TNF-α in Japanese flounder PBL cells was attenuated significantly by inhibition of P2Y6R activity with antagonist MRS 2578. Collectively, we, for the first time, showed the involvement of functional purinergic P2Y6R in fish innate immunity.


Assuntos
Linguado/imunologia , Linguado/metabolismo , Imunidade Inata , Receptores Purinérgicos P2/metabolismo , Difosfato de Uridina/farmacologia , Sequência de Aminoácidos , Animais , Citocinas/genética , Citocinas/metabolismo , Linguado/sangue , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Inflamação/imunologia , Inflamação/patologia , Isotiocianatos/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Filogenia , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Análise de Sequência de Proteína , Tioureia/análogos & derivados , Tioureia/farmacologia
6.
EBioMedicine ; 32: 72-83, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29887330

RESUMO

Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y11 and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.


Assuntos
Depressão/tratamento farmacológico , Fluoxetina/administração & dosagem , Proteínas de Transporte de Nucleotídeos/genética , Receptor A2B de Adenosina/genética , Receptores Purinérgicos P2/genética , Trifosfato de Adenosina/metabolismo , Animais , Antidepressivos/administração & dosagem , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Depressão/genética , Depressão/metabolismo , Depressão/patologia , Exocitose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo
7.
Cell Physiol Biochem ; 46(3): 986-998, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29669327

RESUMO

BACKGROUND/AIMS: Chronic diabetic hyperglycemia can damage various of organ systems and cause serious complications. Although diabetic cardiac autonomic neuropathy (DCAN) is the primary cause of death in diabetic patients, its pathogenesis remains to be fully elucidated. Baicalin is a flavonoid extracted from Scutellaria baicalensis root and has antibacterial, diuretic, anti-inflammatory, anti- metamorphotic, and antispasmodic effects. Our study explored the effects of baicalin on enhancing sympathoexcitatory response induced by DCAN via the P2Y12 receptor. METHODS: A type 2 diabetes mellitus rat model was induced by a combination of diet and streptozotocin. Serum epinephrine was measured by enzyme-linked immunosorbent assay. Blood pressure and heart rate were measured using the indirect tail-cuff method. Heart rate variability was analyzed using the frequency-domain of electrocardiogram recordings. The expression levels of P2Y12, interleukin-1beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and connexin 43 (Cx43) were determined by quantitative real-time reverse transcription-polymerase chain reaction and western blotting. The interaction between baicalin and P2Y12 determined using by molecular docking. RESULTS: Baicalin alleviated elevated blood pressure and heart rate, improved heart rate variability, and decreased the elevated expression levels of P2Y12, IL-1ß, TNF-α, and Cx43 in the stellate ganglia of diabetic rats. Baicalin also reduced the elevated concentration of serum epinephrine and the phosphorylation of p38 mitogen-activated protein kinase in diabetic rats. CONCLUSION: Baicalin decreases sympathetic activity by inhibiting the P2Y12 receptor in stellate ganglia satellite glial cells to maintain the balance between sympathetic and parasympathetic nerves and relieves DCAN in the rat.


Assuntos
Diabetes Mellitus Experimental/patologia , Regulação para Baixo/efeitos dos fármacos , Flavonoides/farmacologia , Receptores Purinérgicos P2/metabolismo , Gânglio Estrelado/metabolismo , Animais , Sítios de Ligação , Pressão Sanguínea/efeitos dos fármacos , Conexina 43/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/metabolismo , Dieta , Ensaio de Imunoadsorção Enzimática , Epinefrina/sangue , Flavonoides/uso terapêutico , Frequência Cardíaca/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Simulação de Acoplamento Molecular , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Gânglio Estrelado/efeitos dos fármacos , Estreptozocina/toxicidade , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Biochim Biophys Acta Mol Basis Dis ; 1864(5 Pt A): 1539-1551, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29454075

RESUMO

Colorectal tumors are immersed in an array of tumor-promoting factors including extracellular nucleotides such as uridine 5'­diphosphate (UDP). UDP is the endogenous agonist of the G protein-coupled P2Y6 receptor (P2Y6R), which may contribute to the formation of a tumor-promoting microenvironment by coordinating resistance to apoptosis. Colorectal cancer (CRC) was chemically induced in P2ry6 knockout (P2ry6-/-) mice using azoxymethane and dextran sulfate sodium challenges. Mice were euthanatized and their tumor load determined. Fixed tissues were stained for histological and immunohistochemistry analysis. Tumoroids were also prepared from CRC tumors resected from P2ry6+/+ mice to determine the role of P2Y6R in resistance to apoptosis, whereas HT29 carcinoma cells were used to elucidate the signaling mechanism involved in P2Y6R anti-apoptotic effect. P2ry6-/- mice developed a reduced number of colorectal tumors with apparent tumors having smaller volumes. Overall dysplastic score was significantly lower in P2ry6-/- animals. Stimulation of P2Y6R with the selective agonist MRS2693 protected HT-29 cells from TNFα-induced apoptosis. This protective effect was mediated by the stabilizing phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP) by AKT. Using CRC-derived tumoroids, P2Y6R activation was found to contribute to chemoresistance since addition of the P2Y6R agonist MRS2693 significantly prevented the cytotoxic effect of 5-fluorouracil. The present study shows that sustained activation of P2Y6R may contribute to intestinal tumorigenesis by blocking the apoptotic process and by contributing to chemoresistance, a substantial concern in the treatment of patients with CRC. These results suggest that P2Y6R may represent a prime target for reducing colorectal carcinogenesis.


Assuntos
Apoptose , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Receptores Purinérgicos P2/genética , Fator de Necrose Tumoral alfa/metabolismo
9.
J Neurosci Res ; 96(2): 253-264, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28752899

RESUMO

Oxidative stress and neural degeneration have been shown to be involved in the pathogenesis of Parkinson's disease (PD). The P2Y6 purinergic receptor (P2Y6R) has been shown to participate in the activation of microglia and the production of pro-inflammatory factors induced by lipopolysaccharide to cause neuronal loss. However, the function of P2Y6R during oxidative stress in neurons is unclear. In the present study, 1-methyl-4-phenylpyridinium (MPP+ ) treatment increased the level of UDP/P2Y6R on neuronal SH-SY5Y cells. Importantly, pharmacological inhibition of P2Y6R or knockdown of P2Y6R using a siRNA exerted an increased protective effect by preventing MPP+ -induced increases in the levels of reactive oxygen species (ROS), superoxide anion, inducible nitric oxide synthase (iNOS), and malondialdehyde (MDA) and down-regulation of superoxide dismutase 1 (SOD1) expression. UDP, an agonist of P2Y6R, enhanced the effects of MPP+ , which was also inhibited by apyrase or MRS2578. Additionally, P2Y6R knockdown also significantly reversed both the loss of cell viability and the increase in the levels of phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) and p38 (p-p38) caused by MPP+ stimulation. However, the inhibition of the ERK1/2 and p38 kinase signaling pathways had no effect on P2Y6R expression. Taken together, these results support the hypothesis that P2Y6R expressed on neuronal SH-SY5Y cell is associated with the progression of oxidative stress and cell death induced by MPP+ , suggesting that P2Y6R may play an important role in the pathogenesis of PD.


Assuntos
Morte Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Herbicidas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , 1-Metil-4-fenilpiridínio/farmacologia , Apirase/farmacologia , Linhagem Celular Tumoral , Humanos , Isotiocianatos/farmacologia , Malondialdeído/metabolismo , Neuroblastoma/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Agonistas do Receptor Purinérgico P2/farmacologia , Antagonistas do Receptor Purinérgico P2/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2/genética , Superóxido Dismutase-1/metabolismo , Tioureia/análogos & derivados , Tioureia/farmacologia , Transfecção , Difosfato de Uridina/farmacologia
10.
Adv Exp Med Biol ; 1051: 107-122, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29134605

RESUMO

The P2Y11 receptor is a G protein-coupled receptor that is stimulated by endogenous purine nucleotides, particularly ATP. Amongst P2Y receptors it has several unique properties; (1) it is the only human P2Y receptor gene that contains an intron in the coding sequence; (2) the gene does not appear to be present in the rodent genome; (3) it couples to stimulation of both phospholipase C and adenylyl cyclase. Its absence in mice and rats, along with a limited range of selective pharmacological tools, has hampered the development of our knowledge and understanding of its properties and functions. Nonetheless, through a combination of careful use of the available tools, suppression of receptor expression using siRNA and genetic screening for SNPs, possible functions of native P2Y11 receptors have been identified in a variety of human cells and tissues. Many are in blood cells involved in inflammatory responses, consistent with extracellular ATP being a damage-associated signalling molecule in the immune system. Thus proposed potential therapeutic applications relate, in the main, to modulation of acute and chronic inflammatory responses.


Assuntos
Trifosfato de Adenosina/metabolismo , Fases de Leitura Aberta , Polimorfismo de Nucleotídeo Único , Receptores Purinérgicos P2 , Trifosfato de Adenosina/genética , Animais , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Íntrons , Camundongos , Especificidade de Órgãos , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Especificidade da Espécie
11.
J Physiol ; 595(23): 7135-7148, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28980705

RESUMO

KEY POINTS: Autologous cardiac progenitor cell (CPC) therapy is a promising approach for treatment of heart failure (HF). There is an unmet need to identify inherent deficits in aged/diseased human CPCs (hCPCs) derived from HF patients in the attempts to augment their regenerative capacity prior to use in the clinical setting. Here we report significant functional correlations between phenotypic properties of hCPCs isolated from cardiac biopsies of HF patients, clinical parameters of patients and expression of the P2Y14 purinergic receptor (P2Y14 R), a crucial detector for extracellular UDP-sugars released during injury/stress. P2Y14 R is downregulated in hCPCs derived from HF patients with lower ejection fraction or diagnosed with diabetes. Augmenting P2Y14 R expression levels in aged/diseased hCPCs antagonizes senescence and improves functional responses. This study introduces purinergic signalling modulation as a potential strategy to rejuvenate and improve phenotypic characteristics of aged/functionally compromised hCPCs prior to transplantation in HF patients. ABSTRACT: Autologous cardiac progenitor cell therapy is a promising alternative approach to current inefficient therapies for heart failure (HF). However, ex vivo expansion and pharmacological/genetic modification of human cardiac progenitor cells (hCPCs) are necessary interventions to rejuvenate aged/diseased cells and improve their regenerative capacities. This study was designed to assess the potential of improving hCPC functional capacity by targeting the P2Y14 purinergic receptor (P2Y14 R), which has been previously reported to induce regenerative and anti-senescence responses in a variety of experimental models. c-Kit+ hCPCs were isolated from cardiac biopsies of multiple HF patients undergoing left ventricular assist device implantation surgery. Significant correlations existed between the expression of P2Y14 R in hCPCs and clinical parameters of HF patients. P2Y14 R was downregulated in hCPCs derived from patients with a relatively lower ejection fraction and patients diagnosed with diabetes. hCPC lines with lower P2Y14 R expression did not respond to P2Y14 R agonist UDP-glucose (UDP-Glu) while hCPCs with higher P2Y14 R expression showed enhanced proliferation in response to UDP-Glu stimulation. Mechanistically, UDP-Glu stimulation enhanced the activation of canonical growth signalling pathways ERK1/2 and AKT. Restoring P2Y14 R expression levels in functionally compromised hCPCs via lentiviral-mediated overexpression improved proliferation, migration and survival under stress stimuli. Additionally, P2Y14 R overexpression reversed senescence-associated morphology and reduced levels of molecular markers of senescence p16INK4a , p53, p21 and mitochondrial reactive oxygen species. Findings from this study unveil novel biological roles of the UDP-sugar receptor P2Y14 in hCPCs and suggest purinergic signalling modulation as a promising strategy to improve phenotypic properties of functionally impaired hCPCs.


Assuntos
Células-Tronco Adultas/metabolismo , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Purinérgicos P2/genética , Adulto , Células-Tronco Adultas/fisiologia , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Células Cultivadas , Senescência Celular , Feminino , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2/metabolismo
12.
Biochem Biophys Res Commun ; 494(1-2): 332-338, 2017 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-29017923

RESUMO

Lysophosphatidylserine (LysoPS) has been shown to have lipid mediator-like actions to induce mast cell degranulation and suppress T lymphocyte proliferation. Recently, three G protein-coupled receptors (GPCRs), LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, were found to react specifically with LysoPS, raising the possibility that LysoPS exerts its roles through these receptors. In this study, we show that LPS3 is expressed in various T cell subtypes and is involved in suppression of Interleukin-2 (IL-2) production in CD4 T cells. We found that LysoPS suppressed the IL-2 production from activated T cells at the mRNA and protein levels. In addition, LysoPS did not have such an effect on the splenocytes and CD4 T cells isolated from LPS3-deficient mice. In LPS3-deficient splenocytes and CD4 T cells, anti-CD3/anti-CD28-triggered IL-2 production is somewhat increased. Interestingly, LysoPS with various fatty acids was up-regulated upon T cell activation. The present study raised the possibility that LysoPS exerts its immunosuppressive roles by down-regulating IL-2 production through a LysoPS-LPS3 axis in T cells.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Interleucina-2/genética , Lisofosfolipídeos/farmacologia , Receptores Acoplados a Proteínas-G/genética , Receptores de Lisofosfolipídeos/genética , Receptores Purinérgicos P2/genética , Animais , Anticorpos/farmacologia , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/genética , Antígenos CD28/imunologia , Complexo CD3/genética , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Separação Celular , Regulação da Expressão Gênica , Interleucina-2/imunologia , Lisofosfolipídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Receptores Acoplados a Proteínas-G/imunologia , Receptores de Lisofosfolipídeos/imunologia , Receptores Purinérgicos P2/imunologia , Transdução de Sinais , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia
13.
J Neuroinflammation ; 14(1): 194, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28962574

RESUMO

BACKGROUND: Neuroinflammation involves cytokine release, astrocyte reactivity and migration. Neuronal Thy-1 promotes DITNC1 astrocyte migration by engaging αVß3 Integrin and Syndecan-4. Primary astrocytes express low levels of these receptors and are unresponsive to Thy-1; thus, inflammation and astrocyte reactivity might be necessary for Thy-1-induced responses. METHODS: Wild-type rat astrocytes (TNF-activated) or from human SOD1G93A transgenic mice (a neurodegenerative disease model) were used to evaluate cell migration, Thy-1 receptor levels, signaling molecules, and reactivity markers. RESULTS: Thy-1 induced astrocyte migration only after TNF priming. Increased expression of αVß3 Integrin, Syndecan-4, P2X7R, Pannexin-1, Connexin-43, GFAP, and iNOS were observed in TNF-treated astrocytes. Silencing of ß3 Integrin prior to TNF treatment prevented Thy-1-induced migration, while ß3 Integrin over-expression was sufficient to induce astrocyte reactivity and allow Thy-1-induced migration. Finally, hSOD1G93A astrocytes behave as TNF-treated astrocytes since they were reactive and responsive to Thy-1. CONCLUSIONS: Therefore, inflammation induces expression of αVß3 Integrin and other proteins, astrocyte reactivity, and Thy-1 responsiveness. Importantly, ectopic control of ß3 Integrin levels modulates these responses regardless of inflammation.


Assuntos
Astrócitos/fisiologia , Movimento Celular/fisiologia , Regulação da Expressão Gênica/genética , Integrina alfaVbeta3/metabolismo , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Conexinas/genética , Conexinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Antígenos Thy-1/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Cicatrização/fisiologia
14.
Pancreas ; 46(10): 1327-1335, 2017 Nov/Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28930866

RESUMO

OBJECTIVES: The aim of this study was to investigate the role of P2X7R (purinergic 2X7 receptor) and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome activation in the process of pancreatic fibrosis in a mouse model of chronic pancreatitis (CP). METHODS: Chronic pancreatitis was induced by repeated intraperitoneal injections of 50 µg/kg cerulein for 6 weeks in mice. P2X7R antagonist oxidized ATP (OxATP) or brilliant blue G (BBG) was administered after the last cerulein injection for 2 weeks. Pancreatic chronic inflammation and fibrosis were evaluated by histological score, Sirius red staining, and alpha-smooth muscle actin immunohistochemical staining. We further determined pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, interleukin 1ß (IL-1ß), and IL-18. RESULTS: The pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, IL-1ß, and IL-18 were all reduced significantly in both the OxATP and BBG groups (P < 0.05). The pancreatic chronic inflammation and the fibrosis indices were all remarkably attenuated (P < 0.05). CONCLUSIONS: P2X7R antagonist OxATP and BBG significantly decreased pancreatic chronic inflammation and fibrosis in a mouse CP model and suggested that blockade of P2X7R-NLRP3 inflammasome signaling pathway may represent a novel therapeutic strategy for CP and its fibrotic process.


Assuntos
Modelos Animais de Doenças , Inflamassomos/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Pâncreas/metabolismo , Pancreatite Crônica/genética , Receptores Purinérgicos P2/genética , Animais , Caspase 1/genética , Caspase 1/metabolismo , Ceruletídeo , Citocinas/metabolismo , Fibrose , Expressão Gênica/efeitos dos fármacos , Humanos , Inflamassomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/metabolismo , Receptores Purinérgicos P2/metabolismo , Corantes de Rosanilina/farmacologia
15.
PLoS One ; 12(8): e0183114, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28800362

RESUMO

Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW) communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme), suramin (broad-spectrum P2 receptor antagonist), 2-APB (IP3 receptor blocker) and thapsigargin (endoplasmic reticulum calcium pump inhibitor) potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 µM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Microglia/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Apirase/farmacologia , Fenômenos Biomecânicos , Compostos de Boro/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Expressão Gênica , Fosfatos de Inositol/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Imagem Molecular , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y12/genética , Suramina/farmacologia , Tapsigargina/farmacologia
16.
Adv Exp Med Biol ; 1051: 139-168, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28815513

RESUMO

The distribution of nucleotide P2Y receptors across different tissues suggests that they fulfil key roles in a number of physiological and pathological conditions. P2Y13 is one of the latest P2Y receptors identified, a novel member of the Gi-coupled P2Y receptor subfamily that responds to ADP, together with P2Y12 and P2Y14. Pharmacological studies drew attention to this new ADP receptor, with a pharmacology that overlaps that of P2Y12 receptors but with unique features and roles. The P2RY12-14 genes all reside on human chromosome 3 at 3q25.1 and their strong sequence homology supports their evolutionary origin through gene duplication. Polymorphisms of P2Y13 receptors have been reported in different human populations, yet their consequences remain unknown. The P2Y13 receptor is versatile in its signalling, extending beyond the canonical signalling of a Gi-coupled receptor. Not only can it couple to different G proteins (Gs/Gq) but the P2Y13 receptor can also trigger several intracellular pathways related to the activation of MAPKs (mitogen-activated protein kinases) and the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 axis. Moreover, the availability of P2Y13 receptor knockout mice has highlighted the specific functions in which it is involved, mainly in the regulation of cholesterol and glucose metabolism, bone homeostasis and aspects of central nervous system function like pain transmission and neuroprotection. This review summarizes our current understanding of this elusive receptor, not only at the pharmacological and molecular level but also, in terms of its signalling properties and specific functions, helping to clarify the involvement of P2Y13 receptors in pathological situations.


Assuntos
Sistema de Sinalização das MAP Quinases , Polimorfismo Genético , Receptores Purinérgicos P2 , Animais , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 3/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Relação Estrutura-Atividade
17.
Ter Arkh ; 89(5): 74-78, 2017.
Artigo em Russo | MEDLINE | ID: mdl-28631703

RESUMO

AIM: To assess the association of CYP2C19 G681A, P2RY12 H1/H2, and ITGB3 T1565C polymorphisms with the extent of platelet aggregation in patients with coronary heart disease (CHD) during antiplatelet therapy. SUBJECTS AND METHODS: 166 male patients with CHD, living in the Western Siberian Region, were examined. All the patients underwent a test for platelet aggregation induced by ADP (2.5 and 5.0 µm) and epinephrine (0.2 µm). Genotyping was performed using an allele-specific polymerase chain reaction technique. RESULTS: The polymorphic variants of the P2RY12 and ITGB3 genes were ascertained to have no impact on the extent of platelet aggregation in patients receiving clopidogrel and acetylsalicylic acid. An association was found between CYP2C19 681A allele carriage and the increased extent of platelet aggregation induced by ADP. CONCLUSION: The carriage of the cytochrome P450 CYP2C19 681A allele rather than platelet receptor gene polymorphisms determines a risk for clopidogrel resistance in patients with CHD.


Assuntos
Aspirina , Doença da Artéria Coronariana , Citocromo P-450 CYP2C19/genética , Agregação Plaquetária/efeitos dos fármacos , Ticlopidina/análogos & derivados , Alelos , Aspirina/administração & dosagem , Aspirina/efeitos adversos , Plaquetas/efeitos dos fármacos , Clopidogrel , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/epidemiologia , Doença da Artéria Coronariana/genética , Resistência a Medicamentos/genética , Humanos , Integrina beta3/genética , Masculino , Pessoa de Meia-Idade , Farmacogenética , Inibidores da Agregação de Plaquetas/administração & dosagem , Inibidores da Agregação de Plaquetas/efeitos adversos , Testes de Função Plaquetária/métodos , Polimorfismo Genético , Receptores Purinérgicos P2/genética , Sibéria/epidemiologia , Ticlopidina/administração & dosagem , Ticlopidina/efeitos adversos
18.
Brain ; 140(6): 1657-1668, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28460015

RESUMO

The sleep disorder narcolepsy with cataplexy is characterized by a highly specific loss of hypocretin (orexin) neurons, leading to the hypothesis that the condition is caused by an immune or autoimmune mechanism. All genetic variants associated with narcolepsy are immune-related. Among these are single nucleotide polymorphisms in the P2RY11-EIF3G locus. It is unknown how these genetic variants affect narcolepsy pathogenesis and whether the effect is directly related to P2Y11 signalling or EIF3G function. Exome sequencing in 18 families with at least two affected narcolepsy with cataplexy subjects revealed non-synonymous mutations in the second exon of P2RY11 in two families, and P2RY11 re-sequencing in 250 non-familial cases and 135 healthy control subjects revealed further six different non-synonymous mutations in the second exon of P2RY11 in seven patients. No mutations were found in healthy controls. Six of the eight narcolepsy-associated P2Y11 mutations resulted in significant functional deficits in P2Y11 signalling through both Ca2+ and cAMP signalling pathways. In conclusion, our data show that decreased P2Y11 signalling plays an important role in the development of narcolepsy with cataplexy.


Assuntos
Narcolepsia/genética , Narcolepsia/fisiopatologia , Receptores Purinérgicos P2/genética , Transdução de Sinais/genética , Adulto , Cataplexia/genética , Cataplexia/fisiopatologia , Éxons , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem
19.
Oncotarget ; 8(23): 37278-37290, 2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28418839

RESUMO

Extracellular ATP-induced Ca2+ signalling is critical in regulating diverse physiological and disease processes. Emerging evidence suggests high concentrations of extracellular ATP in tumour tissues. In this study, we examined the P2 receptor for ATP-induced Ca2+ signalling in human hepatocellular carcinoma (HCC) cells. Fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) showed that extracellular ATP induced an increase in the [Ca2+]i in human HCC Huh-7 and HepG2 cells. NF546, a P2Y11 receptor agonist was equally effective in inducing an increase in the [Ca2+]i. In contrast, agonists for the P2X receptors (αßmeATP and BzATP), P2Y1 receptor (MRS2365) or P2Y2 receptor (MRS2768) were ineffective. In addition, ATP/NF546-induced increases in the [Ca2+]i were strongly inhibited by treatment with NF340, a P2Y11 receptor antagonist. Immunofluorescent confocal imaging and western blotting analysis consistently demonstrated the P2Y11 receptor expression in Huh-7 and HepG2 cells. Transfection with P2Y11-specific siRNA attenuated the P2Y11 receptor protein expression level and also reduced NF546-induced increase in the [Ca2+]i. Importantly, immunohistochemistry revealed that the P2Y11 receptor was expressed at very high level in human HCC tissues and, by contrast, it was barely detected in normal liver tissues. Trans-well cell migration assay demonstrated that ATP and NF546 induced concentration-dependent stimulation of Huh-7 cell migration. Treatment with NF340 prevented ATP-induced stimulation of cell migration. Taken together, our results show carcinoma-specific expression of the P2Y11 receptor and its critical role in mediating ATP-inducing Ca2+ signalling and regulating cell migration in human HCC cells.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Receptores Purinérgicos P2/genética , Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Carcinoma Hepatocelular/patologia , Movimento Celular , Difosfonatos/farmacologia , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Fígado/patologia , Neoplasias Hepáticas/patologia , Naftalenossulfonatos/farmacologia , Purinérgicos , Receptores Purinérgicos P2/metabolismo , Regulação para Cima
20.
Sci Rep ; 7(1): 771, 2017 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-28396595

RESUMO

The role of the P2Y6 receptor in bladder function has recently attracted a great deal of attention in lower urinary tract research. We conducted this study to determine contributions of the P2Y6 receptor in lower urinary tract function of normal phenotypes by comparing P2Y6-deficient mice and wild-type mice. In in vivo experiments, P2Y6-deficient mice had more frequent micturition with smaller bladder capacity compared to wild-type mice; however, there was no difference between these groups in bladder-filling pressure/volume relationships during cystometry under decerebrate, unanaesthetized conditions. Analysis of in vivo bladder contraction revealed significant difference between the 2 groups, with P2Y6-deficient mice presenting markedly shorter bladder contraction duration but no difference in peak contraction pressure. However, analysis of in vitro experiments showed no P2Y6 involvements in contraction and relaxation of bladder muscle strips and in ATP release by mechanical stimulation of primary-cultured urothelial cells. These results suggest that the P2Y6 receptor in the central nervous system, dorsal root ganglion, or both is involved in inhibition of bladder afferent signalling or sensitivity in the pontine micturition centre and that the receptor in the detrusor may be implicated in facilitation to sustain bladder contraction force.


Assuntos
Contração Muscular/genética , Receptores Purinérgicos P2/deficiência , Bexiga Urinária/fisiopatologia , Micção , Trifosfato de Adenosina/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Estimulação Elétrica , Expressão Gênica , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Músculo Liso/fisiopatologia , RNA Mensageiro/genética , Receptores Purinérgicos P2/genética , Reflexo , Bexiga Urinária/metabolismo
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