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1.
Nutrients ; 13(10)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34684530

RESUMO

In hypertensive individuals, platelet morphology and function have been discovered to be altered, and this has been linked to the development of vascular disease, including erectile dysfunction (ED). The impact of nutritional supplementation with Cyperus esculentus (tiger nut, TN) and Tetracarpidium conophorum (walnut, WN) on androgen levels, ectonucleotidases, and adenosine deaminase (ADA) activities in platelets from L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) challenged rats were investigated. We hypothesized that these nuts may show a protective effect on platelets aggregation and possibly enhance the sex hormones, thereby reverting vasoconstriction. Wistar rats (male; 250-300 g; n = 10) were grouped into seven groups as follows: basal diet control group (I); basal diet/L-NAME/Viagra (5 mg/kg/day) as positive control group (II); ED-induced group (basal diet/L-NAME) (III); diet supplemented processed TN (20%)/L-NAME (IV); diet supplemented raw TN (20%)/L-NAME (V); diet supplemented processed WN (20%)/L-NAME (VI); and diet supplemented raw WN (20%)/L-NAME (VII). The rats were given their regular diet for 2 weeks prior to actually receiving L-NAME (40 mg/kg/day) for ten days to induce hypertension. Platelet androgen levels, ectonucleotidases, and ADA were all measured. L-NAME considerably lowers testosterone levels (54.5 ± 2.2; p < 0.05). Supplementing the TN and WN diets revealed improved testosterone levels as compared to the control (306.7 ± 5.7), but luteinizing hormone levels remained unchanged. Compared to control groups, the L-NAME-treated group showed a rise in ATP (127.5%) hydrolysis and ADA (116.7%) activity, and also a decrease in ADP (76%) and AMP (45%) hydrolysis. Both TN and WN supplemented diets resulted in substantial (p < 0.05) reversal effects. Enhanced testosterone levels and modulation of the purinergic system in platelets by TN and WN could be one of the mechanisms by which they aid in vasoconstriction control.


Assuntos
Plaquetas/efeitos dos fármacos , Cyperus , Suplementos Nutricionais , Hipertensão/terapia , Juglans , NG-Nitroarginina Metil Éster/farmacologia , Adenosina Desaminase/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Dieta/métodos , Hidrólise/efeitos dos fármacos , Hipertensão/sangue , Hipertensão/induzido quimicamente , Masculino , Proteínas de Membrana/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Purinérgicos/farmacologia , Ratos , Ratos Wistar , Testosterona/sangue , Vasoconstrição/efeitos dos fármacos
2.
Amino Acids ; 53(3): 359-380, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33586041

RESUMO

The antioxidant and anti-proinflammatory activities of L-leucine were investigated on oxidative testicular injury, ex vivo. In vitro analysis revealed L-leucine to be a potent scavenger of free radicals, while inhibiting acetylcholinesterase activity. Oxidative injury was induced in testicular tissues using FeSO4. Treatment with L-leucine led to depletion of oxidative-induced elevated levels of NO, MDA, and myeloperoxidase activity, with concomitant elevation of reduced glutathione and non-protein thiol levels, SOD and catalase activities. L-leucine caused a significant (p < 0.05) alteration of oxidative-elevated acetylcholinesterase and chymotrypsin activities, while concomitantly elevating the activities of ATPase, ENTPDase and 5'-nucleotidase. L-leucine conferred a protective effect against oxidative induced DNA damage. Molecular docking revealed molecular interactions with COX-2, IL-1 beta and iNOS. Treatment with L-leucine led to restoration of oxidative depleted ascorbic acid-2-sulfate, with concomitant depletion of the oxidative induced metabolites: D-4-Hydroxy-2-oxoglutarate, L-cystine, adenosine triphosphate, maleylacetoacetic acid, cholesteryl ester, and 6-Hydroxy flavin adenine dinucleotide. Treatment with L-leucine reactivated glycolysis while concomitantly deactivating oxidative-induced citrate cycle and increasing the impact-fold of purine metabolism pathway. L-leucine was predicted not to be an inhibitor of CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, with a predicted LD50 value of 5000 mg/Kg and toxicity class of 5. Additionally, L-leucine showed little or no in vitro cytotoxicity in mammalian cells. These results suggest the therapeutic potentials of L-leucine on oxidative testicular injury, as evident by its ability to attenuate oxidative stress and proinflammation, while stalling cholinergic dysfunction and modulating nucleotide hyrolysis; as well as modulate oxidative dysregulated metabolites and their pathways.


Assuntos
Colinérgicos/metabolismo , Leucina/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Purinérgicos/metabolismo , Testículo/lesões , Animais , Anti-Inflamatórios/metabolismo , Antioxidantes/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colinérgicos/química , Dano ao DNA/efeitos dos fármacos , Compostos Ferrosos/toxicidade , Humanos , Leucina/química , Masculino , Simulação de Acoplamento Molecular , Ratos , Testículo/metabolismo
3.
Cardiovasc Res ; 117(5): 1284-1294, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32991685

RESUMO

Adenosine is an endogenous nucleoside that plays a major role in the physiology and physiopathology of the coronary artery system, mainly by activating its A2A receptors (A2AR). Adenosine is released by myocardial, endothelial, and immune cells during hypoxia, ischaemia, or inflammation, each condition being present in coronary artery disease (CAD). While activation of A2AR improves coronary blood circulation and leads to anti-inflammatory effects, down-regulation of A2AR has many deleterious effects during CAD. A decrease in the level and/or activity of A2AR leads to: (i) lack of vasodilation, which decreases blood flow, leading to a decrease in myocardial oxygenation and tissue hypoxia; (ii) an increase in the immune response, favouring inflammation; and (iii) platelet aggregation, which therefore participates, in part, in the formation of a fibrin-platelet thrombus after the rupture or erosion of the plaque, leading to the occurrence of acute coronary syndrome. Inflammation contributes to the development of atherosclerosis, leading to myocardial ischaemia, which in turn leads to tissue hypoxia. Therefore, a vicious circle is created that maintains and aggravates CAD. In some cases, studying the adenosinergic profile can help assess the severity of CAD. In fact, inducible ischaemia in CAD patients, as assessed by exercise stress test or fractional flow reserve, is associated with the presence of a reserve of A2AR called spare receptors. The purpose of this review is to present emerging experimental evidence supporting the existence of this adaptive adenosinergic response to ischaemia or inflammation in CAD. We believe that we have achieved a breakthrough in the understanding and modelling of spare A2AR, based upon a new concept allowing for a new and non-invasive CAD management.


Assuntos
Adenosina/metabolismo , Doença da Artéria Coronariana/metabolismo , Vasos Coronários/metabolismo , Receptor A2A de Adenosina/metabolismo , Animais , Plaquetas/metabolismo , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/patologia , Circulação Coronária , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/patologia , Humanos , Placa Aterosclerótica , Purinérgicos/uso terapêutico , Transdução de Sinais
4.
J Am Heart Assoc ; 9(18): e017404, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32867554

RESUMO

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by increased pulmonary arterial pressure and pulmonary vascular resistance, which result in an increase in afterload imposed onto the right ventricle, leading to right heart failure. Current therapies are incapable of reversing the disease progression. Thus, the identification of novel and potential therapeutic targets is urgently needed. An alteration of nucleotide- and nucleoside-activated purinergic signaling has been proposed as a potential contributor in the pathogenesis of PAH. Adenosine-mediated purinergic 1 receptor activation, particularly A2AR activation, reduces pulmonary vascular resistance and attenuates pulmonary vascular remodeling and right ventricle hypertrophy, thereby exerting a protective effect. Conversely, A2BR activation induces pulmonary vascular remodeling, and is therefore deleterious. ATP-mediated P2X7R activation and ADP-mediated activation of P2Y1R and P2Y12R play a role in pulmonary vascular tone, vascular remodeling, and inflammation in PAH. Recent studies have revealed a role of ectonucleotidase nucleoside triphosphate diphosphohydrolase, that degrades ATP/ADP, in regulation of pulmonary vascular remodeling. Interestingly, existing evidence that adenosine activates erythrocyte A2BR signaling, counteracting hypoxia-induced pulmonary injury, and that ATP release is impaired in erythrocyte in PAH implies erythrocyte dysfunction as an important trigger to affect purinergic signaling for pathogenesis of PAH. The present review focuses on current knowledge on alteration of nucleot(s)ide-mediated purinergic signaling as a potential disease mechanism underlying the development of PAH.


Assuntos
Hipertensão Arterial Pulmonar/metabolismo , Receptores Purinérgicos/metabolismo , Animais , Humanos , Hipertensão Arterial Pulmonar/fisiopatologia , Purinérgicos/farmacologia , Receptores Purinérgicos/efeitos dos fármacos , Receptores Purinérgicos/fisiologia
5.
Dev Comp Immunol ; 113: 103768, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32692996

RESUMO

Neutrophil extracellular trap (NET) formation eliminates/prevents the spread of infectious agents. Platelet activating factor (PAF) is involved in infectious diseases of cattle because it recruits and activates neutrophils. However, its ability to induce NET release and the role of metabolism in this process is not known. We investigated if inhibition of glycolysis, mitochondrial-derived adenosine triphosphate (ATP) synthesis and purinergic signaling though P2X1 purinoceptors interfered with NET formation induced by PAF. We inhibited bovine neutrophils with 2-deoxy-d-glucose, rotenone, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and NF449 to evaluate PAF-mediated NET extrusion. PAF induced mitochondrial hyperpolarization and triggered extracellular ATP release via pannexin-1. Inhibition of mitochondrial metabolism prevented extracellular ATP release. Inhibition of glycolysis, complex-I activity and oxidative phosphorylation prevented NET formation induced by PAF. Inhibition of P2X1 purinergic receptors inhibited mitochondrial hyperpolarization and NET formation. We concluded that PAF-induced NET release is dependent upon glycolysis, mitochondrial ATP synthesis and purinergic signaling.


Assuntos
Trifosfato de Adenosina/metabolismo , Bovinos/fisiologia , Armadilhas Extracelulares/metabolismo , Mitocôndrias/metabolismo , Neutrófilos/imunologia , Fator de Ativação de Plaquetas/metabolismo , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células Cultivadas , Desoxiglucose/farmacologia , Transporte de Elétrons , Glicólise , Imunidade Inata , Ativação de Neutrófilo , Purinérgicos/metabolismo , Purinérgicos/farmacologia , Receptores Purinérgicos P2X1/metabolismo , Rotenona/farmacologia , Transdução de Sinais
7.
Neurosci Res ; 152: 35-43, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31958495

RESUMO

Purinergic transmitters such as adenosine, ADP, ATP, UTP, and UDP-glucose play important roles in a wide range of physiological processes, including the sleep-wake cycle, learning and memory, cardiovascular function, and the immune response. Moreover, impaired purinergic signaling has been implicated in various pathological conditions such as pain, migraine, epilepsy, and drug addiction. Examining the function of purinergic transmission in both health and disease requires direct, sensitive, non-invasive tools for monitoring structurally similar purinergic transmitters; ideally, these tools should have high spatial and temporal resolution in in vivo applications. Here, we review the recent progress with respect to the development and application of new methods for detecting purinergic transmitters, focusing on optical tools; in addition, we provide discussion regarding future perspectives.


Assuntos
Imagem Óptica/métodos , Purinérgicos/metabolismo , Receptores Purinérgicos/metabolismo , Animais , Humanos , Neurotransmissores
8.
Front Immunol ; 10: 1633, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379836

RESUMO

Purine nucleotides and nucleosides are at the center of biologic reactions. In particular, adenosine triphosphate (ATP) is the fundamental energy currency of cellular activity and adenosine has been demonstrated to play essential roles in human physiology and pathophysiology. In this review, we examine the role of purinergic signaling in acute and chronic pulmonary inflammation, with emphasis on ATP and adenosine. ATP is released into extracellular space in response to cellular injury and necrosis. It is then metabolized to adenosine monophosphate (AMP) via ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and further hydrolyzed to adenosine via ecto-5'-nucleotidase (CD73). Adenosine signals via one of four adenosine receptors to exert pro- or anti-inflammatory effects. Adenosine signaling is terminated by intracellular transport by concentrative or equilibrative nucleoside transporters (CNTs and ENTs), deamination to inosine by adenosine deaminase (ADA), or phosphorylation back into AMP via adenosine kinase (AK). Pulmonary inflammatory and hypoxic conditions lead to increased extracellular ATP, adenosine diphosphate (ADP) and adenosine levels, which translates to increased adenosine signaling. Adenosine signaling is central to the pulmonary injury response, leading to various effects on inflammation, repair and remodeling processes that are either tissue-protective or tissue destructive. In the acute setting, particularly through activation of adenosine 2A and 2B receptors, adenosine signaling serves an anti-inflammatory, tissue-protective role. However, excessive adenosine signaling in the chronic setting promotes pro-inflammatory, tissue destructive effects in chronic pulmonary inflammation.


Assuntos
Pneumonia/metabolismo , Purinérgicos/metabolismo , Transdução de Sinais/fisiologia , Adenosina Desaminase/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Humanos
9.
Microb Pathog ; 135: 103649, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31374321

RESUMO

Extracellular adenosine triphosphate (ATP) is as key mediator of immune and inflammatory responses. ATP is normally sequestered in the intracellular milieu and released by apoptotic and necrotic cells, where it acts as a pro-inflammatory mediator in the extracellular milieu. A limited number of studies have explored the involvement of purinergic signaling in oomycete infections, including Saprolegnia parasitica; this is a most destructive oomycete pathogen, associated with high mortality and severe economic losses for fish producers. The aim of this study was to determine whether purinergic signaling exerts anti- or pro-inflammatory effects in spleens of grass carp (Ctenopharyngodon idella) naturally infected by S. parasitica. Animals naturally infected with S. parasitica showed typical gross lesions characterized by cotton-wool tufts on the tail and fins, as well as severe histopathological lesions such as necrosis. Spleen ATP and metabolites of nitric oxide (NOx) levels were higher in fish naturally infected by S. parasitica compared to control on day 7 post-infection (PI). Spleen nucleoside triphosphate diphosphohydrolase (NTPDase) activity (ATP as substrate) was greater in fish naturally infected by S. parasitica than in uninfected on day 7 PI, while no significant differences were observed between groups with respect to NTPDase (adenosine diphosphate as substrate) and 5'-nucleotidase activities. Finally, adenosine deaminase (ADA) activity was lower in fish naturally infected by S. parasitica than in uninfected fish on day 7 PI. In summary, spleen tissue necrosis in the context of saprolegniosis provokes an intense release of ATP into the extracellular milieu, where it interacts with the P2X7 purine receptor and leads to a self-sustained pro-inflammatory deleterious cycle, contributing to an intense inflammatory process. In response to excessive ATP levels in the extracellular milieu, ATP and adenosine hydrolysis were modulated in an attempt to restrict the inflammatory process via upregulation of NTPDase and downregulation of ADA activities. We conclude that the purinergic signaling pathway modulates immune and inflammatory responses during natural infection with S. parasitica.


Assuntos
Trifosfato de Adenosina/metabolismo , Anti-Inflamatórios/metabolismo , Carpas/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/parasitologia , Purinérgicos/metabolismo , Transdução de Sinais , Baço/metabolismo , Adenosina Desaminase/metabolismo , Animais , Carpas/metabolismo , Modelos Animais de Doenças , Doenças dos Peixes/patologia , Proteínas de Peixes/imunologia , Micoses , Necrose , Óxido Nítrico/metabolismo , Saprolegnia/patogenicidade , Baço/patologia
11.
Int J Mol Sci ; 20(12)2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216722

RESUMO

Hearing impairment is the most common sensory deficit, affecting more than 400 million people worldwide. Sensorineural hearing losses currently lack any specific or efficient pharmacotherapy largely due to the insufficient knowledge of the pathomechanism. Purinergic signaling plays a substantial role in cochlear (patho)physiology. P2 (ionotropic P2X and the metabotropic P2Y) as well as adenosine receptors expressed on cochlear sensory and non-sensory cells are involved mostly in protective mechanisms of the cochlea. They are implicated in the sensitivity adjustment of the receptor cells by a K+ shunt and can attenuate the cochlear amplification by modifying cochlear micromechanics. Cochlear blood flow is also regulated by purines. Here, we propose to comprehend this field with the purine-immune interactions in the cochlea. The role of harmful immune mechanisms in sensorineural hearing losses has been emerging in the horizon of cochlear pathologies. In addition to decreasing hearing sensitivity and increasing cochlear blood supply, influencing the immune system can be the additional avenue for pharmacological targeting of purinergic signaling in the cochlea. Elucidating this complexity of purinergic effects on cochlear functions is necessary and it can result in development of new therapeutic approaches in hearing disabilities, especially in the noise-induced ones.


Assuntos
Cóclea/imunologia , Cóclea/metabolismo , Doenças Cocleares/etiologia , Doenças Cocleares/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Cóclea/fisiologia , Cóclea/ultraestrutura , Doenças Cocleares/tratamento farmacológico , Doenças Cocleares/fisiopatologia , Expressão Gênica , Perda Auditiva Neurossensorial/etiologia , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Purinérgicos/metabolismo , Receptores Purinérgicos/genética , Receptores Purinérgicos/metabolismo , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo
12.
J Physiol ; 597(7): 1957-1973, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30746715

RESUMO

KEY POINTS: In the epididymis, elaborate communication networks between epithelial cells are important with respect to establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa, which is essential for male fertility. Proton secretion by epididymal clear cells is achieved via the proton pumping V-ATPase located in their apical membrane. In the present study, we dissect the molecular mechanisms by which clear cells respond to luminal ATP and adenosine to modulate their acidifying activity via the adenosine receptor ADORA2B and the pH-sensitive ATP receptor P2X4. We demonstrate that the hydrolysis of ATP to produce adenosine by ectonucleotidases plays a key role in V-ATPase-dependent proton secretion, and is part of a feedback loop that ensures acidification of the luminal compartment These results help us better understand how professional proton-secreting cells respond to extracellular cues to modulate their functions, and how they communicate with neighbouring cells. ABSTRACT: Cell-cell cross-talk is crucial for the dynamic function of epithelia, although how epithelial cells detect and respond to variations in extracellular stimuli to modulate their environment remains incompletely understood. In the present study, we used the epididymis as a model system to investigate epithelial cell regulation by luminal factors. In the epididymis, elaborate communication networks between the different epithelial cell types are important for establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa. In particular, clear cells (CCs) secrete protons into the lumen via the proton pumping V-ATPase located in their apical membrane, a process that is activated by luminal alkalinization. However, how CCs detect luminal pH variations to modulate their function remains uncharacterized. Purinergic regulation of epithelial transport is modulated by extracellular pH in other tissues. In the present study, functional analysis of the mouse cauda epididymis perfused in vivo showed that luminal ATP and adenosine modulate the acidifying activity of CCs via the purinergic ADORA2B and P2X4 receptors, and that luminal adenosine content is itself regulated by luminal pH. Altogether, our observations illustrate mechanisms by which CCs are activated by pH sensitive P2X4 receptor and ectonucleotidases, providing a feedback mechanism for the maintenance of luminal pH. These novel mechanisms by which professional proton-secreting cells respond to extracellular cues to modulate their functions, as well as how they communicate with neighbouring cells, might be translatable to other acidifying epithelia.


Assuntos
Trifosfato de Adenosina/farmacologia , Adenosina/farmacologia , Epididimo/fisiologia , Purinérgicos , Agonistas Purinérgicos/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Epididimo/efeitos dos fármacos , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Antagonistas Purinérgicos/farmacologia , Receptor A2B de Adenosina/genética , Receptor A2B de Adenosina/metabolismo , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética
13.
Int J Mol Sci ; 19(11)2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30441872

RESUMO

The need for bone grafts is high, due to age-related diseases, such as tumor resections, but also accidents, risky sports, and military conflicts. The gold standard for bone grafting is the use of autografts from the iliac crest, but the limited amount of accessible material demands new sources of bone replacement. The use of mesenchymal stem cells or their descendant cells, namely osteoblast, the bone-building cells and endothelial cells for angiogenesis, combined with artificial scaffolds, is a new approach. Mesenchymal stem cells (MSCs) can be obtained from the patient themselves, or from donors, as they barely cause an immune response in the recipient. However, MSCs never fully differentiate in vitro which might lead to unwanted effects in vivo. Interestingly, purinergic receptors can positively influence the differentiation of both osteoblasts and endothelial cells, using specific artificial ligands. An overview is given on purinergic receptor signaling in the most-needed cell types involved in bone metabolism-namely osteoblasts, osteoclasts, and endothelial cells. Furthermore, different types of scaffolds and their production methods will be elucidated. Finally, recent patents on scaffold materials, as wells as purinergic receptor-influencing molecules which might impact bone grafting, are discussed.


Assuntos
Regeneração Óssea , Regeneração Tecidual Guiada/métodos , Purinérgicos/farmacologia , Receptores Purinérgicos/metabolismo , Tecidos Suporte/química , Animais , Ensaios Clínicos como Assunto , Humanos , Osteogênese/efeitos dos fármacos
14.
J Neuroinflammation ; 15(1): 325, 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30463629

RESUMO

BACKGROUND: The ATP-gated ionotropic P2X7 receptor (P2X7R) has the unusual ability to function as a small cation channel and a trigger for permeabilization of plasmalemmal membranes. In murine microglia, P2X7R-mediated permeabilization is fundamental to microglial activation, proliferation, and IL-1ß release. However, the role of the P2X7R in primary adult human microglia is poorly understood. METHODS: We used patch-clamp electrophysiology to record ATP-gated current in cultured primary human microglia; confocal microscopy to measure membrane blebbing; fluorescence microscopy to demonstrate membrane permeabilization, caspase-1 activation, phosphatidylserine translocation, and phagocytosis; and kit-based assays to measure cytokine levels. RESULTS: We found that ATP-gated inward currents facilitated with repetitive applications of ATP as expected for current through P2X7Rs and that P2X7R antagonists inhibited these currents. P2X7R antagonists also prevented the ATP-induced uptake of large cationic fluorescent dyes whereas drugs that target pannexin-1 channels had no effect. In contrast, ATP did not induce uptake of anionic dyes. The uptake of cationic dyes was blocked by drugs that target Cl- channels. Finally, we found that ATP activates caspase-1 and inhibits phagocytosis, and these effects are blocked by both P2X7R and Cl- channel antagonists. CONCLUSIONS: Our results demonstrate that primary human microglia in culture express functional P2X7Rs that stimulate both ATP-gated cationic currents and uptake of large molecular weight cationic dyes. Importantly, our data demonstrate that hypotheses drawn from work on murine immune cells accurately predict the essential role of P2X7Rs in a number of human innate immune functions such as phagocytosis and caspase-1 activation. Therefore, the P2X7R represents an attractive target for therapeutic intervention in human neuroinflammatory disorders.


Assuntos
Microglia/fisiologia , Receptores Purinérgicos P2X7/metabolismo , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacocinética , Trifosfato de Adenosina/farmacologia , Adulto , Anexina A5/metabolismo , Cálcio/metabolismo , Caspase 1/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Interleucina-1beta/metabolismo , Ionóforos/farmacologia , Masculino , Microglia/efeitos dos fármacos , Nigericina/farmacologia , Fagocitose/efeitos dos fármacos , Purinérgicos/farmacologia
16.
JCI Insight ; 3(17)2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30185656

RESUMO

Invariant natural killer T (iNKT) cells are activated at sites of local tissue injury, or globally during vaso-occlusive episodes of sickle cell disease (SCD). Tissue damage stimulates production of CD1d-restricted lipid antigens that activate iNKT cells to produce Th1- and Th2-type cytokines. Here, we show that circulating iNKT cells in SCD patients express elevated levels of the ectonucleoside triphosphate diphosphosphohydrolase, CD39, as well the adenosine A2A receptor (A2AR). We also investigated the effects of stimulating cultured human iNKT cells on the expression of genes involved in the regulation of purinergic signaling. iNKT cell stimulation caused induction of ADORA2A, P2RX7, CD38, CD39, ENPP1, CD73, PANX1, and ENT1. Transcription of ADA, which degrades adenosine, was reduced. Induction of CD39 mRNA was associated with increased ecto-ATPase activity on iNKT cells that was blocked by POM1. Exposure of iNKT cells to A2AR agonists during stimulation reduced production of IFN-γ and enhanced production of IL-13 and CD39. Based on these findings, we define "purinergic Th2-type cytokine bias" as an antiinflammatory purinergic response to iNKT cell stimulation resulting from changes in the transcription of several genes involved in purine release, extracellular metabolism, and signaling.


Assuntos
Anemia Falciforme/imunologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Transdução de Sinais/genética , 5'-Nucleotidase , ADP-Ribosil Ciclase 1 , Antígenos CD1d , Apirase/metabolismo , Conexinas , Citocinas/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo , Proteínas Ligadas por GPI , Humanos , Imunidade Inata , Interleucina-13 , Proteínas do Tecido Nervoso , Diester Fosfórico Hidrolases , Purinérgicos/metabolismo , Purinas/metabolismo , Pirofosfatases , Receptor A2A de Adenosina , Receptores Purinérgicos P2X7/metabolismo , Fatores de Transcrição
17.
Brain Res ; 1700: 47-55, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29935155

RESUMO

The four kinds of adenosine receptor subtypes (ARs), named as ARA1, ARA2A, ARA2B and ARA3, have multiple biological functions. ARs are differently distributed across the body and have distinguished ability of binding adenosine. We try to figure out how these ARs were expressed in astrocytes and which one has the first priority of utilizing adenosine. Firstly, mRNA expressions and membrane localization of all ARs were evaluated by qPCR and western blot. After the membrane localization of all ARs in astrocytes was being confirmed their individual adenosine binding ability was determined by radio-active ligand binding assay respectively. It was revealed that ARA1 had much superior adenosine binding ability than other AR subtypes. Functional study demonstrated that ARA1 potentially mediated an immune suppressive effect in astrocytes. The activation of ARA1 signaling lead to decreased IL-12 and IL-23 production, and decreased chemokine production, including CCL2, CXCL8 and IP-10. When interacted with CD4 cells ARA1 agonist pre-treated astrocytes showed hindered ability of stimulating CD4 cells to secret IL-17 and IFN-γ and inducing CD4 cells' chemo taxi. Finally, in vivo experiment confirmed that local administration of ARA1agonist ameliorated EAE in wild type B6 recipients, but not Ara1-/- recipients. As a conclusion, this paper suggested that adenosine receptor A1 subtype predominantly binds adenosine in astrocytes and mediates an immunosuppressive effect.


Assuntos
Adenosina/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Receptor A1 de Adenosina/metabolismo , Adenosina/farmacologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Feminino , Imunossupressores/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Purinérgicos/farmacologia , RNA Mensageiro/metabolismo , Receptor A1 de Adenosina/genética
19.
Inflammation ; 41(5): 1648-1660, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29804189

RESUMO

Somatostatin and its analogues are known to have modulatory effects on immune response and their anti-proliferative, anti-angiogenic, and analgesic properties make them attractive candidates for a therapeutic use in immune-mediated diseases, such as rheumatoid arthritis. Here, we demonstrate the ability of the somatostatin analogue octreotide to inhibit interleukin-15 and to increase interleukin-10 production by rheumatoid arthritis fibroblast-like synovial cells maintained in a chronic inflammatory state. We also prove that the inhibitory effect of octreotide on interleukin-15 and tumor necrosis factor-α production depended on the increase in interleukin-10, since neutralizing anti-interleukin-10 antibody was able to partially reverse this inhibition. In addition, our observations suggest an octreotide control on purinergic signaling, with an inhibitory effect on purinergic P2X and P2Y receptors activation. This would have great implications, considering the roles of P2 receptors in the onset of inflammation. Data here reported extend knowledge on the biological action of octreotide and underline its multiple effects on immune response, which could make octreotide an attractive and valid support for the therapy of diseases where several inflammatory mediators are involved, such as rheumatoid arthritis, and in which the simultaneous action on different aspects can be a successful strategy.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Inflamação/tratamento farmacológico , Octreotida/farmacologia , Somatostatina/análogos & derivados , Sinoviócitos/patologia , Artrite Reumatoide/patologia , Humanos , Interleucina-10/agonistas , Interleucina-15/antagonistas & inibidores , Octreotida/uso terapêutico , Purinérgicos , Sinoviócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese
20.
FASEB J ; 32(8): 4356-4369, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29558203

RESUMO

Calcific aortic valve disease is an active disease process with lipoprotein deposition, chronic inflammation, and progressive leaflet degeneration. Expression of ectonucleotidases, a group of membrane-bound enzymes that regulate the metabolism of ATP and its metabolites, may coregulate the degeneration process of valvular interstitial cells (VICs). The aim of this study was to investigate the role of the enzymes of the purinergic system in the degeneration process of VICs. Ovine VICs were cultivated in vitro under different prodegenerative conditions and treated with inhibitors of ectonucleoside triphosphate diphosphohydrolase 1 (CD39)/ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and 5'-nucleotidase (CD73), as well as with adenosine and adenosine receptor agonists. Experiments were performed both in 2-dimensional (2-D) and 3-dimensional (3-D) cell-culture models. Our main findings were that VICs continuously release ATP. Inhibition of ATP hydrolyzing enzymes (CD39 and ENPP1) resulted in profound prodegenerative effects with a vigorous up-regulation of CD39, ENPP1, and CD73, as well as TGF-ß1 and osteopontin at the gene level. In our 3-D model, the effect was more pronounced than in 2-D monolayers. Increasing adenosine levels, as well as stimulating the adenosine receptors A2A and A2B, exhibited strong prodegenerative effects, whereas conversely, lowering adenosine levels by inhibition of CD73 resulted in protective effects against degeneration. Dysregulation of any one of these enzymes plays an important role in the degeneration process of VICs. Stimulation of ATP and adenosine has prodegenerative effects, whereas lowering the adenosine levels exerts a protective effect.-Weber, A., Barth, M., Selig, J. I., Raschke, S., Dakaras, K., Hof, A., Hesse, J., Schrader, J., Lichtenberg, A., Akhyari, P. Enzymes of the purinergic signaling system exhibit diverse effects on the degeneration of valvular interstitial cells in a 3-D microenvironment.


Assuntos
Estenose da Valva Aórtica/metabolismo , Valva Aórtica/patologia , Calcinose/metabolismo , Microambiente Celular/fisiologia , Purinérgicos/metabolismo , Transdução de Sinais/fisiologia , 5'-Nucleotidase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/metabolismo , Valva Aórtica/metabolismo , Apirase/metabolismo , Doença da Válvula Aórtica Bicúspide , Técnicas de Cultura de Células/métodos , Cardiopatias Congênitas/metabolismo , Doenças das Valvas Cardíacas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Ovinos , Regulação para Cima/fisiologia
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