RESUMO
AIMS: It is well established that intracellular cAMP contributes to the relaxation of vas deferens smooth muscle. In many tissues, intracellular cAMP is actively transported to the extracellular space, where it exerts regulatory functions, via its metabolite adenosine. These actions take place through the cAMP conversion to adenosine by ectoenzymes, a process called "extracellular cAMP-adenosine pathway". Herein, we investigated whether, in addition to ATP, extracellular cAMP might be an alternative source of adenosine, influencing the contraction of vas deferens smooth muscle. MAIN METHODS: The effects of cAMP, 8-Br-cAMP and adenosine were analyzed in the isometric contractions of rat vas deferens. cAMP efflux was analyzed by measuring extracellular cAMP levels after exposure of vas deferens segments to isoproterenol and forskolin in the presence or absence of MK-571, an inhibitor of MRP/ABCC transporters. KEY FINDINGS: While 8-Br-cAMP, a cell-permeable cAMP analog, induced relaxation of KCl-precontracted vas deferens, the non-permeant cAMP increased the KCl-induced contractile response, which was mimicked by adenosine, but prevented by inhibitors of ecto-5'-nucleotidase or A1 receptors. Our results also showed that isoproterenol and forskolin increases cAMP efflux via an MRP/ABCC transporter-dependent mechanism, since it is inhibited by MK-571. SIGNIFICANCE: Our data show that activation of ß-adrenoceptors and adenylyl cyclase increases cAMP efflux from vas deferens tissue, which modulates the vas deferens contractile response via activation of adenosine A1 receptors. Assuming that inhibition of vas deferens contractility has been proposed as a strategy for male contraception, the extracellular cAMP-adenosine pathway emerges as a potential pharmacological target that should be considered in studies of male fertility.
Assuntos
5'-Nucleotidase , AMP Cíclico , Contração Muscular , Ratos Wistar , Receptor A1 de Adenosina , Ducto Deferente , Masculino , Animais , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismo , AMP Cíclico/metabolismo , 5'-Nucleotidase/metabolismo , Receptor A1 de Adenosina/metabolismo , Receptor A1 de Adenosina/efeitos dos fármacos , Ratos , Contração Muscular/efeitos dos fármacos , Adenosina/farmacologia , Adenosina/análogos & derivados , Adenosina/metabolismo , Isoproterenol/farmacologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Colforsina/farmacologiaRESUMO
Induction of the adenosine receptor A2B (A2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A2BAR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A2BAR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A2BAR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A2BAR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.
Assuntos
Adesão Celular , Diabetes Mellitus Experimental , Proteína-Tirosina Quinases de Adesão Focal , Podócitos , Proteinúria , Receptor A2B de Adenosina , Animais , Humanos , Masculino , Ratos , Adenosina/metabolismo , Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/tratamento farmacológico , Proteína-Tirosina Quinases de Adesão Focal/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/patologia , Proteinúria/metabolismo , Receptor A2B de Adenosina/efeitos dos fármacos , Receptor A2B de Adenosina/metabolismoRESUMO
The danger of ionizing radiation exposure to human health is a concern. Since its wide use in medicine and industry, the development of radioprotectors has been very significant. Adenosine exerts anti-inflammatory actions and promotes tissue protection and repair, by activating the P1 receptors (A1, A2A, A2B, and A3). Zebrafish (Danio rerio) is an appropriate tool in the fields of toxicology and pharmacology, including the evaluation of radiobiological outcomes and in the search for radioprotector agents. This study aims to evaluate the effect of adenosine in the toxicity induced by radiation in zebrafish. Embryos were treated with 1, 10, or 100 µM adenosine, 30 min before the exposure to 15 Gy of gamma radiation. Adenosine potentiated the effects of radiation in heart rate, body length, and pericardial edema. We evaluated oxidative stress, tissue remodeling and inflammatory. It was seen that 100 µM adenosine reversed the inflammation induced by radiation, and that A2A2 and A2B receptors are involved in these anti-inflammatory effects. Our results indicate that P1R activation could be a promising pharmacological strategy for radioprotection.
Assuntos
Adenosina , Peixe-Zebra , Humanos , Animais , Adenosina/farmacologia , Raios gama/efeitos adversos , Frequência Cardíaca , Anti-InflamatóriosRESUMO
To date, there is insufficient evidence to explain the role of adenosinergic receptors in the reconsolidation of long-term spatial memory. In this work, the role of the adenosinergic receptor family (A1, A2A, A2B, and A3) in this process has been elucidated. It was demonstrated that when infused bilaterally into the hippocampal CA1 region immediately after an early nonreinforced test session performed 24 h posttraining in the Morris water maze task, adenosine can cause anterograde amnesia for recent and late long-term spatial memory. This effect on spatial memory reconsolidation was blocked by A1 or A3 receptor antagonists and mimicked by A1 plus A3 receptor agonists, showing that this effect occurs through A1 and A3 receptors simultaneously. The A3 receptor alone participates only in the reconsolidation of late long-term spatial memory. When the memory to be reconsolidated was delayed (reactivation 5 d posttraining), the amnesic effect of adenosine became transient and did not occur in a test performed 5 d after the reactivation of the mnemonic trace. Finally, it has been shown that the amnesic effect of adenosine on spatial memory reconsolidation depends on the occurrence of protein degradation and that the amnesic effect of inhibition of protein synthesis on spatial memory reconsolidation is dependent on the activation of A3 receptors.
Assuntos
Hipocampo , Memória de Longo Prazo , Ratos , Masculino , Animais , Hipocampo/fisiologia , Memória de Longo Prazo/fisiologia , Memória/fisiologia , Região CA1 Hipocampal , Adenosina/metabolismo , Adenosina/farmacologiaRESUMO
The A1 adenosine receptor is the most widely expressed P1 receptor in vertebrates, performing inhibitory tone of the nervous system. Increased levels of adenosine are crucial to promote tissue protection in threatening situations, such as convulsion and hypoxia. Zebrafish is an established model organism for studies on health and disease. In this study, we evaluated the functionality of A1 adenosine receptor through development of zebrafish (6-7-day-, 3-, 8-, and 24-month-old), assessing: (I) the effects of the agonist N6-cyclopenthyladenosine (CPA) over locomotor parameters, (II) the anticonvulsant properties of CPA and adenosine per se in the pentylenetetrazol-induced seizure, and (III) the gene expression of adora1b through development. CPA promoted decreased distance traveled in the highest concentrations/doses tested (larvae: 75 to 500 µM; adults: 20 mg.kg-1), altered mean velocity (larvae: 50-500 µM; adults: 20 mg.kg-1) and time in the bottom zone of apparatus (adults: decrease in 20 mg.kg-1). Adenosine increased the latency of the larvae to reach stage II at 5 and 10 µM. CPA anticonvulsant effect against convulsive stage II was reached at 75 µM, although it decreased basal locomotor activity in larvae. For adults, CPA 10 mg.kg-1 was effective as anticonvulsant without locomotory effects. Adenosine had minor anticonvulsant effects in the concentration tested (larvae: 5 and 10 µM). The level of gene expression of adora1b was stable in brain from adult animals (8- and 24-month-old animals). These results suggest that zebrafish has similar responses to CPA as mammals. To avoid confounding factors, such as locomotor effects, during any brain function investigation using A1 adenosine receptor as a target, the concentration below 75 µM or below the dose of 20 mg.kg-1 of CPA is ideal for zebrafish at larval and adult stages, respectively.
Assuntos
Anticonvulsivantes , Peixe-Zebra , Animais , Peixe-Zebra/genética , Adenosina/farmacologia , Receptores Purinérgicos P1/genética , Expressão Gênica , MamíferosRESUMO
Human ecto-5'-nucleotidase (h-ecto-5'-NT, CD73) is a homodimeric Zn2+-binding metallophosphoesterase that hydrolyzes adenosine 5'-monophosphate (5'-AMP) to adenosine and phosphate. h-Ecto-5'-NT is a key enzyme in purinergic signaling pathways and has been recognized as a promising biological target for several diseases, including cancer and inflammatory, infectious, and autoimmune diseases. Despite its importance as a biological target, little is known about h-ecto-5'-NT dynamics, which poses a considerable challenge to the design of inhibitors of this target enzyme. Here, to explore h-ecto-5'-NT flexibility, all-atom unbiased molecular dynamics (MD) simulations were performed. Remarkable differences in the dynamics of the open (catalytically inactive) and closed (catalytically active) conformations of the apo-h-ecto-5'-NT were observed during the simulations, and the nucleotide analogue inhibitor AMPCP was shown to stabilize the protein structure in the closed conformation. Our results suggest that the large and complex domain motion that enables the h-ecto-5'-NT open/closed conformational switch is slow, and therefore, it could not be completely captured within the time scale of our simulations. Nonetheless, we were able to explore the faster dynamics of the h-ecto-5'-NT substrate binding site, which is mainly located at the C-terminal domain and well conserved among the protein's open and closed conformations. Using the TRAPP ("Transient Pockets in Proteins") approach, we identified transient subpockets close to the substrate binding site. Finally, conformational states of the substrate binding site with higher druggability scores than the crystal structure were identified. In summary, our study provides valuable insights into h-ecto-5'-NT structural flexibility, which can guide the structure-based design of novel h-ecto-5'-NT inhibitors.
Assuntos
5'-Nucleotidase , Simulação de Dinâmica Molecular , Humanos , Monofosfato de Adenosina/metabolismo , Adenosina/farmacologia , Sítios de LigaçãoRESUMO
ß2-adrenoceptors agonists and phosphodiesterase (PDE) inhibitors are effective bronchodilators, due to their ability to increase intracellular cyclic AMP (cAMP) levels and induce airway smooth muscle (ASM) relaxation. We have shown that increment of intracellular cAMP induced by ß2-adrenoceptors agonist fenoterol is followed by efflux of cAMP, which is converted by ecto-PDE and ecto-5'-nucleotidases (ecto-5'NT) to adenosine, leading to ASM contraction. Here we evaluate whether other classical bronchodilators used to treat asthma and chronic obstructive pulmonary disease (COPD) could induce cAMP efflux and, as consequence, influence the ASM contractility. Our results showed that ß2-adrenoceptor agonists formoterol and PDE inhibitors IBMX, aminophylline and roflumilast induced cAMP efflux and a concentration-dependent relaxation of rat trachea precontracted with carbachol. Pretreatment of tracheas with MK-571 (MRP transporter inhibitor), AMP-CP (ecto-5'NT inhibitor) or CGS-15943 (nonselective adenosine receptor antagonist) potentiated the relaxation induced by ß2-adrenoceptor agonists but did not change the relaxation induced by PDE inhibitors. These data showed that all bronchodilators tested were able to induce cAMP efflux. However, only ß2-adrenoceptor-induced relaxation of tracheal smooth muscle was affected by cAMP efflux and extracellular cAMP-adenosine pathway.
Assuntos
Adenosina , AMP Cíclico , Ratos , Animais , AMP Cíclico/metabolismo , Adenosina/farmacologia , Fumarato de Formoterol/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Broncodilatadores/farmacologia , Relaxamento Muscular , Agonistas Adrenérgicos beta , Traqueia , Receptores AdrenérgicosRESUMO
ATP and adenosine (ADO) are critical players in the context of cancer. In the tumor microenvironment, the signaling dependent on these molecules, and immune cells, is regulated by an enzymatic chain and purinergic receptors called purinome. Primarily, the A2A receptor (A2AR) has a pro-tumor action since it reduces the immune response and favors the growth of malignant melanoma. Therefore, this study aimed to verify the effects of A2AR antagonism with Istradefylline (IST) on the purinergic signaling profile of the melanoma tumor and immunological compartments. We observed reduced tumor growth of melanoma in IST-treated animals. IST inhibited AKT/mTOR pathway, which is involved with tumor growth. In the tumor, spleen, and thymus, the modulation of purinergic enzymes (CD39, CD73, and E-ADA) characterized a pro-inflammatory profile since it favored increased extracellular concentrations of ATP to the detriment of ADO. A2AR inhibition generated a compensatory feedback process with increased A2AR expression at the tumor level. However, there was also an increase in the expression of the P2X7 receptor (P2X7R), which culminated in an increase in pro-inflammatory pathways with the release of IL-1ß and pro-inflammatory cytokines such as IFN-γ and TNF-α. Our data evidence the cross-involvement between expression and action of the A2AR and P2X7R. We suggest that IST is a promising drug for off-label use in cancer since it promotes an anti-tumoral response by producing pro-inflammatory cytokines and blocking of AKT/mTOR tumor growth pathway.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas c-akt , Camundongos , Animais , Citocinas/metabolismo , Adenosina/farmacologia , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Melanoma/tratamento farmacológico , Serina-Treonina Quinases TOR , Microambiente TumoralRESUMO
Alcohol (ethanol) dependence and related disorders are life-threatening conditions and source of suffering for the user, family members and society. Alcohol withdrawal syndrome (AWS) is a little-known dynamic process associated with a high frequency of relapses. A state of hyperglutamatergic neurotransmission and imbalanced GABAergic function is related to an increased susceptibility to seizures during alcohol withdrawal. Adenosine signaling display an important role in endogenous response to decrease seizure and related damages. Here, an intermittent alcohol exposure regimen (1 h daily of 0.5% ethanol solution) for 16 days or 8 days of the same ethanol exposure regimen followed by 1 or 8 days of ethanol withdrawal was used to assess adenosine signaling in the context of seizure susceptibility using adult zebrafish. In both abstainer groups, a sub-convulsant dose of pentylenetetrazol (2.5 mM) was able to increase the frequency of animals reaching a clonic seizure-like state, while continuous-treated animals had no seizure, as did control animals. The total brain mRNA expression of A1 adenosine receptor was decreased in animals with 1 day of ethanol withdrawal. The agonism of A1 adenosine receptor induced an anticonvulsant effect in animals with 1 day of ethanol withdrawal after the injection of the specific agonist (N6-cyclopentyladenosine, 10 mg.Kg- 1; i.p.). These findings reinforce A1 adenosine receptor as a key target in acute alcohol withdrawal syndrome and zebrafish as an excellent platform to study biological mechanism of AWS.