RESUMEN

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.

Asunto(s)

Adhesión Celular , Diabetes Mellitus Experimental , Proteína-Tirosina Quinasas de Adhesión Focal , Podocitos , Proteinuria , Receptor de Adenosina A2B , Animales , Humanos , Masculino , Ratas , Adenosina/metabolismo , Adenosina/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/tratamiento farmacológico , Proteína-Tirosina Quinasas de Adhesión Focal/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/efectos de los fármacos , Podocitos/metabolismo , Podocitos/efectos de los fármacos , Podocitos/patología , Proteinuria/metabolismo , Receptor de Adenosina A2B/efectos de los fármacos , Receptor de Adenosina A2B/metabolismo

RESUMEN

Some chemoattractants and leukocytes such as M1 and M2 macrophages are known to be involved in the development of glomerulosclerosis during diabetic nephropathy (DN). In the course of diabetes, an altered and defective cellular metabolism leads to the increase in adenosine levels, and thus to changes in the polarity (M1/M2) of macrophages. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerulosclerosis and decreased macrophage-myofibroblast transition in DN rats. Therefore, we aimed to investigate the effect of MRS1754 on the glomerular expression/secretion of chemoattractants, the intraglomerular infiltration of leukocytes, and macrophage polarity in DN rats. Kidneys/glomeruli of non-diabetic, DN, and MRS1754-treated DN rats were processed for transcriptomic analysis, immunohistopathology, ELISA, and in vitro macrophage migration assays. The transcriptomic analysis identified an upregulation of transcripts and pathways related to the immune system in the glomeruli of DN rats, which was attenuated using MRS1754. The antagonism of the A2BAR decreased glomerular expression/secretion of chemoattractants (CCL2, CCL3, CCL6, and CCL21), the infiltration of macrophages, and their polarization to M2 in DN rats. The in vitro macrophages migration induced by conditioned-medium of DN glomeruli was significantly decreased using neutralizing antibodies against CCL2, CCL3, and CCL21. We concluded that the pharmacological blockade of the A2BAR decreases the transcriptional expression of genes/pathways related to the immune response, protein expression/secretion of chemoattractants, as well as the infiltration of macrophages and their polarization toward the M2 phenotype in the glomeruli of DN rats, suggesting a new mechanism implicated in the antifibrotic effect of MRS1754.

Asunto(s)

Acetamidas , Antagonistas del Receptor de Adenosina A2 , Polaridad Celular , Factores Quimiotácticos , Nefropatías Diabéticas , Glomérulos Renales , Macrófagos , Purinas , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/inmunología , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Factores Quimiotácticos/antagonistas & inhibidores , Factores Quimiotácticos/genética , Factores Quimiotácticos/metabolismo , Polaridad Celular/efectos de los fármacos , Polaridad Celular/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Antagonistas del Receptor de Adenosina A2/farmacología , Receptor de Adenosina A2B , Acetamidas/farmacología , Purinas/farmacología , Animales , Ratas , Movimiento Celular/efectos de los fármacos , Masculino , Ratas Sprague-Dawley , Transcripción Genética/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Inmunidad/efectos de los fármacos , Inmunidad/genética

RESUMEN

Glioblastoma is the most common and aggressive primary brain tumor, characterized by its high chemoresistance and the presence of a cell subpopulation that persists under hypoxic niches, called glioblastoma stem-like cells (GSCs). The chemoresistance of GSCs is mediated in part by adenosine signaling and ABC transporters, which extrude drugs outside the cell, such as the multidrug resistance-associated proteins (MRPs) subfamily. Adenosine promotes MRP1-dependent chemoresistance under normoxia. However, adenosine/MRPs-dependent chemoresistance under hypoxia has not been studied until now. Transcript and protein levels were determined by RT-qPCR and Western blot, respectively. MRP extrusion capacity was determined by intracellular 5 (6)-Carboxyfluorescein diacetate (CFDA) accumulation. Cell viability was measured by MTS assays. Cell cycle and apoptosis were determined by flow cytometry. Here, we show for the first time that MRP3 expression is induced under hypoxia through the A2B adenosine receptor. Hypoxia enhances MRP-dependent extrusion capacity and the chemoresistance of GSCs. Meanwhile, MRP3 knockdown decreases GSC viability under hypoxia. Downregulation of the A2B receptor decreases MRP3 expression and chemosensibilizes GSCs treated with teniposide under hypoxia. These data suggest that hypoxia-dependent activation of A2B adenosine receptor promotes survival of GSCs through MRP3 induction.

Asunto(s)

Neoplasias Encefálicas , Glioblastoma , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Adenosina/metabolismo , Neoplasias Encefálicas/metabolismo , Resistencia a Antineoplásicos , Glioblastoma/metabolismo , Humanos , Hipoxia/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Células Madre Neoplásicas/metabolismo , Receptor de Adenosina A2B/metabolismo , Receptores Purinérgicos P1/metabolismo

RESUMEN

The purinergic system is fundamental in the tumor microenvironment, since it regulates tumor cell interactions with the immune system, as well as growth and differentiation in autocrine-paracrine responses. Here, we investigated the role of the adenosine A2B receptor (A2BR) in ovarian carcinoma-derived cells' (OCDC) properties. From public databases, we documented that high A2BR expression is associated with a better prognostic outcome in ovarian cancer patients. In vitro experiments were performed on SKOV-3 cell line to understand how A2BR regulates the carcinoma cell phenotype associated with cell migration. RT-PCR and Western blotting revealed that the ADORA2B transcript (coding for A2BR) and A2BR were expressed in SKOV-3 cells. Stimulation with BAY-606583, an A2BR agonist, induced ERK1/2 phosphorylation, which was abolished by the antagonist PSB-603. Pharmacological activation of A2BR reduced cell migration and actin stress fibers; in agreement, A2BR knockdown increased migration and enhanced actin stress fiber expression. Furthermore, the expression of E-cadherin, an epithelial marker, increased in BAY-606583-treated cells. Finally, cDNA microarrays revealed the pathways mediating the effects of A2BR activation on SKOV-3 cells. Our results showed that A2BR contributed to maintaining an epithelial-like phenotype in OCDC and highlighted this purinergic receptor as a potential biomarker.

Asunto(s)

Carcinoma Epitelial de Ovario , Movimiento Celular , Receptor de Adenosina A2B , Actinas , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Femenino , Humanos , Neoplasias Ováricas/genética , Receptor de Adenosina A2B/genética , Receptor de Adenosina A2B/metabolismo , Microambiente Tumoral

RESUMEN

Glioblastoma multiforme (GBM) is a complicated and heterogeneous brain tumor with short-term survival outcomes. Commercial therapies are not practical due to cell infiltration capacity, high proliferative rate, and blood-brain barrier. In this context, recognition of the molecular mechanism of tumor progression might help the development of new cancer therapeutics. Recently, more evidence has supported CD73 and downstream adenosine A2A/A2B receptor signaling playing a crucial role in glioblastoma pathogenesis; therefore, targeting CD73 in murine tumor models can reduce tumor development. CD73 is an ecto-enzyme inducing tumor metastasis, angiogenesis, and immune escape via the production of extracellular adenosine in the tumor microenvironment. In this review, we provided information about clinical characteristics as well as the therapeutic management of glioblastoma. Then, we focused on newly available experimental evidence distinguishing between the essential role of CD73 on this tumor growth and a new method for the treatment of GBM patients.

Asunto(s)

5'-Nucleotidasa/metabolismo , Neoplasias Encefálicas , Glioblastoma , Adenosina , Animales , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Ratones , Receptor de Adenosina A2B , Microambiente Tumoral

RESUMEN

Sickle cell disease (SCD) is a disease resulting from mutation in the globin portion of hemoglobin caused by the replacement of adenine for thymine in the codon of the ß globin gene. In Brazil, SCD affects about 0.3% of the black and Caucasian population. Until now, there is no specific treatment and the available drugs have several serious adverse effects which makes the search for new drugs an emergently need. The use of computational techniques can accelerate the drug development process by prioritization of molecules with affinity against essential targets. Adenosine A2b receptor (rA2b) has been studied in SCD due to its relationship with red blood cells concentration of 2,3-diphosphoglycerate which reduces the hemoglobin affinity for oxygen (O2), facilitating its availability for the tissues. Then, development of rA2b antagonists could be helpful for the treatment of SCD. However, there is still no 3D structure of rA2b and to overcome this limitation, homology modeling should be applied. In this scenario, this study aims to build a suitable 3D model of rA2b by SWISS MODEL and to evaluate the structural aspects of rA2b with known antagonists that may be useful for the identification of new potential antagonists by molecular dynamics on a lipid bilayer environment using GROMACS 5.1.4. The complexes with antagonists ZINC223070016 and ZINC17974526 interacted with key residues by hydrophobic contacts and hydrogen bonds which stabilized them at the rA2b binding site. This intermolecular profile can contribute to the development of more potent rA2b antagonists. Communicated by Ramaswamy H. Sarma.

Asunto(s)

Antagonistas del Receptor de Adenosina A2 , Anemia de Células Falciformes , Humanos , Antagonistas del Receptor de Adenosina A2/química , Receptor de Adenosina A2B/química , Anemia de Células Falciformes/tratamiento farmacológico , Simulación de Dinámica Molecular , Enlace de Hidrógeno

RESUMEN

Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.

Asunto(s)

5'-Nucleotidasa/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Caveolina 1/metabolismo , Células Dendríticas/inmunología , Leishmaniasis/inmunología , Microdominios de Membrana/inmunología , Receptor de Adenosina A2B/metabolismo , Animales , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Células Dendríticas/patología , Inmunidad , Inmunomodulación , Leishmania/inmunología , Leishmaniasis/metabolismo , Leishmaniasis/parasitología , Leishmaniasis/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/parasitología , Macrófagos/patología , Masculino , Microdominios de Membrana/parasitología , Microdominios de Membrana/patología , Ratones , Ratones Endogámicos C57BL

RESUMEN

Diabetic nephropathy (DN) is considered the main cause of kidney disease in which myofibroblasts lead to renal fibrosis. Macrophages were recently identified as the major source of myofibroblasts in a process known as macrophage-myofibroblast transition (MMT). Adenosine levels increase during DN and in vivo administration of MRS1754, an antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A2BAR and MMT in glomerulosclerosis during DN. Kidneys/glomeruli of non-diabetic, diabetic, and MRS1754-treated diabetic (DM+MRS1754) rats were processed for histopathologic, transcriptomic, flow cytometry, and cellular in vitro analyses. Macrophages were used for in vitro cell migration/transmigration assays and MMT studies. In vivo MRS1754 treatment attenuated the clinical and histopathological signs of glomerulosclerosis in DN rats. Transcriptomic analysis demonstrated a decrease in chemokine-chemoattractants/cell-adhesion genes of monocytes/macrophages in DM+MRS1754 glomeruli. The number of intraglomerular infiltrated macrophages and MMT cells increased in diabetic rats. This was reverted by MRS1754 treatment. In vitro cell migration/transmigration decreased in macrophages treated with MRS1754. Human macrophages cultured with adenosine and/or TGF-ß induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A2BAR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.

Asunto(s)

Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Macrófagos/patología , Monocitos/patología , Miofibroblastos/patología , Receptor de Adenosina A2B/metabolismo , Acetamidas/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Biomarcadores/metabolismo , Moléculas de Adhesión Celular/metabolismo , Quimiocinas/metabolismo , Factores Quimiotácticos/farmacología , Fibrosis , Humanos , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Purinas/farmacología , Ratas Sprague-Dawley , Transcripción Genética/efectos de los fármacos

RESUMEN

In cancer, the adenosinergic pathway participates in the generation of an immunosuppressive microenvironment and in the promotion of tumor growth through the generation of adenosine (Ado). The present study analyzed the participation of Ado, generated through the functional activity of the cervical cancer (CeCa) pathway in CeCa cells, to induce the expression and secretion of TGF-ß1, as well as the participation of this factor to maintain CD73 expression. Ado concentrations greater than 10 µM were necessary to induce an increase of over 50% in the production and expression of TGF-ß1 in CeCa tumor cells. Blockade of A2AR and A2BR with the specific antagonists, ZM241385 and MRS1754, respectively, strongly reversed the production of TGF-ß1. TGF-ß1 produced by CeCa cells was necessary to maintain CD73 expression because the addition of anti-TGF-ß neutralizing antibodies or the inhibition of TGF-ßRI strongly reversed the expression of CD73 in the CeCa cells. These results suggested a feedback loop in CeCa cells that favors immunosuppressive activity through the production of TGF-ß1 and Ado as well as the autocrine activity of TGF-ß1 and expression of CD73.

Asunto(s)

5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Comunicación Autocrina/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Acetamidas/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Línea Celular Tumoral , Femenino , Proteínas Ligadas a GPI/metabolismo , Células HeLa , Humanos , Terapia de Inmunosupresión/métodos , Purinas/farmacología , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Triazinas/farmacología , Triazoles/farmacología , Microambiente Tumoral/efectos de los fármacos , Neoplasias del Cuello Uterino/tratamiento farmacológico

RESUMEN

Adenosine receptors are considered as potential targets for drug development against several diseases. The discovery of subtype 2B adenosine receptors role in erythrocyte sickling process proved its importance to neglected diseases such as sickle cell anemia, which affects approximately 29.000 people around the world, but whose treatment is restricted to just one FDA approved drug (hydroxyurea). In order to widen the therapeutic arsenal available to treat sickle cell anemia patients, it is imperative to identify new lead compounds that modify the sickling course and not just its symptoms. In order to accomplish this goal, ligand-based pharmacophore models that differentiate true ligands from decoys and enlighten the structure-activity relationship of known RA2B antagonists were employed screen the lead-like subset of the ZINC database. Following a chemical diversity analysis, 18 compounds were selected for biological evaluation. Among them, one molecule Z1139491704 (pEC50 = 7.77 ± 0.17) has shown better anti-sickling activity than MRS1754 (pEC50 = 7.63 ± 0.12), a commercial RA2B antagonist. Moreover, these compounds exhibited no cytotoxic effect at low micromolar range on mammalian cells. In conclusion, the sound development of validated ligand-based pharmacophore models proved essential to identify novel chemical scaffolds that might be useful to develop anti-sickling drugs.

Asunto(s)

Antagonistas del Receptor de Adenosina A2/farmacología , Anemia de Células Falciformes/tratamiento farmacológico , Receptor de Adenosina A2B/metabolismo , Antagonistas del Receptor de Adenosina A2/síntesis química , Antagonistas del Receptor de Adenosina A2/química , Animales , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Macrófagos/efectos de los fármacos , Ratones , Estructura Molecular , Relación Estructura-Actividad

RESUMEN

Spatiotemporal regulation of cAMP within the cell is required to achieve receptor-specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP-responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein-coupled receptor (Ado-GPCR), bound to either A2A or A2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody-mediated inactivation. A2A -Ado-GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150-tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B -Ado-GPCR stimulation activates D-AKAP2-attached AC5 to generate cAMP, which is channeled to two other D-AKAP2-tethered proteins: guanine-nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or 'signalosomes'. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

Asunto(s)

Adenilil Ciclasas/metabolismo , AMP Cíclico/biosíntesis , Hepatocitos/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Transducción de Señal , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Adenilil Ciclasas/genética , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hepatocitos/citología , Masculino , Cultivo Primario de Células , Ratas , Ratas Wistar , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2B/genética

RESUMEN

The adenosine A2b receptor is a G-protein coupled receptor. Its activation occurs with high extracellular adenosine concentration, for example in inflammation or hypoxia. These conditions are generated in the tumor environment. Studies show that A2b receptor is overexpressed in various tumor lines and biopsies from patients with different cancers. This suggests that A2b receptor can be used by tumor cells to promote progression. Thus A2b participates in different events, such as angiogenesis and metastasis, besides exerting immunomodulatory effects that protect tumor cells. Therefore, adenosine A2b receptor appears as an interesting therapeutic target for cancer treatment.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/patología , Receptor de Adenosina A2B/genética , Regulación hacia Arriba , Adenosina/análisis , Adenosina/genética , Adenosina/inmunología , Antagonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/uso terapéutico , Animales , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Receptor de Adenosina A2B/análisis , Receptor de Adenosina A2B/inmunología , Microambiente Tumoral/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

To investigate the functionality of A2B adenosine receptor (A2BAR) and the nitric oxide (NO) and vascular endothelial growth factor (VEGF) signaling pathway in the endothelial cell proliferation/migration during preeclampsia, we used human umbilical vein endothelial cells (HUVECs) isolated from normal pregnancies (n = 15) or pregnancies with preeclampsia (n = 15). Experiments were performed in presence or absence of the nonselective adenosine receptor agonist NECA, the A2BAR selective antagonist MRS-1754, and the nitric oxide synthase (NOS) inhibitor L-NAME. Results indicated that cells from preeclampsia exhibited a significant higher protein level of A2BAR and logEC50 for NECA-mediated proliferation than normotensive pregnancies. The stimulatory effect of NECA (10 µM, 24 h) on cell proliferation was prevented by MRS-1754 (5 nM) coincubation only in cells from normotensive pregnancies. Nevertheless, L-NAME (100 µM, 24 h) reduced the NECA-induced cell proliferation/migration in HUVEC from normal pregnancy; however in preeclampsia only NECA-induced cell proliferation was reduced by L-NAME. Moreover, NECA increased protein nitration and abundance of VEGF in cells from normal pregnancy and effect prevented by MRS-1754 coincubation. Nevertheless, in preeclampsia NECA did not affect the protein level of VEGF. In conclusion HUVECs from preeclampsia exhibit elevated protein level of A2BAR and impairment of A2BAR-mediated NO/VEGF signaling pathway.

Asunto(s)

Movimiento Celular , Proliferación Celular , Endotelio Vascular/metabolismo , Feto/metabolismo , Preeclampsia/metabolismo , Receptor de Adenosina A2B/metabolismo , Acetamidas/farmacología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Adulto , Antineoplásicos/farmacología , Endotelio Vascular/patología , Femenino , Feto/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Óxido Nítrico/metabolismo , Preeclampsia/patología , Embarazo , Purinas/farmacología , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular

RESUMEN

Adenosine receptors have been considered as potential targets for drug development, but one of the main obstacles to this goal is to selectively inhibit one receptor subtype over the others. This subject is particularly crucial for adenosine A2b receptor antagonists (AdoRA2B). The structure­activity relationships of xanthine derivatives which are AdoRA2B have been comprehensively investigated, but the steric and electronic requirements of deazaxanthine AdoRA2B have not been described from a quantitative standpoint of view. Herein we report our efforts to shorten this knowledge gap through 2D-QSAR (HQSAR) and 3D-QSAR (CoMFA) approaches. The good statistical quality (HQSAR--r(2) = 0.85, q(2)(LOO) = 0.77; CoMFA ­ r(2) = 0.86, q(2) = 0.70) and predictive ability (r(2) = (pred1) = 0.78, r(2)(pred2) = 0.78 and r(2) = (pred1) = 0.70, r(2) = (pred2) = 0.70,respectively) of the models, along with the information provided by contribution and contour maps hints their usefulness to the design of more potent 9-deazaxanthine derivatives.

Asunto(s)

Diseño de Fármacos , Receptor de Adenosina A2B/metabolismo , Xantinas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-Actividad Cuantitativa , Xantinas/síntesis química , Xantinas/química

RESUMEN

A(2B) adenosine receptor antagonists may be beneficial in treating diseases like asthma, diabetes, diabetic retinopathy, and certain cancers. This has stimulated research for the development of potent ligands for this subtype, based on quantitative structure-affinity relationships. In this work, a new ensemble machine learning algorithm is proposed for classification and prediction of the ligand-binding affinity of A(2B) adenosine receptor antagonists. This algorithm is based on the training of different classifier models with multiple training sets (composed of the same compounds but represented by diverse features). The k-nearest neighbor, decision trees, neural networks, and support vector machines were used as single classifiers. To select the base classifiers for combining into the ensemble, several diversity measures were employed. The final multiclassifier prediction results were computed from the output obtained by using a combination of selected base classifiers output, by utilizing different mathematical functions including the following: majority vote, maximum and average probability. In this work, 10-fold cross- and external validation were used. The strategy led to the following results: i) the single classifiers, together with previous features selections, resulted in good overall accuracy, ii) a comparison between single classifiers, and their combinations in the multiclassifier model, showed that using our ensemble gave a better performance than the single classifier model, and iii) our multiclassifier model performed better than the most widely used multiclassifier models in the literature. The results and statistical analysis demonstrated the supremacy of our multiclassifier approach for predicting the affinity of A(2B) adenosine receptor antagonists, and it can be used to develop other QSAR models.

Asunto(s)

Antagonistas del Receptor de Adenosina A2/química , Reconocimiento de Normas Patrones Automatizadas/estadística & datos numéricos , Receptor de Adenosina A2B/química , Máquina de Vectores de Soporte , Árboles de Decisión , Humanos , Ligandos , Redes Neurales de la Computación , Purinas/química , Pirimidinas/química , Relación Estructura-Actividad Cuantitativa , Quinazolinas/química

RESUMEN

UNLABELLED: Ischemia and reperfusion-elicited tissue injury contributes to morbidity and mortality of hepatic surgery and during liver transplantation. Previous studies implicated extracellular adenosine signaling in liver protection. Based on the notion that extracellular adenosine signaling is terminated by uptake from the extracellular towards the intracellular compartment by way of equilibrative nucleoside transporters (ENTs), we hypothesized a functional role of ENTs in liver protection from ischemia. During orthotopic liver transplantation in humans, we observed higher expressional levels of ENT1 than ENT2, in conjunction with repression of ENT1 and ENT2 transcript and protein levels following warm ischemia and reperfusion. Treatment with the pharmacologic ENT inhibitor dipyridamole revealed elevations of hepatic adenosine levels and robust liver protection in a murine model of liver ischemia and reperfusion. Studies in gene-targeted mice for Ent1 or Ent2 demonstrated selective protection from liver injury in Ent1(-/-) mice. Treatment with selective adenosine receptor antagonists indicated a contribution of Adora2b receptor signaling in ENT-dependent liver protection. CONCLUSION: These findings implicate ENT1 in liver protection from ischemia and reperfusion injury and suggest ENT inhibitors may be of benefit in the prevention or treatment of ischemic liver injury.

Asunto(s)

Adenosina/fisiología , Tranportador Equilibrativo 1 de Nucleósido/fisiología , Hígado/irrigación sanguínea , Daño por Reperfusión/prevención & control , Animales , Dipiridamol/farmacología , Transportador Equilibrativo 2 de Nucleósido/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Trasplante de Hígado , Ratones , Ratones Endogámicos C57BL , Receptor de Adenosina A2B/fisiología

RESUMEN

Diabetic nephropathy (DN) continues being the primary cause of chronic hemodialysis and terminal renal disease worldwide. At tissue levels the DN occurs with glomerulopathy affecting the integrity of the filtration barrier and with an extensive glomerular and tubule-interstitial fibrosis. Current available therapeutic approaches have only demonstrated a modest effect on progression of kidney injury. Therefore, more research concerning the pathomechanisms and possible interventions are needed. Interestingly, in the last years it has been documented that DN progresses with growing levels of the nucleoside adenosine. This finding increased the interest in the events controlling the extracellular levels of the nucleoside. While the metabolism of extracellular ATP and cyclic AMP are well recognized sources, evidences regarding the role of the equilibrative nucleoside transporters in controlling adenosine availability and promoting diabetic glomerulopathy have recently acquired a pivotal role. The physiological effects of nucleoside are mediated by the P1 family of adenosine receptors. It has been shown in vivo that the use of an antagonist of the A2B receptor subtype can block the most remarkable early alterations seen in diabetic glomerulopathy. Furthermore, using models of chronic kidney injury it was demonstrated that fibrosis can also be blocked using treatment with the antagonist of A2B receptor subtype. This review highlights these findings that correlate the activity of a low affinity adenosine receptor with an increase in the ligand availability in the pathological state. In addition, we discuss the possible therapeutic interventions of adenosine signaling with regards to DN treatment.

Asunto(s)

Adenosina/metabolismo , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Receptor de Adenosina A2B/metabolismo , Transducción de Señal/efectos de los fármacos , Adenosina/genética , Animales , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Progresión de la Enfermedad , Humanos , Receptor de Adenosina A2B/genética , Transducción de Señal/genética

RESUMEN

Diabetic nephropathy ranks as the most devastating kidney disease worldwide. It characterizes in the early onset by glomerular hypertrophy, hyperfiltration and mesangial expansion. Experimental models show that overproduction of vascular endothelial growth factor (VEGF) is a pathogenic condition for podocytopathy; however the mechanisms that regulate this growth factor induction are not clearly identified. We determined that the adenosine A(2B) receptor (A(2B)AR) mediates VEGF overproduction in ex vivo glomeruli exposed to high glucose concentration, requiring PKCα and Erk1/2 activation. The glomerular content of A(2B)AR was concomitantly increased with VEGF at early stages of renal disease in streptozotocin-induced diabetic rats. Further, in vivo administration of an antagonist of A(2B)AR in diabetic rats blocked the glomerular overexpression of VEGF, mesangial cells activation and proteinuria. In addition, we also determined that the accumulation of extracellular adenosine occurs in glomeruli of diabetic rats. Correspondingly, raised urinary adenosine levels were found in diabetic rats. In conclusion, we evidenced that adenosine signaling at the onset of diabetic kidney disease is a pathogenic event that promotes VEGF induction.

Asunto(s)

Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , Receptor de Adenosina A2B/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Acetamidas/farmacología , Adenosina/metabolismo , Adenosina/orina , Animales , Glucemia/metabolismo , Presión Sanguínea/fisiología , Peso Corporal/fisiología , Diabetes Mellitus Experimental/orina , Nefropatías Diabéticas/orina , Histocitoquímica , Glomérulos Renales/química , Glomérulos Renales/metabolismo , Masculino , Purinas/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología

RESUMEN

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A(2B) adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A(2B) receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.

Asunto(s)

Antígenos CD40/inmunología , Células Dendríticas/inmunología , Leishmania/inmunología , Leishmaniasis/inmunología , Receptor de Adenosina A2B/inmunología , Acetamidas/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Antígeno B7-2/biosíntesis , Antígeno B7-2/inmunología , Células de la Médula Ósea/inmunología , Antígenos CD40/biosíntesis , Células Cultivadas , Antígenos de Histocompatibilidad Clase II/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Nucleotidasas/biosíntesis , Purinas/farmacología , Suramina/farmacología , Linfocitos T/inmunología , Tripanocidas/farmacología

RESUMEN

Human endothelial progenitor cells (hEPC) are recruited to sites of neovascularization where they differentiate into endothelial cells. The signals/factors responsible for hEPC migration and adhesion to sites of injury are not well understood. Elevated levels of adenosine are known to increase mature endothelial cell migration in response to tissue injury. However, the understanding of the role of adenosine in the physiology of hEPC is very limited. Using quantitative polymerase chain reaction and western blot analyses, we detected the expression of the adenosine receptors A2A, A2B, and A3 in hEPC. Stimulation of adenosine receptors using adenosine or the nonselective agonist adenosine-5'-N-ethylcarboxamide (NECA) increased hEPC migration in 1.4-fold and 2.1-fold (P < 0.01), respectively. Stimulation of hEPC using the A2A-specific agonist CGS-21680 resembled the effect observed in migration when using adenosine or NECA. Consequently, NECA and CGS-21680-stimulated migration of hEPC were reverted using the A2A receptor antagonist ZM-241385. NECA-stimulated migration was inhibited in dose-dependent manner using MRS-1523 (Ki of 147 ± 0.016 nM), MRS-1754 (Ki of 1900 ± 0.02 nM), or ZM-241385 (Ki of 0.2 ± 0.01 nM). In conclusion, adenosine stimulates hEPC migration by activating A2A and A3 but not A2B receptors and provides evidence to support a role of adenosine in modulating angiogenic capacity of hEPC.

Asunto(s)

Células Endoteliales/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Receptor de Adenosina A3/metabolismo , Adenosina/metabolismo , Adulto , Western Blotting , Adhesión Celular , Movimiento Celular , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Reacción en Cadena de la Polimerasa , Agonistas del Receptor Purinérgico P1/administración & dosificación , Agonistas del Receptor Purinérgico P1/farmacología , Antagonistas de Receptores Purinérgicos P1/administración & dosificación , Antagonistas de Receptores Purinérgicos P1/farmacología , Células Madre/metabolismo