RESUMO
BACKGROUND AND OBJECTIVES: Equilibrative nucleoside transporter (ENT) 1 is a widely-expressed drug transporter, handling nucleoside analogues as well as endogenous nucleosides. ENT1 has been postulated to be inhibited by some marketed tyrosine kinase inhibitors (TKIs). To obtain insights into this point, the interactions of 24 TKIs with ENT1 activity have been analyzed. METHODS: Inhibition of ENT1 activity was investigated in vitro through quantifying the decrease of [3H]-uridine uptake caused by TKIs in HAP1 ENT2-knockout cells, exhibiting selective ENT1 expression. TKI effects towards ENT1-mediated transport were additionally characterized in terms of their in vivo relevance and of their relationship to TKI molecular descriptors. Putative transport of the TKI lorlatinib by ENT1/ENT2 was analyzed by LC-MS/MS. RESULTS: Of 24 TKIs, 12 of them, each used at 10 µM, were found to behave as moderate or strong inhibitors of ENT1, i.e., they decreased ENT1 activity by at least 35%. This inhibition was concentration-dependent for at least the strongest ones (IC50 less than 10 µM) and was correlated with some molecular descriptors, especially with atom-type E-state indices. Lorlatinib was notably a potent in vitro inhibitor of ENT1/ENT2 (IC50 values around 1.0-2.5 µM) and was predicted to inhibit these nucleoside transporters at relevant clinical concentrations, without, however, being a substrate for them. CONCLUSION: Our data unambiguously add ENT1 to the list of drug transporters inhibited by TKIs, especially by lorlatinib. This point likely merits attention in terms of possible drug-drug interactions, notably for nucleoside analogues, whose ENT1-mediated uptake into their target cells may be hampered by co-administrated TKIs such as lorlatinib.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Aminopiridinas/administração & dosagem , Aminopiridinas/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida , Relação Dose-Resposta a Droga , Transportador Equilibrativo 2 de Nucleosídeo/genética , Técnicas de Inativação de Genes , Humanos , Concentração Inibidora 50 , Lactamas/administração & dosagem , Lactamas/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Espectrometria de Massas em TandemRESUMO
Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.
Assuntos
Antimetabólitos Antineoplásicos/farmacocinética , Clofarabina/farmacocinética , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Testículo/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Humanos , Lamivudina/sangue , Lamivudina/farmacocinética , Lamivudina/farmacologia , Masculino , Ratos Sprague-Dawley , Telomerase/genética , Tioinosina/análogos & derivados , Tioinosina/sangue , Tioinosina/farmacocinética , Tioinosina/farmacologia , Tionucleotídeos/sangue , Tionucleotídeos/farmacocinética , Tionucleotídeos/farmacologiaRESUMO
Pseudomonas aeruginosa infections are increasingly multidrug resistant and cause healthcare-associated pneumonia, a major risk factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Adenosine is a signaling nucleoside with potential opposing effects; adenosine can either protect against acute lung injury via adenosine receptors or cause lung injury via adenosine receptors or equilibrative nucleoside transporter (ENT)-dependent intracellular adenosine uptake. We hypothesized that blockade of intracellular adenosine uptake by inhibition of ENT1/2 would increase adenosine receptor signaling and protect against P. aeruginosa-induced acute lung injury. We observed that P. aeruginosa (strain: PA103) infection induced acute lung injury in C57BL/6 mice in a dose- and time-dependent manner. Using ENT1/2 pharmacological inhibitor, nitrobenzylthioinosine (NBTI), and ENT1-null mice, we demonstrated that ENT blockade elevated lung adenosine levels and significantly attenuated P. aeruginosa-induced acute lung injury, as assessed by lung wet-to-dry weight ratio, BAL protein levels, BAL inflammatory cell counts, pro-inflammatory cytokines, and pulmonary function (total lung volume, static lung compliance, tissue damping, and tissue elastance). Using both agonists and antagonists directed against adenosine receptors A2AR and A2BR, we further demonstrated that ENT1/2 blockade protected against P. aeruginosa -induced acute lung injury via activation of A2AR and A2BR. Additionally, ENT1/2 chemical inhibition and ENT1 knockout prevented P. aeruginosa-induced lung NLRP3 inflammasome activation. Finally, inhibition of inflammasome prevented P. aeruginosa-induced acute lung injury. Our results suggest that targeting ENT1/2 and NLRP3 inflammasome may be novel strategies for prevention and treatment of P. aeruginosa-induced pneumonia and subsequent ARDS.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/metabolismo , Tioinosina/análogos & derivados , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/microbiologia , Lesão Pulmonar Aguda/patologia , Animais , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Masculino , Camundongos , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/patologia , Tioinosina/farmacologiaRESUMO
Intestinal epithelial barrier repair is vital for remission in inflammatory bowel disease (IBD). Extracellular adenosine signaling has been implicated in promoting restoration of epithelial barrier function. Currently, no clinically approved agents target this pathway. Adenosine signaling is terminated by uptake from the extracellular space via equilibrative nucleoside transporters (ENTs). We hypothesized that ENT inhibition could dampen intestinal inflammation. Initial studies demonstrated transcriptional repression of ENT1 and ENT2 in IBD biopsies or in murine IBD models. Subsequent studies in mice with global Ent1 or Ent2 deletion revealed selective protection of Ent2-/- mice. Elevated intestinal adenosine levels in conjunction with abolished protection following pharmacologic blockade of A2B adenosine receptors implicate adenosine signaling as the mechanism of gut protection in Ent2-/- mice. Additional studies in mice with tissue-specific deletion of Ent2 uncovered epithelial Ent2 as the target. Moreover, intestinal protection provided by a selective Ent2 inhibitor was abolished in mice with epithelium-specific deletion of Ent2 or the A2B adenosine receptor. Taken together, these findings indicate that increased mucosal A2B signaling following repression or deletion of epithelial Ent2 coordinates the resolution of intestinal inflammation. This study suggests the presence of a targetable purinergic network within the intestinal epithelium designed to limit tissue inflammation.
Assuntos
Colite Ulcerativa/patologia , Doença de Crohn/patologia , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Mucosa Intestinal/patologia , Receptor A2B de Adenosina/metabolismo , Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/administração & dosagem , Animais , Biópsia , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Doença de Crohn/imunologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/genética , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
Equilibrative nucleoside transporters (ENTs) play a crucial role in the transport of nucleoside and nucleoside analogues, which are important for nucleotide synthesis and chemotherapy. In addition, ENTs regulate extracellular adenosine levels in the vicinity of its receptors and hence influence adenosine-related functions. The clinical applications of ENT inhibitors in the treatment of cardiovascular diseases and cancer therapy have been explored in numerous studies. However, all ENT inhibitors to date are selective for ENT1 but not ENT2. In the present study, we investigated the novel compound 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) as an inhibitor of ENT1 and ENT2. Nucleoside transporter-deficient PK15NTD cells stably expressing ENT1 and ENT2 showed that FPMINT inhibited [3H]uridine and [3H]adenosine transport through both ENT1 and ENT2 in a concentration-dependent manner. The IC50 value of FPMINT for ENT2 was 5-10-fold less than for ENT1, and FPMINT could not be displaced with excess washing. Kinetic studies revealed that FPMINT reduced Vmax of [3H]uridine transport in ENT1 and ENT2 without affecting KM. Therefore, we conclude that FPMINT inhibits ENTs in an irreversible and non-competitive manner. Although already selective for ENT2 over ENT1, further modification of the chemical structure of FPMINT may lead to even better ENT2-selective inhibitors of potential clinical, physiological and pharmacological importance.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Naftalenos/farmacologia , Piperazinas/farmacologia , Triazinas/farmacologia , Transporte Biológico/efeitos dos fármacos , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Naftalenos/química , Piperazinas/química , Triazinas/químicaRESUMO
Extracellular adenosine is a neuromodulator in the central nervous system. Astrocytes mainly participate in adenosine production, and extracellular adenosine accumulates under physiological and pathophysiological conditions. Inhibition of intracellular adenosine metabolism and reduction of the external Ca(2+) concentration ([Ca(2+)]e) participate in adenosine accumulation, but the precise mechanisms remain unclear. This study investigated the mechanisms underlying extracellular adenosine accumulation in cultured rat spinal astrocytes. The combination of adenosine kinase and deaminase (ADK/ADA) inhibition and a reduced [Ca(2+)]e increased the extracellular adenosine level. ADK/ADA inhibitors increased the level of extracellular adenosine but not of adenine nucleotides, which was suppressed by inhibition of equilibrative nucleoside transporter (ENT) 2. Unlike ADK/ADA inhibition, a reduced [Ca(2+)]e increased the extracellular level not only of adenosine but also of ATP. This adenosine increase was enhanced by ENT2 inhibition, and suppressed by sodium polyoxotungstate (ecto-nucleoside triphosphate diphosphohydrolase inhibitor). Gap junction inhibitors suppressed the increases in adenosine and adenine nucleotide levels by reduction of [Ca(2+)]e. These results indicate that extracellular adenosine accumulation by ADK/ADA inhibition is due to the adenosine release via ENT2, while that by reduction of [Ca(2+)]e is due to breakdown of ATP released via gap junction hemichannels, after which ENT2 incorporates adenosine into the cells.
Assuntos
Adenosina/metabolismo , Adenosina/fisiologia , Astrócitos/metabolismo , Cálcio/metabolismo , Medula Espinal/citologia , Adenosina Desaminase , Inibidores de Adenosina Desaminase/farmacologia , Adenosina Quinase/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/fisiologia , Junções Comunicantes/efeitos dos fármacos , Neurotransmissores , Ratos WistarRESUMO
As ENT inhibitors including dilazep have shown efficacy improving oHSV1 targeted oncolytic cancer therapy, a series of dilazep analogues was synthesized and biologically evaluated to examine both ENT1 and ENT2 inhibition. The central diamine core, alkyl chains, ester linkage and substituents on the phenyl ring were all varied. Compounds were screened against ENT1 and ENT2 using a radio-ligand cell-based assay. Dilazep and analogues with minor structural changes are potent and selective ENT1 inhibitors. No selective ENT2 inhibitors were found, although some analogues were more potent against ENT2 than the parent dilazep.
Assuntos
Dilazep/análogos & derivados , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Dilazep/síntese química , Dilazep/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Humanos , Ligação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , SuínosRESUMO
The signaling molecule adenosine has been implicated in attenuating acute lung injury (ALI). Adenosine signaling is terminated by its uptake through equilibrative nucleoside transporters (ENTs). We hypothesized that ENT-dependent adenosine uptake could be targeted to enhance adenosine-mediated lung protection. To address this hypothesis, we exposed mice to high-pressure mechanical ventilation to induce ALI. Initial studies demonstrated time-dependent repression of ENT1 and ENT2 transcript and protein levels during ALI. To examine the contention that ENT repression represents an endogenous adaptive response, we performed functional studies with the ENT inhibitor dipyridamole. Dipyridamole treatment (1 mg/kg; EC50=10 µM) was associated with significant increases in ALI survival time (277 vs. 395 min; P<0.05). Subsequent studies in gene-targeted mice for Ent1 or Ent2 revealed a selective phenotype in Ent2(-/-) mice, including attenuated pulmonary edema and improved gas exchange during ALI in conjunction with elevated adenosine levels in the bronchoalveolar fluid. Furthermore, studies in genetic models for adenosine receptors implicated the A2B adenosine receptor (Adora2b) in mediating ENT-dependent lung protection. Notably, dipyridamole-dependent attenuation of lung inflammation was abolished in mice with alveolar epithelial Adora2b gene deletion. Our newly identified crosstalk pathway between ENT2 and alveolar epithelial Adora2b in lung protection during ALI opens possibilities for combined therapies targeted to this protein set.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Receptor A2B de Adenosina/metabolismo , Transdução de Sinais , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/prevenção & controle , Adenosina/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Dipiridamol/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/genética , Expressão Gênica , Humanos , Immunoblotting , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Alvéolos Pulmonares/metabolismo , Troca Gasosa Pulmonar/efeitos dos fármacos , Troca Gasosa Pulmonar/genética , Receptor A2B de Adenosina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vasodilatadores/farmacologiaRESUMO
The purpose of this study was to clarify the transport characteristics of nucleosides in rat placenta and the changes of functional expression of nucleoside transporters in rat placenta with experimental diabetes mellitus. Placental uptake clearances of [(3)H]adenosine and [(3)H]zidovudine from maternal blood was much higher than that of [(14)C]mannitol. Xenopus oocytes injected with rat ENT1 and ENT2 cRNA took up [(3)H]adenosine with K(m) values of 6.1 and 26 µM, respectively. [(3)H]Adenosine transport by rat placental brush-border membrane vesicles (BBMV) was saturable and was inhibited by nitrobenzylthioinosine (NBMPR), a specific ENT inhibitor, in a manner consistent with involvement of both rat ENT1 and ENT2. [(3)H]Didanosine was modestly taken up by placenta, and the inhibitory effect of 100 µM NBMPR on [(3)H]ddI uptake by BBMV suggested a role of ENT2-mediated transport. Expression of ENT1, ENT2, ENT3, CNT2, and CNT3 mRNAs was detected in placenta of control and streptozotocin (STZ)-induced diabetic pregnant rats, and CNT2 (SLC28A2) expression was significantly increased in STZ-induced diabetic rats. Consistently, Na(+)-dependent adenosine uptake by BBMV from STZ-induced diabetic pregnant rats was higher than that from control rats. These results suggest the involvement of placental ENT2 as well as ENT1 in nucleoside uptake from maternal blood, and the induction of CNT2 in experimental diabetes mellitus.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Gestacional/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Nucleosídeos/metabolismo , Placenta/metabolismo , Adenosina/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Gestacional/induzido quimicamente , Diabetes Gestacional/genética , Transportador Equilibrativo 1 de Nucleosídeo , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Feminino , Troca Materno-Fetal , Microvilosidades/metabolismo , Nucleosídeos/sangue , Nucleosídeos/farmacocinética , Oócitos , Placenta/ultraestrutura , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Trofoblastos/metabolismo , Trofoblastos/ultraestrutura , Regulação para Cima , Xenopus laevis , Zidovudina/metabolismoRESUMO
BACKGROUND: Adenosine uptake into cells by nucleoside transporters plays a significant role in governing extracellular adenosine concentration. Extracellular adenosine is an important signaling molecule that modulates many cellular functions via 4 G-protein-coupled receptor subtypes (A(1), A(2A), A(2B), and A(3)). Previously, we demonstrated that adenosine is critical in maintaining airway homeostasis and airway repair and that airway host defenses are impaired by alcohol. Taken together, we hypothesized that ethanol impairs adenosine uptake via the nucleoside transport system. METHODS: To examine ethanol-induced alteration on adenosine transport, we used a human bronchial epithelial cell line (BEAS-2B). Cells were preincubated for 10 minutes in the presence and absence of varying concentrations of ethanol (EtOH). In addition, some cells were pretreated with S-(4-Nitrobenzyl)-6-thioinosine (100 microM: NBT), a potent adenosine uptake inhibitor. Uptake was then determined by addition of [(3)H]-adenosine at various time intervals. RESULTS: Increasing EtOH concentrations resulted in increasing inhibition of adenosine uptake when measured at 1 minute. Cells pretreated with NBT effectively blocked adenosine uptake. In addition, short-term EtOH revealed increased extracellular adenosine concentration. Conversely, adenosine transport became desensitized in cells exposed to EtOH (100 mM) for 24 hours. To determine the mechanism of EtOH-induced desensitization of adenosine transport, cAMP activity was assessed in response to EtOH. Short-term EtOH exposure (10 minutes) had little or no effect on adenosine-mediated cAMP activation, whereas long-term EtOH exposure (24 hours) blocked adenosine-mediated cAMP activation. Western blot analysis of lysates from unstimulated BEAS-2B cells detected a single 55 kDa band indicating the presence of hENT1 and hENT2, respectively. Real-time RT-PCR of RNA from BEAS-2B revealed transcriptional expression of ENT1 and ENT2. CONCLUSIONS: Collectively, these data reveal that acute exposure of cells to EtOH inhibits adenosine uptake via a nucleoside transporter, and chronic exposure of cells to EtOH desensitizes the adenosine transporter to these inhibitory effects of ethanol. Furthermore, our data suggest that inhibition of adenosine uptake by EtOH leads to an increased extracellular adenosine accumulation, influencing the effect of adenosine at the epithelial cell surface, which may alter airway homeostasis.
Assuntos
Adenosina/antagonistas & inibidores , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Etanol/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Adenosina/metabolismo , Linhagem Celular Transformada , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , HumanosRESUMO
The placenta requires nucleosides as nutrients for fetal growth, so it is important to examine potential interactions between placental transports of nucleosides and drugs to ensure the safety of pharmacotherapy during pregnancy. The purposes of this study are to clarify the uptake mechanisms of nucleosides from the maternal side of the syncytiotrophoblast and to investigate the inhibitory effect of various drugs on nucleoside uptake, using the rat syncytiotrophoblast cell line TR-TBT 18d-1, which shows syncytial-like morphology and functional expression of several transporters. Initial uptake of [(3)H]uridine or [(3)H]adenosine from the apical side of TR-TBT 18d-1 was markedly reduced by an excess of the respective unlabelled compound, and was slightly reduced by replacement of Na(+) with N-methyl-d-glucamine, indicating that both uptakes were Na(+)-independent. [(3)H]Uridine and [(3)H]adenosine uptakes in the absence of Na(+) were significantly and concentration-dependently inhibited by both 0.1 microM and 100 microM nitrobenzylthioinosine, suggesting the involvement of equilibrative nucleoside transporters (ENTs, SLC29). Kinetic analysis of adenosine uptake yielded a K(m) value of approximately 17 microM. These results are consistent with the reported uptake characteristics of uridine and adenosine by ENT1 and ENT2. The uptakes were significantly reduced by high concentrations of several nucleoside drugs, including cytarabine, vidarabine, zidovudine, mizoribine, caffeine and amitriptyline, but the effects were small within the therapeutic concentration ranges. In summary, our results suggest that ENTs are involved in apical uptake of uridine and adenosine in the syncytiotrophoblast. However, therapeutic concentrations of the drugs tested in this study might have little influence on maternal-to-fetal nucleoside transfer.
Assuntos
Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Nucleosídeos/farmacocinética , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Animais , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Imunossupressores/farmacologia , Nucleosídeos/antagonistas & inibidores , Ratos , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Trítio/farmacocinéticaRESUMO
To investigate the mechanism of cellular resistance to 6-MP, we established a 6-MP resistant cell line (CEM-MP5) by stepwise selection of the human T-lymphoblastic leukemia cell line (CEM). CEM-MP5 cells were about 100-fold resistant to 6-MP compared with parental CEM cells. Western blot analysis demonstrated that multidrug resistant protein 4 (MRP4) was increased in CEM-MP5 cells, whereas the levels of the nucleoside transporters hENT1, hCNT2 and hCNT3 were decreased compared with those of parental CEM cells. Consistent with the operation of an efflux pump, accumulation of [14C]6-MP and/or its metabolites was reduced, and ATP-dependent efflux was increased in CEM-MP5 cells. Taken together these results showed that up-regulation of MRP4 and down-regulation of influx transporters played a major role in 6-MP resistance of CEM-MP5 cells.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Leucemia/tratamento farmacológico , Mercaptopurina/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Proteínas de Transporte de Nucleosídeos/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Humanos , Hipoxantina Fosforribosiltransferase/metabolismo , Leucemia/metabolismo , Proteínas de Membrana Transportadoras/análise , Mercaptopurina/farmacocinética , Metiltransferases/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Proteínas de Neoplasias/análise , Proteínas de Transporte de Nucleosídeos/análise , Regulação para CimaRESUMO
The cardioprotective actions of adenosine are terminated by its uptake into endothelial cells with subsequent metabolism through hypoxanthine to uric acid. This process involves xanthine oxidase-mediated generation of reactive oxygen species (ROS), which have been implicated in the vascular dysfunction observed in ischemia-reperfusion injury. The equilibrative nucleoside transporter, ENT2, mediates the transfer of hypoxanthine into cells. We hypothesize that ENT2 also mediates the cellular release of hypoxanthine, which would limit the amount of intracellular hypoxanthine available for xanthine oxidase-mediated ROS production. Rat microvascular endothelial cells (MVECs) were isolated from skeletal muscle by lectin-affinity purification. The transport of [(3)H]hypoxanthine was assessed using an oil-stop method, and hypoxanthine metabolites were identified by thin-layer chromatography. MVECs accumulated hypoxanthine with a K(m) of 300 microM and a V(max) of 2.8 pmol microl(-1) s(-1). ATP-depleted cells loaded with [(3)H]hypoxanthine released the radiolabel with kinetics similar to that obtained for [(3)H]hypoxanthine influx. The uptake and release of [(3)H]hypoxanthine were both blocked by ENT2 inhibitors with similar order of potency. Thus, ENT2 mediates both the influx and efflux of hypoxanthine. Inhibition of ENT2 in MVECs might be expected to increase the amount of intracellular hypoxanthine available for metabolism by xanthine oxidase and enhance the intracellular production of ROS.
Assuntos
Endotélio Vascular/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Hipoxantina/metabolismo , Músculo Esquelético/irrigação sanguínea , Alopurinol/farmacologia , Animais , Antimetabólitos/farmacologia , Transporte Biológico , Células Cultivadas , Cromatografia em Camada Fina , Desoxiglucose/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Hipoxantina/análise , Masculino , Microcirculação/citologia , Microcirculação/metabolismo , Ratos , Ratos Wistar , Rotenona/farmacologia , Trítio , Desacopladores/farmacologiaRESUMO
Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.
Assuntos
Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Purinas/metabolismo , 2-Cloroadenosina/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Células Cultivadas , Pré-Escolar , Vasos Coronários/efeitos dos fármacos , Dilazep/farmacologia , Dipiridamol/farmacologia , Cães , Células Endoteliais/efeitos dos fármacos , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Feminino , Humanos , Hipoxantina/metabolismo , Cinética , Microcirculação/metabolismo , Proteínas de Transporte de Nucleobases/antagonistas & inibidores , Piperazinas/farmacologia , Ligação Proteica , Purinas/farmacologia , Ratos , Suínos , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Transfecção , TrítioRESUMO
In C6 glioma cells, adenine nucleotides, especially AMP, and adenosine inhibited cell proliferation in time- and concentration-dependent manners. alpha,beta-methylene-ADP, an ecto-5'-nucleotidase inhibitor, suppressed the hydrolysis of AMP and reversed the inhibition of cell growth induced by AMP but not by adenosine. Adenosine deaminase eliminated both AMP- and adenosine-mediated growth inhibitions. 5'-N-ethylcarboxamidoadenosine, an adenosine receptor agonist, had little effect on the cell growth. Equilibrative nucleoside transporters, ENT-1 and ENT-2, were expressed in C6 cells by determining their mRNAs. ENT inhibitors, nitrobenzylthioinosine and dipyridamole, suppressed the uptake of [(3)H]adenosine into C6 cells, and attenuated AMP- or adenosine-mediated growth inhibition. Furthermore, an adenosine kinase inhibitor 5-iodotubercidin reversed the growth inhibition induced by AMP and adenosine. When uridine was added in the extracellular space, AMP- or adenosine-induced cell growth inhibition was completely reversed, suggesting that intracellular pyrimidine starvation would be involved in their cytostatic effects. These results indicate that extracellular adenine nucleotides inhibit C6 cell growth via adenosine, which is produced by ecto-nucleotidases including CD73 at the extracellular space and then incorporated into cells by ENT2. Intracellular AMP accumulation by adenosine kinase after adenosine uptake would induce C6 cell growth inhibition through pyrimidine starvation.
Assuntos
Adenosina/metabolismo , Neoplasias Encefálicas/patologia , Glioma/patologia , 5'-Nucleotidase/metabolismo , Nucleotídeos de Adenina/metabolismo , Nucleotídeos de Adenina/farmacologia , Adenosina Desaminase/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Adenosina Quinase/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Dipiridamol/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Hidrólise , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio , Tiazóis , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Uridina/farmacologiaRESUMO
Dihydropyridine-type calcium channel antagonists, in addition to having a vasodilatory effect, are known to inhibit cellular uptake of nucleosides such as adenosine. However, the nucleoside transporter subtypes involved and the mechanism by which this occurs are not known. Therefore, we have studied the inhibitory effects of dihydropyridines on both human equilibrative nucleoside transporters, hENT-1 and hENT-2, which are the major transporters mediating nucleoside transport in most tissues. Among the dihydropyridines tested, nimodipine proved to be the most potent inhibitor of hENT-1, with an IC(50) value of 60+/-31 muM, whereas nifedipine, nicardipine, nitrendipine, and felodipine exhibited 100-fold less effective inhibitory activity. Nifedipine, nitrendipine, and nimodipine inhibited hENT-2 with IC(50) values in the micromolar range; however, nicardipine and felodipine had no significant effect on hENT-2. Removal of the 4-aryl ring or changing the nitro group at the 4-aryl ring proved not to be detrimental to the inhibitory effects of dihydropyridines on hENT-1, but resulted in a drastic decrease in their inhibitory effects on hENT-2. Kinetic studies revealed that nimodipine and nifedipine reduced V(max) of [(3)H]uridine transport without affecting K(m). The inhibitory effects of nimodipine and nifedipine could be washed out. In addition, nimodipine and nifedipine inhibited the rate of NBTGR-induced dissociation of [(3)H]NBMPR from hENT-1 cell membrane. We conclude that dihydropyridines are non-competitive inhibitors of hENT-1 and hENT-2, probably working through reversible interactions with the allosteric sites. The inhibitory potencies of dihydropyridines may be associated with the structure of the 4-aryl ring, as well as the ester groups at the C-3 and C-5 positions.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Di-Hidropiridinas/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Canais de Cálcio/metabolismo , Linhagem Celular , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Humanos , Transfecção , Uridina/metabolismoRESUMO
Thiazolidinediones are a new class of anti-diabetic agents which increase insulin sensitivity by binding to the peroxisome proliferator-activated receptor gamma (PPAR(gamma)) and stimulating the expression of insulin-responsive genes involved in glucose and lipid metabolism. These drugs also have vasodilatory and anti-proliferative effects on vascular smooth muscle cells. However the mechanisms for these actions are not fully understood. Adenosine is a vasodilator and a substrate of equilibrative nucleoside transporters (ENT). The present study studied the effects of three thiazolidinediones, troglitazone, pioglitazone and ciglitazone, on ENT1 in the human aortic smooth muscle cells (HASMCs). Although incubating HASMCs for 48h with thiazolidinediones had no effect on ENT1 mRNA and protein levels, troglitazone acutely inhibited [3H]adenosine uptake and [3H]NBMPR binding of HASMCs with IC50 values of 2.35+/-0.35 and 3.99+/-0.57microM, respectively. The effect of troglitazone on ENT1 was PPAR(gamma)-independent and kinetic studies revealed that troglitazone was a competitive inhibitor of ENT1. In contrast, pioglitazone and ciglitazone had minimal effects on [3H]adenosine uptake by HASMCs. Troglitazone differs from pioglitazone and ciglitazone in that its side-chain contains a Vitamin E moiety. The difference in structure of troglitazone did not account for its inhibitory effect on ENT1 because Vitamin E did not inhibit [3H]adenosine uptake by HASMCs. Using the nucleoside transporter deficient PK15NTD cells stably expressing ENT1 and ENT2, it was found that troglitazone inhibited ENT1 but had no effect on ENT2. From these results, it is suggested that troglitazone may enhance the vasodilatory effect of adenosine by inhibiting ENT1. Pharmacologically, troglitazone is a novel inhibitor of ENT1.
Assuntos
Aorta/efeitos dos fármacos , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Aorta/citologia , Aorta/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismoRESUMO
Continuous in vitro cultivation of human lymphoid H9 cells in the presence of 0.5microM arabinosyl-cytosine (araC) resulted in cell variant, H9-araC cells, that was >600-fold resistant to the drug and cross resistant to its analogs and other unrelated nucleosides, e.g. dideoxycytidine (5-fold), thiacytidine (2-fold), 2-fluoro-adenine arabinoside (8.3-fold), and 2-chloro-deoxyadenosine (2.1-fold). Compared to the parental cell line, the resistant cells accumulated <1% araCTP, and had reduced deoxycytidine kinase (dCK) activity (31.4%) and equilibrative nucleoside transporter 1 (ENT1) protein. The expression of the dCK gene in araC resistant cells was reduced to 60% of H9 cells, which correlated with lower dCK protein and activity. Whereas, there was no difference in the expression of ENT1 mRNA between the cell lines, ENT1 protein content was much lower in the resistant cells than in H9 cells. The concentrative nucleoside transporter (CNT3) was slightly increased in H9-araC cells, but CNT2, and MDR1 remained unaffected. Although a definitive correlation remains to be established, the amount of Sp1 protein, a transcription factor, that regulates the expressions of dCK, nucleoside transporters and other cellular proteins, was found reduced in H9-araC cells. Like ENT1, the Sp1 mRNA levels remained unaffected in H9-araC whereas protein contents were reduced. These observations are indicative of differences in the production and/or turnover of ENT1 and Sp1 proteins in H9-araC cells. Since nucleoside transporters and dCK play an important role in the activity of potential antiviral and anticancer deoxynucleoside analogs, understanding of their regulation is important. These studies show that the exposure of cells to araC, in vitro, is capable of simultaneously affecting more than one target site to confer resistance. The importance of this observation in the clinical use of araC remains to be determined.
Assuntos
Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linhagem Celular , Desoxicitidina Quinase/antagonistas & inibidores , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 1 de Nucleosídeo/biossíntese , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/genética , Humanos , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/metabolismoRESUMO
The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile429 in TM 11 of CeENT1 and Met33 in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC50 value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu442 of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport.
Assuntos
Proteínas de Caenorhabditis elegans/química , Dipiridamol/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/química , Transportador Equilibrativo 2 de Nucleosídeo/química , Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Relação Dose-Resposta a Droga , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 2 de Nucleosídeo/antagonistas & inibidores , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Relação Estrutura-Atividade , Uridina/metabolismoRESUMO
hENT1 and hENT2 are members of the human equilibrative nucleoside transporter family. hENT1 is ubiquitously expressed and plays an important role in the disposition and pharmacological activity of nucleoside drugs and nucleosides, such as adenosine. hENT2 is expressed in only a few tissues (e.g. muscle). hENT1 and hENT2 differ in their affinity for nucleoside substrates and in their sensitivity to inhibitors, such as nitrobenzylthioinosine (NBMPR). hENT1 has higher (or equal) affinity to hENT2 for all natural nucleosides except inosine. hENT1 is also more sensitive to NBMPR inhibition (IC50 approximately 0.4-8 nM) when compared with hENT2 (IC50 approximately 2.8 microM). This difference in inhibition potency is substantially dependent on the difference in amino acid at position 154 in hENT1 (glycine) and hENT2 (serine). Since NBMPR competitively inhibits nucleoside transporter activity, we hypothesized that G154 may also play a role in the transport of natural nucleosides and in the inhibition by other hENT1 inhibitors, dipyridamole (DP), and dilazep (DZ). Our results, using a yeast expression system, demonstrate that substituting glycine 154 of hENT1 with serine of hENT2 converts hENT1 to a transporter that exhibits partial characteristics of hENT2. For example, this conversion reduces sensitivity of hENT1 to the inhibitors NBMPR, DP, and DZ and reduces its transport affinity for the natural nucleosides cytidine and adenosine. However, this conversion renders hENT1 less sensitive to inhibition by anti-HIV drugs azidothymidine, dideoxyinosine, and the nucleobase, hypoxanthine. Collectively, these results suggest that glycine 154 plays an important role in the transport of nucleosides and in sensitivity to the inhibitors NBMPR, DP, and DZ.