RESUMEN

Adenosine is a well-known endogenous modulator of neuronal excitability with anticonvulsant properties. Thus, the modulation exerted by adenosine might be an effective tool to control seizures. In this study, we investigated the effects of drugs that are able to modulate adenosinergic signaling on pentylenetetrazole (PTZ)-induced seizures in adult zebrafish. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) decreased the latency to the onset of the tonic-clonic seizure stage. The adenosine A1 receptor agonist cyclopentyladenosine (CPA) increased the latency to reach the tonic-clonic seizure stage. Both the adenosine A2A receptor agonist and antagonist, CGS 21680 and ZM 241385, respectively, did not promote changes in seizure parameters. Pretreatment with the ecto-5'nucleotidase inhibitor adenosine 5'-(α,ß-methylene) diphosphate (AMPCP) decreased the latency to the onset of the tonic-clonic seizure stage. However, when pretreated with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), or with the nucleoside transporter (NT) inhibitors, dipyridamole and S-(4-Nitrobenzyl)-6-thioinosine (NBTI), animals showed longer latency to reach the tonic-clonic seizure status. Finally, our molecular analysis of the c-fos gene expression corroborates these behavioral results. Our findings indicate that the activation of adenosine A1 receptors is an important mechanism to control the development of seizures in zebrafish. Furthermore, the actions of ecto-5'-nucleotidase, ADA, and NTs are directly involved in the control of extracellular adenosine levels and have an important role in the development of seizure episodes in zebrafish.

Asunto(s)

Adenosina/metabolismo , Pentilenotetrazol/toxicidad , Convulsiones/inducido químicamente , Transducción de Señal/efectos de los fármacos , Pez Cebra , Adenina/análogos & derivados , Adenina/farmacología , Adenosina/análogos & derivados , Adenosina/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Compuestos de Bencilo/farmacología , Convulsivantes/toxicidad , Dipiridamol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos/genética , Genes fos/fisiología , Fenetilaminas/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Convulsiones/metabolismo , Tioinosina/análogos & derivados , Tioinosina/farmacología , Xantinas/farmacología

RESUMEN

Adenosine causes vasodilation of human placenta vasculature by increasing the transport of arginine via cationic amino acid transporters 1 (hCAT-1). This process involves the activation of A(2A) adenosine receptors (A(2A)AR) in human umbilical vein endothelial cells (HUVECs). Insulin increases hCAT-1 activity and expression in HUVECs, and A(2A)AR stimulation increases insulin sensitivity in subjects with insulin resistance. However, whether A(2A)AR plays a role in insulin-mediated increase in L-arginine transport in HUVECs is unknown. To determine this, we first assayed the kinetics of saturable L-arginine transport (1 minute, 37°C) in the absence or presence of nitrobenzylthioinosine (NBTI, 10 µmol/L, adenosine transport inhibitor) and/or adenosine receptors agonist/antagonists. We also determined hCAT-1 protein and mRNA expression levels (Western blots and quantitative PCR), and SLC7A1 (for hCAT-1) reporter promoter activity. Insulin and NBTI increased the extracellular adenosine concentration, the maximal velocity for L-arginine transport without altering the apparent K(m) for L-arginine transport, hCAT-1 protein and mRNA expression levels, and SLC7A1 transcriptional activity. An A2AAR antagonist ZM-241385 blocked these effects. ZM241385 inhibited SLC7A1 reporter transcriptional activity to the same extent in cells transfected with pGL3-hCAT-1(-1606) or pGL3-hCAT-1(-650) constructs in the presence of NBTI + insulin. However, SLC7A1 reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1(-1606), and the ZM-241385 sensitive fraction of the NBTI response was similar in the absence or in the presence of insulin. Thus, insulin modulation of hCAT-1 expression and activity requires functional A(2A)AR in HUVECs, a mechanism that may be applicable to diseases associated with fetal insulin resistance, such as gestational diabetes.

Asunto(s)

Arginina/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Insulina/farmacología , Receptor de Adenosina A2A/metabolismo , Adenosina/metabolismo , Adolescente , Adulto , Sistema de Transporte de Aminoácidos y+/genética , Transporte Biológico/efectos de los fármacos , Transportador de Aminoácidos Catiónicos 1/genética , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Cinética , Masculino , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Tioinosina/análogos & derivados , Tioinosina/farmacología , Transcripción Genética/efectos de los fármacos , Adulto Joven

RESUMEN

Extracellular adenosine removal is via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) in the endothelium, thus regulating adenosine-induced revascularization and angiogenesis. Since human endothelial progenitor cells (hEPCs) promote revascularization, we hypothesize differential expression of nucleoside transporters in hEPCs. hEPCs were cultured 3 (hEPC-3d) or 14 (hEPC-14d) days. RT-PCR for prominin 1, CD34, octamer-4, kinase insert domain receptor, oxidized low-density lipoprotein (lectin-like) receptor 1 and tyrosine endothelial kinase was used to evaluate phenotypic differentiation. Flow cytometry was used to estimate CD34(+)/KDR(-) (non-differentiated), CD34(-)/KDR(+) (differentiated) or CD34(+)/KDR(+) (mixed) cell populations. Adenosine transport was measured in absence or presence of sodium, S-(4-nitrobenzyl)-6-thio-inosine (NBTI, 1-10 µM), inosine, hypoxanthine or guanine (0.1-5 mM), hENTs protein abundance by western blot, and hENTs, hCNT1, hCNT2 and hCNT3 mRNA expression by real time RT-PCR. hEPC-3d cells were CD34(+)/KDR(-) compared with hEPC-14d cells that were CD34(-)/KDR(+). hEPC-3d cells exhibit hENT1-like adenosine transport (NBTI-sensitive, Na(+)-independent), which is absent in hEPC-14d cells. hEPC-14d cells exhibit two transport components: component 1 (NBTI insensitive, Na(+)-independent) and component 2 (NBTI insensitive, Na(+)-dependent, Hill coefficient ∼1.8), the latter resembling CNT3-like transport. hEPC-3d cells express hENT1 protein and mRNA, which is reduced (∼90%) in hEPC-14d cells, but instead only hCNT3 mRNA is expressed in this cell type. hENT2, hCNT1 and hCNT2 were undetectable in hEPCs. Thus, hEPCs exhibit a differential expression of hENT1 and hCNT3 functional nucleoside transporters, which could be related with its differentiation stage.

Asunto(s)

Células Endoteliales/fisiología , Tranportador Equilibrativo 1 de Nucleósido/biosíntesis , Transportador Equilibrativo 2 de Nucleósido/biosíntesis , Células Madre/fisiología , Adenosina/metabolismo , Transporte Biológico , Western Blotting , Diferenciación Celular/fisiología , Células Endoteliales/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/genética , Transportador Equilibrativo 2 de Nucleósido/genética , Humanos , Cinética , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Tioinosina/análogos & derivados , Tioinosina/farmacología

RESUMEN

Tubercidin (TUB) is an adenosine analog with potent antiparasite action, unfortunately associated with severe host toxicity. Prevention of TUB toxicity can be reached associating nitrobenzylthioinosine (NBMPR), an inhibitor of the purine nucleoside transport, specifically target to the mammal cells. It was demonstrated that this nucleoside transport inhibitor has no significant effect in the in vitro uptake of TUB by Schistosoma mansoni and Trypanosoma gambiense. Seeking to evaluate if the association of these compounds is also effective against leishmania, we analyzed the TUB-NBMPR combined treatment in in vitro cultures of promastigote forms of Leishmania (L.) amazonensis, Leishmania (L.) chagasi, Leishmania (L.) major, and Leishmania (V.) braziliensis as well as in cultures of amastigote forms of L. (L.) amazonensis, mice macrophages infected with L. (L.) amazonensis, and in vivo tests in BALB/c mice infected with L. (L.) amazonensis. We demonstrated that TUB-NBMPR combined treatment can be effective against leishmania cells protecting mammalian cells from TUB toxicity.

Asunto(s)

Antiparasitarios/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Leishmania/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Tioinosina/análogos & derivados , Tionucleótidos/uso terapéutico , Tubercidina/uso terapéutico , Animales , Antiparasitarios/farmacología , Antiparasitarios/toxicidad , Células Cultivadas , Quimioterapia Combinada , Inhibidores Enzimáticos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Schistosoma mansoni/efectos de los fármacos , Tioinosina/farmacología , Tioinosina/uso terapéutico , Tionucleótidos/farmacología , Trypanosoma brucei gambiense/efectos de los fármacos , Tubercidina/farmacología , Tubercidina/toxicidad

RESUMEN

Malignant gliomas are the most common and devastating primary tumors in the brain and, despite treatment, patients with these tumors have a poor prognosis. The participation of ecto-5'-NT/CD73 per se as a proliferative factor, being involved in the control of cell growth, differentiation, invasion, migration and metastasis processes has been previously proposed. In the present study, we evaluated the activity and functions of ecto-5'-NT/CD73 during the proliferation process of rat C6 and human U138MG glioma cell lines. Increasing confluences and culture times led to an increase in ecto-5'-NT/CD73 activity in both C6 and U138MG glioma cells. RT-PCR analysis and flow cytometry analysis showed a significant increase in ecto-5'-NT/CD73 mRNA and protein levels, respectively, comparing confluent with sub-confluent cultures in human U138MG glioma cells. Ecto-5'-nucleotidase/CD73 may regulate the extracellular adenosine 5'-monophosphate (AMP) and adenosine levels. Treatment with 1 microM APCP, a competitive ecto-5'-NT/CD73 inhibitor, caused a significant reduction of 30% in glioma cell proliferation. In addition, 100 microM adenosine increases cell proliferation by 36%, and the treatment with adenosine plus NBTI and dipyridamole, produced an additional and significant increase of on cell proliferation. The inhibitory effect on cell proliferation caused by APCP was reverted by co-treatment with NBTI and dipyridamole. AMP (1 mM and 3 mM) decreased U138MG glioma cell proliferation by 29% and 42%, respectively. Taken together, these results suggest the participation of ecto-5'-NT/CD73 in cell proliferation and that this process is dependent upon the enzyme's production of adenosine, a proliferative factor, and removal of AMP, a toxic molecule for gliomas.

Asunto(s)

5'-Nucleotidasa/biosíntesis , Neoplasias Encefálicas/enzimología , Proliferación Celular , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/enzimología , 5'-Nucleotidasa/antagonistas & inhibidores , Adenosina/metabolismo , Adenosina/farmacología , Adenosina Monofosfato/metabolismo , Marcadores de Afinidad/farmacología , Animales , Neoplasias Encefálicas/patología , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Glioma/patología , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , Ratas , Tioinosina/análogos & derivados , Tioinosina/farmacología

RESUMEN

L-Arginine transport and nitric oxide (NO) synthesis (L-arginine/NO pathway) are stimulated by insulin, adenosine or elevated extracellular D-glucose in human umbilical vein endothelial cells (HUVEC). Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) has been proposed as a mechanism regulating adenosine plasma concentration, and therefore its vascular effects in human umbilical veins. Thus, altered expression and/or activity of hENT1 or hENT2 could lead to abnormal physiological plasma adenosine level. We have characterized insulin effect on adenosine transport in HUVEC cultured in normal (5 mM) or high (25 mM) D-glucose. Insulin (1 nM) increased overall adenosine transport associated with higher hENT2-, but lower hENT1-mediated transport in normal D-glucose. Insulin increased hENT2 protein abundance in normal or high D-glucose, but reduced hENT1 protein abundance in normal D-glucose. Insulin did not alter the reduced hENT1 protein abundance, but blocked the reduced hENT1 and hENT2 mRNA expression induced by high D-glucose. Insulin effect on hENT1 mRNA expression in normal D-glucose was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and mimicked by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor). L-NAME did not block insulin effect on hENT2 expression. In conclusion, insulin stimulation of overall adenosine transport results from increased hENT2 expression and activity via a NO-independent mechanism. These findings could be important in hyperglycemia-associated pathological pregnancies, such as gestational diabetes, where plasma adenosine removal by the endothelium is reduced, a condition that could alter the blood flow from the placenta to the fetus affecting fetus growth and development.

Asunto(s)

Adenosina/metabolismo , Células Endoteliales/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Transportador Equilibrativo 2 de Nucleósido/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Venas Umbilicales/anatomía & histología , Marcadores de Afinidad/metabolismo , Transporte Biológico , Células Cultivadas , Citrulina/metabolismo , Células Endoteliales/citología , Tranportador Equilibrativo 1 de Nucleósido/genética , Transportador Equilibrativo 2 de Nucleósido/genética , Humanos , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tioinosina/análogos & derivados , Tioinosina/metabolismo

RESUMEN

Reduced oxygen level (hypoxia) induces endothelial dysfunction and release of the endogenous nucleoside adenosine. Human umbilical vein endothelium (HUVEC) function in an environment with 3% to 5% O2 and exhibit efficient adenosine membrane transport via human equilibrative nucleoside transporters 1 (hENT1). We studied whether adenosine transport and hENT1 expression are altered by hypoxia in HUVEC. Hypoxia (0 to 24 hours, 2% and 1% O2) reduced maximal hENT1-adenosine transport velocity (V(max)) and maximal nitrobenzylthionosine (NBMPR, a high-affinity hENT1 protein ligand) binding, but increased extracellular adenosine concentration. Hypoxia also reduced hENT1 protein and mRNA levels, effects unaltered by N(omega)-nitro-l-arginine methyl ester (l-NAME, nitric oxide synthase [NOS] inhibitor) or PD-98059 (inhibitor of mitogen-activated protein kinase kinase 1 and 2 [MEK1/2]). Hypoxia reduced endothelial NOS (eNOS) activity and eNOS phosphorylation at Ser(1177), but increased eNOS protein level. Hypoxia increased (1 to 3 hours), but reduced (24 hours) p42/44(mapk) phosphorylation. Thus, hypoxia-increased extracellular adenosine may result from reduced hENT1-adenosine transport in HUVEC. Hypoxia effect seems not to involve NO, but p42/44(mapk) may be required for the relatively rapid effect (1 to 3 hours) of hypoxia. These results could be important in diseases where the fetus is exposed to intrauterine environments poor in oxygen, such as intrauterine growth restriction, or where adenosine transport is altered, such as gestational diabetes.

Asunto(s)

Hipoxia de la Célula , Células Endoteliales/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/genética , Regulación de la Expresión Génica , Adenosina/metabolismo , Transporte Biológico , Células Cultivadas , Regulación hacia Abajo , Tranportador Equilibrativo 1 de Nucleósido/fisiología , Transportador Equilibrativo 2 de Nucleósido/fisiología , Retardo del Crecimiento Fetal/etiología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Fosforilación , ARN Mensajero/análisis , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Venas Umbilicales/metabolismo

RESUMEN

Extracellular purines are involved in the regulation of a wide range of physiological processes, including cytoprotection, ischemic preconditioning, and cell death. These actions are usually mediated via triggering of membrane purinergic receptors, which may activate antioxidant enzymes, conferring cytoprotection. Recently, it was demonstrated that the oxidative stress induced by cisplatin up-regulated A1 receptor expression in rat testes, suggesting an involvement of purinergic signaling in the response of testicular cells to oxidant injury. In this article, we report the effect of hydrogen peroxide on purinergic agonist release by cultured Sertoli cells. Extracellular inosine levels are strongly increased in the presence of H2O2, suggesting an involvement of this nucleoside on Sertoli cells response to oxidant treatment. Inosine was observed to decrease H2O2-induced lipoperoxidaton and cellular injury, and it also preserved cellular ATP content during H2O2 exposure. These effects were abolished in the presence of nucleoside uptake inhibitors, indicating that nucleoside internalisation is essential for its action in preventing cell damage.

Asunto(s)

Adenina/análogos & derivados , Peróxido de Hidrógeno/farmacología , Inosina/metabolismo , Peroxidación de Lípido/fisiología , Oxidantes/farmacología , Células de Sertoli/metabolismo , Tioinosina/análogos & derivados , Adenina/farmacología , Inhibidores de la Adenosina Desaminasa , Adenosina Trifosfato/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dipiridamol/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Peroxidación de Lípido/efectos de los fármacos , Masculino , Niacinamida/farmacología , Estrés Oxidativo , Ratas , Células de Sertoli/efectos de los fármacos , Tioinosina/farmacología

RESUMEN

It has been long postulated that extracellular purines can modulate the function of the male reproductive system by interacting with different purinergic receptors of Sertoli and germinative cells. Many authors have described the biological changes induced by extracellular ATP and/or adenosine in these cells, and some hypothetical models for paracrine communication mediated by purines were proposed; however, the cellular source(s) of these molecules in seminiferous tubules remains unknown. In this study, we demonstrated for the first time that Sertoli cells are able to release ATP (0.3 nmol/mg protein) and adenosine (0.1 nmol/mg protein) in the extracellular medium, while germinative and myoid peritubular cells are able to secrete adenosine (0.02 and 0.37 nmol/mg protein, respectively). Indeed, all the three types of cells were able to release inosine at significant concentrations (about 0.4 nmol/mg protein). This differential secretion depending on the cellular type suggests that these molecules may be involved in the paracrine regulation and/or control of the maturation processes of these cells.

Asunto(s)

Adenina/análogos & derivados , Adenosina Difosfato/análogos & derivados , Espacio Extracelular/metabolismo , Purinas/metabolismo , Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismo , Tioinosina/análogos & derivados , 5'-Nucleotidasa/antagonistas & inhibidores , Adenina/farmacología , Adenosina/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacocinética , Adenosina Trifosfato/farmacología , Marcadores de Afinidad/farmacología , Animales , Células Cultivadas , Dipiridamol/farmacología , Inhibidores Enzimáticos/farmacología , Espacio Extracelular/enzimología , Células Germinativas/metabolismo , Cinética , Masculino , Comunicación Paracrina , Inhibidores de Fosfodiesterasa/farmacología , Purinas/farmacocinética , Purinas/farmacología , Ratas , Ratas Wistar , Receptores Purinérgicos/metabolismo , Túbulos Seminíferos/citología , Células de Sertoli/efectos de los fármacos , Tioinosina/farmacología

RESUMEN

Chronic incubation with elevated D-glucose reduces adenosine transport in endothelial cells. In this study, exposure of human umbilical vein endothelial cells to 25 mmol/L D-glucose or 100 micromol/L ATP, ATP-gamma-S, or UTP, but not ADP or alpha,beta-methylene ATP, reduced adenosine transport with no change in transport affinity. Inhibition of transport by D-glucose, ATP, and ATP-gamma-S was associated with reduced maximal binding, with no changes in the apparent dissociation constant for nitrobenzylthioinosine (NBMPR). A significant reduction (approximately 60+/-10%, P<0.05; n=6) in the number of human equilibrative NBMPR-sensitive nucleoside transporters (hENT1s) per cell (1.8+/-0.1x10(6) in 5 mmol/L D-glucose) and in hENT1 mRNA levels was observed in cells exposed to D-glucose or ATP-gamma-S. Incubation with elevated D-glucose, but not with D-mannitol, increased the ATP release by 3+/-0.2-fold. The effects of D-glucose and nucleotides on the number and activity of hENT1 and hENT1 mRNA were blocked by reactive blue 2 (nonspecific P2Y purinoceptor antagonist), suramin (Galpha(s) protein inhibitor), or hexokinase but not by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (nonselective P2 purinoceptor antagonist). Our findings demonstrate that inhibition of adenosine transport via hENT1 in endothelial cells cultured in 25 mmol/L D-glucose could be due to stimulation of P2Y2 purinoceptors by ATP, which is released from these cells in response to D-glucose. This could be a mechanism to explain in part the vasodilatation observed in the early stages of diabetes mellitus or in response to D-glucose infusion.

Asunto(s)

Adenosina Trifosfato/análogos & derivados , Adenosina/metabolismo , Endotelio Vascular/metabolismo , Glucosa/farmacología , Receptores Purinérgicos P2/metabolismo , Tioinosina/análogos & derivados , Tioinosina/farmacología , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Unión Competitiva/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Tranportador Equilibrativo 1 de Nucleósido , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , ARN Mensajero/metabolismo , Receptores Purinérgicos P2Y2 , Venas Umbilicales , Uridina Trifosfato/farmacología

RESUMEN

Previous work showed the presence of adenosine receptors as well as adenosine uptake and release mechanisms in developing chick retinal neurons in culture. In the present work we show that exogenous glutamate or kainate promotes extensive cell death in these cultures which is blocked when the cultures are previously incubated with adenosine. Addition of glutamate or kainate to purified cultures of retinal neurons and photoreceptors induced massive death of cultured cells which was inhibited in both cases by preincubation with MK801, an NMDA antagonist, or DNQX, an AMPA/kainate antagonist. Cell death was also greatly attenuated by preincubation with adenosine plus EHNA, an adenosine deaminase inhibitor, NBI, an adenosine uptake blocker, the permeable cAMP analogs 8-Br cAMP and Sp cAMP and the A(2a) agonists CGS 21680 and DPMA, but not with the A(1) receptor agonist CHA. Kinetic studies performed determining the intracellular LDH activity showed that maximal death was observed after 8 h and in concentrations of glutamate as low as 50 microM. We also observed a time-dependent protective effect of adenosine beginning after 1 h of preincubation and reaching a maximal effect after 24 h, indicating its association with changes in cellular metabolism induced by long-term exposure of cells to the nucleoside. The results show that adenosine inhibits glutamate toxicity in retinal neurons through a long-term activation of A(2a) receptors and elevation of intracellular cyclic AMP levels.

Asunto(s)

Embrión de Pollo/fisiología , Ácido Glutámico/efectos de los fármacos , Neuronas/fisiología , Neurotoxinas/antagonistas & inhibidores , Receptores Purinérgicos P1/fisiología , Retina/fisiología , Adenosina/agonistas , Adenosina/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/análogos & derivados , Sinergismo Farmacológico , Ácido Glutámico/farmacología , L-Lactato Deshidrogenasa/metabolismo , Neuronas/efectos de los fármacos , Neurotoxinas/farmacología , Agonistas del Receptor Purinérgico P1 , Receptor de Adenosina A2A , Retina/citología , Retina/efectos de los fármacos , Tioinosina/análogos & derivados , Tioinosina/farmacología , Factores de Tiempo

RESUMEN

The effects of elevated D-glucose on adenosine transport were investigated in human cultured umbilical vein endothelial cells isolated from normal pregnancies. Elevated D-glucose resulted in a time- (8-12 h) and concentration-dependent (half-maximal at 10+/-2 mM) inhibition of adenosine transport, which was associated with a reduction in the Vmax for nitrobenzylthioinosine (NBMPR)-sensitive (es) saturable nucleoside with no significant change in Km. d-Fructose (25 mM), 2-deoxy-D-glucose (25 mM) or D-mannitol (20 mM) had no effect on adenosine transport. Adenosine transport was inhibited following incubation of cells with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 100 nM, 30 min to 24 h). D-Glucose-induced inhibition of transport was abolished by calphostin C (100 nM, an inhibitor of PKC), and was not further reduced by PMA. Increased PKC activity in the membrane (particulate) fraction of endothelial cells exposed to D-glucose or PMA was blocked by calphostin C but was unaffected by NG-nitro-L-arginine methyl ester (L-NAME; 100 microM, an inhibitor of nitric oxide synthase (NOS)) or PD-98059 (10 microM, an inhibitor of mitogen-activated protein kinase kinase 1). D-Glucose and PMA increased endothelial NOS (eNOS) activity, which was prevented by calphostin C or omission of extracellular Ca2+ and unaffected by PD-98059. Adenosine transport was inhibited by S-nitroso-N-acetyl-l, d-penicillamine (SNAP; 100 microM, an NO donor) but was increased in cells incubated with L-NAME. The effect of SNAP on adenosine transport was abolished by PD-98059. Phosphorylation of mitogen-activated protein kinases p44mapk (ERK1) and p42mapk (ERK2) was increased in endothelial cells exposed to elevated D-glucose (25 mM for 30 min to 24 h) and the NO donor SNAP (100 microM, 30 min). The effect of D-glucose was blocked by PD-98059 or L-NAME, which also prevented the inhibition of adenosine transport mediated by elevated D-glucose. Our findings provide evidence that D-glucose inhibits adenosine transport in human fetal endothelial cells by a mechanism that involves activation of PKC, leading to increased NO levels and p42-p44mapk phosphorylation. Thus, the biological actions of adenosine appear to be altered under conditions of sustained hyperglycaemia.

Asunto(s)

Adenosina/metabolismo , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Glucosa/farmacología , Tioinosina/análogos & derivados , Adenosina/antagonistas & inhibidores , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Feto/fisiología , Flavonoides/farmacología , Humanos , Proteínas Quinasas Activadas por Mitógenos/fisiología , NG-Nitroarginina Metil Éster/farmacología , Naftalenos/farmacología , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Penicilamina/análogos & derivados , Penicilamina/farmacología , Proteína Quinasa C/fisiología , S-Nitroso-N-Acetilpenicilamina , Acetato de Tetradecanoilforbol/farmacología , Tioinosina/metabolismo

RESUMEN

Adenosine transport was characterized in human umbilical artery smooth muscle cells isolated from non-diabetic and diabetic pregnant subjects. Transport of adenosine was mediated by a Na+-independent transport system inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR) in both cell types. Diabetes increased adenosine transport, an effect that was associated with a higher maximal velocity (Vmax) for NBMPR-sensitive (es) saturable nucleoside transport (18 +/- 2 vs. 61 +/- 3 pmol (microgram protein)-1 min-1, P < 0.05) and the maximal number of binding sites (Bmax) for specific [3H]NBMPR binding (74 +/- 4 vs. 156 +/- 10 pmol (microgram protein)-1, P < 0.05), with no significant changes in the Michaelis-Menten (Km) and dissociation (Kd) constants, respectively. Adenosine transport was unaltered by inhibition of nitric oxide (NO) synthase (with 100 microM NG-nitro-L-arginine methyl ester, L-NAME) or protein synthesis (with 1 microM cycloheximide), but was increased by inhibition of adenylyl cyclase activity (with 100 microM, SQ-22536) in non-diabetic cells. Diabetes-induced adenosine transport was blocked by L-NAME and associated with an increase in L-[3H]citrulline formation from L-[3H]arginine and intracellular cGMP, but with a decrease in intracellular cAMP compared with non-diabetic cells. Expression of inducible NO synthase (iNOS) was unaltered by diabetes. Dibutyryl cGMP (dbcGMP) increased, but dibutyryl cAMP (dbcAMP) decreased, adenosine transport in non-diabetic cells. dbcGMP or the NO donor S-nitrosoacetylpenicillamine (SNAP, 100 microM) did not alter the diabetes-elevated adenosine transport. However, activation of adenylyl cyclase with forskolin (1 microM), directly or after incubation of cells with dbcAMP, inhibited adenosine transport in both cell types. Our findings provide the first evidence that adenosine transport in human umbilical artery smooth muscle cells is mediated by the NBMPR-sensitive transport system es, and that its activity is upregulated by gestational diabetes.

Asunto(s)

Adenosina/metabolismo , Diabetes Gestacional/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiología , Nucleótidos Cíclicos/fisiología , Tioinosina/análogos & derivados , Arterias Umbilicales/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Células Cultivadas , AMP Cíclico/fisiología , GMP Cíclico/fisiología , Diabetes Gestacional/patología , Femenino , Humanos , Membranas Intracelulares/metabolismo , Cinética , Músculo Liso Vascular/patología , Embarazo , Valores de Referencia , Tioinosina/farmacología , Arterias Umbilicales/patología

RESUMEN

The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells.

Asunto(s)

Proteínas Portadoras/efectos de los fármacos , Endotelio Vascular/metabolismo , Hipoxantinas/metabolismo , Proteínas de la Membrana/efectos de los fármacos , Tioinosina/análogos & derivados , Adenosina/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , Línea Celular , Endotelio Vascular/enzimología , Humanos , Hipoxantina , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Nucleósidos , Tioinosina/farmacología

RESUMEN

Uptake and metabolism of adenosine by human placenta were studied using the single-circulation paired-tracer technique. When isolated cotyledons were perfused through the fetal (basal) circulation at mean pressures of 36 +/- 3.3 mmHg and mean flow rates of 6.6 +/- 0.3 ml/min the maximal [3H]adenosine uptake was 51.3 +/- 3.9 per cent. The uptake was not changed when the vascular resistance was pharmacologically increased. Adenosine uptake was significantly inhibited by adenosine, inosine and nitrobenzylthioinosine (NBMPR), but was unaffected by hypoxanthine. The kinetic analysis of adenosine transport showed it to be a saturable and, Na(+)-independent process, with a Km of 60.8 microM and a Jmax of 0.148 mumol/min. Thin layer chromatographic analysis showed that about 65 per cent of [3H]adenosine was metabolized (10-30 sec) in a single passage through the fetoplacental circulation. [3H]hypoxanthine and [3H]adenine were the major products recovered in the venous perfusate. In the presence of NBMPR the fractional recovery of [3H]adenine and [3H]phosphorylated derivatives was reduced while that of [3H]hypoxanthine was increased. These overall results show that the uptake of adenosine is a Na(+)-independent, NBMPR-sensitive, carrier-mediated process, which appears to be specific for nucleosides, and suggests that metabolization of adenosine proceeds both intra- and extracellularly.

Asunto(s)

Adenosina/metabolismo , Placenta/metabolismo , Transporte Biológico/fisiología , Femenino , Humanos , Hipoxantina , Hipoxantinas/farmacología , Técnicas In Vitro , Perfusión , Embarazo , Nucleósidos de Purina/farmacología , Sodio/farmacología , Tioinosina/análogos & derivados , Tioinosina/farmacología , Vasoconstricción/fisiología

RESUMEN

Pregnancy complicated by diabetes is a relatively frequent event and may result in fetal embriopathy. However, little is known regarding whether placental transport functions are altered. In this study, we have compared the activity of the nitrobenzylthioinosine (NBMPR)-sensitive adenosine transporter and adenosine metabolism in human placental brush-border- and basal-membrane vesicles from placentas of normal and diabetic pregnancies. Neither [3H]NBMPR binding, a marker of the facilitative-diffusion nucleoside transporter in the human placenta, nor adenosine metabolism exhibited a significant difference in either the brush-border- or the basal-membrane vesicles between the normal and diabetic group, except for an increased affinity in [3H]NBMPR binding at the basal side in diabetic placenta. This result contrasts with an earlier finding using the same group of patients that adenosine transport is downregulated in umbilical vein endothelial cells from diabetic pregnancies. It is concluded that adenosine transport is modulated selectively in different tissues in diabetic pregnancies.

Asunto(s)

Adenosina/metabolismo , Placenta/metabolismo , Embarazo en Diabéticas/metabolismo , Marcadores de Afinidad , Transporte Biológico/efectos de los fármacos , Femenino , Humanos , Microvellosidades/metabolismo , Embarazo , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Tritio