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Epilepsy is a chronic nervous system disease. Excessive increase of the excitatory neurotransmitter glutamate in the body results in an imbalance of neurotransmitters and excessive excitation of neurons, leading to epileptic seizures. Long‑term recurrent seizures lead to behavior and cognitive changes, and even increase the risk of death by 2‑ to 3‑fold relative to the general population. Adenosine A1 receptor (A1R), a member of the adenosine system, has notable anticonvulsant effects, and adenosine levels are controlled by the type 1 equilibrative nucleoside transporter (ENT1); in addition the p38 MAPK signaling pathway is involved in the regulation of ENT1, although the effect of its inhibitors on the expression levels of A1R and ENT1 is unclear. Therefore, in the present study, SB203580 was used to inhibit the p38 MAPK signaling pathway in rats, and the expression levels of A1R and ENT1 in the brain tissue of rats with acute LiCl‑pilocarpine‑induced status epilepticus was detected. SB203580 decreased pathological damage of hippocampal neurons, prolonged seizure latency, reduced the frequency of seizures, and decreased levels of A1R and ENT1 protein in rats.

Disruptions in circadian rhythms are risk factors for excessive alcohol drinking. The ethanol-sensitive adenosine equilibrative nucleoside transporter type 1 (ENT1, slc29a1) regulates ethanol-related behaviors, sleep, and entrainment of circadian rhythms. However, the mechanism underlying the increased ethanol consumption in ENT1 knockout (KO) mice in constant light (LL) and whether there are sex differences in ethanol consumption in ENT1 mice are less studied. Here, we investigated the effects of loss of ENT1, LL, and sex on ethanol drinking using two-bottle choice. In addition, we monitored the locomotor activity rhythms. We found that LL increased ethanol drinking and reduced accumbal ENT1 expression and adenosine levels in male but not female mice, compared with control mice. Interestingly, only LL-exposed male, not female, ENT1 KO mice exhibited higher ethanol drinking and a longer circadian period with a higher amplitude compared with wild-type (WT) mice. Furthermore, viral-mediated rescue of ENT1 expression in the NAc of ENT1 KO mice reduced ethanol drinking, demonstrating a possible causal link between ENT1 expression and ethanol drinking in males. Together, our findings indicate that deficiency of ENT1 expression contributes to excessive ethanol drinking in a sex-dependent manner., (© 2019 Society for the Study of Addiction.)

Slc29a1 encodes for equilibrative nucleoside transporter subtype 1 (ENT1), the primary mechanism of adenosine transfer across cell membranes. Previous studies showed that tissues isolated from Slc29a1-null mice are relatively resistant to injury caused by vascular ischemia-reperfusion. To determine if there are similar changes in the microvasculature, and investigate underlying mechanism, we examined aortas isolated from wildtype and Slc29a1-null mice. Aorta macrostructure and gene expression were examined histologically and by qPCR, respectively. Wire myography was used to assess the contractile properties of isolated thoracic aortic rings and their response to adenosine under both normoxic and hypoxic conditions. In vivo haemodynamic parameters were assessed using the tail-cuff method. Slc29a1-null mice had significantly (P<0.05) increased plasma adenosine (2.75-fold) and lower blood pressure (~15% ↓) than wild-type mice. Aortas from Slc29a1-null mice were stiffer with a smaller circumference (11% ↓), and had an enhanced contractile response to KCl and receptor-mediated stimuli. Blockade of ENT1 with nitrobenzylthioinosine significantly enhanced (by ~3.5-fold) the response of aorta from wild-type mice to phenylephrine, but had minimal effect on aortas from Slc29a1-null mice. Adenosine enhanced phenylephrine-mediated constriction in the wild-type tissue under both normoxic (11.7-fold) and hypoxic (3.6-fold) conditions, but had no effect on the Slc29a1-null aortic aorta. In conclusion, aortas from Slc29a1-null mice respond to hypoxic insult in a manner comparable to wild-type tissues that have been pharmacologically preconditioned with adenosine. These data also support a role for ENT1 in the regulation of the protective effects of adenosine on contractile function in elastic conduit arteries such as thoracic aorta., Competing Interests: The authors have declared that no competing interests exist.

The human equilibrative nucleoside transporter-1 (hENT1) is important for the entry of anti-cancer and anti-viral nucleoside analog therapeutics into the cell, and thus for their efficacy. Understanding of hENT1 structure-function relationship could assist with development of nucleoside analogs with better cellular uptake properties. However, structural and biophysical studies of hENT1 remain challenging as the hydrophobic nature of the protein leads to complete aggregation upon detergent-based membrane isolation. Here we report detergent-free reconstitution of the hENT1 transporter into styrene maleic acid co-polymer lipid particles (SMALPs) that form a native lipid disc. SMALP-purified hENT1, expressed in Sf9 insect cells binds a variety of ligands with a similar affinity as the protein in native membrane, and exhibits increased thermal stability compared to detergent-solubilized hENT1. hENT1-SMALPs purified using FLAG affinity M2 resin yielded ~0.4mg of active and homogenous protein per liter of culture as demonstrated by ligand binding, size-exclusion chromatography and SDS-PAGE analyses. Electrospray ionization mass spectrometry (ESI-MS) analysis showed that each hENT1 lipid disc contains 16 phosphatidylcholine (PC) and 2 phosphatidylethanolamine (PE) lipid molecules. Polyunsaturated lipids are specifically excluded from the hENT1 lipid discs, possibly reflecting a functional requirement for a dynamic lipid environment. Our work demonstrates that human nucleoside transporters can be extracted and purified without removal from their native lipid environment, opening up a wide range of possibilities for their biophysical and structural studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Human equilibrative nucleoside transporter 1 (hENT1) is a major route of entry of nucleosides and nucleoside analog drugs. The regulation of hENT1 is poorly understood in spite of its clinical importance as a drug transporter. Immunofluorescence microscopy and fluorescence-activated cell sorting suggested that cytidine pre-treatment (40 μM, 6 h) promotes hENT1 internalization in a way that does not affect either hENT1-mediated nucleoside uptake or gemcitabine-induced cytotoxicity. The Scatchard plot analyses of our NBTI binding data support previous speculations that hENT1 proteins exist as two sub-populations, and suggest that cytidine pre-treatment leads to the internalization of one population.

The high-prevalence antigen, Ata, was first identified in 1967, but it was not until 2015 that Ata became AUG1 of a new blood group system, Augustine (AUG). The new system was established after the identification of the gene encoding Ata and the recognition of a null phenotype (AUG:–1,–2) in an At(a–) patient with an antibody (anti-AUG2) reactive with At(a–) red blood cells. The At(a–) phenotype is very rare and, with the exception of the one family with the null phenotype, has only been found in individuals of African origin. Anti-Ata has been implicated in immediate and delayed hemolytic transfusion reactions, but not in severe hemolytic disease of the fetus and newborn. The Augustine gene is SLC29A1, which encodes the equilibrative nucleoside transporter ENT1. At(a–) (AUG:–1,2) results from homozygosity for c.1171G>A, encoding Glu391Lys, whereas the AUGnull (AUG:–1,–2) phenotype results from homozygosity for a splice site mutation, c.589+1G>C, in the only family where it has been found. Absence of ENT1 in that family may be associated with pseudogout and abnormal bone calcification.

We studied regulation of hypoxanthine transport depending on its concentration in the culture medium. Caco-2 cells were differentiated on membrane filters to create a model of the intestine. Different hypoxanthine uptake on the apical and basolateral cell membranes was observed. The expression of SLC29 family genes encoding passive nucleoside transporters increased upon changes in hypoxanthine concentration in the medium Localization of the transporters and their influence on the effect of pharmacological preparations are discussed.

The type 1 equilibrative nucleoside transporter (ENT1) is implicated in regulating levels of extracellular adenosine ([AD]ex). In the basal forebrain (BF) levels of [AD]ex increase during wakefulness and closely correspond to the increases in the electroencephalogram (EEG) delta (0.75-4.5Hz) activity (NRδ) during subsequent non-rapid eye movement sleep (NREMS). Thus in the BF, [AD]ex serves as a biochemical marker of sleep homeostasis. Waking EEG activity in theta range (5-9Hz, Wθ) is also described as a marker of sleep homeostasis. An hour-by-hour temporal relationship between the Wθ and NRδ is unclear. In this study we examined the relationship between these EEG markers of sleep homeostasis during spontaneous sleep-wakefulness and during sleep deprivation (SD) and recovery sleep in the ENT1 gene knockout (ENT1KO) mouse. We observed that baseline NREMS amount was decreased during the light period in ENT1KO mice, accompanied by a weak correlation between Wθ of each hour and NRδ of its subsequent hour when compared to their wild-type (WT) littermates. Perfusion of low dose of adenosine into BF not only strengthened the Wθ-NRδ relationship, but also increased NREMS to match with the WT littermates suggesting decreased [AD]ex in ENT1KO mice. However, the SD-induced [AD]ex increase in the BF and the linear correlation between the EEG markers of sleep homeostasis were unaffected in ENT1KO mice suggesting that during SD, sources other than ENT1 contribute to increase in [AD]ex. Our data provide evidence for a differential regulation of wakefulness-associated [AD]ex during spontaneous vs prolonged waking., (Published by Elsevier Ltd.)

Type 1 equilibrative nucleoside transporter (ENT1) promotes glutamate release by inhibition of adenosine signaling. However, whether ENT1 plays a role in epileptic seizure that involves elevated glutamatergic neurotransmission is unknown. Here, we report that both seizure rats and patients show increased expression of ENT1. Intrahippocampal injection of a specific inhibitor of ENT1, nitrobenzylthioinosine (NBTI), attenuates seizure severity and prolongs onset latency. In order to examine whether NBTI would be effective as antiepileptic after peripheral application, we injected NBTI intraperitoneally, and the results were similar to those obtained after intrahippocampal injection. NBTI administration leads to suppressed neuronal firing in seizure rats. In addition, increased mEPSC in seizure are inhibited by NBTI. Finally, NBTI results in deactivation of phosphorylated cAMP-response element-binding protein in the seizure rats. These results indicate that ENT1 plays an important role in the development of seizure. Inhibition of ENT1 might provide a novel therapeutic approach toward the control of epileptic seizure.

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., (© 2013 by the American Association for the Study of Liver Diseases.)

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1(-/-) mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro-computed tomography (µCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1(-/-) mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1(-/-) mice than in wild-type, consistent with loss of ENT1--a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1(-/-) mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1(-/-) mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1(-/-) mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders., (Copyright © 2013 American Society for Bone and Mineral Research.)

The fluorinated analog of deoxycytidine, Gemcitabine (Gemzar), is the main chemotherapeutic drug in pancreatic cancer, but survival remains weak mainly because of the high resistance of tumors to the drug. Recent works have shown that the mucin MUC4 may confer an advantage to pancreatic tumor cells by modifying their susceptibility to drugs. However, the cellular mechanism(s) responsible for this MUC4-mediated resistance is unknown. The aim of this work was to identify the cellular mechanisms responsible for gemcitabine resistance linked to MUC4 expression. CAPAN-2 and CAPAN-1 adenocarcinomatous pancreatic cancer (PC) cell lines were used to establish stable MUC4-deficient clones (MUC4-KD) by shRNA interference. Measurement of the IC50 index using tetrazolium salt test indicated that MUC4-deficient cells were more sensitive to gemcitabine. This was correlated with increased Bax/BclXL ratio and apoptotic cell number. Expression of Equilibrative/Concentrative Nucleoside Transporter (hENT1, hCNT1/3), deoxycytidine kinase (dCK), ribonucleotide reductase (RRM1/2) and Multidrug-Resistance Protein (MRP3/4/5) was evaluated by quantitative RT-PCR (qRT-PCR) and western blotting. Alteration of MRP3, MRP4, hCNT1 and hCNT3 expression was observed in MUC4-KD cells, but only hCNT1 alteration was correlated to MUC4 expression and sensitivity to gemcitabine. Decreased activation of MAPK, JNK and NF-κB pathways was observed in MUC4-deficient cells, in which the NF-κB pathway was found to have an important role in both sensitivity to gemcitabine and hCNT1 regulation. Finally, and in accordance with our in vitro data, we found that MUC4 expression was conversely correlated to that of hCNT1 in tissues from patients with pancreatic adenocarcinoma. This work describes a new mechanism of PC cell resistance to gemcitabine, in which the MUC4 mucin negatively regulates the hCNT1 transporter expression via the NF-κB pathway. Altogether, these data point out to MUC4 and hCNT1 as potential targets to ameliorate the response of pancreatic tumors to gemcitabine treatment.

A(1) adenosine receptors (A1AR) are required for the modulation of afferent arteriolar tone by changes in luminal NaCl concentration implying that extracellular adenosine concentrations need to change in synchrony with NaCl. The present experiments were performed in mice with a null mutation in the gene for the major equilibrative nucleoside transporter ENT1 to test whether interference with adenosine disposition by cellular uptake of adenosine may modify TGF characteristics. Responses of stop flow pressure (P(SF)) to maximum flow stimulation were measured in mice with either C57Bl/6 or SWR/J genetic backgrounds. Maximum flow stimulation reduced P(SF) in ENT1(-/-) compared with wild-type (WT) mice by 1.6 ± 0.4 mmHg (n = 28) and 5.8 ± 1.1 mmHg (n = 17; P < 0.001) in C57Bl/6 and by 1.4 ± 0.4 mmHg (n = 15) and 9 ± 1.5 mmHg (n = 9; P < 0.001) in SWR/J. Plasma concentrations of adenosine and inosine were markedly higher in ENT1(-/-) than WT mice (ado: 1,179 ± 78 and 225 ± 48 pmol/ml; ino: 179 ± 24 and 47.5 ± 9 pmol/ml). Renal mRNA expressions of the four adenosine receptors, ENT2, and adenosine deaminase were not significantly different between WT and ENT1(-/-) mice. No significant differences of glomerular filtration rate or mean arterial blood pressure were found while plasma renin concentration, and heart rates were significantly lower in ENT1(-/-) animals. In conclusion, TGF responsiveness is significantly attenuated in the absence of ENT1, pointing to a role of nucleoside transport in the NaCl-synchronous changes of extracellular adenosine levels in the juxtaglomerular apparatus interstitium.

Neotyphodium coenophialum-infected tall fescue contains ergopeptines. Except for interactions with biogenic amine receptors (e.g., dopamine type-2 receptor, D2R), little is known about how ergopeptines affect animal metabolism. The effect of ergopeptines on bovine nucleoside transporters (NT) was evaluated using Madin-Darby bovine kidney (MDBK) cells. Equilibrative NT1 (ENT1)-like activity accounted for 94% of total NT activity. Inhibitory competition (IC(50)) experiments found that this activity was inhibited by both bromocriptine (a synthetic model ergopeptine and D2R agonist) and ergovaline (a predominant ergopeptine of tall fescue). Kinetic inhibition analysis indicated that bromocriptine inhibited ENT1-like activity through a competitive and noncompetitive mechanism. Domperidone (a D2R antagonist) inhibited ENT1 activity more in the presence than in the absence of bromocriptine and displayed an IC(50) value lower than that of bromocriptine or ergovaline, suggesting that inhibition was not through D2R-mediated events. These novel mechanistic findings imply that cattle consuming endophyte-infected tall fescue have reduced ENT1 activity and, thus, impaired nucleoside metabolism.

To better understand the role of equilibrative nucleoside transporters (ENT) in purine nucleoside-dependent physiology of the cardiovascular system, we investigated whether the ENT1-null mouse heart was cardioprotected in response to ischemia (coronary occlusion for 30 min followed by reperfusion for 2 h). We observed that ENT1-null mouse hearts showed significantly less myocardial infarction compared with wild-type littermates. We confirmed that isolated wild-type adult mouse cardiomyocytes express predominantly ENT1, which is primarily responsible for purine nucleoside uptake in these cells. However, ENT1-null cardiomyocytes exhibit severely impaired nucleoside transport and lack ENT1 transcript and protein expression. Adenosine receptor expression profiles and expression levels of ENT2, ENT3, and ENT4 were similar in cardiomyocytes isolated from ENT1-null adult mice compared with cardiomyocytes isolated from wild-type littermates. Moreover, small interfering RNA knockdown of ENT1 in the cardiomyocyte cell line, HL-1, mimics findings in ENT1-null cardiomyocytes. Taken together, our data demonstrate that ENT1 plays an essential role in cardioprotection, most likely due to its effects in modulating purine nucleoside-dependent signaling and that the ENT1-null mouse is a powerful model system for the study of the role of ENTs in the physiology of the cardiomyocyte.

Background: Plasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine transporters have been an area of focus in the search for anti-malarial drugs. Although the uptake of purines through the human equilibrative nucleoside transporter (hENT1), the human facilitative nucleobase transporter (hFNT1) and the parasite-induced new permeation pathway (NPP) has been studied, no information appears to exist on the relative contribution of these three transporters to the uptake of adenosine and hypoxanthine. Using the appropriate transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated., Methods: The transport of adenosine, hypoxanthine and adenine into uninfected and P. falciparum-infected human erythrocytes was investigated in the presence or absence of classical inhibitors of the hFNT1, hENT1 and NPP. The effective inhibition of the various transporters by the classical inhibitors was verified using appropriate known substrates. The ability of high concentration of unlabelled substrates to saturate these transporters was also studied., Results: Transport of exogenous purine into infected or uninfected erythrocytes occurred primarily through saturable transporters rather than through the NPP. Hypoxanthine and adenine appeared to enter erythrocytes mainly through the hFNT1 nucleobase transporter whereas adenosine entered predominantly through the hENT1 nucleoside transporter. The rate of purine uptake was approximately doubled in infected cells compared to uninfected erythrocytes. In addition, it was found that the rate of adenosine uptake was considerably higher than the rate of hypoxanthine uptake in infected human red blood cells (RBC). It was also demonstrated that furosemide inhibited the transport of purine bases through hFNT1., Conclusion: Collectively, the data obtained in this study clearly show that the endogenous host erythrocyte transporters hENT1 and hFNT1, rather than the NPP, are the major route of entry of purine into parasitized RBC. Inhibitors of hENT1 and hFNT1, as well as the NPP, should be considered in the development of anti-malarials targeted to purine transport.

The functionality of human erythrocyte nucleoside transporter ENT1 was examined at ice-cold temperatures (ICT; measured temperature, 0.5-0.7 degrees C) using rightside-out membrane vesicles (ROVs). The uptake of uridine, an ENT1 substrate, showed saturation kinetics and was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), a specific ENT1 inhibitor, at both 23 degrees C and ICT. [3H]Uridine uptake was markedly trans-stimulated by preloading ROVs with unlabeled uridine or ribavirin, another ENT1 substrate, and the overshoot phenomenon was observed at ICT. Similarly, [3H]ribavirin uptake was markedly trans-stimulated by unlabeled ribavirin or uridine at ICT. The trans-stimulated uptake of [3H]uridine at ICT was inhibited by ENT1 inhibitors/substrates such as NBMPR, dipyridamole, adenosine, and ribavirin in a concentration-dependent manner. The inhibition of [3H]uridine uptake by NBMPR and dipyridamole at ICT was also observed in intact red blood cells. Like uridine uptake, [3H]D-glucose uptake by ROVs, which is mediated by facilitative glucose transporter GLUT1, was trans-stimulated by unlabeled D-glucose at ICT, and the overshoot phenomenon was observed. In contrast, the ability of ATP-dependent transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein via multidrug resistance-associated protein 5 in inside-out membrane vesicles disappeared at ICT. These results clearly indicate that human erythrocyte transporters such as ENT1 function even at very low temperatures near 0 degrees C. The significance of these findings in transporter research is discussed.

Transgenic mice that express human equilibrative nucleoside transporter subtype 1 (hENT1) under the control of a neuron-specific enolase promoter have been generated. Southern blot and PCR revealed the presence of the transgene in five founder mice. Mice from each founder line were examined by reverse transcriptase (RT)-PCR and found to express hENT1 in RNA isolated from whole brain, cerebral cortex, striatum, hippocampus, and cerebellum but not liver, kidney, heart, lung or skeletal muscle. Cortical synaptosomes prepared from transgenic mice had significantly increased [(3)H]adenosine uptake and [(3)H]nitrobenzylthioinosine binding, relative to samples from wild-type mice. In behavioral tests, transgenic mice had altered responses to caffeine and ethanol, two drugs that inhibit and enhance, respectively, adenosine receptor activity. Caffeine-induced locomotor stimulation was attenuated whereas the hypnotic effect of ethanol was enhanced in transgenic mice. Caffeine was more potent in inhibiting ethanol-induced motor incoordination in wild-type than in transgenic mice. No differences in expression of mouse genes for adenosine receptors, nucleoside transporters, or purine metabolizing enzymes were detected by RT-PCR analyses. These data indicate that expression of hENT1 in neurons does not trigger adaptive changes in expression of adenosine-related genes. Instead, hENT1 expression affects dynamic changes in endogenous adenosine levels, as revealed by altered behavioral responses to drugs that affect adenosine receptor signalling.

Pre-eclampsia is associated with elevated maternal blood pressure and proteinuria, altered fetal growth, and increased plasma adenosine concentration in the mother and the fetus. Human equilibrative nucleoside transporters 1 (hENT1) and hENT2 are crucial to maintain physiological plasma levels of adenosine, thus modulating its several biological effects through adenosine receptor activation. However, it is unknown whether hENTs and adenosine receptors are expressed in human placental microvascular endothelium (hPMEC). To assay whether the increased fetal plasma adenosine concentration in pre-eclampsia results from altered hENT-mediated transport, and the potential involvement of adenosine receptors in this phenomenon, we investigated hENTs and A2A and A2B adenosine receptors expression and function in hPMEC. Cells were isolated and cultured from normal pregnancies (n=17) or pre-eclampsia with adequate-for-gestational age fetuses (n=7). hENT1, hENT2, A2A and A2B adenosine receptors were expressed and functional in hPMEC. Extracellular adenosine concentration was higher (4-fold) in pre-eclampsia versus normal pregnancies. hPMEC from pre-eclampsia exhibit increased total transport (hENT1+hENT2), and maximal velocity (Vmax) for hENT2- (2-fold), but reduced Vmax for hENT1-mediated adenosine transport (75%), with no changes in apparent Km. hENT2 expression was increased (4.5-fold), but hENT1 protein abundance was reduced (80%) in pre-eclampsia. Equally, A2A expression was reduced (50-80%) in pre-eclampsia. CGS-21680 (A2A agonist) did not alter hENTs expression or activity, but ZM-241385 (A2A antagonist) blocked pre-eclampsia effects and increased hENT1-mediated transport in normal pregnancies. Thus, A2B adenosine receptors may differentially modulate hENTs in hPMEC, which could be a mechanism attempting to re-establish physiological extracellular adenosine levels in pre-eclampsia.

Several mammalian nucleoside transporters have been identified at the molecular level. Human and rat equilibrative nucleoside transporter 2 (hENT2 and rENT2, respectively) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we characterized the transport of a variety of nucleosides and nucleobases via recombinant mouse ENT2 (mENT2). Cloned mENT2 mediated the uptake of nucleosides and purine nucleobases, but not pyrimidine nucleobases. The mENT2-mediated uptake of adenosine was significantly inhibited by nucleosides and nucleobases, irrespective of purine and pyrimidine. The K(m) values for the uptake of nucleosides and purine nucleobases mediated by mENT2 varied between 1.24 and 16.3 microM, and the transport clearances of adenosine and hypoxanthine via the transporter were greater than those of other substrates. Therefore, we concluded that mENT2 is nucleoside and purine nucleobase transporter, and pyrimidine nucleobases are blockers for the transporter, differing from hENT2 and rENT2 that were reported to also transport pyrimidine nucleobases.

Human erythrocyte glucose sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions ([sugar](intracellular) = [sugar](extracellular), where brackets indicate concentration). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Biphasic exchange at 20 mM 3MG eliminates the possibility that the rapid exchange phase represents ATP-dependent 3MG binding to the glucose transport protein (GLUT1; cellular [GLUT1] of

Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=-0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=-0.30; P=0.04), decitabine (rp=-0.29; P=0.04) and gemcitabine (rp=-0.33; P=0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (rp=-0.31; P=0.03) and decitabine (rp=0.33; P=0.03), respectively. The dCK/PN-I ratio correlated inversely with LC50 values for gemcitabine (rp=-0.45, P=0.001) and the dCK/CDA ratio seemed to correlate with LC50 values for decitabine (rp=-0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML.

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.

The human equilibrative nucleoside transporter 1 (hENT1) is an important modulator of the physiological action of adenosine. We identified amino acid residues involved in adenosine transport using a yeast-based assay to rapidly screen and identify randomly generated hENT1 mutants that exhibited decreased sensitivity to inhibition of adenosine transport by various hENT1 competitive inhibitors. We identified Met89 and Ser160 as important in the affinity of hENT1 for various substrates and inhibitors. Mutation to Met89Cys or Ser160Cys significantly (p < 0.05) increased the S6-(4-nitrobenzyl)-mercaptopurine riboside (NBMPR) IC50 values by approximately 4- and 6-fold, respectively (42 +/- 13 and 65 +/- 1.6 nM) compared with the wild-type transporter (11 +/- 0.7 nM). The double mutant Met89Cys/Ser160Cys synergistically increased the NBMPR IC50 value to approximately 19-fold of that of the wild-type transporter. In contrast, compared with wild-type hENT1, the sensitivity to dipyridamole inhibition was significantly (p < 0.05) increased by only the Ser160Cys (approximately 2.6-fold) or the double mutant Met89Cys/Ser160Cys (approximately 4.7-fold) but not by the Met89Cys mutant. Mutation to Met89Cys or Ser160Cys increased the Km of adenosine (approximately 8- and 3-fold) and the Ki of guanosine (approximately 6- and 2-fold). The double mutant increased both the Km value of adenosine and the Ki value of guanosine by approximately 8-fold and seemed to confer no additional reduction in adenosine or guanosine affinity than that by mutation of Met89 alone. Together, these data indicate that transmembrane domains (TMDs) 2 (Met89) and 4 (Ser160) of hENT1 interact and are important in conferring sensitivity to NBMPR. In contrast, Ser160 and Met89 of hENT1, respectively, play a dominant role in conferring sensitivity to dipyridamole and adenosine/guanosine affinity.

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive alpha' and alpha subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2alpha' (decreased CK2alpha' activity) increased the number of binding sites (approximately 1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (approximately 1.8-fold) the V(max) for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the V(max) of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2alpha' activity had no effect, however, on either the K(D) of [3H]NBMPR binding or the K(m) of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4-12 h of induction of the inactive CK2alpha' subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2alpha' reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2alpha'-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

The blood-brain barrier (BBB) transport of synthetic A(1) receptor agonists was studied in an in situ brain perfusion model in the presence and absence of the selective nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI). For 8-methylamino-N(6)cyclopentyladenosine (MCPA), N(6)-cyclopentyladenosine (CPA), 2'deoxy-N(6)-cyclopentyladenosine (2'dCPA) and 5'deoxy-N(6)-cyclopentyl adenosine (5'dCPA) the brain uptake clearance was low with values of 0.0045+/-0.0012, 0.018+/-0.0020, 0.022+/-0.0028 and 0.12+/-0.054 ml min(-1)g(-1), respectively. In the presence of an average NBTI plasma concentration of 2.6+/-0.3 microg ml(-1) (NBTI dose: 3 mg kg(-1) i.v.) the values of the brain uptake clearance were 0.0062+/-0.0012, 0.013+/-0.0017, 0.014+/-0.0030 and 0.13+/-0.066 ml min(-1)g(-1), respectively and not significantly different from the values in the absence of NBTI. In a separate experiment the brain uptake of MCPA from phosphate buffered saline (PBS) and whole blood were compared. The brain uptake clearance from whole blood (0.0012+/-0.001 ml min(-1)g(-1)) was significantly lower than from PBS (0.0045+/-0.0012 ml min(-1)g(-1)). The results of these studies show that the rENT1 nucleoside transporter does not contribute significantly to the transport of synthetic A(1) receptor agonists across the BBB and that binding to blood constituents restricts the brain uptake.