Anticonvulsant effect of equilibrative nucleoside transporters 1 inhibitor in a mouse model of Dravet syndrome.

There are limited therapeutic options for patients with Dravet syndrome (DS). The equilibrative nucleoside transporters 1 (ENT1) mediate both the influx and efflux of adenosine across the cell membrane exerted beneficial effects in the treatment of epilepsy. This study aimed to evaluate the anticonvulsant effect of the ENT1 inhibitor in an animal model of DS (Scn1aE1099X/+ mice). J7 (5 mg/kg) treatment was efficacious in elevating seizure threshold in Scn1aE1099X/+ mice after hyperthermia exposure. Moreover, the J7 treatment significantly reduced the frequency of spontaneous excitatory post-synaptic currents (sEPSCs, ~35% reduction) without affecting the amplitude in dentate gyrus (DG) granule cells. Pretreatment with the adenosine A1 receptor (A1R) antagonist, DPCPX, abolished the J7 effects on sEPSCs. These observations suggest that the J7 shows an anticonvulsant effect in hyperthermia-induced seizures in Scn1aE1099X/+ mice. This effect possibly acts on presynaptic A1R-mediated signaling modulation in granule cells.

Erythrocyte type 1 equilibrative nucleoside transporter expression in sickle cell disease and sickle cell trait.

Hypoxia-mediated red blood cell (RBC) sickling is central to the pathophysiology of sickle cell disease (SCD). The signalling nucleoside adenosine is thought to play a significant role in this process. This study investigated expression of the erythrocyte type 1 equilibrative nucleoside transporter (ENT1), a key regulator of plasma adenosine, in adult patients with SCD and carriers of sickle cell trait (SCT). Relative quantitative expression analysis of erythrocyte ENT1 was carried out by Western blot and flow cytometry. Patients with SCD with steady state conditions, either with SS or SC genotype, untreated or under hydroxycarbamide (HC) treatment, exhibited a relatively high variability of erythrocyte ENT1, but with levels not significantly different from normal controls. Most strikingly, expression of erythrocyte ENT1 was found to be significantly decreased in patients with SCD undergoing painful vaso-occlusive episode and, unexpectedly, also in healthy SCT carriers. Promoting hypoxia-induced adenosine signalling, the reduced expression of erythrocyte ENT1 might contribute to the pathophysiology of SCD and to the susceptibility of SCT individuals to altitude hypoxia or exercise to exhaustion.

Augustine Blood Group System and Equilibrative Nucleoside Transporter 1.

Augustine (AUG) is a blood group system comprising four antigens: AUG1, AUG2 (Ata), and AUG4 are of very high frequency; AUG3 is of very low frequency. These antigens are located on ENT1, an equilibrative nucleoside transporter encoded by SLC19A1. AUG antibodies are of clinical relevance in blood transfusion and pregnancy: anti-AUG2 have caused haemolytic transfusion reactions; the only anti-AUG3 was associated with severe haemolytic disease of the fetus and newborn. ENT1 is present in almost all human tissues. It facilitates the transfer of purine and pyrimidine nucleosides and is responsible for the majority of adenosine transport across plasma membranes. Adenosine transport appears to be an important factor in the regulation of bone metabolism. The AUGnull phenotype (AUG:-1,-2,-3,-4) has been found in three siblings, who are homozygous for an inactivating splice-site mutation in SLC29A1. Although ENT1 is very likely to be absent from all cells in these three individuals, they were apparently healthy with normal lifestyles. However, they suffered frequent attacks of pseudogout, a form of arthritis, in various joints with multiple calcifications around their hand joints. Ectopic calcification in the hips, pubic symphysis, and lumbar discs was present in the propositus. The three AUGnull individuals had misshapen red cells with deregulated protein phosphorylation, but no anaemia or shortening of red cell lifespan. Defective in vitro erythropoiesis in the absence of ENT1 was confirmed by shRNA-mediated knockdown of ENT1 during in vitro erythropoiesis of CD34+ progenitor cells from individuals with normal ENT1. Nucleoside transporters, such as ENT1, are vital in the uptake of synthetic nucleoside analogue drugs, used in cancer and viral chemotherapy. It is feasible that the efficacy of these drugs would be compromised in patients with the extremely rare AUGnull phenotype.

Adaptative mechanism of the equilibrative nucleoside transporter 1 (ENT-1) and blood adenosine levels in elite freedivers.

PURPOSE: Long static or intense dynamic apnoea-like high-altitude exposure is inducing hypoxia. Adenosine is known to participate to the adaptive response to hypoxia leading to the control of heart rate, blood pressure and vasodilation. Extracellular adenosine level is controlled through the equilibrative nucleoside transporter 1 (ENT-1) and the enzyme adenosine deaminase (ADA). The aim of this study was to determine the control of adenosine blood level (ABL) via ENT-1 and ADA during apnoea-induced hypoxia in elite freedivers was similar to high-altitude adaptation. METHODS: Ten freediver champions and ten controls were studied. Biological (e.g. ENT-1, ADA, ABL, PaO2, PaCO2 and pH) and cardiovascular (e.g. heart rate, arterial pressure) parameters were measured at rest and after a submaximal dry static apnoea. RESULTS: In freedivers, ABL was higher than in control participants in basal condition and increased more in response to apnoea. Also, freedivers showed an ADA increased in response to apnoea. Finally, ENT-1 level and function were reduced for the free divers. CONCLUSION: Our results suggest in freedivers the presence of an adaptive mechanism similar to the one observed in human exposed to chronic hypoxia induced by high-altitude environment.

Nicotinic α7 receptor-induced adenosine release from perisynaptic Schwann cells controls acetylcholine spillover from motor endplates.

Acetylcholine (ACh) spillover from motor endplates occurs after neuronal firing bursts being potentiated by cholinesterase inhibitors (e.g., neostigmine). Nicotinic α7 receptors (α7nAChR) on perisynaptic Schwann cells (PSCs) can control ACh spillover by unknown mechanisms. We hypothesized that adenosine might be the gliotransmitter underlying PSCs-nerve terminal communication. Rat isolated hemidiaphragm preparations were used to measure (1) the outflow of [3 H]ACh, (2) real-time transmitter exocytosis by video-microscopy with the FM4-64 fluorescent dye, and (3) skeletal muscle contractions during high-frequency (50 Hz) nerve stimulation bursts in the presence of a selective α7nAChR agonist, PNU 282987, or upon inhibition of cholinesterase activity with neostigmine. To confirm our prediction that α7nAChR-mediated effects require direct activation of PSCs, we used fluorescence video-microscopy in the real-time mode to measure PNU 282987-induced [Ca2+ ]i transients from Fluo-4 NW loaded PSCs in non-stimulated preparations. The α7nAChR agonist, PNU 282987, decreased nerve-evoked diaphragm tetanic contractions. PNU 282987-induced inhibition was mimicked by neostigmine and results from the reduction of ACh exocytosis measured as decreases in [3 H]ACh release and FM4-64 fluorescent dye unloading. Methyllycaconitine blockage of α7nAChR and the fluoroacetate gliotoxin both prevented inhibition of nerve-evoked ACh release and PSCs [Ca2+ ]i transients triggered by PNU 282987 and neostigmine. Adenosine deamination, inhibition of the ENT1 nucleoside outflow, and blockage of A1 receptors prevented PNU 282987-induced inhibition of transmitter release. Data suggest that α7nAChR controls tetanic-induced ACh spillover from the neuromuscular synapse by promoting adenosine outflow from PSCs via ENT1 transporters and retrograde activation of presynaptic A1 inhibitory receptors.

Dynamic-related protein 1 inhibitor eases epileptic seizures and can regulate equilibrative nucleoside transporter 1 expression.

BACKGROUND: Dynamic-related protein 1 (Drp1) is a key protein involved in the regulation of mitochondrial fission, and it could affect the dynamic balance of mitochondria and appears to be protective against neuronal injury in epileptic seizures. Equilibrative nucleoside transporter 1 (ENT1) is expressed and functional in the mitochondrial membrane that equilibrates adenosine concentration across membranes. Whether Drp1 participates in the pathogenesis of epileptic seizures via regulating function of ENT1 remains unclear. METHODS: In the present study, we used pilocarpine to induce status epilepticus (SE) in rats, and we used mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor to Drp1, to suppress mitochondrial fission in pilocarpine-induced SE model. Mdivi-1administered by intraperitoneal injection before SE induction, and the latency to firstepileptic seizure and the number of epileptic seizures was thereafter observed. The distribution of Drp1 was detected by immunofluorescence, and the expression patterns of Drp1 and ENT1 were detected by Western blot. Furthermore, the mitochondrial ultrastructure of neurons in the hippocampal CA1 region was observed by transmission electron microscopy. RESULTS: We found that Drp1 was expressed mainly in neurons and Drp1 expression was significantly upregulated in the hippocampal and temporal neocortex tissues at 6 h and 24 h after induction of SE. Mitochondrial fission inhibitor 1 attenuated epileptic seizures after induction of SE, reduced mitochondrial damage and ENT1 expression. CONCLUSIONS: These data indicate that Drp1 is upregulated in hippocampus and temporal neocortex after pilocarpine-induced SE and the inhibition of Drp1 may lead to potential therapeutic target for SE by regulating ENT1 after pilocarpine-induced SE.

Type 1 equilibrative nucleoside transporter (ENT1) regulates sex-specific ethanol drinking during disruption of circadian rhythms.

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.

Contribution of adenosine and ATP to the carotid body chemosensory activity in ageing.

KEY POINTS: Adenosine and ATP are excitatory neurotransmitters involved in the carotid body (CB) response to hypoxia. During ageing the CB exhibits a decline in its functionality, demonstrated by decreased hypoxic responses. In aged rats (20-24 months old) there is a decrease in: basal and hypoxic release of adenosine and ATP from the CB; expression of adenosine and ATP receptors in the petrosal ganglion; carotid sinus nerve (CSN) activity in response to hypoxia; and ventilatory responses to ischaemic hypoxia. There is also an increase in SNAP25, ENT1 and CD73 expression. It is concluded that, although CSN activity and ventilatory responses to hypoxia decrease with age, adjustments in purinergic metabolism in the CB in aged animals are present aiming to maintain the contribution of adenosine and ATP. The possible significance of the findings in the context of ageing and in CB-associated pathologies is considered. ABSTRACT: During ageing the carotid body (CB) exhibits a decline in its functionality. Here we investigated the effect of ageing on functional CB characteristics as well as the contribution of adenosine and ATP to CB chemosensory activity. Experiments were performed in 3-month-old and 20- to 24-month-old male Wistar rats. Ageing decreased: the number of tyrosine hydroxylase immune-positive cells, but not type II cells or nestin-positive cells in the CB; the expression of P2X2 and A2A receptors in the petrosal ganglion; and the basal and hypoxic release of adenosine and ATP from the CB. Ageing increased ecto-nucleotidase (CD73) immune-positive cells and the expression of synaptosome associated protein 25 (SNAP25) and equilibrative nucleoside transporter 1 (ENT1) in the CB. Additionally, ageing did not modify basal carotid sinus nerve (CSN) activity or the activity in response to hypercapnia, but decreased CSN activity in hypoxia. The contribution of adenosine and ATP to stimuli-evoked CSN chemosensory activity in aged animals followed the same pattern of 3-month-old animals. Bilateral common carotid occlusions during 5, 10 and 15 s increased ventilation proportionally to the duration of ischaemia, an effect decreased by ageing. ATP contributed around 50% to ischaemic-ventilatory responses in young and aged rats; the contribution of adenosine was dependent on the intensity of ischaemia, being maximal in ischaemias of 5 s (50%) and much smaller in 15 s ischaemias. Our results demonstrate that both ATP and adenosine contribute to CB chemosensory activity in ageing. Though CB responses to hypoxia, but not to hypercapnia, decrease with age, the relative contribution of both ATP and adenosine for CB activity is maintained.

Loss of ENT1 increases cell proliferation in the annulus fibrosus of the intervertebral disc.

Mice lacking equilibrative nucleoside transporter 1 (ENT1 -/- ) demonstrate progressive calcification of spinal tissues including the annulus fibrosus (AF) of the intervertebral disc (IVD). We previously established ENT1 as the primary nucleoside transporter in the AF and demonstrated dysregulation of biomineralization pathways. To identify cellular pathways altered by loss of ENT1, we conducted microarray analysis of AF tissue from wild-type (WT) and ENT1 -/- mice before calcification (2 months of age) and associated with calcification (6 months of age). Bioinformatic analyses identified cell cycle dysregulation in ENT1 -/- AF tissues and implicated the E2f family of transcription factors as potential effectors. Quantitative polymerase chain reaction analysis confirmed increased expression of multiple E2f transcription factors and E2f interacting proteins ( Rb1 and Cdk2) in ENT1 -/- AF cells compared with WT at 6 months of age. At this time point, ENT1 -/- AF tissues showed increased JNK MAPK pathway activation, CDK1, minichromosome maintenance complex component 5 (Mcm5), and proliferating cell nuclear antigen (PCNA) protein expression, and PCNA-positive proliferating cells compared with WT controls. The current study demonstrates that loss of ENT1-mediated adenosine transport leads to increased cell proliferation in the AF of the IVD.

Regulation of hepatic glucose production and AMPK by AICAR but not by metformin depends on drug uptake through the equilibrative nucleoside transporter 1 (ENT1).

AIM: Recently we have observed differences in the ability of metformin and AICAR to repress glucose production from hepatocytes using 8CPT-cAMP. Previous results indicate that, in addition to activating protein kinase A, 8CPT-modified cAMP analogues suppress the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter ENT1. We aimed to exploit 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR, which is highly selective for a high-affinity binding-site on ENT1, to investigate the role of ENT1 in the liver-specific glucose-lowering properties of AICAR and metformin. METHODS: Primary mouse hepatocytes were incubated with AICAR and metformin in combination with cAMP analogues, glucagon, forskolin and NBMPR. Hepatocyte glucose production (HGP) and AMPK signalling were measured, and a uridine uptake assay with supporting LC-MS was used to investigate nucleoside depletion from medium by cells. RESULTS: AICAR and metformin increased AMPK pathway phosphorylation and decreased HGP induced by dibutyryl cAMP and glucagon. HGP was also induced by 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR; however, in each case this was resistant to suppression by AICAR but not by metformin. Cross-validation of tracer and mass spectrometry studies indicates that 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR inhibited the effects of AICAR, at least in part, by impeding its uptake into hepatocytes. CONCLUSIONS: We report for the first time that suppression of ENT1 induces HGP. ENT1 inhibition also impedes uptake and the effects of AICAR, but not metformin, on HGP. Further investigation of nucleoside transport may illuminate a better understanding of how metformin and AICAR each regulate HGP.

Label-Free Neuroproteomics of the Hippocampal-Accumbal Circuit Reveals Deficits in Neurotransmitter and Neuropeptide Signaling in Mice Lacking Ethanol-Sensitive Adenosine Transporter.

The neural circuit of the dorsal hippocampus (dHip) and nucleus accumbens (NAc) contributes to cue-induced learning and addictive behaviors, as demonstrated by the escalation of ethanol-seeking behaviors observed following deletion of the adenosine equilibrative nucleoside transporter 1 (ENT1-/-) in mice. Here we perform quantitative LC-MS/MS neuroproteomics in the dHip and NAc of ENT1-/- mice. Using Ingenuity Pathway Analysis, we identified proteins associated with increased long-term potentiation, ARP2/3-mediated actin cytoskeleton signaling and protein expression patterns suggesting deficits in glutamate degradation, GABAergic signaling, as well as significant changes in bioenergetics and energy homeostasis (oxidative phosphorylation, TCA cycle, and glycolysis). These pathways are consistent with previously reported behavioral and biochemical phenotypes that typify mice lacking ENT1. Moreover, we validated decreased expression of the SNARE complex protein VAMP1 (synaptobrevin-1) in the dHip as well as decreased expression of pro-dynorphin (PDYN), neuroendocrine convertase (PCSK1), and Leu-Enkephalin (dynorphin-A) in the NAc. Taken together, our proteomic approach provides novel pathways indicating that ENT1-regulated signaling is essential for neurotransmitter release and neuropeptide processing, both of which underlie learning and reward-seeking behaviors.

A semi-quantitative translational pharmacology analysis to understand the relationship between in vitro ENT1 inhibition and the clinical incidence of dyspnoea and bronchospasm.

Adenosine contributes to the pathophysiology of respiratory disease, and adenosine challenge leads to bronchospasm and dyspnoea in patients. The equilibrative nucleoside transporter 1 (ENT1) terminates the action of adenosine by removal from the extracellular environment. Therefore, it is proposed that inhibition of ENT1 in respiratory disease patients leads to increased adenosine concentrations, triggering bronchospasm and dyspnoea. This study aims to assess the translation of in vitro ENT1 inhibition to the clinical incidence of bronchospasm and dyspnoea in respiratory disease, cardiovascular disease and healthy volunteer populations. Four marketed drugs with ENT1 activity were assessed; dipyridamole, ticagrelor, draflazine, cilostazol. For each patient population, the relationship between in vitro ENT1 [3H]-NBTI binding affinity (Ki) and [3H]-adenosine uptake (IC50) to the incidence of: (1) bronchospasm/severe dyspnoea; (2) tolerated dyspnoea and; (3) no adverse effects, was evaluated. A high degree of ENT1 inhibition (≥13.3x Ki, ≥4x IC50) associated with increased incidence of bronchospasm/severe dyspnoea for patients with respiratory disease only, whereas a lower degree of ENT1 inhibition (≥0.1x Ki, ≥0.05x IC50) associated with a tolerable level of dyspnoea in both respiratory and cardiovascular disease patients. ENT1 inhibition had no effect in healthy volunteers. Furthermore, physicochemical properties correlative with ENT1 binding were assessed using a set of 1625 diverse molecules. Binding to ENT1 was relatively promiscuous (22% compounds Ki<1µM) especially for neutral or basic molecules, and greater incidence tracked with higher lipophilicity (clogP >5). This study rationalises inclusion of an assessment of ENT1 activity during early safety profiling for programs targeting respiratory disorders.

Molecular Basis of Nucleobase Transport Systems in Mammals.

Nucleobases are water-soluble compounds that need specific transporters to cross biological membranes. Cumulative evidence based on studies using animal tissues and cells indicates that the carrier-mediated transport systems for purine and pyrimidine nucleobases can be classified into the following two types: concentrative transport systems that mediate nucleobase transport depending on the sodium ion concentration gradient; and other systems that mediate facilitated diffusion depending on the concentration gradient of the substrate. Recently, several molecular transporters that are involved in both transport systems have been identified. The function and activity of these transporters could be of pharmacological significance considering the roles that they play not only in nucleotide synthesis and metabolism but also in the pharmacokinetics and delivery of a variety of nucleobase analogues used in anticancer and antiviral drug therapy. The present review provides an overview of the recent advances in our understanding of the molecular basis of nucleobase transport systems, focusing on the transporters that mediate purine nucleobases, and discusses the involvement of intracellular metabolism in purine nucleobase transport and chemotherapy using ganciclovir.

Caffeine exposure alters adenosine system and neurochemical markers during retinal development.

Evidence points to beneficial properties of caffeine in the adult central nervous system, but teratogenic effects have also been reported. Caffeine exerts most of its effects by antagonizing adenosine receptors, especially A1 and A2A subtypes. In this study, we evaluated the role of caffeine on the expression of components of the adenosinergic system in the developing avian retina and the impact of caffeine exposure upon specific markers for classical neurotransmitter systems. Caffeine exposure (5-30 mg/kg by in ovo injection) to 14-day-old chick embryos increased the expression of A1 receptors and concomitantly decreased A2A adenosine receptors expression after 48 h. Accordingly, caffeine (30 mg/kg) increased [(3) H]-8-cyclopentyl-1,3-dipropylxanthine (A1 antagonist) binding and reduced [(3) H]-ZM241385 (A2A antagonist) binding. The caffeine time-response curve demonstrated a reduction in A1 receptors 6 h after injection, but an increase after 18 and 24 h. In contrast, caffeine exposure increased the expression of A2A receptors from 18 and 24 h. Kinetic assays of [(3) H]-S-(4-nitrobenzyl)-6-thioinosine binding to the equilibrative adenosine transporter ENT1 revealed an increase in Bmax with no changes in Kd , an effect accompanied by an increase in adenosine uptake. Immunohistochemical analysis showed a decrease in retinal content of tyrosine hydroxylase, calbindin and choline acetyltransferase, but not Brn3a, after 48 h of caffeine injection. Furthermore, retinas exposed to caffeine had increased levels of phosphorylated extracellular signal-regulated kinase and cAMP-response element binding protein. Overall, we show an in vivo regulation of the adenosine system, extracellular signal-regulated kinase and cAMP-response element binding protein function and protein expression of specific neurotransmitter systems by caffeine in the developing retina. The beneficial or maleficent effects of caffeine have been demonstrated by the work of different studies. It is known that during animal development, caffeine can exert harmful effects, impairing the correct formation of CNS structures. In this study, we demonstrated cellular and tissue effects of caffeine's administration on developing chick embryo retinas. Those effects include modulation of adenosine receptors (A1 , A2 ) content, increasing in cAMP response element-binding protein (pCREB) and extracellular signal-regulated kinase phosphorylation (pERK), augment of adenosine equilibrative transporter content/activity, and a reduction of some specific cell subpopulations. ENT1, Equilibrative nucleoside transporter 1.

Equilibrative nucleoside transporter ENT1 as a biomarker of Huntington disease.

The initial goal of this study was to investigate alterations in adenosine A2A receptor (A2AR) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an A2AR antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [3H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease.

Low expression of equilibrative nucleoside transporter 1 is associated with poor prognosis in chemotherapy-naïve pT2 gallbladder adenocarcinoma patients.

AIMS: Equilibrative nucleoside transporter 1 (ENT1) is the major transporter of the chemotherapeutic drug gemcitabine, the current therapy for advanced gallbladder cancer (GBC). ENT1 expression has been proposed as a predictive marker for gemcitabine-treated pancreatic cancer patients. The aim of study was to explore the value of ENT1 measurement in chemotherapy-naïve patients with advanced GBC. MATERIALS AND RESULTS: Immunohistochemistry for ENT1 was performed on 214 GBC samples from patients who had never undergone co-adjuvant or neo-adjuvant chemotherapy. Advanced GBC cases were divided into groups with low or high ENT1 expression. Kaplan-Meier tests were used for survival analyses. The Cox regression method was used to assess the association of ENT1 expression with overall survival (OS). Low ENT1 expression was associated with younger patient age (P = 0.03) and moderate-to-poor histological differentiation (P = 0.01). pT2 patients with low ENT1 expression had shorter median survival (17.3 versus 28.7 months) and lower OS (17.3% versus 33.3%, P < 0.05) than patients with high ENT1 expression. Low ENT1 expression was an independent prognostic factor for OS (P = 0.036). CONCLUSIONS: ENT1 is a prognostic marker for pT2 GBC patients. Additional studies are needed to determine whether ENT1 has predictive value for gemcitabine response in GBC.

Adenosinergic system and nucleoside transporters in attention deficit hyperactivity disorder: Current findings.

Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder with high heterogeneity that can affect individuals of any age. It is characterized by three main symptoms: inattention, hyperactivity, and impulsivity. These neurobehavioral alterations and neurochemical and pharmacological findings are mainly attributed to unbalanced catecholaminergic signaling, especially involving dopaminergic pathways within prefrontal and striatal areas. Dopamine receptors and transporters are not solely implicated in this imbalance, as evidence indicates that the dopaminergic signaling is modulated by adenosine activity. To this extent, alterations in adenosinergic signaling are probably involved in ADHD. Here, we review the current knowledge about adenosine's role in the modulation of chemical, behavioral and cognitive parameters of ADHD, especially regarding dopaminergic signaling. Current literature usually links adenosine receptors signaling to the dopaminergic imbalance found in ADHD, but there is evidence that equilibrative nucleoside transporters (ENTs) could also be implicated as players in dopaminergic signaling alterations seen in ADHD, since their involvement in other neurobehavioral impairments.

Deep Brain Stimulation Inhibits Epileptic Seizures via Increase of Adenosine Release and Inhibition of ENT1, CD39, and CD73 Expression.

Deep brain stimulation (DBS) of the anterior nucleus of the thalamus is an efficacious treatment option for patients with refractory epilepsy. Our previous study demonstrates that adenosine is a potential target of DBS for the treatment of epilepsy. Equilibrative nucleoside transporters-1 (ENT1) and ectonucleotidases (CD39, CD73) function as regulators of extracellular adenosine in the brain. It is unclear whether ENT1, CD39, and CD73 are involved in the mechanism of DBS for epilepsy. A total of 48 SD male rats were divided into four groups: control (naïve rats), Pilo (pilocarpine induced rats with epilepsy), DBS (rats with epilepsy treated with DBS for 8 weeks), and sham. In the present study, video electroencephalogram monitoring, Morris water maze assays, in vivo measurements of adenosine using fiber photometry, histochemistry, and western blot were performed on the hippocampus. DBS markedly attenuated spontaneous recurrent seizures (SRSs) and enhanced spatial learning in rats with epilepsy, assessed through video-EEG and water maze assays. Fibred photometry measurements of an adenosine sensor revealed dynamic increase in extracellular adenosine during DBS. The expressions of ENT1, CD39, and CD73 in Pilo group and sham group increased compared with the control group, while the expressions of ENT1, CD39, and CD73 in DBS group decreased compared to that of Pilo group and sham group. The findings indicate that DBS reduces the number of SRSs and improves spatial memory in rats with epilepsy with concomitant decrease of ENT1, CD39, and CD73 expressions. Adenosine-modulating enzymes might be the potential targets of DBS for the treatment of epilepsy.

Characterization of Equilibrative Nucleoside Transport of the Pancreatic Cancer Cell Line: Panc-1.

Objectives: Gemcitabine, a first-line chemotherapeutic nucleoside analog drug (NAD) for pancreatic cancer, faces limitations due to drug resistance. Characterizing pancreatic cancer cells' transport characteristics may help identify the mechanisms behind drug resistance, and develop more effective therapeutic strategies. Therefore, in this study, we aimed to determine the nucleoside transport properties of Panc-1 cells, one of the commonly used pancreatic adenocarcinoma cell lines. Materials and Methods: To assess the presence of equilibrative nucleoside transporter-1 (ENT-1) in Panc-1 cells, we performed immunofluorescence staining, western blot analysis, and S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding assays. We also conducted standard uptake assays to measure the sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine. In addition, we determined the half-maximal inhibitory concentration (IC50) of gemcitabine. Statistical analyses were performed using GraphPad Prism version 8.0 for Windows. Results: The sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine was measured using standard uptake assays, and the transport rates were determined as 111.1 ± 3.4 pmol/mg protein/10 s, 62.5 ± 4.8 pmol/mg protein/10 s, and 101.3 ± 2.5 pmol/mg protein/10 s, respectively. Furthermore, the presence of ENT-1 protein was confirmed using NBTI binding assays (Bmax 1.52 ± 0.1 pmol/mg protein; equilibrium dissociation constant 0.42 ± 0.1 nM). Immunofluorescence assays and western blot analysis also revealed ENT-1 in Panc-1 cells. The determined IC50 of gemcitabine in Panc-1 cells was 2 µM, indicating moderate sensitivity. Conclusion: These results suggest that Panc-1 is a suitable preclinical cellular model for studying NAD transport properties and potential therapies in pancreatic cancer and pharmaceutical research.

Potential roles of adenosine deaminase-2 in diabetic retinopathy.

The early activation of microglia that induces retinal inflammation in DR may serve as a target for therapeutic intervention of DR. Our demonstration that retinal inflammation is attenuated via adenosine receptor A(2A)AR supports the hypothesis that a mechanism to maintain extracellular concentrations of adenosine important in normal physiology is impaired in DR. Extracellular concentrations of adenosine are regulated by the interplay of equiliberative nucleoside transporter (ENT)s with enzymes of adenosine metabolism including adenosine deaminase-1 (ADA1), adenosine kinase (AK) and CD73. In the vertebrates but not rodents, a macrophage-associated ADA2 is identified. The role of ADA2 is, therefore, understudied as the sequencing probes or antibodies to mouse ADA2 are not available. We identified increased ADA2 expression and activity in human and porcine retinas with diabetes, and in Amadori glycated albumin (AGA)- or hyperglycemia-treated porcine and human microglia. In rodent as well as porcine cells, modulation of TNF-α release is mediated by A(2A)AR. Quantitative analysis of normal and diabetic porcine retinas reveals that while the expression levels of ADA2, A2AAR, ENT1, TNF-α and MMP9 are increased, the levels of AK are reduced during inflammation as an endogenous protective mechanism. To determine the role of ADA2, we found that AGA induces ADA2 expression, ADA2 activity and TNF-α release, and that TNF-α release is blocked by ADA2-neutralizing antibody or ADA2 siRNA, but not by scrambled siRNA. These results suggest that retinal inflammation in DR is mediated by ADA2, and that the anti-inflammatory activity of A(2A)AR signaling is impaired in diabetes due to increased ADA2 activity.