Development of a Novel HEK293 Cell Model Lacking SLC29A1 to Study the Pharmacology of Endogenous SLC29A2-Encoded Equilibrative Nucleoside Transporter Subtype 2.

Equilibrative nucleoside transporters (ENTs) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogs used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of coexpressed ENT1. We created a cell model where ENT1 was removed from human embryonic kidney (HEK293) cells using CRISPR/cas9 [ENT1 knockout (KO) cells]; this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [3H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [3H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by quantitative polymerase chain reaction. Wild-type HEK293 cells and ENT1KO cells had a similar expression of SLC29A2/ENT2 transcript/protein and ENT2-mediated [3H]2-chloroadenosine transport activity (Vmax values of 1.02 ± 0.06 and 1.50 ± 0.22 pmol/µl/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (Ki) for ENT2 (2.6 µM), while hypoxanthine was the only nucleobase with a submillimolar affinity (320 µM). A range of nucleoside/nucleobase analogs were also tested for their affinity for ENT2 in this model, with affinities (Ki) ranging from 8.6 µM for ticagrelor to 2,300 µM for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. SIGNIFICANCE STATEMENT: We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function.

Kinetic profiling of novel spirobenzo-oxazinepiperidinone derivatives as equilibrative nucleoside transporter 1 inhibitors.

Evaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand's duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds' chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1.

NBTI attenuates neuroinflammation and apoptosis partly by ENT1/NLRP3/Bcl2 pathway after subarachnoid hemorrhage in rats.

OBJECTIVES: Neuroinflammation and apoptosis are two key factors contributing to early brain injury (EBI) after subarachnoid hemorrhage (SAH) and are strongly associated with a poor prognosis. Recently, equilibrative nucleoside transporter 1 (ENT1) was emerged to accelerate the severity of inflammation and cell apoptosis in several nervous system diseases, including cerebral ischemia, neurodegeneration and epilepsy. However, no study has yet elaborated the expression levels and effects of ENT1 in EBI after SAH. METHODS: Sprague-Dawley rats were subjected to SAH by endovascular perforation. Nitrobenzylthioinosine (NBTI) was intranasally administered at 0.5 h after SAH. The protein expression levels of ENT1, NLRP3, Bcl2, Bax, ACS, Caspase-1, IL-1 were detected by western blot. The modified Garcia score and beam balance score were employed to evaluate the neurologic function of rats following SAH. In addition, hematoxylin-eosin, fluoro-jade C and TdT-mediated dUTP nick-end labeling staining were then used to evaluate brain tissue damage and neuronal apoptosis. RESULTS: Analysis indicated that endogenous levels of ENT1 were significantly upregulated at 24-hour post-SAH, accompanied by NLRP3 inflammasome activation and Bcl2 decline. The administration of NBTI, an inhibitor of ENT1, at a dose of 15 mg/kg, ameliorated neurologic deficits and morphologic lesions at both 24 and 72 h after SAH. Moreover, ENT1 inhibition efficiently mitigated neuronal degeneration and cell apoptosis. In addition, NBTI at 15 mg/kg observably increased Bcl2 content and decreased Bax level. Furthermore, suppression of ENT1 notably reduced the expression levels of NLRP3, apoptosis associated speck like protein containing CARD, caspase-1 and IL-1ß. CONCLUSIONS: NBTI relieved SAH-induced EBI partly through ENT1/NLRP3/Bcl2 pathway.

Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.

S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) is Not a Selective Inhibitor of Equilibrative Nucleoside Transporters but Also Blocks Efflux Activity of Breast Cancer Resistance Protein.

PURPOSE: S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) is routinely used at concentrations of 0.10 µM and 0.10 mM to specifically inhibit transport of nucleosides mediated by equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2), respectively. We recently showed that NBMPR (0.10 mM) might also inhibit placental active efflux of [3H]zidovudine and [3H]tenofovir disoproxil fumarate. Here we test the hypothesis that NBMPR abolishes the activity of P-glycoprotein (ABCB1) and/or breast cancer resistance protein (ABCG2). METHODS: We performed accumulation assays with Hoechst 33342 (a model dual substrate of ABCB1 and ABCG2) and bi-directional transport studies with the ABCG2 substrate [3H]glyburide in transduced MDCKII cells, accumulation studies in choriocarcinoma-derived BeWo cells, and in situ dual perfusions of rat term placenta with glyburide. RESULTS: NBMPR inhibited Hoechst 33342 accumulation in MDCKII-ABCG2 cells (IC50 = 53 µM) but not in MDCKII-ABCB1 and MDCKII-parental cells. NBMPR (0.10 mM) also inhibited bi-directional [3H]glyburide transport across monolayers of MDCKII-ABCG2 cells and blocked ABCG2-mediated [3H]glyburide efflux by rat term placenta in situ. CONCLUSION: NBMPR at a concentration of 0.10 mM abolishes ABCG2 activity. Researchers using NBMPR to evaluate the effect of ENTs on pharmacokinetics must therefore interpret their results carefully if studying compounds that are substrates of both ENTs and ABCG2.

Blockade of equilibrative nucleoside transporter 1/2 protects against Pseudomonas aeruginosa-induced acute lung injury and NLRP3 inflammasome activation.

Pseudomonas aeruginosa infections are increasingly multidrug resistant and cause healthcare-associated pneumonia, a major risk factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Adenosine is a signaling nucleoside with potential opposing effects; adenosine can either protect against acute lung injury via adenosine receptors or cause lung injury via adenosine receptors or equilibrative nucleoside transporter (ENT)-dependent intracellular adenosine uptake. We hypothesized that blockade of intracellular adenosine uptake by inhibition of ENT1/2 would increase adenosine receptor signaling and protect against P. aeruginosa-induced acute lung injury. We observed that P. aeruginosa (strain: PA103) infection induced acute lung injury in C57BL/6 mice in a dose- and time-dependent manner. Using ENT1/2 pharmacological inhibitor, nitrobenzylthioinosine (NBTI), and ENT1-null mice, we demonstrated that ENT blockade elevated lung adenosine levels and significantly attenuated P. aeruginosa-induced acute lung injury, as assessed by lung wet-to-dry weight ratio, BAL protein levels, BAL inflammatory cell counts, pro-inflammatory cytokines, and pulmonary function (total lung volume, static lung compliance, tissue damping, and tissue elastance). Using both agonists and antagonists directed against adenosine receptors A2AR and A2BR, we further demonstrated that ENT1/2 blockade protected against P. aeruginosa -induced acute lung injury via activation of A2AR and A2BR. Additionally, ENT1/2 chemical inhibition and ENT1 knockout prevented P. aeruginosa-induced lung NLRP3 inflammasome activation. Finally, inhibition of inflammasome prevented P. aeruginosa-induced acute lung injury. Our results suggest that targeting ENT1/2 and NLRP3 inflammasome may be novel strategies for prevention and treatment of P. aeruginosa-induced pneumonia and subsequent ARDS.

Role of equilibrative nucleoside transporter 1 (ENT1) in the disposition of cytarabine in mice.

Cytarabine (Ara-C) is a nucleoside analog used in the treatment of acute myeloid leukemia (AML). Despite the many years of clinical use, the identity of the transporter(s) involved in the disposition of Ara-C remains poorly studied. Previous work demonstrated that concurrent administration of Ara-C with nitrobenzylmercaptopurine ribonucleoside (NBMPR) causes an increase in Ara-C plasma levels, suggesting involvement of one or more nucleoside transporters. Here, we confirmed the presence of an NMBPR-mediated interaction with Ara-C resulting in a 2.5-fold increased exposure. The interaction was unrelated to altered blood cell distribution, and subsequent studies indicated that the disposition of Ara-C was unaffected in mice with a deficiency of postulated candidate transporters, including ENT1, OCTN1, OATP1B2, and MATE1. These studies indicate the involvement of an unknown NBMPR-sensitive Ara-C transporter that impacts the pharmacokinetic properties of this clinically important agent.

Structures of human ENT1 in complex with adenosine reuptake inhibitors.

The human equilibrative nucleoside transporter 1 (hENT1), a member of the SLC29 family, plays crucial roles in adenosine signaling, cellular uptake of nucleoside for DNA and RNA synthesis, and nucleoside-derived anticancer and antiviral drug transport in humans. Because of its central role in adenosine signaling, it is the target of adenosine reuptake inhibitors (AdoRI), several of which are used clinically. Despite its importance in human physiology and pharmacology, the molecular basis of hENT1-mediated adenosine transport and its inhibition by AdoRIs are limited, owing to the absence of structural information on hENT1. Here, we present crystal structures of hENT1 in complex with two chemically distinct AdoRIs: dilazep and S-(4-nitrobenzyl)-6-thioinosine (NBMPR). Combined with mutagenesis study, our structural analyses elucidate two distinct inhibitory mechanisms exhibited on hENT1 and provide insight into adenosine recognition and transport. Our studies provide a platform for improved pharmacological intervention of adenosine and nucleoside analog drug transport by hENT1.

Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1).

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.

FSCPX, a Chemical Widely Used as an Irreversible A1 Adenosine Receptor Antagonist, Modifies the Effect of NBTI, a Nucleoside Transport Inhibitor, by Reducing the Interstitial Adenosine Level in the Guinea Pig Atrium.

Based on in silico results, recently we have assumed that FSCPX, an irreversible A1 adenosine receptor antagonist, inhibits the action of NBTI that is apparent on E/c curves of adenosine receptor agonists. As a mechanism for this unexpected effect, we hypothesized that FSCPX might modify the equilibrative and NBTI-sensitive nucleoside transporter (ENT1) in a way that allows ENT1 to transport adenosine but impedes NBTI to inhibit this transport. This assumption implies that our method developed to estimate receptor reserve for agonists with short half-life such as adenosine, in its original form, overestimates the receptor reserve. In this study, therefore, our goals were to experimentally test our assumption on this effect of FSCPX, to improve our receptor reserve-estimating method and then to compare the original and improved forms of this method. Thus, we improved our method and assessed the receptor reserve for the direct negative inotropic effect of adenosine with both forms of this method in guinea pig atria. We have found that FSCPX inhibits the effects of NBTI that are mediated by increasing the interstitial concentration of adenosine of endogenous (but not exogenous) origin. As a mechanism for this action of FSCPX, inhibition of enzymes participating in the interstitial adenosine production can be hypothesized, while modification of ENT1 can be excluded. Furthermore, we have shown that, in comparison with the improved form, the original version of our method overestimates receptor reserve but only to a small extent. Nevertheless, use of the improved form is recommended in the future.

Flow Cytometric Analysis of Nucleoside Transporters Activity in Chemoresistant Prostate Cancer Model.

BACKGROUND: Nucleoside analogues represent a relevant class of antimetabolites used for therapy of various types of cancer. However, their effectivity is limited by drug resistance. The nucleoside transport capability of tumour cells is considered to be a determinant of the clinical outcome of treatment regimens using antimetabolites. Due to hydrophilic properties of antimetabolites, their transport across the plasma membrane is mediated by two families of transmembrane proteins, the SLC28 family of cation-linked concentrative nucleoside transporters (hCNTs) and SLC29 family of energy-independent equilibrative nucleoside transporters (hENTs). Loss of functional nucleoside transporters has been associated with reduced efficacy of antimetabolites and their derivatives and treatment failure in diverse malignancies including solid tumours, such as pancreatic adenocarcinoma. MATERIAL AND METHODS: The effectivity and kinetics of antimetabolite uptake were analysed using control and docetaxel-resistant PC3 cells. For this purpose, fluorescent nucleoside analogue probe uridine-furane and inhibitor of nucleoside transporters, S-(4-nitrobenzyl) -6-thioinosine were exploited. Combination of flow cytometry, confocal microscopy and real-time quantitative polymerase chain reaction methodology were used for the analysis. RESULTS: Here we utilized flow cytometric assay for analysis of nucleoside transporters activity employing fluorescent nucleoside analogue, uridine-furane. We have determined the long-time kinetics of uridine-furane incorporation and quantified its levels in the parental prostate cancer cell line PC3 and its chemoresistant derivative. Finally, we have shown an association between the activity and mRNA expression of nucleoside transporters and sensitivity to various nucleoside analogues. CONCLUSION: Fluorescent techniques can serve as an effective tool for the detection of nucleoside transporter activity which has the potential for application in clinical oncology.Key words: nucleoside transporter proteins - drug resistance - prostatic neoplasm - chemotherapy.

Contribution of equilibrative nucleoside transporter(s) to intestinal basolateral and apical transports of anticancer trifluridine.

Trifluridine (FTD) exhibits anticancer activities after its oral administration despite its hydrophilic nature. It was previously reported that concentrative nucleoside transporter (CNT) 1 mediates the apical uptake of FTD in human small intestinal epithelial cells (HIECs). In the present study, FTD was also identified as a substrate for equilibrative nucleoside transporter (ENT) 1 and ENT2 in transporter gene-transfected cells. An immunocytochemical analysis revealed that ENT1 was expressed at the basolateral and apical membranes of HIECs. Cellular accumulation increased in the presence of S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an ENT selective inhibitor. Cytotoxicity in HIEC monolayers at low FTD concentrations was increased by NBMPR, and this may have been due to inhibition of the ENT-mediated basolateral transport of FTD by NBMPR. These results suggest that ENTs reduce the intestinal cytotoxicity of FTD by facilitating its basolateral efflux. On the other hand, the intracellular accumulation and cytotoxicity of FTD in HIECs were decreased at higher concentrations of FTD by NBMPR, and this may have been due to the NBMPR inhibition of the apical uptake of FTD, which has been suggested to be mediated by CNTs and ENTs. In conclusion, ENTs were responsible for intestinal transepithelial permeation by mediating the basolateral efflux of FTD after its uptake by CNT1 from the apical side, resulting in decreases in its intracellular accumulation and intestinal toxicity in humans. Equilibrative nucleoside transporters may also partially contribute to the low-affinity uptake of FTD across the apical membrane along with high-affinity CNT1.

Enhancement of inosine-mediated A2AR signaling through positive allosteric modulation.

Inosine is an endogenous nucleoside that is produced by metabolic deamination of adenosine. Inosine is metabolically more stable (half-life 15h) than adenosine (half-life <10s). Inosine exerts anti-inflammatory and immunomodulatory effects similar to those observed with adenosine. These effects are mediated in part through the adenosine A2A receptor (A2AR). Relative to adenosine inosine exhibits a lower affinity towards the A2AR. Therefore, it is generally believed that inosine is incapable of activating the A2AR through direct engagement, but indirectly activates the A2AR upon metabolic conversion to higher affinity adenosine. A handful of studies, however, have provided evidence for direct inosine engagement at the A2AR leading to activation of downstream signaling events and inhibition of cytokine production. Here, we demonstrate that under conditions devoid of adenosine, inosine as well as an analog of inosine 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine selectively and dose-dependently activated A2AR-mediated cAMP production and ERK1/2 phosphorylation in CHO cells stably expressing the human A2AR. Inosine also inhibited LPS-stimulated TNF-α, CCL3 and CCL4 production by splenic monocytes in an A2AR-dependent manner. In addition, we demonstrate that a positive allosteric modulator (PAM) of the A2AR enhanced inosine-mediated cAMP production, ERK1/2 phosphorylation and inhibition of pro-inflammatory cytokine and chemokine production. The cumulative effects of allosteric enhancement of adenosine-mediated and inosine-mediated A2AR activation may be the basis for the sustained anti-inflammatory and immunomodulatory effects observed in vivo and thereby provide insights into potential therapeutic interventions for inflammation- and immune-mediated diseases.

Vector-independent transmembrane transport of oligodeoxyribonucleotides involves p38 mitogen activated protein kinase phosphorylation.

The main roles of equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs) are to transfer single nucleosides and analogues for the nucleic acid salvage pathway. Oligodeoxyribonucleotides (ODNs) can be transported into the cytoplasm or nucleus of cells under certain conditions. Among ODNs composed of a single type of nucleotide, the transport efficiency differs with the length and nucleotide composition of the ODNs and varies in different types of leukaemia cells; among the 5 tested random sequence ODNs and 3 aptamers with varying sequences, the data showed that some sequences were associated with significantly higher transport efficiency than others. The transport of ODNs was sodium, energy, and pH-independent, membrane protein-dependent, substrate nonspecific for ODNs and 4-nitrobenzylthioinosine (NBMPR)-insensitive, but it showed a low sensitivity to dipyridamole (IC50 = 35.44 µmol/L), distinguishing it from ENT1-4 and CNTs. The delivery efficiency of ODNs was superior to that of Lipofection and Nucleofection, demonstrating its potential applications in research or therapeutics. Moreover, this process was associated with p38 mitogen activated protein kinase (p38MAPK) instead of c-Jun N-terminal kinase (JNK) signalling pathways. We have denoted ODN transmembrane transport as equilibrative nucleic acid transport (ENAT). Overall, these findings indicate a new approach and mechanism for transmembrane transport of ODNs.

Methodical Challenges and a Possible Resolution in the Assessment of Receptor Reserve for Adenosine, an Agonist with Short Half-Life.

The term receptor reserve, first introduced and used in the traditional receptor theory, is an integrative measure of response-inducing ability of the interaction between an agonist and a receptor system (consisting of a receptor and its downstream signaling). The underlying phenomenon, i.e., stimulation of a submaximal fraction of receptors can apparently elicit the maximal effect (in certain cases), provides an opportunity to assess the receptor reserve. However, determining receptor reserve is challenging for agonists with short half-lives, such as adenosine. Although adenosine metabolism can be inhibited several ways (in order to prevent the rapid elimination of adenosine administered to construct concentration-effect (E/c) curves for the determination), the consequent accumulation of endogenous adenosine biases the results. To address this problem, we previously proposed a method, by means of which this bias can be mathematically corrected (utilizing a traditional receptor theory-independent approach). In the present investigation, we have offered in silico validation of this method by simulating E/c curves with the use of the operational model of agonism and then by evaluating them using our method. We have found that our method is suitable to reliably assess the receptor reserve for adenosine in our recently published experimental setting, suggesting that it may be capable for a qualitative determination of receptor reserve for rapidly eliminating agonists in general. In addition, we have disclosed a possible interference between FSCPX (8-cyclopentyl-N³-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-N¹-propylxanthine), an irreversible A1 adenosine receptor antagonist, and NBTI (S-(2-hydroxy-5-nitrobenzyl)-6-thioinosine), a nucleoside transport inhibitor, i.e., FSCPX may blunt the effect of NBTI.

Nitrobenzylthioinosine mimics adenosine to attenuate the epileptiform discharge of hippocampal neurons from epileptic rats.

Nitrobenzylthioinosine (NBTI), a specific inhibitor of type 1 equilibrative nucleoside transporter, could regulate the extracellular adenosine concentration and have protective roles in seizures. However, the protection mechanism of NBTI in seizures remains poorly understood. Here, the expression pattern and subcellular distribution of adenosine A1 receptor were detected by Western blot analysis and double-labeling immunofluorescence staining in Lithium Chloride-Pilocarpine induced epileptic rat model. At 24 h after pilocarpine induced rat seizures, hippocampal slices were prepared and the evoked excitatory postsynaptic currents (eEPSCs) amplitude of pyramidal neurons in hippocampus CA1 region was recorded using whole-cell patch clamp. In vivo, compared to control group, Western blotting analysis showed that the expression of adenosine A1 receptor protein was increased at 24 h and 72 h after seizure, didn't change at 0 min and 1 w, and decreased at 2 w. Double-label immunofluorescence revealed that adenosine A1 receptor was mainly expressed in the membrane and cytoplasm of neurons. In Vitro, adenosine decreased the eEPSCs amplitude of pyramidal neurons in hippocampus CA1 region, NBTI also had the same effect. Meantime, NBTI could further inhibit eEPSCs amplitude on the basis of lower concentration adenosine (50µM), and adenosine A1 receptor inhibitor DPCPX partially reversed this effect. Taken together, we confirmed that the expression of adenosine A1 receptor protein was increased in the early seizures and decreased in the late seizures. At the same time, NBTI mimics adenosine to attenuate the epileptiform discharge through adenosine A1 receptor, which might provide a novel therapeutic approach toward the control of epilepsy.

Intracellular acidification increases adenosine transport in human umbilical vein endothelial cells.

INTRODUCTION: Adenosine is taken up via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) at a physiological extracellular pH (pHo ∼7.4) in human umbilical vein endothelial cells (HUVECs). Acidic pHo increases the uptake of adenosine and 5-hydroxytryptamine (5HT) via hENT4 in this cell type. However, modulation of hENT1 and hENT2 transport activity by the pHi is unknown. We investigated whether hENT1 and hENT2-adenosine transport was regulated by acidic pHi. METHODS: HUVECs loaded with a pH sensitive probe were subjected to 0.1-20 mmol/L NH4Cl pulse assay to generate 6.9-6.2 pHi. Before pHi started to recover, adenosine transport kinetics (0-500 µmol/L, 37 °C) in the absence or presence 1 or 10 µmol/L S-(4-nitrobenzyl)-6-thio-inosine (NBTI), 2 mmol/L hypoxanthine, 2 mmol/L adenine, 100 µmol/L 5HT, or 500 µmol/L adenosine, was measured. RESULTS: Overall adenosine transport (i.e., hENT1+hENT2) was semisaturable and partially inhibited by 1 µmol/L, but abolished by 10 µmol/L NBTI in cells non-treated or treated with NH4Cl. The initial velocity and non-saturable, lineal component for overall transport were increased after NH4Cl pulse. hENT1 and hENT2-mediated adenosine transport maximal capacity was increased by acidic pHi. hENT1 activity was more sensitive than hENT2 activity to acidic pHi. DISCUSSION: hENT1 and hENT2-adenosine transport is differentially regulated by acidic pHi in HUVECs. These findings are important in pathologies associated with pHi alterations such as gestational diabetes mellitus.

Paracrine effect of carbon monoxide - astrocytes promote neuroprotection through purinergic signaling in mice.

The neuroprotective role of carbon monoxide (CO) has been studied in a cell-autonomous mode. Herein, a new concept is disclosed - CO affects astrocyte-neuron communication in a paracrine manner to promote neuroprotection. Neuronal survival was assessed when co-cultured with astrocytes that had been pre-treated or not with CO. The CO-pre-treated astrocytes reduced neuronal cell death, and the cellular mechanisms were investigated, focusing on purinergic signaling. CO modulates astrocytic metabolism and extracellular ATP content in the co-culture medium. Moreover, several antagonists of P1 adenosine and P2 ATP receptors partially reverted CO-induced neuroprotection through astrocytes. Likewise, knocking down expression of the neuronal P1 adenosine receptor A2A-R (encoded by Adora2a) reverted the neuroprotective effects of CO-exposed astrocytes. The neuroprotection of CO-treated astrocytes also decreased following prevention of ATP or adenosine release from astrocytic cells and inhibition of extracellular ATP metabolism into adenosine. Finally, the neuronal downstream event involves TrkB (also known as NTRK2) receptors and BDNF. Pharmacological and genetic inhibition of TrkB receptors reverts neuroprotection triggered by CO-treated astrocytes. Furthermore, the neuronal ratio of BDNF to pro-BDNF increased in the presence of CO-treated astrocytes and decreased whenever A2A-R expression was silenced. In summary, CO prevents neuronal cell death in a paracrine manner by targeting astrocytic metabolism through purinergic signaling.

Cellular Uptake of Decitabine by Equilibrative Nucleoside Transporters in HCT116 Cells.

DNA hypermethylation, an epigenetic change that silences gene expression without altering nucleotide sequences, plays a critical role in the formation and progression of colorectal cancers as well as in the acquisition of drug resistance. Decitabine (DAC), a DNA methyltransferase 1 inhibitor of nucleoside analogues, has been shown to restore gene expression silenced by hypermethylation. In the present study, the mechanisms underlying both uridine and DAC uptake were examined in the human colon cancer cell line HCT116. Real-time polymerase chain reaction analysis revealed that ENT1 mRNA was the most abundant among the nucleoside transporters examined in HCT116 cells. The ENT1 protein was detected in the membrane fraction, as determined by Western blotting. The uptake of uridine or DAC was time- and concentration-dependent, but also Na(+)-independent. The uptake of these agents was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENTs), and was also decreased in cells treated with ENT1 small interfering RNA. The uptake of both uridine and DAC was inhibited by uridine, cytidine, adenosine, or inosine, while that of DAC was also inhibited by thymidine. The expression of MAGEA1 mRNA, the DNA of which was methylated in HCT116 cells, was increased by DAC treatment, and this increment was attenuated by concomitant treatment with NBMPR. The IC50 value of DAC was also increased in the presence of NBMPR. These results suggest that DAC is mainly taken up by ENT1 and that this uptake is one of the key determinants of the activity of DAC in HCT116 cells.

Saturable Hepatic Extraction of Gemcitabine Involves Biphasic Uptake Mediated by Nucleoside Transporters Equilibrative Nucleoside Transporter 1 and 2.

Hepatic arterial infusion (HAI) chemotherapy with gemcitabine (GEM) is expected to be more effective and safer method to treat hepatic metastasis of pancreatic cancer compared with intravenous administration, because it affords higher tumor exposure with lower systemic exposure. Thus, a key issue for dose selection is the saturability of hepatic uptake of GEM. Therefore, we investigated GEM uptake in rat and human isolated hepatocytes. Hepatic GEM uptake involved high- and low-affinity saturable components with Km values of micromolar and millimolar order, respectively. The uptake was inhibited concentration dependently by S-(4-nitrobenzyl)-6-thioinosine (NBMPR) and was sodium-ion-independent, suggesting a contribution of equilibrative nucleoside transporters (ENTs). The concentration dependence of uptake in the presence of 0.1 µM NBMPR showed a single low-affinity binding site. Therefore, the high- and low-affinity sites correspond to ENT1 and ENT2, respectively. Our results indicate hepatic extraction of GEM is predominantly mediated by the low-affinity site (hENT2), and at clinically relevant hepatic concentrations of GEM, hENT2-mediated uptake would not be completely saturated. This is critical for HAI, because saturation of hepatic uptake would result in a marked increase of GEM concentration in the peripheral circulation, abrogating the advantage of HAI over intravenous administration in terms of severe adverse events.