Inhibitors Of Equilibrative Nucleoside Transporters Research Articles

Suppression of Ehrlich ascites tumor cell proliferation via G1 arrest induced by dietary nucleic acid-derived nucleosides.

The nucleic acids found in food play a crucial role in maintaining various bodily functions. This study investigated the potential anticancer effects of dietary nucleic acids, an area that is still not fully understood. By utilizing an in vivo mouse model and an in vitro cell model, we discovered an anti-proliferative impact of RNA in both systems. DNA exhibited anti-proliferative effects in the mouse model, while this phenomenon wasn't observed in the in vitro cell model using Ehrlich ascites tumor (EAT) cells. Conversely, DNA hydrolysate demonstrated distinct anti-proliferative effects in EAT cells, suggesting that nucleotides or nucleosides generated during nucleic acid digestion act as active constituents. Furthermore, we examined various nucleosides and two sodium-independent equilibrative nucleoside transporter inhibitors (ENTs), identifying guanosine and 2'-deoxyguanosine as pivotal in the anti-proliferative effect. We also found that the anti-proliferation activity with both nucleosides was suppressed by the treatment of dipyridamole, a non-selective inhibitor for ENT1 and ENT2, but not nitrobenzylthioinosine, a low inhibitor for ENT2. The uptake of these compounds into cells is likely facilitated by ENT2. These nucleotides impeded the progression of cancer cells from the G1 phase to the S phase in the cell cycle. Another significant finding is the increased expression of CCAAT/enhancer-binding protein (C/EBPβ) induced by guanosine and 2'-deoxyguanosine. Furthermore, immunostaining revealed that C/EBPβ diffuses into the nucleus, indicating its presence. This suggests that guanosine or 2-deoxyguanosine induces G1 arrest in cancer cells via the activation of C/EBPβ. Encouraged by these promising results, guanosine and 2'-deoxyguanosine show potential applications in cancer prevention.

Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK.

Sunitinib is associated with cardiotoxicity through inhibition of AMP-protein kinase (AMPK) signaling. By contrast, the common antidiabetic agent metformin has demonstrated cardioprotection through indirect AMPK activation. In this study, we investigate the effects of metformin during sunitinib-induced cytotoxicity. Left ventricular developed pressure, coronary flow, heart rate, and infarct size were measured in Langendorff-perfused rat hearts treated with 1 µM sunitinib ±50 µM metformin ±1 µM human equilibrative nucleoside transporter inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI). Western blot analysis was performed for p-AMPKα levels. Primary isolated cardiac myocytes from the left ventricular tissue were used to measure live cell population levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess adjunctive treatment of and metformin in human hepatoma G2 and promyelocytic leukemia (HL-60) cells treated with 0.1-100 µM sunitinib ±50 µM metformin. In the perfused hearts, coadministration of metformin attenuated the sunitinib-induced changes to left ventricular developed pressure, infarct size, and cardiac myocyte population. Western blot analysis revealed a significant decrease in p-AMPKα during sunitinib treatment, which was attenuated after coadministration with metformin. All metformin-induced effects were attenuated, and NBTI was coadministered. The MTT assay demonstrated an increase in the EC50 value during coadministration of metformin with sunitinib compared with sunitinib monotherapy in hepatoma G2 and HL-60 cell lines, demonstrating the impact and complexity of metformin coadministration and the possible role of AMPK signaling. This study highlights the novel cardioprotective properties of metformin and AMPK activation during sunitinib-induced cardiotoxicity when administered together in the Langendorff heart model.

Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters.

Equilibrative nucleoside transporters (ENTs) play a vital role in nucleotide synthesis, regulation of adenosine function and chemotherapy. Current inhibitors of ENTs are mostly ENT1-selective. Our previous study has demonstrated that 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) is a novel inhibitor of ENTs, which is more selective to ENT2 than to ENT1. The present study aimed to screen a series of FPMINT analogues and study their structure-activity relationship. Nucleoside transporter-deficient cells transfected with cloned human ENT1 and ENT2 were used as in vitro models. The results of the [3H]uridine uptake study showed that the replacement of the naphthalene moiety with the benzene moiety could abolish the inhibitory effects on ENT1 and ENT2. The addition of chloride to the meta position of this benzene moiety could restore only the inhibitory effect on ENT1 but had no effect on ENT2. However, the addition of the methyl group to the meta position or the ethyl or oxymethyl group to the para position of this benzene moiety could regain the inhibitory activity on both ENT1 and ENT2. The presence of a halogen substitute, regardless of the position, in the fluorophenyl moiety next to the piperazine ring was essential for the inhibitory effects on ENT1 and ENT2. Among the analogues tested, compound 3c was the most potent inhibitor. Compound 3c reduced V max of [3H]uridine uptake in ENT1 and ENT2 without affecting K m. The inhibitory effect of compound 3c could not be washed out. Compound 3c did not affect cell viability, protein expression and internalization of ENT1 and ENT2. Therefore, similar to FPMINT, compound 3c was an irreversible and non-competitive inhibitor. Molecular docking analysis also showed that the binding site of compound 3c in ENT1 may be different from that of other conventional inhibitors. It is expected that structural modification may further improve its potency and selectivity and lead to the development of useful pharmacological agents.

A Randomized, Placebo-Controlled Crossover Study with Dipyridamole for Restless Legs Syndrome.

ABSTRACTBackgroundNew pharmacological targets are needed for restless legs syndrome. Preclinical data suggest that a hypoadenosinergic state plays an important pathogenetic role.ObjectiveThe objective of this study was to determine whether inhibitors of equilibrative nucleoside transporters, for example, dipyridamole, could provide effective symptomatic treatment.MethodsA 2‐week double‐blind, placebo‐controlled crossover study assessed the efficacy of dipyridamole (possible up‐titration to 300 mg) in untreated patients with idiopathic restless legs syndrome. Multiple suggested immobilization tests and polysomnography were performed after each treatment phase. Severity was assessed weekly using the International Restless Legs Rating Scale, Clinical Global Impression, and the Medical Outcomes Study Sleep scale. The primary end point was therapeutic response.ResultsTwenty‐eight of 29 patients recruited were included. International Restless Legs Rating Scale scores improved from a mean ± standard deviation of 24.1 ± 3.1 at baseline to 11.1 ± 2.3 at the end of week 2, versus 23.7 ± 3.4 to 18.7 ± 3.2 under placebo (P < 0.001). Clinical Global Impression, Medical Outcomes Study Sleep, and Multiple Suggested Immobilization Test scores all improved (P < 0.001). The mean effective dose of dipyridamole was 217.8 ± 33.1 mg/d. Sleep variables improved. The mean periodic leg movement index at the end of treatment with dipyridamole was 8.2 ± 3.5 versus. 28.1 ± 6.7 under placebo. Side effects (dipyridamole vs placebo) included abdominal distension (18% vs. 7%), dizziness (10.7% vs 7.1%), diarrhea, and asthenia (each 7.1% vs 3.6%).ConclusionsDipyridamole has significant therapeutic effects on both sensory and motor symptoms of restless legs syndrome and on sleep. Our findings confirm the efficacy of dipyridamole in restless legs syndrome predicted from preclinical studies and support a key role of adenosine in restless legs syndrome. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

The Dihydroorotate Dehydrogenase Inhibitor Brequinar Is Synergistic with ENT1/2 Inhibitors.

The dihydroorotate dehydrogenase (DHODH) inhibitor brequinar failed all clinical trials for solid tumors. To investigate mechanisms to increase brequinar's efficacy, we employed a combination strategy to simultaneously inhibit the nucleotide salvage pathways. Brequinar is synergistic with the equilibrative nucleoside transporter (ENT) inhibitor dipyridamole, but not the concentrative nucleoside transporter inhibitor phlorizin. This synergy carries over to ENT1/2 inhibition, but not ENT4. Our previously described brequinar analogue 41 was also synergistic with dipyridamole as were the FDA-approved DHODH inhibitors leflunomide and teriflunomide but the latter required much higher concentrations than brequinar. Therefore, a combination of brequinar and ENT inhibitors presents a potential anti-cancer strategy in select tumors.

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.

S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) is routinely used at concentrations of 0.10μM and 0.10mM to specifically inhibit transport of nucleosides mediated by equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2), respectively. We recently showed that NBMPR (0.10mM) 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). 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. NBMPR inhibited Hoechst 33342 accumulation in MDCKII-ABCG2 cells (IC50 = 53μM) but not in MDCKII-ABCB1 and MDCKII-parental cells. NBMPR (0.10mM) 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. NBMPR at a concentration of 0.10mM 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.

Mutual role of ecto-5'-nucleotidase/CD73 and concentrative nucleoside transporter 3 in the intestinal uptake of dAMP.

2'-Deoxyadenosine 5'-monophosphate (dAMP), a deoxyribonucleotide found in DNA, affects intestinal cell growth. The molecular mechanisms underlying gastrointestinal absorption of foreign DNA ingested along with food has hardly been investigated. The aim of this study was to investigate the mechanism underlying intestinal absorption of dAMP. The uptake of [3H]dAMP by Caco-2 cells was Na+- and pH-dependent and was inhibited by various nucleosides. In contrast, nitrobenzylthioinosine (NMBPR), an equilibrative nucleoside transporter inhibitor, showed little inhibitory effects on [3H]dAMP uptake. Additionally, human concentrative nucleoside transporter (CNT) 3, transiently expressed in COS-7 cells, mediated the uptake of [3H]dAMP. A kinetic study revealed that the Km value of CNT3-mediated uptake of dAMP (59.6 μM) was close to that of 2'-deoxyadenosine (dAdo) (56.3 μM), whereas the dAMP Vmax (15.6 pmol·mg protein–1min–1) was 500-fold lesser than the dAdo Vmax (7782 pmol·mg protein–1min–1). Further, [3H]dAMP uptake was greater in COS-7 cells expressing ecto-5'-nucleotidase/CD73 with CNT3 than in those expressing CNT3 alone. These data suggest that, although dAMP is a substrate of CNT3, it is dephosphorylated to dAdo by CD73 and is efficiently absorbed as dAdo from the intestinal lumen.

Contribution of equilibrative nucleoside transporters 1 and 2 to gemcitabine uptake in pancreatic cancer cells.

Hepatic arterial infusion (HAI) chemotherapy is expected to be a more effective and safer method to treat the hepatic metastasis of pancreatic cancer than intravenous (iv) administration because of higher tumor exposure and lower systemic exposure. To clarify the uptake mechanism of nucleoside anticancer drugs, including gemcitabine (GEM), in pancreatic cancer, we investigated the uptakes of radiolabeled uridine (a general substrate of nucleoside transporters) and GEM in pancreatic cancer cell lines MIA-PaCa2 and As-PC1. Uridine uptake was inhibited by non-labeled GEM and also by S-(4-nitrobenzyl)-6-thioinosine (NBMPR; an inhibitor of equilibrative nucleoside transporters, ENTs) in a concentration-dependent manner, suggesting that ENTs contribute to uridine uptake in pancreatic cancer cells. As for GEM, saturable uptake was mediated by high- and low-affinity components with Km values of micromolar and millimolar orders, respectively. Uptake was inhibited in a concentration-dependent manner by NBMPR and was sodium ion-independent. Moreover, the concentration dependence of uptake in the presence of 0.1μM NBMPR showed a single low-affinity site. These results indicated that the high- and low-affinity sites correspond to hENT1 and hENT2, respectively. The results indicated that at clinically relevant hepatic concentrations of GEM in GEM-HAI therapy, the metastatic tumor exposure of GEM is predominantly determined by hENT2 under unsaturated conditions, suggesting that hENT2 expression in metastatic tumor would be a candidate biomarker for indicating anticancer therapy with GEM-HAI.

Gemcitabine: Selective cytotoxicity, induction of inflammation and effects on urothelial function

Intravesical gemcitabine has recently been introduced for the treatment of superficial bladder cancer and has a favourable efficacy and toxicity profile in comparison to mitomycin c (MMC), the most commonly used chemotherapeutic agent. The aim of this study was to assess the cytotoxic potency of gemcitabine in comparison to MMC in urothelial cell lines derived from non-malignant (UROtsa) and malignant (RT4 and T24) tissues to assess selectivity. Cells were treated with gemcitabine or mitomycin C at concentrations up to the clinical doses for 1 or 2h respectively (clinical duration). Treatment combined with hyperthermia was also examined. Cell viability, ROS formation, urothelial function (ATP, acetylcholine and PGE2 release) and secretion of inflammatory cytokines were assessed. Gemcitabine displayed a high cytotoxic selectivity for the two malignant cell lines (RT4, T24) compared to the non-malignant urothelial cells (UROtsa, proliferative and non-proliferative). In contrast, the cytotoxic effects of MMC were non-selective with equivalent potency in each of the cell lines. The cytotoxic effect of gemcitabine in the malignant cell lines was associated with an elevation in free radical formation and was significantly decreased in the presence of an equilibrative nucleoside transporter inhibitor. Transient changes in urothelial ATP and PGE2 release were observed, with significant increase in release of interleukin-6, interleukin-8 and interleukin-1β from urothelial cells treated with gemcitabine. The selectivity of gemcitabine for malignant urothelial cells may account for the less frequent adverse urological effects with comparison to other commonly used chemotherapeutic agents.

An adenosine kinase inhibitor, ABT-702, inhibits spinal nociceptive transmission by adenosine release via equilibrative nucleoside transporters in rat

Adenosine kinase (AK) inhibitor is a potential candidate for controlling pain, but some AK inhibitors have problems of adverse effects such as motor impairment. ABT-702, a non-nucleoside AK inhibitor, shows analgesic effect in animal models of pain. Here, we investigated the effects of ABT-702 on synaptic transmission via nociceptive and motor reflex pathways in the isolated spinal cord of neonatal rats. The release of adenosine from the spinal cord was measured by HPLC. ABT-702 inhibited slow ventral root potentials (sVRPs) in the nociceptive pathway more potently than monosynaptic reflex potentials (MSRs) in the motor reflex pathway. The inhibitory effects of ABT-702 were mimicked by exogenously applied adenosine, blocked by 8CPT (8-cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist, and augmented by EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), an adenosine deaminase (ADA) inhibitor. Equilibrative nucleoside transporter (ENT) inhibitors reversed the effects of ABT-702, but not those of adenosine. ABT-702 released adenosine from the spinal cord, an effect that was also reversed by ENT inhibitors. The ABT-702-facilitated release of adenosine by way of ENTs inhibits nociceptive pathways more potently than motor reflex pathways in the spinal cord via activation of A1 receptors. This feature is expected to lead to good analgesic effects, but, caution may be required for the use of AK inhibitors in the case of ADA dysfunction or a combination with ENT inhibitors.

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.

In vitro analysis of transport and metabolism of 4′-thiothymidine in human tumor cells

IntroductionThe use of thymidine (TdR) and thymidine analogs such as 3′-fluoro-3′-deoxythymidine (FLT) as positron emission tomography (PET)-based proliferation markers can provide information on tumor response to treatment. Studies on another TdR analog, 4′-thiothymidine (4DST), suggest that it might be a better PET-based proliferation tracer than either TdR or FLT. 4DST is resistant to the catabolism that complicates analysis of TdR in PET studies, but unlike FLT, 4DST is incorporated into DNA. MethodsTo further evaluate 4DST, the kinetics of 4DST transport and metabolism were determined and compared to FLT and TdR. Transport and metabolism of FLT, TdR and 4DST were examined in the human adenocarcinoma cell line A549 under exponential-growth conditions. Single cell suspensions were incubated in buffer supplemented with radiolabeled tracer in the presence or absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENT). Kinetics of tracer uptake was determined in whole cells and tracer metabolism measured by high performance liquid chromatography of cell lysates. ResultsTdR and 4DST were qualitatively similar in terms of ENT-dependent transport, shapes of uptake curves, and relative levels of DNA incorporation. FLT did not incorporate into DNA, showed a significant temperature effect for uptake, and its transport had a significant NBMPR-resistant component. Overall 4DST metabolism was significantly slower than either TdR or FLT. Conclusions4DST provides a good alternative for TdR in PET and has advantages over FLT in proliferation measurement. However, slow 4DST metabolism and the short half-life of the 11C label might limit widespread use in PET.

Intracellular Adenosine Inhibits IgE-Dependent Degranulation of Human Skin Mast Cells

Adenosine (ADO) can enhance and inhibit mast cell degranulation. Potentiation of degranulation occurs at relatively low concentrations of ADO (10−6–10−5 M) through triggering of A3AR, whereas, inhibition occurs at higher concentrations of ADO reportedly through triggering of A2aAR. However, the discrepancy in the concentration of ADO that inhibits degranulation and that required to trigger ADORs suggests a different mechanism. The purpose of this study is to determine the mechanism by which ADO inhibits human mast cell degranulation. We compare the effectiveness of A2aAR specific antagonist ZM241385 and equilibrative nucleoside transporter inhibitors Dipyridamole and NBMPR in preventing ADO-mediated inhibition of FcεRI-induced degranulation of human skin mast cells (hSMCs). Western blotting is done to analyze the effect of ADO on FcεRI-induced Syk phosphorylation. Dipyridamole and NBMPR completely and dose-dependently prevented ADO from inhibiting FcεRI-induced degranulation in all hSMC preparations. In contrast, ZM241385 at 10−5 M was effective in only 3 of 10 hSMC preparations. Moreover, NBMPR was effective even in those hSMC preparations not responsive to ZM241385. ADO inhibited degranulation induced by FcεRI crosslinking, but not that induced by complement component 5a (C5a), Substance P or calcium ionophore. Accordingly, ADO significantly attenuated FcεRI-induced phosphorylation of Syk at the critical activating tyrosine (Y525). Blocking the influx of ADO, but not A2aAR signals, is necessary and sufficient to prevent ADO from inhibiting FcεRI-induced mast cell degranulation. Thus, ADO specifically inhibits FcεRI-induced degranulation of hSMCs primarily by an intracellular mechanism that requires its influx via equilibrative nucleoside transporter 1 (ENT1).

Equilibrative nucleoside transporter (ENT)-1-dependent elevation of extracellular adenosine protects the liver during ischemia and reperfusion

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. 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.

Dipyridamole analogs as pharmacological inhibitors of equilibrative nucleoside transporters. Identification of novel potent and selective inhibitors of the adenosine transporter function of human equilibrative nucleoside transporter 4 (hENT4)

To identify needed human equilibrative nucleoside transporter 4 (hENT4) inhibitors, we cloned and stably expressed the recombinant protein in PK15NTD (nucleoside transporter deficient) cells, and, investigated its interaction with a series of dipyridamole analogs synthesized in our laboratory. Compounds were tested in this newly established hENT4 expressing system as well in previous stably expressed hENT1 and hENT2 expressing systems. Of the dipyridamole analogs evaluated, about one fourth of the compounds inhibited hENT4 with higher potencies than dipyridamole. The most potent of them, Compound 30 displayed an IC50 of 74.4nM, making it about 38 times more potent than dipyridamole (IC50=2.8μM), and selectivities of about 80-fold and 20-fold relative to ENT1 and ENT2, respectively. Structure–activity relationship showed nitrogen-containing monocyclic rings and noncyclic substituents at the 4- and 8-positions of the pyrimido[5,4-d]pyrimidine were important for the inhibitory activity against hENT4. The most potent and selective hENT4 inhibitors tended to have a 2,6-di(N-monohydroxyethyl) substitution on the pyrimidopyrimidine ring system. The inhibitors of hENT4 identified in this study are the most selective and potent inhibitors of hENT4 adenosine transporter function to date, and should serve as useful pharmacological/biochemical tools and/or potential leads for ENT4-based therapeutics. Also, the new hENT4-expressing PK15 cell line established will serve as a useful screening tool for the discovery and design of hENT4 ligands.

Residue Ile89 in human plasma membrane monoamine transporter influences its organic cation transport activity and sensitivity to inhibition by dilazep

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter belonging to the equilibrative nucleoside transporter (ENT) family. Despite its distinct substrate specificity from the classic nucleoside transporters ENT1 and 2, PMAT appears to share similar protein architecture with ENT1/2 and retains low affinity binding to classic ENT inhibitors such as nitrobenzylmercaptopurine riboside (NBMPR) and the coronary vasodilators dilazep and dipyridamole. Here we investigated the role of residue Ile89, a position known to be important for ENT interaction with dilazep, dipyridamole, and nucleoside substrates, in PMAT transport function and its interaction with classic ENT inhibitors using Madin–Darby canine kidney (MDCK) cells stably expressing human PMAT. Substitution of Ile89 in PMAT with Met, the counterpart residue in ENT1, resulted in normal plasma membrane localization and protein expression. Transport kinetic analysis revealed that I89M mutant had a 2.7-fold reduction in maximal transport velocity (Vmax) with no significant change in apparent binding affinity (Km) towards the prototype PMAT substrate 1-methyl-4-phenylpyridinium (MPP+), suggesting that I89 is an important determinant for the catalytic activity of PMAT. Dose-dependent inhibition studies further showed that the I89M mutation significantly increased PMAT's sensitivity to dilazep by 2.5-fold without affecting its sensitivity to dipyridamole and NBMPR. Located at the extracellular end of transmembrane domain 1 of PMAT, I89 may occupy an important position close to the substrate permeation pathway and may be involved in direct interaction with the vasodilator dilazep.

Physiological and Pharmacological Roles of Vascular Nucleoside Transporters

Adenosine modulates various vascular functions such as vasodilatation and anti-inflammation. The local concentration of adenosine in the vicinity of adenosine receptors is fine tuned by 2 classes of nucleoside transporters: equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs). In vascular smooth muscle cells, 95% of adenosine transport is mediated by ENT-1 and the rest by ENT-2. In endothelial cells, 60%, 10%, and 30% of adenosine transport are mediated by ENT-1, ENT-2, and CNT-2, respectively. In vitro studies show that glucose per se increases the expression level of ENT-1 via mitogen-activating protein kinase-dependent pathways. Similar results have been demonstrated in diabetic animal models. Hypertension is associated with the increased expression of CNT-2. It has been speculated that the increase in the activities of ENT-1 and CNT-2 may reduce the availability of adenosine to adenosine receptors, thereby weakening the vascular functions of adenosine. This may explain why patients with diabetes and hypertension suffer greater morbidity from ischemia and atherosclerosis. No oral hypoglycemic agents can inhibit ENTs, but an exception is troglitazone (a thiazolidinedione that has been withdrawn from the market). ENTs are also sensitive to dihydropyridine-type calcium-channel blockers, particularly nimodipine, which can inhibit ENT-1 in the nanomolar range. Those calcium-channel blockers are noncompetitive inhibitors of ENTs, probably working through the reversible interactions with allosteric sites. The nonsteroidal anti-inflammatory drug sulindac sulfide is a competitive inhibitor of ENT-1. In addition to their original pharmacological actions, it is believed that the drugs mentioned above may regulate vascular functions through potentiation of the effects of adenosine.

Differential adenosine uptake in mixed neuronal/glial or purified glial cultures of avian retinal cells: Modulation by adenosine metabolism and the ERK cascade

Adenosine is an important modulator of neuronal survival and differentiation in the CNS. Our previous work showed that nucleoside transporters (NTs) are present in cultures of chick retinal cells, but little is known about the mechanisms regulating adenosine transport in these cultures. Our aim in the present work was to study the participation of the adenosine metabolism as well as the ERK pathway on adenosine uptake in different types of retinal cultures (mixed and purified glial cultures). Kinetic analysis in both cultures revealed that the uptake reached equilibrium after 30min and presented two components. Incubation of cultures with S-(p-nitrobenzyl)-6-thioinosine (NBTI) or dipyridamole, different inhibitors of equilibrative nucleoside transporters (ENTs), produced a significant and concentration-dependent uptake reduction in both cultures. However, while dipyridamole presented similar maximal inhibitory effects in both cultures (although in different concentrations), the inhibition by NBTI was smaller in glial cultures than in mixed cultures, suggesting the presence of different transporters. Moreover, pre-incubation of [3H]-adenosine with adenosine deaminase (ADA) or adenosine kinase (ADK) inhibition with iodotubercidin promoted significant uptake inhibition in both cultures, indicating that the uptake is predominantly for adenosine and not inosine, and that taken up adenosine is preferentially directed to the synthesis of adenine nucleotides. In both cultures, the MEK inhibitors PD98059 or UO126, but not the inactive analog U0124, induced a significant and concentration-dependent uptake decrease. We have not observed any change in adenosine metabolism induced by MEK inhibitors, suggesting that this pathway is mediating a direct effect on NTs. Our results show the expression of different NTs in retinal cells in culture and that the activity of these transporters can be regulated by the ERK pathway or metabolic enzymes such as ADK which are then potential targets for regulation of Ado levels in normal or pathological conditions.

Mechanism of action of ribavirin in a novel hepatitis C virus replication cell system

Ribavirin (RBV) is a potential partner of interferon (IFN)-based therapy for patients with chronic hepatitis C. However, to date, its anti-hepatitis C virus (HCV) mechanism remains ambiguous due to the marginal activity of RBV on HCV RNA replication in HuH-7-derived cells, which are currently used as the only cell culture system for robust HCV replication. We investigated the anti-HCV activity of RBV using novel cell assay systems. The recently discovered human hepatoma cell line, Li23, which enables robust HCV replication, and the recently developed Li23-derived drug assay systems (ORL8 and ORL11), in which the genome-length HCV RNA (O strain of genotype 1b) encoding renilla luciferase efficiently replicates, were used for this study. At clinically achievable concentrations, RBV unexpectedly inhibited HCV RNA replication in ORL8 and ORL11 systems, but not in OR6 (an HuH-7-derived assay system). The anti-HCV activity of RBV was almost cancelled by an inhibitor of equilibrative nucleoside transporters. The evaluation of the anti-HCV mechanisms of RBV proposed to date using ORL8 ruled out the possibility that RBV induces error catastrophe, the IFN-signaling pathway or oxidative stress. However, we found that the anti-HCV activity of RBV was efficiently cancelled with guanosine, and demonstrated that HCV RNA replication was notably suppressed in inosine monophosphate dehydrogenase (IMPDH)-knockdown cells, suggesting that the antiviral activity of RBV is mediated through the inhibition of IMPDH. In conclusion, we demonstrated for the first time that inhibition of IMPDH is a major antiviral target by which RBV at clinically achievable concentrations inhibits HCV RNA replication.

Cannabinoids inhibit the synaptic uptake of adenosine and dopamine in the rat and mouse striatum

Adenosine, dopamine and endocannabinoids strictly modulate the release of one another in the dorsolateral striatum thereby controlling synaptic plasticity. As a second level of interaction, they regulate the action of one another via receptor heteromer formation. Here we investigated a putative third level of interaction, i.e. the possible control by cannabinoids of synaptic dopamine and adenosine reuptake. We found that a large number of endo- and exogenous cannabinoid ligands inhibit the uptake of [ 3H]adenosine and [ 3H]dopamine in rat sriatal nerve terminals. Maximal effects were often comparable to those of the dopamine transporter inhibitor, GBR12783 and the equilibrative nucleoside transporter inhibitor, dipyridamole. Cannabinoid ligands were generally more potent to inhibit the uptake of adenosine than that of dopamine. The inhibitory effect was: (1) unrelated to the pharmacological profile(s) of the ligands at the cannabinoid CB 1, CB 2, GPR55 and at the vanilloid TRPV 1 receptors; (2) not prevented by the cannabinoid CB 1 receptor antagonist/inverse agonist, LY320135; and (3) maintained in the cannabinoid CB 1 receptor knockout mice. In the same experiments, only O-2050, cannabidiol, and WIN55212-3 inhibited the simultaneously measured DL-TBOA-sensitive uptake of [ 14C]glutamate. In summary, many cannabinoid ligands are able to inhibit the synaptic uptake of adenosine and dopamine. These effects are not mediated by cannabinoid CB 1 receptors, and should be an additional mechanism to consider when interpreting synaptic effects of cannabinoids.