Molecular Characterization of Equilibrative Nucleoside Transporters in the Rat Carotid Body and Their Regulation by Chronic Hypoxia.

The mammalian carotid body (CB) is the main peripheral arterial chemoreceptor organ that is excited by decreases in blood PO2 (hypoxia) and increases in blood PCO2/H+. An increase in CB afferent carotid sinus nerve (CSN) discharge results in respiratory and cardiovascular reflex responses that help maintain homeostasis. The CB consists mainly of innervated clusters of the chemoreceptive type I (glomus) cells that are associated with the processes of glial-like type II cells. Extracellular ATP and adenosine (ADO) levels increase in response to acute hypoxia and there is evidence that during chronic sustained hypoxia ADO elevation plays a major role in regulating CB chemosensitivity and CSN discharge. We recently characterized the molecular identities of ectonucleotidase enzymes involved in regulating extracellular ATP hydrolysis to produce ADO in the rat CB. In the present study, we focus on a molecular characterization of the equilibrative nucleoside transporter (ENT) system that is known to regulate extracellular ADO concentrations in the rat CB based on pharmacological studies. Examination of ENT expression using quantitative PCR (qPCR) analysis revealed the expression of both ENT1 and ENT2 mRNAs in whole CB extracts from ~2-week-old juvenile rats. In dissociated rat CB cultures, both ENT1 and ENT2 immunoreactivity was localized to type I cell clusters. Furthermore, we show that ENT1 and ENT2 mRNA expression is downregulated in CBs isolated from rat pups exposed to chronic hypobaric hypoxia (~1 week). These findings reveal the molecular identities of the ENT system expressed in the rat CB and are consistent with the proposed shift to ADO signaling during chronic hypoxia.

Demonstration of Nucleoside Transporter Activity in the Nose-to-Brain Distribution of [18F]Fluorothymidine Using PET Imaging.

To evaluate the role of nucleoside transporters in the nose-to-brain uptake of [18F]fluorothymidine (FLT), an equilibrative nucleoside transporter (ENT1,2) and concentrative nucleoside transporter (CNT1-3) substrate, using PET to measure local tissue concentrations. Anesthetized Sprague-Dawley rats were administered FLT by intranasal (IN) instillation or tail-vein injection (IV). NBMPR (nitrobenzylmercaptopurine riboside), an ENT1 inhibitor, was administered either IN or intraperitoneally (IP). Dynamic PET imaging was performed for up to 40 min. A CT was obtained for anatomical co-registration and attenuation correction. Time-activity curves (TACs) were generated for the olfactory bulb (OB) and remaining brain, and the area-under-the-curve (AUC) for each TAC was calculated to determine the total tissue exposure of FLT. FLT concentrations were higher in the OB than in the rest of the brain following IN administration. IP administration of NBMPR resulted in increased OB and brain FLT exposure following both IN and IV administration, suggesting that NBMPR decreases the clearance rate of FLT from the brain. When FLT and NBMPR were co-administered IN, there was a decrease in the OB AUC while an increase in the brain AUC was observed. The decrease in OB exposure was likely the result of inhibition of ENT1 uptake activity in the nose-to-brain transport pathway. FLT distribution patterns show that nucleoside transporters, including ENT1, play a key role in the distribution of transporter substrates between the nasal cavity and the brain via the OB.

Gemcitabine versus FOLFIRINOX in patients with advanced pancreatic adenocarcinoma hENT1-positive: everything was not too bad back when everything seemed worse

Purpose: hENT1 is a transmembrane protein which acts as a nucleoside transporter and is the main mediator of Gemcitabine (GEM) uptake into human cells. In this retrospective study we compared GEM versus FOLFIRINOX in patients with metastatic pancreatic cancer in which hENT1 evaluation was available. Methods: 149 patients affected by unresectable metastatic pancreatic cancer, treated in our institution from 2009 to 2013, have been screened for inclusion in this retrospective study. Seventy patients, treated with GEM or FOLFIRINOX in first-line therapy, fulfilled clinical inclusion criteria for survival analysis. Thirty-one patients were available and contained sufficient quality/quantity RNA for evaluation of hENT1 expression by RT-PCR. The primary endpoint was OS and the secondary endpoint was PFS. Results: The survival analysis, carried out on 70 patients regardless of hENT1 expression, showed a statistically longer OSandPFS in the group treated with FOLFIRINOX compared to GEM. Within the exploratory analysis, which included 31 patients, no differences were found in hENT1 positive patients treated with FOLFIRINOX compared to GEM in terms of OS (8.5 vs 7 months, HR: 0.89; 95 % CI 0.3-2.5; p = 0.8) and PFS (5.5 vs 5 months, HR: 0.8, 95 % CI 0.2-2.2; p = 0.61). GEM-treated hENT1 positive patients showed a statistically significant improvement both of OS (8 vs 2 months; p = 0.0012) and PFS (5 vs 1 months; p = 0.0004) in comparison to GEM-treated hENT1 negative patients. Conclusions: In our exploratory analysis GEM seems as effective as FOLFIRINOX in terms of survival with a better safety profile in hENT1 positive metastatic pancreatic cancer (AU)

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Nucleoside transporters in the purinome.

The purinome is a rich complex of proteins and cofactors that are involved in fundamental aspects of cellular homeostasis and cellular responses. The purinome is evolutionarily ancient and is made up of thousands of members. Our understanding of the mechanisms linking some parts of this complex network and the physiological relevance of the various connections is well advanced. However, our understanding of other parts of the purinome is less well developed. Our research focuses on the adenosine or nucleoside transporters (NTs), which are members of the membrane purinome. Nucleoside transporters are integral membrane proteins that are responsible for the flux of nucleosides, such as adenosine, and nucleoside analog drugs, used in a variety of anti-cancer, anti-viral and anti-parasite therapies, across cell membranes. Nucleoside transporters form the SLC28 and SLC29 families of solute carriers and the protein members of these families are widely distributed in human tissues including the central nervous system (CNS). NTs modulate purinergic signaling in the CNS primarily through their effects on modulating prevailing adenosine levels inside and outside the cell. By clearing the extracellular milieu of adenosine, NTs can terminate adenosine receptor-dependent signaling and this raises the possibility of regulatory feedback loops that tie together receptor signaling with transporter function. Despite the important role of NTs as modulators of purinergic signaling in the human body, very little is known about the nature or underlying mechanisms of regulation of either the SLC28 or SLC29 families, particularly within the context of the CNS purinome. Here we provide a brief overview of our current understanding of the regulation of members of the SLC29 family and highlight some interesting avenues for future research.

Intracellular adenosine inhibits IgE-dependent degranulation of human skin mast cells.

PURPOSE: Adenosine (ADO) can enhance and inhibit mast cell degranulation. Potentiation of degranulation occurs at relatively low concentrations of ADO (10−6­10−5 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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).

Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus.

The neuromodulator adenosine plays an important role in many physiological and pathological processes within the mammalian CNS. However, the precise mechanisms of how the concentration of extracellular adenosine increases following neural activity remain contentious. Here we have used microelectrode biosensors to directly measure adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. Adenosine release was both action potential and Ca²âº dependent and could be evoked with low stimulation frequencies and small numbers of stimuli. Adenosine release required the activation of ionotropic glutamate receptors and could be evoked by local application of glutamate receptor agonists. Approximately 40% of stimulated-adenosine release occurred by translocation of adenosine via equilibrative nucleoside transporters (ENTs). This component of release persisted in the presence of the gliotoxin fluoroacetate and thus results from the direct release of adenosine from neurons. A reduction of adenosine release in the presence of NTPDase blockers, in slices from CD73(-/-) and dn-SNARE mice, provides evidence that a component of adenosine release arises from the extracellular metabolism of ATP released from astrocytes. This component of release appeared to have slower kinetics than the direct ENT-mediated release of adenosine. These data suggest that activity-dependent adenosine release is surprisingly complex and, in the hippocampus, arises from at least two distinct mechanisms with different cellular sources.

Na+-independent nucleoside transporters regulate adenosine and hypoxanthine levels in Müller cells and the inner blood-retinal barrier.

PURPOSE: To elucidate the mechanism(s) of hypoxanthine production in Müller cells and the elimination of hypoxanthine across the inner blood-retinal barrier (BRB). METHODS: The hypoxanthine biosynthesis and adenosine transport in Müller cells were investigated using a conditionally immortalized rat Müller cell line, TR-MUL5 cells. The elimination of hypoxanthine across the inner BRB was assessed by an in vivo microdialysis method and an in vitro transport study using a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB2 cells. RESULTS: [(3)H]Hypoxanthine was detected in TR-MUL5 cells and TR-MUL5 cell-cultured medium 3 hours after [(3)H]adenosine incubation, indicating that the hypoxanthine is produced in TR-MUL5 cells. [(3)H]Adenosine was taken up into TR-MUL5 cells, which express mRNAs of nucleoside transporters (ENT1-2 and CNT1-2), in an Na(+)-independent and concentration-dependent manner (Km = 20 µM). Moreover, 100 µM nitrobenzylmercaptopurine riboside (NBMPR) and azidothymidine, which are inhibitors of ENT2, inhibited [(3)H]adenosine uptake, suggesting that ENT2 is a major contributor to adenosine transport in Müller cells. [(3)H]Hypoxanthine was eliminated from the rat vitreous humor and this process was inhibited in the presence of NBMPR. [(3)H]Hypoxanthine uptake by TR-iBRB2 cells took place in an Na(+)-independent and concentration-dependent manner with Km values of 4.3 µM and 2.9 mM, and was inhibited by 100 µM NBMPR. CONCLUSIONS: Our findings suggest that hypoxanthine is produced from adenosine in Müller cells and ENT2 plays a major role in adenosine uptake in Müller cells. Hypoxanthine in the retina is eliminated via Na(+)-independent equilibrative nucleoside transporters.

Functional outcome of a novel SLC29A3 mutation identified in a patient with H syndrome.

The H syndrome (OMIM 612391) is an autosomal recessive disorder characterized by hyperpigmentation, hypertrichosis, histiocytosis and short stature. It is caused by mutations in the SLC29A3 gene, which encodes for the equilibrative nucleoside transporter 3 protein (ENT3), of still uncertain subcellular localisation. Here we report a new case of H syndrome with the novel mutation c.243delA, which has been concomitantly described by others [A. Bolze, A. Abhyankar, A.V. Grant, B. Patel, R. Yadav, M. Byun, D. Caillez, J.F. Emile, M. Pastor-Anglada, L. Abel, A. Puel, R. Govindarajan, L. de Pontual, J.L. Casanova, A mild form of SLC29A3 disorder: a frameshift deletion leads to the paradoxical translation of an otherwise noncoding mRNA splice variant, PLoS ONE 7 (2012) e29708]. Patient-derived primary skin fibroblasts and B-lymphoblastoid cell lines (B-LCL) were obtained and, although no differences were found in mRNA levels of ENT3, a significant increase in plasma membrane equilibrative transport activity was found in fibroblasts from the patient. Loss of function of key proteins implicated in nucleoside metabolism can lead to mitochondrial DNA (mtDNA) depletion syndromes (MDS). Measurement of respiratory chain complex activity revealed that mitochondrial function was unaltered. Neither fibroblasts nor B-LCL showed mtDNA depletion when compared with controls. Fibroblasts and B-LCL from the patient were not particularly protected when mitochondrial damage was induced using nucleoside-derived drugs susceptible to being transported by ENT3. Analysis of mtDNA amounts in tissues obtained at autopsy proved inconclusive with respect to mitochondrial involvement in the pathogenesis of this syndrome. Overall, the data do not support the inclusion of H syndrome among the MDS and these findings are compatible with its recent inclusion among the lysosomal storage diseases.

Equilibrative nucleoside transporter 3 deficiency perturbs lysosome function and macrophage homeostasis.

Lysosomal storage diseases (LSDs) are a group of heterogeneous disorders caused by defects in lysosomal enzymes or transporters, resulting in accumulation of undegraded macromolecules or metabolites. Macrophage numbers are expanded in several LSDs, leading to histiocytosis of unknown pathophysiology. Here, we found that mice lacking the equilibrative nucleoside transporter 3 (ENT3) developed a spontaneous and progressive macrophage-dominated histiocytosis. In the absence of ENT3, defective apoptotic cell clearance led to lysosomal nucleoside buildup, elevated intralysosomal pH, and altered macrophage function. The macrophage accumulation was partly due to increased macrophage colony-stimulating factor and receptor expression and signaling secondary to the lysosomal defects. These studies suggest a cellular and molecular basis for the development of histiocytosis in several human syndromes associated with ENT3 mutations and potentially other LSDs.

Metabolic network of nucleosides in the brain.

Brain relies on circulating nucleosides, mainly synthesised de novo in the liver, for the synthesis of nucleotides, RNA, nuclear and mitochondrial DNA, coenzymes, and pyrimidine sugar- and lipid-conjugates. Essentially, the paths of nucleoside salvage in the brain include a two step conversion of inosine and guanosine to IMP and GMP, respectively, and a one step conversion of adenosine, uridine, and cytidine, to AMP, UMP, and CMP, respectively. With the exception of IMP, the other four nucleoside monophosphates are converted to their respective triphosphates via two successive phosphorylation steps. Brain ribonucleotide reductase converts nucleoside diphosphates to their deoxy counterparts. The delicate qualitative and quantitative balance of intracellular brain nucleoside triphosphates is maintained by the relative concentrations of circulating nucleosides, the specificity and the K(m) values of the transport systems and of cytosolic and mitochondrial nucleoside kinases and 5'-nucleotidases, and the relative rates of nucleoside triphosphate extracellular release. A cross talk between extra- and intra-cellular nucleoside metabolism exists, in which released nucleoside triphosphates, utilised as neuroactive signals, are catabolised by a membrane bound ectonucleotidase cascade system to their respective nucleosides, which are uptaken into brain cytosol, and converted back to nucleoside triphosphates by the salvage enzymes. Finally, phosphorolysis of brain nucleosides generates pentose phosphates, which are utilised for nucleoside interconversion, 5-phosphoribosyl-1-pyrophosphate synthesis, and energy repletion. This review focuses on these aspects of brain nucleoside metabolism, with the aim of giving a comprehensive picture of the metabolic network of nucleosides in normoxic conditions, with some hints on the derangements in anoxic/ischemic conditions.

Immune cell regulation by autocrine purinergic signalling.

Stimulation of almost all mammalian cell types leads to the release of cellular ATP and autocrine feedback through a diverse array of purinergic receptors. Depending on the types of purinergic receptors that are involved, autocrine signalling can promote or inhibit cell activation and fine-tune functional responses. Recent work has shown that autocrine signalling is an important checkpoint in immune cell activation and allows immune cells to adjust their functional responses based on the extracellular cues provided by their environment. This Review focuses on the roles of autocrine purinergic signalling in the regulation of both innate and adaptive immune responses and discusses the potential of targeting purinergic receptors for treating immune-mediated disease.

Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement.

We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A(1) or A(3) antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A(2A) and A(2B), respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A(2A) and A(2B) receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A(2A)/A(2B) receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A(2A) and A(2B) agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A(2A) and A(2B) receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by alpha-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the development of edema before ALI but also partially reversed edema after ALI. The data suggest that adenosine deaminase inhibition may be useful in treatment of pulmonary edema in settings of ALI.

The role of nucleoside transporters in the erythrocyte disposition and oral absorption of ribavirin in the wild-type and equilibrative nucleoside transporter 1-/- mice.

Ribavirin [1-(beta-d-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide] is the treatment of choice for hepatitis C virus infection. Ribavirin is a substrate of several nucleoside transporters, including the equilibrative nucleoside transporter (Ent) and the concentrative nucleoside transporter 2. To determine the role of Ent1 in ribavirin absorption and erythrocyte distribution, we examined its pharmacokinetics in Ent1-null mice. After intravenous administration, we found that the erythrocyte area under the curve (AUC(0-12 h)) was reduced 3.05-fold along with 2.63-fold reduction of erythrocyte versus plasma AUC ratio in the Ent1(-/-) mice, whereas there was no significant difference in the plasma AUC(0-12 h) between Ent1(+/+) and Ent1(-/-) mice. After 48 h, we found a similar fraction of ribavirin or total radioactivity excreted in the urine between the Ent1(+/+) and Ent1(-/-) mice. After oral administration of three different doses, 0.024, 0.24, and 6.1 mg/kg, we found that the dose-normalized plasma AUC(0-12 h) of ribavirin was 69.7 +/- 12.0, 20.7 +/- 1.5, and 18.3 +/- 2.7 min/l, respectively, in the Ent1(+/+) mice and 18.9 +/- 2.8, 13.0 +/- 0.5, and 12.2 +/- 1.0 min/l, respectively, in the Ent1(-/-) mice. It is interesting that at the highest dose, the dose-normalized plasma AUC(0-30 min), AUC(0-12 h), and C(max) in the Ent1(+/+) mice were decreased 4.0-, 3.8-, and 3.4-fold, respectively, compared with the lowest dose, suggesting absorption was saturated at the highest dose we used. The dose-normalized plasma AUC(0-12 h) was 3.7- and 1.5-fold lower at the lowest and the highest dose, respectively, in the Ent1(-/-) mice compared with those of the Ent1(+/+) mice. Our findings indicate that Ent1 plays a significant role in the oral absorption and erythrocyte distribution of ribavirin.

Transport of nucleoside analogs across the plasma membrane: a clue to understanding drug-induced cytotoxicity.

Nucleoside analogs are widely used in the treatment of cancer and viral-induced diseases. Efficacy of treatments relies upon a variety of events, including transport across tissue and target barriers, which determine drug pharmacokinetics and target cell bioavailability. To exert their action, nucleosides have to be chemically modified, thus compromising cellular uptake by those routes which are responsible for the uptake of natural nucleosides and nucleobases. In this review we will focus on established knowledge and recent advances in the understanding of nucleoside- and nucleobase-derived drug uptake mechanisms. Basically, these drug uptake processes involve the gene families SLC22, SLC28 and SLC29. These gene families encode Organic Anion Transporter (OAT)/Organic Cation Transporter (OCT), Concentrative Nucleoside Transporter (CNT) and Equilibrative Nucleoside Transporter (ENT) proteins, respectively. The pharmacological profiles of these plasma membrane carriers as well as their basic physiological and regulatory properties, including their tissue and subcellular distribution will be reviewed. This knowledge is crucial for the understanding of nucleoside- and nucleobase-derived drug bioavailability and therapeutic action. Moreover, changes in both transporter expression and/or transporter function (for instance as a consequence of gene variability) might also modulate response to treatment, thereby anticipating a putative diagnostic and predictive added value to the analysis of transporter expression and their corresponding genetic variants.

Cloning and pharmacological characterization of a novel gene encoding human nucleoside transporter 1 (hNT1) from a human breast cancer cDNA library.

AIMS: We isolated a novel gene encoding human nucleoside transporter 1 (hNT1), from a human breast cancer cDNA library. MAIN METHODS: A nondirectional cDNA library was screened by an EST clone (GenBanktrade mark/EMBL/DDBJ: BU944345). A Xenopus laevis oocyte expression system was used for functional characterization. Membrane localization in the human breast was determined by immunohistochemistry. KEY FINDINGS: Isolated hNT1 cDNA consisted of 246 base pairs that encoded an 82-amino acid protein. By RT-PCR analysis, hNT1 mRNA was strongly detected in the breast cancer tissues. When expressed in X. oocytes, hNT1 mediated the high affinity transport of [(3)H]5-fluorouracil (5-FU) with a K(m) value of 69.2+/-24.5 nM in time- and pH-dependent, and Na(+)-independent manners. A cis-inhibition experiment revealed that hNT1 mediated transport of [(3)H]5-FU is strongly inhibited by various nucleosides such as pyrimidine, uracil, uridine, guanosine, inosine, thymidine, adenosine, cytidine and purine suggesting that hNT1 may be involved in the trans epithelial transport of these endogenous substrates. Immunohistochemical analysis revealed that the hNT1 protein is localized in the lactiferous duct epithelium. SIGNIFICANCE: Our present results indicate that a newly isolated cDNA clone, hNT1, is a key molecule for the breast handling of 5-FU in humans.

Cyclic ADP-ribose mediates formyl methionyl leucyl phenylalanine (fMLP)-induced intracellular Ca(2+) rise and migration of human neutrophils.

Although cyclic ADP-ribose (cADPR), a novel Ca(2+)-mobilizing mediator, is suggested to be involved in the functions of neutrophils in rodents, its role in human neutrophils remains unclear. The present study examined the ability of cADPR to mobilize Ca(2+) and mediate formyl methionyl leucyl phenylalanine (fMLP)-stimulated increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and migration in human neutrophils. cADPR induced Ca(2+) release from digitonin-permeabilized neutrophils, and the release was blocked by 8Br-cADPR, an antagonist of cADPR. Immunophilin ligands, FK506 and rapamycin, but not cyclosporine A, inhibited cADPR-induced Ca(2+) release. 8Br-cADPR partially reduced fMLP-induced [Ca(2+)](i) rise and abolished the rise in combination with 2APB, an IP(3)-receptor antagonist. Anti-CD38Ab and NADase that interfere with cADPR formation, reduced the fMLP-induced [Ca(2+)](i) rise. When beta-NAD(+), a substrate of ADP-ribosyl cyclase, and cADPR were added to the medium, the former gradually increased [Ca(2+)](i) and the latter potentiated the fMLP-induced [Ca(2+)](i) rise. The beta-NAD(+)-induced [Ca(2+)](i) rise in Ca(2+)-free medium was inhibited by anti-CD38Ab, 8Br-cADPR, FK506, ruthenium red, and thapsigargin. mRNAs of nucleoside transporter (NT), ENT1, ENT2, CNT, and CNT3 were expressed in neutrophils; and their inhibitors, inosine, uridine, and s-(4-nitrobenzyl)-6-thioinosine, reduced the [Ca(2+)](i) rise induced by beta-NAD(+) and fMLP. fMLP-timulated migration was inhibited by the removal of Ca(2+) from the medium or by the addition of 8Br-cADPR, anti-CD38Ab, NADase, and NT inhibitors. These results suggest that cADPR was synthesized extracellularly by CD38, transported into the cells through NTs, and then Ca(2+) was mobilized by FK506-binding protein-dependent process. This process may be involved in fMLP-induced intracellular Ca(2+) signaling and migration in human neutrophils.

Functional characterization of a nucleoside-derived drug transporter variant (hCNT3C602R) showing altered sodium-binding capacity.

A novel cloned polymorphism of the human concentrative nucleoside transporter hCNT3 was described and functionally characterized. This variant consists of a T/C transition leading to the substitution of cysteine 602 by an arginine residue in the core of transmembrane domain 13. The resulting hCNT3(C602R) protein has the same selectivity and affinity for natural nucleosides and nucleoside-derived drugs as hCNT3 but much lower concentrative capacity. The insertion of the transporter into a polarized membrane seems unaffected in the variant. In a preliminary survey of a typical Spanish population, this variant showed an allelic frequency of 1%. The functional impairment of the hCNT3(C602R) polymorphism is attributable to the presence of an arginine rather than the loss of a cysteine at position 602, because an engineered hCNT3 protein with a serine residue at this position (hCNT3(C602S)) and hCNT3 have similar kinetic parameters. The sodium activation kinetic analysis of both transporters revealed a variation in the affinity for sodium and a shift in the Hill coefficient that could be consistent with a stoichiometry of 2:1 and 1:1 sodium/nucleoside, for hCNT3 and hCNT3(C602R), respectively. In conclusion, the presence of an arginine residue in the core of transmembrane domain 13 is responsible for the different sodium affinity showed by the polymorphic transporter compared with the reference transporter. Individuals with the hCNT3(C602R) variant might show a lower nucleoside and nucleoside analog concentrative capacity, which could be clinically relevant.

Up-regulation of MRP4 and down-regulation of influx transporters in human leukemic cells with acquired resistance to 6-mercaptopurine.

To investigate the mechanism of cellular resistance to 6-MP, we established a 6-MP resistant cell line (CEM-MP5) by stepwise selection of the human T-lymphoblastic leukemia cell line (CEM). CEM-MP5 cells were about 100-fold resistant to 6-MP compared with parental CEM cells. Western blot analysis demonstrated that multidrug resistant protein 4 (MRP4) was increased in CEM-MP5 cells, whereas the levels of the nucleoside transporters hENT1, hCNT2 and hCNT3 were decreased compared with those of parental CEM cells. Consistent with the operation of an efflux pump, accumulation of [14C]6-MP and/or its metabolites was reduced, and ATP-dependent efflux was increased in CEM-MP5 cells. Taken together these results showed that up-regulation of MRP4 and down-regulation of influx transporters played a major role in 6-MP resistance of CEM-MP5 cells.