Search

This is the first report of constrictive pericarditis (CP) in a 16-year-old boy with H syndrome with pericardial involvement predominantly over the right ventricle with favorable response to anti-inflammatory treatment. H syndrome, first reported in 2008, is a new auto-inflammatory syndrome with multiorgan involvement due to mutation in the SLC29A3 gene. We described the echocardiographic characteristics of asymmetric pericardial involvement and presented the cardiac computed tomography angiographic and magnetic resonance imaging findings. We reviewed the echocardiographic signs of CP, introduced tricuspid E/A respiratory alternans as a novel echocardiographic sign of right ventricular dominant CP, and explained the underlying mechanism.

The emphasis of our study was to determine the physiological function of miR-1224-5p in rectal cancer (RC) and its in-depth mechanism. First, the expression of miR-1224-5p in RC tissues was analyzed using public data from the TCGA database. Then, miR-1224-5p expression in RC cell lines SW480 and SW837 was measured using the qRT-PCR assay. The subsequent CCK-8 assay was executed to assess the function of miR-1224-5p in the viability of the RC cell. Bioinformatics prediction prompted that SLC29A3 may be a potential target gene for miR-1224-5p. Western blotting and dual-luciferase reporter assays were performed to affirm the above forecasting. Kaplan-Meier analysis and Cox multivariate analysis were carried out to assess the relationship between SLC29A3 and prognosis. Finally, CCK-8, colony formation assay, and transwell assay were used for functional analysis of miR-1224-5p/ SLC29A3 axis in vitro. MiR-1224-5p was expressed at low levels in RC tissues and cell lines. Up-regulation of miR-1224-5p inhibited SW480 cell viability, while inhibition of miR-1224-5p enhanced the viability of SW837 cells. What is more, we affirmed that miR-1224-5p could direct target SLC29A3, which was expressed at high levels in RC tissues. In addition, SLC29A3 could be used as an independent predictive factor of prognosis in patients with RC, and the higher SLC29A3 expression, the lower survival rate. Finally, cellular functional experiments evidenced that miR-1224-5p mimic can reduce the cell viability, invasion, and migration, while overexpression of SLC29A3 presented an opposite effect. Importantly, co-transfection experiments indicated that SLC29A3 can reverse miR-1224-5p-mediated inhibition in the malignant progression of RC cells. Our work raised the possibility that miR-1224-5p functioned as a tumor suppressor in RC, which achieved its function via targeting SLC29A3.

Elevated proliferation rates in cancer can be visualized with positron emission tomography (PET) using 3′-deoxy-3′-l-[18F]fluorothymidine ([18F]FLT). This study investigates whether [18F]FLT transp...

Aim To examine the extent to which discriminatory testing using antibodies and Type 1 diabetes genetic risk score, validated in European populations, is applicable in a non‐European population. Methods We recruited 127 unrelated children with diabetes diagnosed between 9 months and 5 years from two centres in Iran. All children underwent targeted next‐generation sequencing of 35 monogenic diabetes genes. We measured three islet autoantibodies (islet antigen 2, glutamic acid decarboxylase and zinc transporter 8) and generated a Type 1 diabetes genetic risk score in all children. Results We identified six children with monogenic diabetes, including four novel mutations: homozygous mutations in WFS1 (n=3), SLC19A2 and SLC29A3, and a heterozygous mutation in GCK. All clinical features were similar in children with monogenic diabetes (n=6) and in the rest of the cohort (n=121). The Type 1 diabetes genetic risk score discriminated children with monogenic from Type 1 diabetes [area under the receiver‐operating characteristic curve 0.90 (95% CI 0.83–0.97)]. All children with monogenic diabetes were autoantibody‐negative. In children with no mutation, 59 were positive to glutamic acid decarboxylase, 39 to islet antigen 2 and 31 to zinc transporter 8. Measuring zinc transporter 8 increased the number of autoantibody‐positive individuals by eight. Conclusions The present study provides the first evidence that Type 1 diabetes genetic risk score can be used to distinguish monogenic from Type 1 diabetes in an Iranian population with a large number of consanguineous unions. This test can be used to identify children with a higher probability of having monogenic diabetes who could then undergo genetic testing. Identification of these individuals would reduce the cost of treatment and improve the management of their clinical course., What's new? Studies in white European populations have recently shown that a genetic risk score for Type 1 diabetes has a high ability to discriminate between Type 1 diabetes and monogenic diabetes.The diagnostic utility of this genetic risk score in non‐European populations is unknown.This study provides the first evidence that the Type 1 diabetes genetic risk score discriminates children with monogenic diabetes from those with Type 1 diabetes in the Iranian population with a large number of consanguineous unions.The Type 1 diabetes genetic risk score can be used to improve the selection of non‐European children for monogenic diabetes testing, resulting in the correct diagnosis, improving their clinical management and providing families with recurrence risk information.

The gastrointestinal tract is the absorptive organ for nutrients found in foods after digestion. Nucleosides and, to a lesser extent nucleobases, are the late products of nucleoprotein digestion. These metabolites are absorbed by nucleoside (and nucleobase) transporter (NT) proteins. NTs are differentially distributed along the gastrointestinal tract showing also polarized expression in epithelial cells. Concentrative nucleoside transporters (CNTs) are mainly located at the apical side of enterocytes, whereas equilibrative nucleoside transporters (ENTs) facilitate the basolateral efflux of nucleosides and nucleobases to the bloodstream. Moreover, selected nucleotides and the bioactive nucleoside adenosine act directly on intestinal cells modulating purinergic signaling. NT-polarized insertion is tightly regulated. However, not much is known about the modulation of intestinal NT function in humans, probably due to the lack of appropriate cell models retaining CNT functional expression. Thus, the possibility of nutritional regulation of intestinal NTs has been addressed using animal models. Besides the nutrition-related role of NT proteins, orally administered drugs also need to cross the intestinal barrier, this event being a major determinant of drug bioavailability. In this regard, NT proteins might also play a role in pharmacology, thereby allowing the absorption of nucleoside- and nucleobase-derived drugs. The relative broad selectivity of these membrane transporters also suggests clinically relevant drug-drug interactions when using combined therapies. This review focuses on all these physiological and pharmacological aspects of NT protein biology. © 2017 American Physiological Society. Compr Physiol 8:1003-1017, 2018.

This is the peer reviewed version of the following article: Low, D., Tang, M.M., Surana, U., Lee, J.Y., Pramano, Z.A.D. and Leong, K.F. (2019), H syndrome – the first report in Malaysia. Int J Dermatol, 58: e190-e193. doi:10.1111/ijd.14518, which has been published in final form at https://doi.org/10.1111/ijd.14518. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.

We screened small-molecule compounds that inhibit osteoclast differentiation to find new anti-osteoporosis agents and found that a novel compound, SUKU-1, suppressed osteoclastogenesis. We also synthesized 38 derivatives of SUKU-1 and discovered that nine of them had inhibitory effects on osteoclastogenesis and that SUKU-33 was the most potent inhibitor. Next, we investigated the mechanisms by which SUKU-33 suppressed osteoclast differentiation. By measuring the uptake of [(3) H]-uridine in cells, we found that SUKU-33 suppressed both equilibrative nucleoside transporters and concentrative nucleoside transporters. These results suggest that SUKU-33 inhibits osteoclast differentiation by suppressing nucleoside transporters.

Idiopathic pulmonary fibrosis is a devastating lung disease with limited treatment options. The signaling molecule adenosine is produced in response to injury and serves a protective role in early stages of injury and is detrimental during chronic stages of disease such as seen in lung conditions such as pulmonary fibrosis. Understanding the association of extracellular adenosine levels and the progression of pulmonary fibrosis is critical for designing adenosine based approaches to treat pulmonary fibrosis. The goal of this study was to use various models of experimental lung fibrosis to understand when adenosine levels are elevated during pulmonary fibrosis and whether these elevations were associated with disease progression and severity. To accomplish this, extracellular adenosine levels, defined as adenosine levels found in bronchioalveolar lavage fluid, were determined in mouse models of resolvable and progressive pulmonary fibrosis. We found that relative bronchioalveolar lavage fluid adenosine levels are progressively elevated in association with pulmonary fibrosis and that adenosine levels diminish in association with the resolution of lung fibrosis. In addition, treatment of these models with dipyridamole, an inhibitor of nucleoside transporters that potentiates extracellular adenosine levels, demonstrated that the resolution of lung fibrosis is blocked by the failure of adenosine levels to subside. Furthermore, exacerbating adenosine levels led to worse fibrosis in a progressive fibrosis model. Increased adenosine levels were associated with elevation of IL-6 and IL-17, which are important inflammatory cytokines in pulmonary fibrosis. These results demonstrate that extracellular adenosine levels are closely associated with the progression of experimental pulmonary fibrosis and that this signaling pathway may mediate fibrosis by regulating IL-6 and IL-17 production.—Luo, F., Le, N.-B., Mills, T., Chen, N.-Y., Karmouty-Quintana, H., Molina, J. G., Davies, J., Philip, K., Volcik, K. A., Liu, H., Xia, Y., Eltzschig, H. K., Blackburn, M. R. Extracellular adenosine levels are associated with the progression and exacerbation of pulmonary fibrosis.

Nucleoside transporter (NT) and nucleic-related enzyme (NRE) play key roles in the physiology of nucleosides and the pharmacology of its analogs in mammals. In this study, we examined the effect of fluoxetine, a selective serotonin reuptake inhibitor, and pergolide, a dopamine D receptor agonist, on the expression of NTs and NREs in mouse brain. It was confirmed by the detection of corresponding mRNAs that three equilibrative nucleoside transporter (ENT1-3) isoforms, concentrative nucleoside transporter 2 (CNT2), CNT3, adenosine kinase (AK), and apyrase, but not CNT1, were expressed in brain tissue. Based on an assessment by mRNA determination, the cerebral expression of CNT2 was found to be increased by administration of fluoxetine and pergolide to mice. Furthermore, pergolide increased the expression of ENT2. However, fluoxetine and pergolide had no significant effect on the expression of mRNA for other NTs, AK, and apyrase. Therefore, we concluded that the expression of several NT isoforms, but not NREs, in mouse brain was affected by treatment with fluoxetine and pergolide.

The H syndrome is a recently defined autosomal recessive genodermatosis caused by mutations in the nucleoside transporter hENT3. It is characterized by cutaneous changes of progressive sclerosis, hyperpigmentation, and hypertrichosis that follow a specific pattern and are associated with multiple systemic manifestations. A case is presented and a brief review is introduced based on the few related reports.

BACKGROUND Lesch-Nyhan (LN) syndrome is associated with deficient hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity, but the connection between the aberrant purine metabolism and the neurological symptoms remains largely unknown. The aim of this study was to determine adenosine transporter subtypes affected by HPRT deficiency and by the associated hypoxanthine excess. MATERIALS AND METHODS Nucleoside transporter types (depending on their sodium dependence and 10 microm nitrobenzylthioinosine, NBTI, sensitivity) involved in adenosine transport were compared between peripheral blood lymphocytes (PBL) obtained from nine LN patients, PBL(LN) (2-21 years) and from nine controls, PBL(C) (2-23 years) under basal conditions and after 25 microm hypoxanthine incubation. RESULTS We found four types of adenosine transporters in PBL: equilibrative and concentrative transporters that are either sensitive (ENT1 or cs) or insensitive (ENT2 or ci) to NBTI. Adenosine ENT1 uptake was the predominant transporter in both PBL(C) (55%) and PBL(LN) (46%). Under basal conditions no significant differences were found in adenosine transport between PBL(C) and PBL(LN). Incubation of PBL with 25 microm hypoxanthine markedly decreased total adenosine transport in both cell types. Hypoxanthine affected equilibrative transport (mainly ENT2 type) in PBL(LN) and PBL(C). Only in PBL(C) was concentrative transport affected by hypoxanthine. Expressions of human (h) ENT1 and hENT2 mRNA were not significantly modified by hypoxanthine incubation in PBL(C). CONCLUSIONS This study contributes to further knowledge of the defective adenosine transport found in PBL(LN). Increased hypoxanthine levels, similar to those reported in HPRT deficient patients, reduced adenosine uptake by 32% in PBL(LN) as compared to normal transport.

The proliferative response of T lymphocytes is a crucial step in cell-mediated immunity. This study was undertaken to investigate the mechanisms leading to the impaired proliferative response of diabetic T lymphocytes. T cells that had been isolated from the spleen of normal rats and cultured in medium containing 20 mm glucose and no insulin displayed the same degree of proliferative impairment as cells isolated from diabetic rats. The rate of T-cell proliferation, when induced with concanavalin A or anti-CD3 and anti-CD28 antibodies, was not affected by the inhibition of nucleoside transporters. T cells cultured at high glucose concentrations in the absence of insulin displayed decreased expression of adenosine kinase, and released measurable extracellular quantities of adenosine. Under resting conditions, the level of cAMP was 5.9-fold higher in these cells compared to cells grown in low glucose and in the presence of insulin. Experiments with specific adenosine receptor agonists and antagonists showed that adenosine-induced suppression of diabetic T cell proliferation was mediated by the A2A adenosine receptor, but not by the A2B receptor. Treatment of diabetic T cells with 10 microm H-89, a specific protein kinase A inhibitor, restored T-cell proliferation. These results show that suppressed proliferation of diabetic T lymphocytes is evoked by the decreased expression of adenosine kinase, leading to the outflow of adenosine from the cell. Extracellular adenosine then stimulates the A2A receptor and induces cAMP production, leading to the activation of protein kinase A, and suppression of T-cell proliferation.

Nucleoside transport processes regulate the levels of adenosine available to modulate neurotransmission, vascular tone and other physiological events. However, although equilibrative transporter transcripts or proteins have been mapped in the central nervous system of rats and humans, little is known about the presence and distribution of the complete family of nucleoside transporters in brain. In this study, we analysed the distribution of the transcript encoding the high affinity adenosine-preferring concentrative transporter CNT2 in the rat central nervous system and compared it with that of the equilibrative transporter ENT1. Furthermore, we evaluated the changes in expression of these two transporters in a situation of increased extracellular levels of adenosine, such as sleep deprivation. CNT2 mRNA was widespread in rat brain, although most prevalent in the amygdala, the hippocampus, specific neocortical regions and the cerebellum. The distribution of CNT2 mRNA only partially overlapped that of ENT1. Most of the cells labelled were neurones. Total sleep deprivation dramatically diminished the amounts of CNT2 mRNA, whereas ENT1 mRNA remained unchanged. This specific decrease in CNT2 transcript suggests a new physiological role for the transporter in the modulation of extracellular adenosine levels and the sleep/wakefulness cycle.

Tsx is a nucleoside-specific outer membrane (OM) transporter of Gram-negative bacteria. We present crystal structures of Escherichia coli Tsx in the absence and presence of nucleosides. These structures provide a mechanism for nucleoside transport across the bacterial OM. Tsx forms a monomeric, 12-stranded beta-barrel with a long and narrow channel spanning the outer membrane. The channel, which is shaped like a keyhole, contains several distinct nucleoside-binding sites, two of which are well defined. The base moiety of the nucleoside is located in the narrow part of the keyhole, while the sugar occupies the wider opening. Pairs of aromatic residues and flanking ionizable residues are involved in nucleoside binding. Nucleoside transport presumably occurs by diffusion from one binding site to the next.

The effect of hypoxia on the release of adenosine was studied in vitro in the rat whole carotid body (CB) and compared with the effect of hypoxia (2%, 5% and 10% O(2)) on adenosine concentrations in superior cervical ganglia (SCG) and carotid arteries. Moderate hypoxia (10% O(2)) increased adenosine concentrations released from the CBs by 44%, but was not a strong enough stimulus to evoke adenosine release from SCG and arterial tissue. The extracellular pathways of adenosine production in rat CBs in normoxia and hypoxia were also investigated. S-(p-nitrobenzyl)-6-thioinosine (NBTI) and dipyridamole were used as pharmacological tools to inhibit adenosine equilibrative transporters (ENT) and alpha,beta-methylene ADP (AOPCP) to inhibit ecto-5'-nucleotidase. Approximately 40% of extracellular adenosine in the CB came from the extracellular catabolism of ATP, under both normoxic and hypoxic conditions. Low pO(2) triggers adenosine efflux through activation of NBTI-sensitive ENT. This effect was only apparent in hypoxia and when adenosine extracellular concentrations were reduced by the blockade of ecto-5'-nucleotidase. We concluded that CB chemoreceptor sensitivity could be related to its low threshold for the release of adenosine in response to hypoxia here quantified for the first time.

Adenosine modulates nociceptive processing in the superficial dorsal horn of the spinal cord. In other tissues, membrane transporters influence profoundly the extracellular levels of adenosine. To investigate the putative role of nucleoside transporters in the regulation of excitatory synaptic transmission in the dorsal horn, we employed immunohistochemistry and whole-cell patch-clamp recording of substantia gelatinosa neurons in slices of rat spinal cord in vitro. The rat equilibrative nucleoside transporter (rENT1) was revealed by antibody staining to be abundant in neonatal and mature dorsal horn, especially within laminae I-III. This was confirmed by immunoblots of dorsal horn homogenate. Nitrobenzylthioinosine (NBMPR), a potent non-transportable inhibitor of rENT1, attenuated synaptically evoked EPSCs onto lamina II neurons in a concentration-dependent manner. Application of an adenosine A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine produced a parallel rightward shift in the NBMPR concentration-effect curve. The effects of NBMPR were partially reversed by adenosine deaminase, which facilitates the metabolic degradation of adenosine. The modulation by NBMPR of evoked EPSCs was mimicked by exogenous adenosine or the selective A1 receptor agonist, 2-chloro-N6-cyclopentyl adenosine. NBMPR reduced the frequency but not the amplitude of spontaneous miniature EPSCs and increased the paired-pulse ratio of evoked currents, an effect that is consistent with presynaptic modulation. These data provide the first direct evidence that nucleoside transporters are able to critically modulate glutamatergic synaptic transmission.

Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with L-[3H]adenosine, our objectives here were to determine (1) if L-[3H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological D-adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. L-[3H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. L-[3H]Adenosine release was inhibited dose-dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 microM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with L-[3H]adenosine alone or I-[3H]adenosine plus unlabeled L-adenosine, D-adenosine, or uridine, L-[3H]adenosine release was inhibited 42% by L-adenosine, 69% by uridine, and 81% by D-adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged high-frequency orthodromic stimulation of superior cervical ganglia is known to result in increased acetylcholine (ACh) synthesis and ACh content after the period of stimulation. In a previous study, we provided evidence to suggest that adenosine acts as an extracellular signal to activate this increased ACh synthesis and we proposed that the source of that adenosine might be postsynaptic. Thus, the purpose of the present study was to test whether direct stimulation of the post-ganglionic nerves could affect ganglionic ACh content. Antidromic conditioning of ganglia (15 Hz, 45 min) did not affect significantly their ACh content. However, if ganglia were allowed a 15-min rest period after this antidromic conditioning, their ACh stores were increased by 20% ; a similar increase was induced by 4-Hz stimulation before the rest period. During the 15-Hz antidromic stimulation, ACh release was not clearly increased above the basal level, suggesting that preganglionic nerve endings were not stimulated to an extent that could explain the increased ACh content. Orthodromic stimulation (5 Hz) of ganglia 15 min after they had been subjected to antidromic conditioning (15 Hz, 45 min) showed increased ACh release in comparison with that from control unconditioned ganglia. Moreover, the extra ACh released by the conditioned ganglia was quantitatively similar to the increase in the ACh stores, as if most, or all, of the additional ACh was released by preganglionic stimulation. If the antidromic conditioning and the rest period were done during perfusion with Ca 2+ -free medium, the ganglia did not accumulate extra ACh. The ACh content was also not changed if ganglia were conditioned in the absence of Ca 2+ but rested with normal Ca 2+ . However, ACh content was increased by 23% when the antidromic stimulation was done with normal Ca 2+ but the rest period was without Ca 2+ . To test the role of adenosine in this retrograde effect, the effect of nucleoside transport inhibitors was tested. Dipyridamole blocked the antidromic stimulation-induced increase, but nitrobenzylthioinosine did not. Overall, these results are consistent with the idea that a diffusible retrograde messenger activates ACh synthesis. The sensitivity to blockade by dipyridamole suggests that adenosine might be that signal.

1. The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. 2. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km = 177 +/- 36 microM and Vmax = 1.9 +/- 0.2 nmol x ml(-1) x s(-1) (0.7 nmol (mg protein)(-1) x s(-1)). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72% inhibition) or dipyridamol (64% inhibition; P < 0.05). 3. In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57% larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. 4. Inhibition of ecto-5'-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70% lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. 5. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the skeletal muscle cells during contraction.

The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter. 5-(SAENTA-x8)-fluorescein inhibited adenosine transport and [3H]NBTI binding with a Ki of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5-(SAENTA-x8)-fluorescein specifically interacted with the NBTI-sensitive nucleoside transporters with high affinity (KD=6 nM). Activation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside transport in single cells from heterogeneous neural cell populations.

BACKGROUND AND PURPOSE Inhibitors of DNA methyltransferases (DNMTs), such as azacytidine, decitabine and zebularine, are used for the epigenetic treatment of cancer. Their action may depend upon their translocation across the plasma membrane. The aim of this study was to identify transporter proteins contributing to DNMT inhibitor action. EXPERIMENTAL APPROACH Drug interactions with selected hCNT and hENT proteins were studied in transiently transfected HeLa and MDCK cells. Interaction with human organic cation transporters (hOCTs) was assessed in transiently transfected HeLa cells and Xenopus laevis oocytes. KEY RESULTS Zebularine uptake was mediated by hCNT1, hCNT3 and hENT2. Decitabine interacted with but was not translocated by any nucleoside transporter (NT) type. hCNT expression at the apical domain of MDCK cells promoted net vectorial flux of zebularine. Neither hOCT1 nor hOCT2 transported decitabine, but both were involved in the efflux of zebularine, suggesting these proteins act as efflux transporters. hOCT1 polymorphic variants, known to alter function, decreased zebularine efflux. CONCLUSIONS AND IMPLICATIONS This study highlights the influence of human NTs and hOCTs on the pharmacokinetics and pharmacodynamics of selected DNMT inhibitors. As hOCTs may also behave as efflux transporters, they could contribute either to chemoresistance or to chemosensitivity, depending upon the nature of the drug or combination of drugs being used in cancer therapy.

1. Draflazine, a nucleoside transport inhibitor, was administered as a 15 min i.v. infusion of 2.5 mg to eight healthy male subjects. Plasma and whole blood concentrations were measured up to 32 h post-dose, and were related to adenosine breakdown inhibition (ABI) measured ex vivo, which served as a pharmacodynamic endpoint. 2. The red blood cell/plasma distribution of draflazine was non-linear and characterized as a capacity-limited specific binding to the nucleoside transporter on the red blood cells. The binding (dissociation) constant Kd was 0.87 ng ml-1 plasma and the maximal specific binding capacity (Bmax) was 164 ng ml-1 RBC, which corresponds to about 14,000 specific binding sites per erythrocyte. Non-specific binding amounted to less than 15% of the total binding. 3. The pharmacokinetics of draflazine in blood were determined in each subject and characterized by a two-compartment pharmacokinetic model. The pharmacokinetic parameters (mean +/- s.d.) were: clearance 22.0 +/- 8.0 ml mm-1, volume of distribution at steady-state 39.8 +/- 4.7 l and terminal half-life 24.0 +/- 9.4 h. Concentrations in plasma were much lower, and could only be determined accurately in pooled plasma samples with a red blood cell binding assay. The pharmacokinetic parameters in pooled plasma were: clearance 551 ml min-1, volume of distribution at steady-state 349 l and terminal half-life 10.7 h. 4. A non-linear relationship was observed between the plasma or blood concentration of draflazine and the ABI determined ex vivo. This relationship was characterized by the sigmoidal Emax pharmacodynamic model. Based on concentrations in pooled plasma, values of the pharmacodynamic parameters were Emax 100%, IC50 10.5 ng ml-1 and Hill factor 0.9. When using whole blood concentrations, the relationship was much steeper with values (mean +/- s.d.) Emax 92.4 +/- 5.6%, IC50 76.0 +/- 15.3 ng ml-1 and Hill factor 3.5 +/- 0.9. 5. Binding to the nucleoside transporter on red blood cells is an important determinant of the pharmacokinetics of draflazine and a high degree of occupancy of the transporter by draflazine is required to inhibit adenosine breakdown ex vivo. It is suggested that red blood cell nucleoside transporter occupancy may serve as a useful pharmacodynamic endpoint in dose ranging studies with draflazine.

Uridine transport was investigated in cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue. In intact cells, the kinetic parameters for uridine uptake were Km 150 +/- 45 microM, and Vmax 414 +/- 17 pmol . 10(6) cells-1 . min-1. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (Ki 3 nM) and dipyridamole (Ki 54 nM) are consistent with a facilitated diffusion nucleoside transport system. The IC50 value for the adenosine transport inhibition by uridine was very high (240 microM), agreeing with the relative affinities of these nucleosides in the chromaffin cell nucleoside transport system, which was 150-fold higher for adenosine than for uridine. Uridine was significantly metabolized in chromaffin cells but not in plasma membrane vesicles. The affinity of uridine transport measured in these membrane vesicles was reproducible and similar to the affinity found for intact cells with a Km value of 185 +/- 11 microM and a Vmax value of 4.24 +/- 0.10 pmol . mg protein-1 . s-1. These membrane preparations were employed to investigate the regulatory action of ATP and other nucleotide analogues on nucleoside transport. ATP increased the Vmax value but the Km value was not significantly modified. Adenosine 5'-[beta,gamma-imino]triphosphate, 1,N6-ethenoadenosine 5'-triphosphate, and adenosine(5')-tetraphospho(5')adenosine(Ap4A) at 100 microM were able to mimic the ATP effect. These results agree with a regulatory role of ATP, and the uridine transport on chromaffin plasma membrane vesicles is a good model for analyzing the nucleoside-transporter function and regulation.

1. The aim of the present study was to determine the effect of the nucleoside transporter inhibitor, draflazine, on the force of contraction in human myocardium and the affinity of the compound for the nucleoside transporter. Nucleoside transport inhibitors, like draflazine, are of potential importance for cardiopreservation of donor hearts for heart transplantation. 2. Functional experiments were performed in isolated electrically driven (1 Hz, 1.8 mmol l-1 Ca2+) human atrial trabeculae and ventricular papillary muscle strips. The affinity of draflazine for the myocardial nucleoside transporter was studied in isolated membranes from human ventricular myocardium and human erythrocytes in radioligand binding experiments using [3H]-nitrobenzylthioinosine ([3H]-NBTI). Dipyridamole was studied for comparison. 3. In membranes from human myocardium and erythrocytes, [3H]-NTBI labelled 1.18 pmol mg-1 protein and 23.0 pmol mg-1 protein, respectively, nucleoside transporter molecules with a KD value of 0.8 nmol l-1. Draflazine concentration-dependently inhibited binding of [3H]-NBTI to myocardial and erythrocyte membranes with a K(i)-value of 4.5 nmol l-1. The potency as judged from the K(i) values was ten times greater than that of dipyridamole in both myocardial and erythrocyte membranes. 4. Draflazine, at concentrations up to 100 mumol l-1, did not produce negative inotropic effects in atrial and ventricular myocardium. (-)-N6-phenylisopropyladenosine (R-PIA) and carbachol did not reduce force of contraction in ventricular myocardium, but exerted concentration-dependent direct negative inotropic effects in atrial myocardium. 5. The data provide evidence that draflazine specifically binds to the nucleoside transporter of the human heart and erythrocytes with high affinity. The compound does not produce negative inotropic effects at concentrations as high as 100 micromol 1-1.6. Draflazine could be a useful agent for cardio preservation because it does not produce cardio depressant effects. Thus, it may be possible to perfuse explanted hearts directly with this agent without the hazard of cardiodepression.

Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein-labeled ligand for the es nucleoside transporter, 5-(SAENTA-x2)-fluorescein. This paper reports the synthesis of 5-(SAENTA-x8)-fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 +/- 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5-(SAENTA-x2)-fluorescein. The fluorescence signal arising from 5-(SAENTA-x8)-fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell-bound fluorescence output of 5-(SAENTA-x8)-fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5-(SAENTA-x8)-fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples.

S49 murine lymphoma cells were examined for expression of various nucleoside transport systems using a non-metabolized nucleoside, formycin B, as substrate. Nitrobenzylthioinosine (NBTI)-sensitive, facilitated transport was the primary nucleoside transport system of the cells. The cells also expressed very low levels of NBTI-resistant, facilitated nucleoside transport as well as of Na(+)-dependent, concentrative formycin B transport. Concentrative transport was specific for uridine and purine nucleosides, just as the concentrative nucleoside transporters of other mouse and rat cells. A nucleoside transport mutant of S49 cells, AE-1, lacked both the NBTI-sensitive, facilitated and Na(+)-dependent, concentrative formycin B transport activity, but Na(+)-dependent, concentrative transport of alpha-aminoisobutyrate was not affected.

The nucleoside transporter of cerebral microvessels and choroid plexus was identified and characterized using [3H]nitrobenzylthioinosine (NBMPR) as a specific probe. [3H]NBMPR bound reversibly and with high affinity to a single specific site in particulate fractions of cerebral microvessels, choroid plexus, and cerebral cortex of the rat and the pig. The dissociation constants (KD 0.1-0.7 nM) were similar in the various tissue preparations from each species, but the maximal binding capacities (Bmax) were about fivefold higher in cerebral microvessels and choroid plexus than in the cerebral cortex. Nitrobenzylthioguanosine and dipyridamole were the most potent competitors for [3H]NBMPR binding. Several naturally occurring nucleosides displaced specific [3H]NBMPR binding to cerebral microvessels in vitro, in a rank order that correlated well with their ability to cross the blood-brain barrier in vivo. Adenosine analogues and theophylline were less effective in displacing [3H]NBMPR binding than in displacing adenosine receptor ligands. Photoactivation of cerebral microvessels and choroid plexus bound with [3H]NBMPR followed by solubilization and polyacrylamide gel electrophoresis labeled a protein(s) with a molecular weight of approximately 60,000. These results indicate that cerebral microvessels and choroid plexus have a much higher density of the nucleoside transporter moiety than the cerebral cortex and that this nucleoside transporter has pharmacological properties and a molecular weight similar to those of erythrocytes and other mammalian tissues.

BOT-2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome-mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa-65 cells to the BOT-2 cells, correcting the permease deficiency. Poly-L-ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 x 10(-5) and 1 x 10(-5), respectively. Transferants MGP-1 and MGL-1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone-induced alkaline phosphatase activity. MGP-1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL-1 remains thymidine transport-deficient and surveys on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL-1 is similar to HeLa-65, 2% of that in BOT-2, and in addition, is inducible 25-30-fold by 3 micro M hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.