Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters.

The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells' ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.

Interaction Profiles of Central Nervous System Active Drugs at Human Organic Cation Transporters 1-3 and Human Plasma Membrane Monoamine Transporter.

Many psychoactive compounds have been shown to primarily interact with high-affinity and low-capacity solute carrier 6 (SLC6) monoamine transporters for norepinephrine (NET; norepinephrine transporter), dopamine (DAT; dopamine transporter) and serotonin (SERT; serotonin transporter). Previous studies indicate an overlap between the inhibitory capacities of substances at SLC6 and SLC22 human organic cation transporters (SLC22A1-3; hOCT1-3) and the human plasma membrane monoamine transporter (SLC29A4; hPMAT), which can be classified as high-capacity, low-affinity monoamine transporters. However, interactions between central nervous system active substances, the OCTs, and the functionally-related PMAT have largely been understudied. Herein, we report data from 17 psychoactive substances interacting with the SLC6 monoamine transporters, concerning their potential to interact with the human OCT isoforms and hPMAT by utilizing radiotracer-based in vitro uptake inhibition assays at stably expressing human embryonic kidney 293 cells (HEK293) cells. Many compounds inhibit substrate uptake by hOCT1 and hOCT2 in the low micromolar range, whereas only a few substances interact with hOCT3 and hPMAT. Interestingly, methylphenidate and ketamine selectively interact with hOCT1 or hOCT2, respectively. Additionally, 3,4-methylenedioxymethamphetamine (MDMA) is a potent inhibitor of hOCT1 and 2 and hPMAT. Enantiospecific differences of R- and S-α-pyrrolidinovalerophenone (R- and S-α-PVP) and R- and S-citalopram and the effects of aromatic substituents are explored. Our results highlight the significance of investigating drug interactions with hOCTs and hPMAT, due to their role in regulating monoamine concentrations and xenobiotic clearance.

Model Analysis of the Apparent Saturation Kinetics of Purine Nucleobase Uptake in Cells co-Expressing Transporter and Metabolic Enzyme.

PURPOSE: This study aims to understand the effect of salvage enzyme activity on the saturable kinetics of facilitated cellular uptake of purine nucleobase by developing a cellular kinetic model incorporating equilibrative nucleobase transporter 1 (ENBT1) and adenine phosphoribosyltransferase (APRT), with adenine as a model nucleobase. METHODS: A cellular kinetic model incorporating the functions of ENBT1 and APRT was developed using Napp software and employed for model-based analysis of the cellular disposition of adenine. RESULTS: Simulation analysis using the developed cellular kinetic model could account for the experimentally observed time-dependent changes in the Km(app) value of adenine for ENBT1-mediated uptake. At a long experimental time, the model shows that uptake of adenine is rate-limited by APRT, enabling determination of the Km value for APRT. At early time, the rate-limiting step for adenine uptake is ENBT1-mediated transport, enabling determination of the Km value for ENBT1. Further simulations showed that the effect of experimental time on the Km(app) value for ENBT1-mediated uptake is dependent on the APRT expression level. CONCLUSION: Our findings indicate that both enzyme expression levels and experimental time should be considered when using cellular uptake studies to determine the Km values of purine nucleobases for facilitated transporters.

α-PPP and its derivatives are selective partial releasers at the human norepinephrine transporter: A pharmacological characterization of interactions between pyrrolidinopropiophenones and high and low affinity monoamine transporters.

While classical cathinones, such as methcathinone, have been shown to be monoamine releasing agents at human monoamine transporters, the subgroup of α-pyrrolidinophenones has thus far solely been characterized as monoamine transporter reuptake inhibitors. Herein, we report data from previously undescribed α-pyrrolidinopropiophenone (α-PPP) derivatives and compare them with the pharmacologically well-researched α-PVP (α-pyrrolidinovalerophenone). Radiotracer-based in vitro uptake inhibition assays in HEK293 cells show that the investigated α-PPP derivatives inhibit the human high-affinity transporters of dopamine (hDAT) and norepinephrine (hNET) in the low micromolar range, with α-PVP being ten times more potent. Similar to α-PVP, no relevant pharmacological activity was found at the human serotonin transporter (hSERT). Unexpectedly, radiotracer-based in vitro release assays reveal α-PPP, MDPPP and 3Br-PPP, but not α-PVP, to be partial releasing agents at hNET (EC50 values in the low micromolar range). Furthermore, uptake inhibition assays at low-affinity monoamine transporters, i.e., the human organic cation transporters (hOCT) 1-3 and human plasma membrane monoamine transporter (hPMAT), bring to light that all compounds inhibit hOCT1 and 2 (IC50 values in the low micromolar range) while less potently interacting with hPMAT and hOCT3. In conclusion, this study describes (i) three new hybrid compounds that efficaciously block hDAT while being partial releasers at hNET, and (ii) highlights the interactions of α-PPP-derivatives with low-affinity monoamine transporters, giving impetus to further studies investigating the interaction of drugs of abuse with OCT1-3 and PMAT.

Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells.

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

Serotonin Transporter and Plasma Membrane Monoamine Transporter Are Necessary for the Antidepressant-Like Effects of Ketamine in Mice.

Major depressive disorder is typically treated with selective serotonin reuptake inhibitors (SSRIs), however, SSRIs take approximately six weeks to produce therapeutic effects, if any. Not surprisingly, there has been great interest in findings that low doses of ketamine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, produce rapid and long-lasting antidepressant effects. Preclinical studies show that the antidepressant-like effects of ketamine are dependent upon availability of serotonin, and that ketamine increases extracellular serotonin, yet the mechanism by which this occurs is unknown. Here we examined the role of the high-affinity, low-capacity serotonin transporter (SERT), and the plasma membrane monoamine transporter (PMAT), a low-affinity, high-capacity transporter for serotonin, as mechanisms contributing to ketamine's ability to increase extracellular serotonin and produce antidepressant-like effects. Using high-speed chronoamperometry to measure real-time clearance of serotonin from CA3 region of hippocampus in vivo, we found ketamine robustly inhibited serotonin clearance in wild-type mice, an effect that was lost in mice constitutively lacking SERT or PMAT. As expected, in wild-type mice, ketamine produced antidepressant-like effects in the forced swim test. Mapping onto our neurochemical findings, the antidepressant-like effects of ketamine were lost in mice lacking SERT or PMAT. Future research is needed to understand how constitutive loss of either SERT or PMAT, and compensation that occurs in other systems, is sufficient to void ketamine of its ability to inhibit serotonin clearance and produce antidepressant-like effects. Taken together with existing literature, a critical role for serotonin, and its inhibition of uptake via SERT and PMAT, cannot be ruled out as important contributing factors to ketamine's antidepressant mechanism of action. Combined with what is already known about ketamine's action at NMDA receptors, these studies help lead the way to the development of drugs that lack ketamine's abuse potential but have superior efficacy in treating depression.

Identification of the Uptake Transporter Responsible for Distribution of Acotiamide into Stomach Tissue.

The acetylcholinesterase inhibitor, acotiamide, improves gastric motility and is clinically used to treat functional dyspepsia. The present study aimed to identify the transporters involved in the distribution of acotiamide in stomach tissue. Acotiamide uptake by the gastric cancer-derived model cell line, Hs746 T, was Na+- and pH-independent. The initial uptake velocity of acotiamide was saturable with increasing concentrations of acotiamide and was inhibited by selective serotonin reuptake inhibitors, which are potent inhibitors of the plasma membrane monoamine transporter (PMAT). The uptake of acotiamide by PMAT gene-transfected HEK293 cells was saturable, with similar Km (197.9 µM) values to those of uptake by Hs 746T cells (106 µM). Moreover, immunoreactivity of PMAT was found in the gastric smooth muscle and vascular endothelial cells. These results suggest that PMAT contributes to the distribution of acotiamide in the stomach, where it exerts its pharmacological effects.

Serotonin uptake via plasma membrane monoamine transporter during myocardial ischemia-reperfusion in the rat heart in vivo.

Serotonin (5-HT) accumulates in the heart during myocardial ischemia and induces deleterious effects on the cardiomyocytes through receptor-dependent and monoamine oxidase-dependent pathways. We aimed to clarify the involvement of extra-neuronal monoamine transporters in the clearance of 5-HT during ischemia and reperfusion in the heart. Using a microdialysis technique in the anesthetized Wistar rat heart, we monitored myocardial interstitial 5-HT and 5-hydroxyindole acetic acid (5-HIAA) concentration by means of electro-chemical detection coupled with high-performance liquid chromatography (HPLC-ECD). Effects of inhibitors of the plasma membrane monoamine transporter (PMAT) and the organic cation transporter 3 (OCT3) (decynium-22 and corticosterone) on the 5-HT and 5-HIAA concentrations during baseline, coronary occlusion, and reperfusion were investigated. Basal dialysate 5-HT concentration were increased by local administration of decynium-22, but not by corticosterone. Addition of fluoxetine, a serotonin transporter (SERT) inhibitor further increased the 5-HT concentration upon during administration of decynium-22. Decynium-22 elevated the background level of 5-HT during coronary occlusion and maintained 5-HT concentration at a high level during reperfusion. Production of 5-HIAA in the early reperfusion was significantly suppressed by decynium-22. These results indicate that PMAT and SERT independently regulate basal level of interstitial 5-HT, and PMAT plays a more important role in the clearance of 5-HT during reperfusion. These data suggest the involvement of PMAT in the monoamine oxidase-dependent deleterious pathway in the heart.

Bidirectional transport of 2-chloroadenosine by equilibrative nucleoside transporter 4 (hENT4): Evidence for allosteric kinetics at acidic pH.

Adenosine has been reported to be transported by equilibrative nucleoside transporter 4 (ENT4), encoded by the SLC29A4 gene, in an acidic pH-dependent manner. This makes hENT4 of interest as a therapeutic target in acidic pathologies where adenosine is protective (e.g. vascular ischaemia). We examined the pH-sensitivity of nucleoside influx and efflux by hENT4 using a recombinant transfection model that lacks the confounding influences of other nucleoside transporters (PK15-NTD). We established that [3H]2-chloroadenosine, which is resistant to metabolism by adenosine deaminase, is a substrate for hENT4. Transport of [3H]2-chloroadenosine at a pH of 6.0 in PK15-NTD cells stably transfected with SLC29A4 was biphasic, with a low capacity (Vmax ~ 30 pmol/mg/min) high-affinity component (Km ~ 50 µM) apparent at low substrate concentrations, which shifted to a high capacity (Vmax ~ 500 pmol/mg/min) low affinity system (Km > 600 µM) displaying positive cooperativity at concentrations above 200 µM. Only the low affinity component was observed at a neutral pH of 7.5 (Km ~ 2 mM). Efflux of [3H]2-chloroadenosine from these cells was also enhanced by more than 4-fold at an acidic pH. Enhanced influx and efflux of nucleosides by hENT4 under acidic conditions supports its potential as a therapeutic target in pathologies such as ischaemia-reperfusion injury.

Mechanisms of gemcitabine oral absorption as determined by in situ intestinal perfusions in mice.

Gemcitabine is a widely used chemotherapeutic drug that is administered via intravenous infusion due to a low oral bioavailability of only 10%. This low oral bioavailability is believed to be the result of gemcitabine's low intestinal permeability and oral absorption, followed by significant presystemic metabolism. In the present study, we sought to define the mechanisms of gemcitabine intestinal permeability, the potential for saturation of intestinal uptake, and the transporter(s) responsible for mediating the oral absorption of drug using in situ single-pass intestinal perfusions in mice. Concentration-dependent studies were performed for gemcitabine over 0.5-2000 µM, along with studies of 5 µM gemcitabine in a sodium-containing buffer ±â€¯thymidine (which can inhibit concentrative (i.e., CNT1 and CNT3) and equilibrative (i.e., ENT1 and ENT2) nucleoside transporters) or dilazep (which can inhibit ENT1 and ENT2), or in a sodium-free buffer (which can inhibit CNT1 and CNT3). Our findings demonstrated that gemcitabine was, in fact, a high-permeability drug in the intestine at low concentrations, that jejunal uptake of gemcitabine was saturable and mediated almost exclusively by nucleoside transporters, and that jejunal flux was mediated by both high-affinity, low-capacity (Km = 27.4 µM, Vmax = 3.6 pmol/cm2/s) and low-affinity, high-capacity (Km = 700 µM, Vmax = 35.9 pmol/cm2/s) transport systems. Thus, CNTs and ENTs at the apical membrane allow for gemcitabine uptake from the lumen to enterocyte, whereas ENTs at the basolateral membrane allow for gemcitabine efflux from the enterocyte to portal venous blood.

Metformin Triggers PYY Secretion in Human Gut Mucosa.

CONTEXT: The antidiabetic drug metformin causes weight loss, but the underlying mechanisms are unclear. Recent clinical studies show that metformin increases plasma levels of the anorectic gut hormone, peptide YY (PYY), but whether this is through a direct effect on the gut is unknown. OBJECTIVE: We hypothesized that exposure of human gut mucosal tissue to metformin would acutely trigger PYY secretion. DESIGN, SETTING, PARTICIPANTS, AND INTERVENTIONS: Mucosal tissue was prepared from 46 human colonic and 9 ileal samples obtained after surgical resection and ex vivo secretion assays were performed. Tissue was exposed to metformin, as well as a series of other compounds as part of our mechanistic studies, in static incubations. Supernatant was sampled after 15 minutes. MAIN OUTCOME MEASURES: PYY levels in supernatant, measured using ELISA. RESULTS: Metformin increased PYY secretion from both ileal (P < 0.05) and colonic (P < 0.001) epithelia. Both basal and metformin-induced PYY secretion were unchanged across body mass index or in tissues obtained from individuals with type 2 diabetes. Metformin-dependent PYY secretion was blocked by inhibitors of the plasma membrane monoamine transporter (PMAT) and the serotonin reuptake transporter (SERT), as well as by an inhibitor of AMP kinase (AMPK). CONCLUSIONS: This is a report of a direct action of metformin on the gut epithelium to trigger PYY secretion in humans, occurring via cell internalization through PMAT and SERT and intracellular activation of AMPK. Our results provide further support that the role of metformin in the treatment of metabolic syndrome has a gut-based component.

Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter.

Growing evidence supports involvement of low-affinity/high-capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium-22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for α1-adrenoceptors than D22 and showed antidepressant-like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [3H]methyl-4-phenylpyridinium acetate in overexpressed HEK cells and [3H]citalopram, [3H]nisoxetine and [3H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant-like effects in mice and help account for our previous findings that D22 lacks antidepressant-like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.

Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche.

BACKGROUND: Trypanosoma cruzi, the causative agent of Chagas' disease is unable to synthesise its own purines and relies on salvage from the host. In other protozoa, purine uptake has been shown to be mediated by Equilibrative Nucleoside Transporters (ENTs). METHODS: To investigate the functionality of T. cruzi-encoded ENT transporters, its four putative ENT genes (TcrNB1, TcrNB2, TcrNT1 and TcrNT2) were cloned and expressed in genetically adapted Trypanosoma brucei procyclic cells from which the nucleobase transporter locus was deleted. RESULTS: TcrNB1 displayed very high affinity for hypoxanthine (Km 93.8 ±â€¯4.7 nM for) and guanine, and moderate affinity for adenine. TcrNT1 was found to be a high-affinity guanosine/inosine transporter (inosine Km is 1.0 ±â€¯0.03 µM; guanosine Ki is 0.92 ±â€¯0.2 µM). TcrNT2 encoded a high-affinity thymidine transporter (Km = 223.5 ±â€¯7.1 nM) with a clear preference for 2'-deoxypyrimidines. TcrNB2, whose activity could not be determined in our system, could be a low-affinity purine nucleobase transporter, given its sequence and predicted structural similarities to Leishmania major NT4. All 4 transporter genes were highly expressed in the amastigote forms, with much lower expression in the non-dividing stages. CONCLUSIONS: The data appear to show that, surprisingly, T. cruzi has a preference for oxopurines over aminopurines and efficiently transports 2'-deoxypyrimidines. The T. cruzi ENTs display exceptionally high substrate affinity as an adaptation to their intracellular localisation. GENERAL SIGNIFICANCE: This study reports the first cloning of T. cruzi purine and pyrimidine transporters, including the first gene encoding a pyrimidine-selective protozoan transporter.

[Analysis of brain histamine clearance using genetically engineered mice].

Histamine acts as a neurotransmitter to regulate various physiological functions in CNS. Recent reports showed the involvement of histaminergic dysfunction in neurological disorders. Neurotransmitter clearance is essential to determine brain neurotransmitter concentration. However, molecular mechanism of brain histamine clearance remains largely unknown. First, we examined the molecular mechanism of histamine clearance in primary human astrocytes. We demonstrated that extracellular histamine was transported through organic cation transporter (OCT) 3 and plasma membrane monoamine transporter (PMAT), and subsequently intracellular histamine was inactivated by histamine N-methyltransferase (HNMT) in cytosol. Next, we generated HNMT knockout (HNMT KO) mice to investigate the role of HNMT in vivo. HNMT deficiency dramatically enhanced brain histamine concentration, indicating the important role of HNMT in histamine inactivation. HNMT KO mice showed high aggression via abnormal histamine H2 receptor (H2R) activation and the disrupted sleep-wake cycle via excessive H1R activation. These observations show that HNMT plays a pivotal role in regulating brain histamine concentration, and modulates aggression as well as the sleep-wake cycle. Although importance of OCT3 and PMAT in histaminergic nervous system remains still unknown, our preliminary data show the contribution of PMAT to brain histamine concentration. We also try to find novel inhibitors targeting brain histamine clearance. We hope our study could lead a better understanding of neuropsychiatric disorders and the development of new drugs inhibiting HNMT, OCT3 and PMAT activity.

Role of adenosine signaling in coordinating cardiomyocyte function and coronary vascular growth in chronic fetal anemia.

Fetal anemia causes rapid and profound changes in cardiac structure and function, stimulating proliferation of the cardiac myocytes, expansion of the coronary vascular tree, and impairing early contraction and relaxation. Although hypoxia-inducible factor-1α is sure to play a role, adenosine, a metabolic byproduct that increases coronary flow and growth, is implicated as a major stimulus for these adaptations. We hypothesized that genes involved in myocardial adenosine signaling would be upregulated in chronically anemic fetuses and that calcium-handling genes would be downregulated. After sterile surgical instrumentation under anesthesia, gestationally timed fetal sheep were made anemic by isovolumetric hemorrhage for 1 wk (16% vs. 35% hematocrit). At 87% of gestation, necropsy was performed to collect heart tissue for PCR and immunohistochemical analysis. Anemia increased mRNA expression levels of adenosine receptors ADORA 1, ADORA2A, and ADORA2B in the left and right ventricles (adenosine receptor ADORA3 was unchanged). In both ventricles, anemia also increased expression of ectonucleoside triphosphate diphosphohydrolase 1 and ecto-5'-nucleotidase. The genes for both equilibrative nucleoside transporters 1 and 2 were expressed more abundantly in the anemic right ventricle but were not different in the left ventricle. Neither adenosine deaminase nor adenosine kinase cardiac levels were significantly changed by chronic fetal anemia. Chronic fetal anemia did not significantly change cardiac mRNA expression levels of the voltage-dependent L-type calcium channel, ryanodine receptor 1, sodium-calcium exchanger, sarcoplasmic/endoplasmic reticulum calcium transporting ATPase 2, phospholamban, or cardiac calsequestrin. These data support local metabolic integration of vascular and myocyte function through adenosine signaling in the anemic fetal heart.

Expression of Concentrative Nucleoside Transporters ( SLC28A) in the Human Placenta: Effects of Gestation Age and Prototype Differentiation-Affecting Agents.

Equilibrative ( SLC29A) and concentrative ( SLC28A) nucleoside transporters contribute to proper placental development and mediate uptake of nucleosides/nucleoside-derived drugs. We analyzed placental expression of SLC28A mRNA during gestation. Moreover, we studied in choriocarcinoma-derived BeWo cells whether SLC29A and SLC28A mRNA levels can be modulated by activity of adenylyl cyclase, retinoic acid receptor activation, CpG islands methylation, or histone acetylation, using forskolin, all- trans-retinoic acid, 5-azacytidine, and sodium butyrate/sodium valproate, respectively. We found that expression of SLC28A1, SLC28A2, and SLC28A3 increases during gestation and reveals considerable interindividual variability. SLC28A2 was shown to be a dominant subtype in the first-trimester and term human placenta, while SLC28A1 exhibited negligible expression in the term placenta only. In BeWo cells, we detected mRNA of SLC28A2 and SLC28A3. Levels of the latter were affected by 5-azacytidine and all- trans-retinoic acid, while the former was modulated by sodium valproate (but not sodium butyrate), all- trans-retinoic acid, 5-azacytidine, and forskolin that caused 25-fold increase in SLC28A2 mRNA; we documented by analysis of syncytin-1 that the observed changes in SLC28A expression do not correlate with the morphological differentiation state of BeWo cells. Upregulated SLC28A2 mRNA was reflected in elevated uptake of [3H]-adenosine, high-affinity substrate of concentrative nucleoside transporter 2. Using KT-5720 and inhibitors of phosphodiesterases, we subsequently confirmed importance of cAMP/protein kinase A pathway in SLC28A2 regulation. On the other hand, SLC29A genes exhibited constitutive expression and none of the tested compounds increased SLC28A1 expression to detectable levels. In conclusion, we provide the first evidence that methylation status and activation of retinoic acid receptor affect placental SLC28A2 and SLC28A3 transcription and substrates of concentrative nucleoside transporter 2 might be taken up in higher extent in placentas with overactivated cAMP/protein kinase A pathway and likely in the term placenta.

Functional Characterization of the Saccharomyces cerevisiae Equilibrative Nucleoside Transporter 1 (ScENT1).

Equilibrative nucleoside transporters (ENTs) are polytopic membrane transporters responsible for the translocation of nucleosides, nucleobases-to a lesser extent-and nucleoside analog therapeutics across cellular membranes. ENTs function in a diffusion controlled bidirectional manner and are thought to utilize an alternating access transport mechanism. However, a detailed understanding of ENT function at the molecular level has remained elusive. ScENT1 (formerly known as Function Unknown Now 26 or FUN26) is the only known ENT ortholog endogenously expressed in S. cerevisiae, and a proteoliposome assay system was used to study homogenously overexpressed and purified ScENT1 (wildtype relative to L390A and F249I mutants). L390 and F249 are highly conserved residues and were found to alter transporter function. L390A produced a reduction of mean transport activity while F249I increased mean substrate translocation relative to wildtype protein. However, both mutations resulted in transport of UTP-a novel gain of function for any ENT. These residues were then mapped onto an ab initio model of FUN26 which suggests they function in substrate translocation (L390) or cytoplasmic gating (F249). Furthermore, wildtype, L390A, and F249I were found to be sensitive to the presence of alcohols. Ethanol attenuated ScENT1-mediated transport of uridine by ~50%. These findings further demonstrate functional similarities between ScENT1 and human ENT isoforms and support identification of FUN26 as ScENT1, the first ENT isoform in S. cerevisiae.

Role of nucleoside transporters in transplacental pharmacokinetics of nucleoside reverse transcriptase inhibitors zidovudine and emtricitabine.

INTRODUCTION: Zidovudine (AZT) and emtricitabine (FTC) are effective and well tolerated antiretroviral drugs, routinely used in the prevention of perinatal HIV transmission. However, precise mechanism(s) involved in their transfer from mother to fetus are not fully elucidated. Since both drugs are nucleoside analogues, we hypothesized that the mechanisms of their transplacental passage might include equilibrative nucleoside transporters, ENT1 and/or ENT2. METHODS: To address this issue, we performed in vitro accumulation assays in the BeWo placental trophoblast cell line, ex vivo uptake studies in fresh villous fragments isolated from human placenta and in situ dually perfused rat term placenta experiments. RESULTS: Applying this complex array of methods, we did not prove that ENTs play a significant role in transfer of AZT or FTC across the placenta. DISCUSSION: We conclude that the transplacental passage of AZT and FTC is independent of ENTs. Disposition of either compound into the fetal circulation should thus not be affected by ENT-mediated drug-drug interactions or placental expression of the transporters.

Pharmacological and physiological assessment of serotonin formation and degradation in isolated preparations from mouse and human hearts.

Using transgenic (TG) mice that overexpress the human serotonin (5-HT)4a receptor specifically in cardiomyocytes, we wanted to know whether 5-HT can be formed and degraded in the mammalian heart and whether this can likewise lead to inotropic and chronotropic effects in this TG model. We noted that the 5-HT precursor 5-hydroxy-tryptophan (5-HTP) can exert inotropic and chronotropic effects in cardiac preparations from TG mice but not from wild-type (WT) mice; similar results were found in human atrial preparations as well as in intact TG animals using echocardiography. Moreover, by immunohistochemistry we could detect 5-HT metabolizing enzymes and 5-HT transporters in mouse hearts as well as in human atria. Hence, in the presence of an inhibitor of aromatic l-amino acid decarboxylase, the positive inotropic effects of 5-HTP were absent in TG and isolated human atrial preparations, and, moreover, inhibitors of enzymes involved in 5-HT degradation enhanced the efficacy of 5-HT in TG atria. A releaser of neurotransmitters increased inotropy in the isolated TG atrium, and this effect could be blocked by a 5-HT4a receptor antagonist. Fluoxetine, an inhibitor of 5-HT uptake, elevated the potency of 5-HT to increase contractility in the TG atrium. In addition, inhibitors of organic cation and monoamine transporters apparently reduced the positive inotropic potency of 5-HT in the TG atrium. Hence, we tentatively conclude that a local production and degradation of 5-HT in the mammalian heart and more specifically in mammalian myocytes probably occurs. Conceivably, this formation of 5-HT and possibly impaired degradation may be clinically relevant in cases of unexplained tachycardia and other arrhythmias.NEW & NOTEWORTHY The present work suggests that inotropically active serotonin (5-HT) can be formed in the mouse and human heart and probably by cardiomyocytes themselves. Moreover, active degradation of 5-HT seems to occur in the mammalian heart. These findings may again increase the interest of researchers for cardiac effects of 5-HT.

Molecular Basis of Nucleobase Transport Systems in Mammals.

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