Nucleoside transporters and human organic cation transporter 1 determine the cellular handling of DNA-methyltransferase inhibitors.

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.

Organic cation transporters.

Over the last 15 years, a number of transporters that translocate organic cations were characterized functionally and also identified on the molecular level. Organic cations include endogenous compounds such as monoamine neurotransmitters, choline, and coenzymes, but also numerous drugs and xenobiotics. Some of the cloned organic cation transporters accept one main substrate or structurally similar compounds (oligospecific transporters), while others translocate a variety of structurally diverse organic cations (polyspecific transporters). This review provides a survey of cloned organic cation transporters and tentative models that illustrate how different types of organic cation transporters, expressed at specific subcellular sites in hepatocytes and renal proximal tubular cells, are assembled into an integrated functional framework. We briefly describe oligospecific Na(+)- and Cl(-)-dependent monoamine neurotransmitter transporters ( SLC6-family), high-affinity choline transporters ( SLC5-family), and high-affinity thiamine transporters ( SLC19-family), as well as polyspecific transporters that translocate some organic cations next to their preferred, noncationic substrates. The polyspecific cation transporters of the SLC22 family including the subtypes OCT1-3 and OCTN1-2 are presented in detail, covering the current knowledge about distribution, substrate specificity, and recent data on their electrical properties and regulation. Moreover, we discuss artificial and spontaneous mutations of transporters of the SLC22 family that provide novel insight as to the function of specific protein domains. Finally, we discuss the clinical potential of the increasing knowledge about polymorphisms and mutations in polyspecific organic cation transporters.

Organic cation transporter capable of transporting serotonin is up-regulated in serotonin transporter-deficient mice.

The serotonin (5HT) transporter (5HTT) regulates serotonergic neurotransmission by mediating the reuptake of 5HT from the synaptic cleft. Although lacking the high affinity and selectivity of the 5HTT, the brain expresses a large number of other transporters, including the polyspecific organic cation transporters (OCTs). OCT1 and OCT3, members of the potential-sensitive organic cation transporter gene family, physiologically transport a wide spectrum of organic cations. In addition, both transporters mediate low-affinity 5HT transport and, therefore, may participate in the clearance of excessive 5HT. Because concentrations of extracellular 5HT are increased in the brain of 5HTT-deficient mice, they are a model for investigating the role of OCTs in 5HT system homeostasis. Here, we analyzed OCT1 and OCT3 gene expression in the brain of 5HTT knockout mice by semiquantitative competitive polymerase chain reaction and in situ hybridization. We demonstrate that, in 5HTT-deficient mice, OCT3 mRNA concentrations were significantly increased in the hippocampus, but not in other brain regions, including cortex, striatum, cerebellum, and brainstem. In contrast, no difference in OCT1 expression was detected between 5HTT knockout and control mice. Up-regulation of OCT3 expression and enhanced low-affinity 5HT uptake may limit the adverse effects of elevated extracellular 5HT and may play a critical role in maintaining 5HT-dependent functions of the hippocampus in the absence of 5HTT.

Downregulation of the Na(+)- D-glucose cotransporter SGLT1 by protein RS1 (RSC1A1) is dependent on dynamin and protein kinase C.

We have previously shown that the regulatory protein RS1, cloned from pig, rabbit and human (RSC1A1), is localized intracellularly and inhibits the transcription of the Na(+)- D-glucose cotransporter SGLT1 in LLC-PK(1) cells. We also reported that transport activities of human SGLT1 (hSGLT1) and human organic cation transporter hOCT2 expressed in Xenopus oocytes were decreased upon co-expression of human RS1 (hRS1). The present paper indicates that the glucose transporter GLUT1 and the peptide transporter PEPT1 are not influenced by hRS1. Voltage-clamp experiments in oocytes expressing hSGLT1 demonstrated that hRS1 reduced the maximal substrate-induced currents but did not change substrate activation, membrane potential dependence, Na(+) dependence or substrate selectivity of hSGLT1. Co-expression experiments with a dominant-negative dynamin mutant showed that the posttranslational inhibition of hSGLT1 by hRS1 was dependent on the function of dynamin. Finally, we observed that hRS1 changed the short-term effect of protein kinase C (PKC) on hSGLT1. Whereas the PKC activators phorbol-12-myristate-13-acetate (PMA) and sn-1,2-dioctanoyl glycerol (DOG) increased alpha-methyl glucose (AMG) uptake expressed by hSGLT1 alone as described earlier, PMA and DOG decreased AMG uptake mediated by hSGLT1 when hRS1 was co-expressed. Taken together, these data indicate that hRS1 modulates dynamin-dependent trafficking of intracellular vesicles containing hSGLT1 in Xenopus oocytes, and modulates PKC-dependent short-term regulation of this transporter.

The plasma membrane-associated protein RS1 decreases transcription of the transporter SGLT1 in confluent LLC-PK1 cells.

Previously we cloned RS1, a 67-kDa polypeptide that is associated with the intracellular side of the plasma membrane. Upon co-expression in Xenopus laevis oocytes, human RS1 decreased the concentration of the Na(+)-D-glucose co-transporter hSGLT1 in the plasma membrane (Valentin, M., Kühlkamp, T., Wagner, K., Krohne, G., Arndt, P., Baumgarten, K., Weber, W.-M., Segal, A., Veyhl, M., and Koepsell, H. (2000) Biochim. Biophys. Acta 1468, 367-380). Here, the porcine renal epithelial cell line LLC-PK1 was used to investigate whether porcine RS1 (pRS1) plays a role in transcriptional up-regulation of SGLT1 after confluence and in down-regulation of SGLT1 by high extracellular D-glucose concentrations. Western blots indicated a dramatic decrease of endogenous pRS1 protein at the plasma membrane after confluence but no significant effect of D-glucose. In confluent LLC-PK1 cells overexpressing pRS1, SGLT1 mRNA, protein, and methyl-alpha-D-glucopyranoside uptakes were drastically decreased; however, the reduction of methyl-alpha-D-glucopyranoside uptake after cultivation with 25 mm D-glucose remained. In confluent pRS1 antisense cells, the expression of SGLT1 mRNA and protein was strongly increased, whereas the reduction of SGLT1 expression during cultivation with high D-glucose was not influenced. Nuclear run-on assays showed that the transcription of SGLT1 was 10-fold increased in the pRS1 antisense cells. The data suggest that RS1 participates in transcriptional up-regulation of SGLT1 after confluence but not in down-regulation by D-glucose.

Interaction of cations, anions, and weak base quinine with rat renal cation transporter rOCT2 compared with rOCT1.

The rat organic cation transporter (rOCT)-2 was characterized by electrical and tracer flux measurements compared with rOCT1. By applying choline gradients to voltage-clamped Xenopus oocytes expressing rOCT2, potential-dependent currents could be induced in both directions. Tracer flux measurements with seven organic cations revealed similar Michaelis-Menten constant values for both transporters, with the exception of guanidine. In parallel experiments with rOCT2 and rOCT1, inhibition of tetraethylammonium transport by 12 cations, 2 weak bases, corticosterone, and the anions para-amminohippurate, alpha-ketoglutarate, and probenecid was characterized. The IC(50) values of many inhibitors were similar for both transporters, whereas others were significantly different. Mepiperphenidol and O-methylisoprenaline showed an approximately 70-fold lower and corticosterone a 38-fold higher affinity for rOCT2. With the use of these inhibitors together with previous information on cation transporters, experimental protocols are proposed to dissect out the individual contributions of rOCT2 and rOCT1 in intact proximal tubule preparations. Inhibition experiments at different pH levels strongly suggest that the weak base quinine passively permeates the plasma membrane at physiological pH and inhibits rOCT2 from the intracellular side.

Localization of organic cation transporters OCT1 and OCT2 in rat kidney.

Renal excretion and reabsorption of organic cations are mediated by electrogenic and electroneutral organic cation transporters, which belong to a recently discovered family of polyspecific transporters. These transporters are electrogenic and exhibit differences in substrate specificity. In rat, the renal expression of the polyspecific cation transporters rOCT1 and rOCT2 was investigated. By in situ hybridization, significant amounts of both rOCT1 and rOCT2 mRNA were detected in S1, S2, and S3 segments of proximal tubules. By immunohistochemistry, expression of the rOCT1 protein was mainly observed in S1 and S2 segments of proximal tubules, with lower expression levels in the S3 segments. At variance, rOCT2 protein was mainly expressed in the S2 and S3 segments. Both transporters were localized to the basolateral cell membrane. Neither rOCT1 nor rOCT2 was detected in the vasculature, the glomeruli, and nephron segments other than proximal tubules. The data suggest that rOCT1 and rOCT2 are responsible for basolateral cation uptake in the proximal tubule, which represents the first step in cation secretion.

Selectivity of the polyspecific cation transporter rOCT1 is changed by mutation of aspartate 475 to glutamate.

After site-directed mutagenesis, the organic cation transporter rOCT1 was expressed in Xenopus laevis oocytes or human embryonic kidney cells and functionally characterized. rOCT1 belongs to a new family of polyspecific transporters that includes transporters for organic cations and anions and the Na(+)-carnitine cotransporter. When glutamate was substituted for Asp475 (middle of the proposed 11th transmembrane alpha-helix), the V(max) values for choline, tetraethylammonium (TEA), N(1)-methylnicotinamide, and 1-methyl-4-phenylpyridinium were reduced by 89 to 98%. The apparent K(m) values were also decreased (choline by 15-fold, TEA by 8-fold, N(1)-methylnicotinamide by 4-fold) or remained constant (1-methyl-4-phenylpyridinium). After the mutation, the membrane potential dependence of the K(m) value for [(3)H]choline uptake was abolished. The affinity of n-tetraalkyl ammonium compounds to inhibit TEA uptake was increased. This affinity and its increase by the D475E mutation were increased with the length of the n-alkyl chains. After expression in X. laevis oocytes, the IC(50) ratios of wild-type and D475E mutant were 1.7 (tetramethylammonium), 4.3 (TEA), 5.0 (tetrapropylammonium), 5.0 (tetrabutylammonium), and 65 (tetrapentylammonium). Cationic inhibitors with ring structures were differentially affected: the IC(50) value for TEA inhibition by cyanine 863 remained unchanged, whereas it was increased for quinine. The data suggest that rOCT1 contains a large cation-binding pocket with several interaction domains that may be responsible for high-affinity binding of structurally different cations and that Asp475 is located close to one of these interaction domains.

Membrane localization of the electrogenic cation transporter rOCT1 in rat liver.

The polyspecific cation transporter rOCT1 in the rat was the first identified member of a new protein family with 12 presumed membrane-spanning alpha-helices and two large hydrophilic loops. Previous studies showed that rOCT1 is mainly expressed in liver and mediates electrogenic uptake of small organic cations into cells. Antibodies against partial sequences of rOCT1 were raised and their specificity was verified. Immunohistochemistry with rat liver and Western blots with isolated membranes showed that rOCT1 is localized within sinusoidal membranes of hepatocytes. Antibody reactions were also performed with intact and permeabilized human embryonic kidney cells that were stably transfected with rOCT1. They showed that the large hydrophilic loop after the first alpha-helix of rOCT1 is located extracellularly, while the C-terminus is located intracellularly. Translational regulation is suggested since the message of rOCT1 was distributed throughout the liver lobuli, whereas rOCT1 protein was observed only in hepatocytes surrounding the central veins.

Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine.

Recently, we cloned the human cation transporter hOCT2, a member of a new family of polyspecific transporters from kidney, and demonstrated electrogenic uptake of tetraethylammonium, choline, N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. Using polymerase chain reaction amplification, cDNA sequencing, in situ hybridization, and immunohistochemistry, we now show that hOCT2 message and protein are expressed in neurons of the cerebral cortex and in various subcortical nuclei. In Xenopus laevis oocytes expressing hOCT2, electrogenic transport of norepinephrine, histamine, dopamine, serotonin, and the antiparkinsonian drugs memantine and amantadine was demonstrated by tracer influx, tracer efflux, electrical measurements, or a combination. Apparent Km values of 1.9 +/- 0.6 mM (norepinephrine), 1.3 +/- 0.3 mM (histamine), 0.39 +/- 0.16 mM (dopamine), 80 +/- 20 microM (serotonin), 34 +/- 5 microM (memantine), and 27 +/- 3 microM (amantadine) were estimated. Measurement of trans-effects in depolarized oocytes and human embryonic kidney cells expressing hOCT2 suggests that there were different rates and specificities for cation influx and efflux. The hypothesis is raised that hOCT2 plays a physiological role in the central nervous system by regulating interstitial concentrations of monoamine neurotransmitters that have evaded high affinity uptake mechanisms. We show that amantadine does not interact with the expressed human Na+/Cl- dopamine cotransporter. However, concentrations of amantadine that are effective for the treatment of Parkinson's disease may increase the interstitial concentrations of dopamine and other aminergic neurotransmitters by competitive inhibition of hOCT2.

Transport of the new chemotherapeutic agent beta-D-glucosylisophosphoramide mustard (D-19575) into tumor cells is mediated by the Na+-D-glucose cotransporter SAAT1.

For beta-D-glucosylisophosphoramide mustard (beta-D-Glc-IPM), a new alkylating drug in which isophosphoramide mustard is stabilized, a higher selectivity and lower myelotoxicity was observed than for the currently used cytostatic ifosfamide. Because beta-D-Glc-IPM is hydrophilic and does not diffuse passively through the lipid bilayer, we investigated whether a transporter may be involved in the cellular uptake. A variety of cloned Na+-sugar cotransporters were expressed in Xenopus oocytes, and uptake measurements were performed. By tracer uptake and electrical measurements it was found that beta-D-Glc-IPM was transported by the low-affinity Na+-D-glucose cotransporter SAAT1, which had been cloned from pig and is also expressed in humans. At membrane potentials between -50 and -150 mV, a 10-fold higher substrate affinity (Km approximately 0.25 mM) and a 10-fold lower Vmax value were estimated for beta-D-Glc-IPM transport than for the transport of D-glucose or methyl-alpha-D-glucopyranoside (AMG). Transport of beta-D-Glc-IPM and glucose by SAAT1 is apparently performed by the same mechanism because similar sodium dependence, dependence on membrane potential, electrogenicity, and phlorizin inhibition were determined for beta-D-Glc-IPM, D-glucose, and AMG. Transcription of human SAAT1 was demonstrated in various human carcinomas and tumor cell lines. In one of these, the human carcinoma cell line T84, phlorizin inhibitable uptake of beta-D-Glc-IPM was demonstrated with substrate saturation and an apparent Km of 0.4 mM. The data suggest that the Na+-D-glucose cotransporter SAAT1 transports beta-D-Glc-IPM into human tumor cells and may accumulate the drug in the cells. They provide an example for drug targeting by employing a plasma membrane transporter.

Cloning and characterization of two human polyspecific organic cation transporters.

Previously we cloned a polyspecific transporter from rat (rOCT1) that is expressed in renal proximal tubules and hepatocytes and mediates electrogenic uptake of organic cations with different molecular structures. Recently a homologous transporter from rat kidney (rOCT2) was cloned but not characterized in detail. We report cloning and characterization of two homologous transporters from man (hOCT1 and hOCT2) displaying approximately 80% amino acid identity to rOCT1 and rOCT2, respectively. Northern blots showed that hOCT1 is mainly transcribed in liver, while hOCT2 is found in kidney. Using in situ hybridization and immunohistochemistry, expression of hOCT2 was mainly detected in the distal tubule where the transporter is localized at the luminal membrane. After expression in Xenopus laevis oocytes, hOCT1 and hOCT2 mediate tracer influx of N-1-methylnicotinamide (NMN), tetraethylammonium (TEA), and 1-methyl-4-phenylpyridinium (MPP). For cation transport by hOCT2 apparent K(m) and K(i) values were determined in tracer flux measurements. In addition, electrical measurements were performed with voltage-clamped oocytes. Similar to rOCT1, cation transport by hOCT2 was pH independent, electrogenic, and polyspecific; however, the cation specificity was different. In voltage-clamped hOCT2-expressing oocytes, inward currents were induced by superfusion with MPP, TEA, choline, quinine, d-tubocurarine, pancuronium, and cyanine863. Cation transport in distal tubules is indicated for the first time. Here hOCT2 mediates the first step in cation reabsorption. hOCT1 may participate in hepatic excretion of organic cations.

The two human organic cation transporter genes SLC22A1 and SLC22A2 are located on chromosome 6q26.

Polyspecific transporters for organic cations (OCT) belong to a new protein family which also include organic anion transporters. The first human transporters from this family (OCT1, OCT2) have been recently cloned. They translocate small cations like tetraethylammonium, choline and monoamine neurotransmitters and are involved in hepatic and renal cation excretion, respectively. We have localized the OCT1 and OCT2 genes (SLC22A1, SLC22A2) on chromosome 6q26.

Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1.

The previously cloned rat cation transporter rOCT1 detected in renal proximal tubules and hepatocytes (Gründemann, D., Gorboulev, V., Gambaryan, S., Veyhl, M., and Koepsell, H. (1994) Nature 372, 549-552) was expressed in Xenopus oocytes, and transport properties were analyzed using tracer uptake studies and electrophysiological measurements. rOCT1 induced highly active transport of a variety of cations, including the classical substrates for cation transport, such as N-1-methylnicotinamide, 1-methyl-4-phenylpyridinium (MPP), and tetraethylammonium (TEA), but also the physiologically important choline. In oocytes rOCT1 also mediated efflux of MPP, which could be trans-stimulated by MPP and TEA. Cation transport via rOCT1 was electrogenic. In voltage-clamped oocytes, transport of TEA and choline via rOCT1 produced inwardly directed currents, which were independent of extracellular ion composition or pH. The choline- and TEA-induced currents were voltage-dependent at nonsaturating concentrations, and the apparent affinity of these cations was decreased at depolarized voltages. Other substrates transported by rOCT1 were the polyamines spermine and spermidine. Interestingly, the previously described potent inhibitors of rOCT1, cyanine 863, quinine, and D-tubocurarine were substrates themselves. The data indicate that rOCT1 is an effective transport system that is responsible for electrogenic uptake of a wide variety of organic cations into epithelial cells of renal proximal tubules and hepatocytes.

Monoamine neurotransmitter transport mediated by the polyspecific cation transporter rOCT1.

The polyspecific cation transporter rOCT,1 which is localized in the basolateral membrane of rat renal proximal tubules and in sinusoidal membranes of hepatocytes, was analyzed for transport of monoamine neurotransmitters. In voltage-clamp experiments with rOCT1-expressing Xenopus oocytes, superfusion with dopamine, serotonin, noradrenaline, histamine and the permanent cation acetylcholine induced saturable inwardly directed currents with apparent Km values ranging from 20 to 100 microM. Transport of dopamine was also demonstrated by uptake measurements in oocytes and in the mammalian cell line (HEK 293) which was permanently transfected with rOCT1. The high uptake rates measured in rOCT1-expressing oocytes and in transfected HEK 293 cells suggest that rOCT1 is a high capacity transporter which mediates the first step in the excretion of monoamine neurotransmitters.

Drug excretion mediated by a new prototype of polyspecific transporter.

Cationic drugs of different types and structures (antihistaminics, antiarrhythmics, sedatives, opiates, cytostatics and antibiotics, for example) are excreted in mammals by epithelial cells of the renal proximal tubules and by hepatocytes in the liver. In the proximal tubules, two functionally disparate transport systems are involved which are localized in the basolateral and luminal plasma membrane and are different from the previously identified neuronal monoamine transporters and ATP-dependent multidrug exporting proteins. Here we report the isolation of a complementary DNA from rat kidney that encodes a 556-amino-acid membrane protein, OCT1, which has the functional characteristics of organic cation uptake over the basolateral membrane of renal proximal tubules and of organic cation uptake into hepatocytes. OCT1 is not homologous to any other known protein and is found in kidney, liver and intestine. As OCT1 translocates hydrophobic and hydrophilic organic cations of different structures, it is considered to be a new prototype of polyspecific transporters that are important for drug elimination.