Role of SLC22A1 polymorphic variants in drug disposition, therapeutic responses, and drug-drug interactions.

The SCL22A1 gene encodes the broad selectivity transporter hOCT1. hOCT1 is expressed in most epithelial barriers thereby contributing to drug pharmacokinetics. It is also expressed in different drug target cells, including immune system cells and others. Thus, this membrane protein might also contribute to drug pharmacodynamics. Up to 1000 hOCT1 polymorphisms have been identified so far, although only a small fraction of those have been mechanistically studied. A paradigm in the field of drug transporter pharmacogenetics is the impact of hOCT1 gene variability on metformin clinical parameters, affecting area under the concentration-time curve, Cmax and responsiveness. However, hOCT1 also mediates the translocation of a variety of drugs used as anticancer, antiviral, anti-inflammatory, antiemetic agents as well as drugs used in the treatment of neurological diseases among. This review focuses exclusively on those drugs for which some pharmacogenetic data are available, and aims at highlighting the need for further clinical research in this area.

Human organic cation transporter 1 (hOCT1) as a mediator of bendamustine uptake and cytotoxicity in chronic lymphocytic leukemia (CLL) cells.

Bendamustine is used in the treatment of chronic lymphocytic leukemia (CLL). Routes for bendamustine entry into target cells are unknown. This study aimed at identifying transporter proteins implicated in bendamustine uptake. Our results showed that hOCT1 is a bendamustine transporter, as bendamustine could cis-inhibit the uptake of a canonical hOCT1 substrate, with a Ki in the micromolar range, consistent with the EC50 values of the cytotoxicity triggered by this drug in HEK293 cells expressing hOCT1. hOCT1 polymorphic variants determining impaired bendamustine-transporter interaction, consistently reduced bendamustine cytotoxicity in HEK293 cells stably expressing them. Exome genotyping of the SLC22A1 gene, encoding hOCT1, was undertaken in a cohort of 241 CLL patients. Ex vivo cytotoxicity to bendamustine was measured in a subset of cases and shown to correlate with SLC22A1 polymorphic variants. In conclusion, hOCT1 is a suitable bendamustine transporter, thereby contributing to its cytotoxic effect depending upon the hOCT1 genetic variants expressed.

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.

Predominant role of active versus facilitative glucose transport for glucagon-like peptide-1 secretion.

AIMS/HYPOTHESIS: Several glucose-sensing pathways have been implicated in glucose-triggered secretion of glucagon-like peptide-1 (GLP-1) from intestinal L cells. One involves glucose metabolism and closure of ATP-sensitive K(+) channels, and another exploits the electrogenic nature of Na(+)-coupled glucose transporters (SGLTs). This study aimed to elucidate the role of these distinct mechanisms in glucose-stimulated GLP-1 secretion. METHODS: Glucose uptake into L cells (either GLUTag cells or cells in primary cultures, using a new transgenic mouse model combining proglucagon promoter-driven Cre recombinase with a ROSA26tdRFP reporter) was monitored with the FLII(12)Pglu-700 µÎ´6 glucose sensor. Effects of pharmacological and genetic interference with SGLT1 or facilitative glucose transport (GLUT) on intracellular glucose accumulation and metabolism (measured by NAD(P)H autofluorescence), cytosolic Ca(2+) (monitored with Fura2) and GLP-1 secretion (assayed by ELISA) were assessed. RESULTS: L cell glucose uptake was dominated by GLUT-mediated transport, being abolished by phloretin but not phloridzin. NAD(P)H autofluorescence was glucose dependent and enhanced by a glucokinase activator. In GLUTag cells, but not primary L cells, phloretin partially impaired glucose-dependent secretion, and suppressed an amplifying effect of glucose under depolarising high K(+) conditions. The key importance of SGLT1 in GLUTag and primary cells was evident from the impairment of secretion by phloridzin or Sglt1 knockdown and failure of glucose to trigger cytosolic Ca(2+) elevation in primary L cells from Sglt1 knockout mice. CONCLUSIONS/INTERPRETATION: SGLT1 acts as the luminal glucose sensor in L cells, but intracellular glucose concentrations are largely determined by GLUT activity. Although L cell glucose metabolism depends partially on glucokinase activity, this plays only a minor role in glucose-stimulated GLP-1 secretion.

Nitric oxide-induced regulation of renal organic cation transport after renal ischemia-reperfusion injury.

Renal organic cation transporters are downregulated by nitric oxide (NO) in rat endotoxemia. NO generated by inducible NO synthase (iNOS) is substantially increased in the renal cortex after renal ischemia-reperfusion (I/R) injury. Therefore, we investigated the effects of iNOS-specific NO inhibition on the expression of the organic cation transporters rOct1 and rOct2 (Slc22a1 and Slc22a2, respectively) after I/R injury both in vivo and in vitro. In vivo, N(6)-(1-iminoethyl)-L-lysine (L-NIL) completely inhibited NO generation after I/R injury. Moreover, L-NIL abolished the ischemia-induced downregulation of rOct1 and rOct2 as determined by qPCR and Western blotting. Functional evidence was obtained by measuring the fractional excretion (FE) of the endogenous organic cation serotonin. Concordant with the expression of the rate-limiting organic cation transporter, the FE of serotonin decreased after I/R injury and was totally abolished by L-NIL. In vitro, ischemia downregulated both rOct1 and rOct2, which were also abolished by L-NIL; the same was true for the uptake of the organic cation MPP. We showed that renal I/R injury downregulates rOct1 and rOct2, which is most probably mediated via NO. In principle, this may be an autocrine effect of proximal tubular epithelial cells. We conclude that rOct1, or rOct1 and rOct2 limit the rate of the renal excretion of serotonin.

ABC-transporter gene-polymorphisms are potential pharmacogenetic markers for mitoxantrone response in multiple sclerosis.

Escalation therapy with mitoxantrone (MX) in highly active multiple sclerosis is limited by partially dose-dependent side-effects. Predictors of therapeutic response may result in individualized risk stratification and MX dosing. ATP-binding cassette-transporters ABCB1 and ABCG2 represent multi-drug resistance mechanisms involved in active cellular MX efflux. Here, we investigated the role of ABC-gene single nucleotide polymorphisms (SNPs) for clinical MX response, corroborated by experimental in vitro and in vivo data. Frequencies of ABCB1 2677G>T, 3435C>T and five ABCG2-SNPs were analysed in 832 multiple sclerosis patients (Germany, Spain) and 264 healthy donors. Using a flow-cytometry-based in vitro assay, MX efflux in leukocytes from individuals with variant alleles in both ABC-genes (designated genotype ABCB1/ABCG2-L(ow), 22.2% of patients) was 37.7% lower than from individuals homozygous for common alleles (ABCB1/ABCG2-H(igh), P < 0.05, 14.8% of patients), resulting in genotype-dependent MX accumulation and cell death. Addition of glucocorticosteroids (GCs) inhibited MX efflux in vitro. ABC-transporters were highly expressed in leukocyte subsets, glial and neuronal cells as well as myocardium, i.e. cells/tissues potentially affected by MX therapy. In vivo significance was further corroborated in experimental autoimmune encephalomyelitis in Abcg2(-/-) animals. Using a MX dose titrated to be ineffective in wild-type animals, disease course and histopathology in Abcg2(-/-) mice were strongly ameliorated. Retrospective clinical analysis in MX monotherapy patients (n = 155) used expanded disability status scale, relapse rate and multiple sclerosis functional composite as major outcome parameters. The clinical response rate [overall 121 of 155 patients (78.1%)] increased significantly with genotypes associated with decreasing ABCB1/ABCG2-function [ABCB1/ABCG2-H 15/24 (62.5%) responders, ABCB1/ABCG2-I(ntermediate) 78/98 (79.6%), ABCB1/ABCG2-L 28/33 (84.8%), exact Cochran-Armitage test P = 0.039]. The odds ratio for response was 1.9 (95% CI 1.0-3.5) with each increase in ABCB1/ABCG2 score (from ABCB1/ABCG2-H to -I-, and -I to -L). In 36 patients with severe cardiac or haematological side effects no statistically relevant difference in genotype frequency was observed. However, one patient with biopsy proven cardiomyopathy only after 24 mg/m2 MX exhibited a rare genotype with variant, partly homozygous alleles in 3 ABC-transporter genes. In conclusion, SNPs in ABC-transporter genes may serve as pharmacogenetic markers associated with clinical response to MX therapy in multiple sclerosis. Combined MX/GC-treatment warrants further investigation.

The effects of genetic polymorphisms in the organic cation transporters OCT1, OCT2, and OCT3 on the renal clearance of metformin.

Organic cation transporters (OCTs) can mediate metformin transmembrane transport. We explored metformin pharmacokinetics in relation to genetic variations in OCT1, OCT2, OCT3, OCTN1, and MATE1 in 103 healthy male Caucasians. Renal clearance varied 3.8-fold and was significantly dependent on creatinine clearance (r(2) = 0.42, P < 0.0001), age (r(2) = 0.09, P = 0.002), and OCT1 polymorphisms. Carriers of zero, one, and two low-activity OCT1 alleles (Arg61Cys, Gly401Ser, 420del, or Gly465Arg) had mean renal clearances of 30.6, 33.1, and 37.1 l/h, respectively (P = 0.04, after adjustment for creatinine clearance and age). Immunohistochemical staining of human kidneys demonstrated OCT1 expression on the apical side of proximal and distal tubules. Increased renal clearance, in parallel with the known decreased hepatic uptake, may contribute to reduced metformin efficacy in low-activity genotypes. Renal OCT1 expression may be important not only in relation to metformin but with respect to other drugs as well.

Protein kinase C-dependent trafficking of glutamate transporters excitatory amino acid carrier 1 and glutamate transporter 1b in cultured cerebellar granule cells.

Previous data showed that cell surface expression of the glutamate transporters GLT1a and excitatory amino acid carrier 1 (EAAC1), localized in glia and neurons of the CNS, can be regulated by protein kinase C (PKC). Regulation and physiological importance of GLT1b, a splice variant of GLT1a, is not understood. In the present study we used cultured cerebellar granule cells (CGCs) from mice to investigate PKC dependent trafficking of GLT1b in comparison to GLT1a and EAAC1 using immunohistochemistry and subcellular fractionation followed by Western blotting. In neurites of CGCs, GLT1b and EAAC1 were localized to different aggregates of vesicles that were different from vesicle aggregates containing vesicular glutamate transporters. In CGCs cultured with low-potassium medium, stimulation of PKC by phorbol ester enhanced the formation of large varicosities in neurites that exhibited immunoreactivity for GLT1a, GLT1b, and EAAC1. Stimulation of PKC leads to a significant increase of GLT1b and EAAC1 in the plasma membrane whereas GLT1a in the plasma membrane was decreased. Following PKC stimulation, also a significant increase of transporter-mediated glutamate uptake representing sodium dependent glutamate uptake, was observed. Similarly, the fraction of glutamate uptake, that was sensitive to the inhibitor WAY-213613 and represents uptake by GLT1a and GLT1b, was increased after stimulation by PKC. The findings suggest that PKC is similarly involved in regulation of surface trafficking of GLT1b and EAAC1 and that PKC stimulated increase in surface location of GLT1b and EAAC1 in glutamatergic CGCs.

Calmodulin-associated post-translational regulation of rat organic cation transporter 2 in the kidney is gender dependent.

In this work, regulation of organic cation transporter type 2 from rat (rOCT2) stably transfected in HEK293 cells was investigated by microfluorimetry with 4-(4-(dimethylamino)styryl)-N-methylpyridinium as substrate. The transport mediated by rOCT2 was specifically stimulated by PKA, phosphatidylinositol-3-kinase, p56(lck) tyrosine kinase, mitogen-extracellular-signal-regulated-kinase-1/2, calmodulin (CaM), and CaM-kinase-II. The regulatory pattern of rOCT2 differs markedly quantitatively and qualitatively from that of other OCT isoforms. Only CaM-dependent upregulation is conserved throughout the OCT family. For this reason, CaM regulation of rOCT2 was also investigated in isolated S3-segments (known to express only rOCT2) of male and female rat proximal tubules. Inhibition of CaM by calmidazolium significantly decreased rOCT2 activity (-49.0 +/- 13.6%, n = 4) in male but not female (9.0 +/- 13.0%, n = 4) rats. Real-time PCR and Western blot investigations of CaM expression in rat kidneys showed that male animals have significantly higher CaM expression. This is the first study describing post-translational gender-dependent rOCT2 regulation.

In vivo two-photon fluorescence microscopy opens a new area for investigation of the excretion of cationic drugs in the kidney.

Polyspecific transporters mediate excretion and reabsorption of organic cations in kidney. With in vivo two-photon fluorescence microscopy, excretion and reabsorption of a fluorescent cation in rat renal proximal tubules was resolved. In combination with specific inhibitors, the contribution of individual cation transporters can be determined.

Molecular evidence of organic ion transporters in the rat adrenal cortex with adrenocorticotropin-regulated zonal expression.

Experimental evidence suggested that secretion of steroid hormones from adrenocortical cells involves carrier-mediated transport: Cortisol release from, and uptake of p-[3H]aminohippurate into, bovine adrenocortical cells showed properties of the renal p-[3H]aminohippurate/anion exchanger OAT1. Other poly-specific transporters such as organic anion-transporting polypeptides (oatps) and organic cation transporters (OCTs) could also be involved in steroid hormone release. A homology-cloning procedure was established to detect these transporters in rat adrenal gland cDNA. PCR revealed the presence of OAT1, oatp1, oatp2, and oatp3. In situ hybridization localized OAT1 in the outer zona fasciculata, oatp3 in the zona glomerulosa, and oatp1 and oatp2 in the inner zona fasciculata and outer zona reticularis. An OCT2-specific probe produced signals in the zona glomerulosa and outer zona fasciculata. Pretreatment of rats with ACTH increased the expression of OAT1 mRNA that spread to all zones, and hypophysectomy strongly decreased it. A less pronounced regulation was detected for OCT2 and oatp3. Specific antibodies confirmed the localization of OAT1 in the outer zona fasciculata, supporting a possible role of OAT1 in cortisol release. The zonated distribution of transporters furthermore suggest that oatp1-3 and OCT2 may be important for the endocrine function of rat adrenocortical cells.

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.

Drug uptake systems in liver and kidney.

The hepatobiliary system and the kidneys are the main routes by which drugs and their metabolites leave the body. Compounds that are mainly excreted into bile in general have relatively high molecular weights, are amphipathic and highly bound to plasma proteins. In contrast, compounds that are predominantly excreted into urine have relatively low molecular weights, are more hydrophilic and generally less protein bound. The first step in drug elimination in liver and kidney is uptake into hepatocytes or into proximal tubular cells. The substrate specificity and affinity of the uptake carriers expressed at the basolateral membranes of hepatocytes and proximal tubular cells could therefore play an important role for the determination of the main elimination route of a compound. This review discusses the tissue distribution, substrate specificity, transport mechanism, and regulation of the members of the organic anion transporting polypeptide (Oatp/OATP) superfamily (solute carrier family SLC21A) and the SLC22A family containing transporters for organic cations (OCTs) and organic anions (OATs). The Oatps/OATPs are mainly important for the hepatic uptake of large amphipathic organic anions, organic cations and uncharged substrates, whereas OCTs and OATs mediate uptake of predominantly small organic cations and anions in liver and kidney.

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 organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP.

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.

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.

Maintenance of serotonin in the intestinal mucosa and ganglia of mice that lack the high-affinity serotonin transporter: Abnormal intestinal motility and the expression of cation transporters.

The enteric serotonin reuptake transporter (SERT) has been proposed to play a critical role in serotonergic neurotransmission and in the initiation of peristaltic and secretory reflexes. We analyzed potential compensatory mechanisms and enteric function in the bowels of mice with a targeted deletion of SERT. The guts of these animals were found to lack mRNA encoding SERT; moreover, high-affinity uptake of 5-HT into epithelial cells, mast cells, and enteric neurons was present in the SERT +/+ bowel but absent in the SERT -/- bowel. However, both the SERT +/+ gut and the -/- gut expressed molecules capable of transporting 5-HT, but with affinities and selectivity much lower than those of SERT. These included the dopamine transporter (DAT) and polyspecific organic cation transporters OCT-1 and OCT-3. DAT and OCT immunoreactivities were present in both the submucosal and myenteric plexuses, and the OCTs were also located in the mucosal epithelium. 5-HT was found in all of its normal sites in the SERT -/- bowel, which contained mRNA encoding tryptophan hydroxylase, but no 5-HT was present in the blood of SERT -/- animals. Stool water and colon motility were increased in most SERT -/- animals; however, the increase in motility (diarrhea) occasionally alternated irregularly with decreased motility (constipation). The watery diarrhea is probably attributable to the potentiation of serotonergic signaling in SERT -/- mice, whereas the transient constipation may be caused by episodes of enhanced 5-HT release leading to 5-HT receptor desensitization.

Reduced hepatic uptake and intestinal excretion of organic cations in mice with a targeted disruption of the organic cation transporter 1 (Oct1 [Slc22a1]) gene.

The polyspecific organic cation transporter 1 (OCT1 [SLC22A1]) mediates facilitated transport of small (hydrophilic) organic cations. OCT1 is localized at the basolateral membrane of epithelial cells in the liver, kidney, and intestine and could therefore be involved in the elimination of endogenous amines and xenobiotics via these organs. To investigate the pharmacologic and physiologic role of this transport protein, we generated Oct1 knockout (Oct1(-/-)) mice. Oct1(-/-) mice appeared to be viable, healthy, and fertile and displayed no obvious phenotypic abnormalities. The role of Oct1 in the pharmacology of substrate drugs was studied by comparing the distribution and excretion of the model substrate tetraethylammonium (TEA) after intravenous administration to wild-type and Oct1(-/-) mice. In Oct1(-/-) mice, accumulation of TEA in liver was four to sixfold lower than in wild-type mice, whereas direct intestinal excretion of TEA was reduced about twofold. Excretion of TEA into urine over 1 h was 53% of the dose in wild-type mice, compared to 80% in knockout mice, probably because in Oct1(-/-) mice less TEA accumulates in the liver and thus more is available for rapid excretion by the kidney. In addition, we found that absence of Oct1 leads to decreased liver accumulation of the anticancer drug metaiodobenzylguanidine and the neurotoxin 1-methyl-4-phenylpyridium. In conclusion, our data show that Oct1 plays an important role in the uptake of organic cations into the liver and in their direct excretion into the lumen of the small intestine.