Organic Cation/carnitine Transporter 1 Research Articles

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Effects of 1α,25-dihydroxyvitamin D3 on the pharmacokinetics and biodistribution of ergothioneine, an endogenous organic cation/carnitine transporter 1 substrate, in rats.

PurposeThis study aimed to investigate the effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression levels of organic cation/carnitine transporter 1 (OCTN1) as well as the pharmacokinetics and biodistribution of ergothioneine, an OCTN1 substrate, in rats.MethodsRats pretreated with 1,25(OH)2D3 (2.56 nmol/kg/day) for four days were administered ergothioneine (2 mg/kg) intravenously. The expression levels of rat OCTN1 (rOCTN1) in organs were determined using real-time quantitative polymerase chain reaction. Ergothioneine levels in plasma, urine, and organs (with and without intravenous injection of exogenous ergothioneine) were determined using liquid chromatography-tandem mass spectrometry.Results1,25(OH)2D3 pretreatment resulted in a significant decrease in rOCTN1 mRNA expression levels in the kidney and brain, a significant increase in basal plasma levels of ergothioneine (from 48 h), and a significant decrease in the tissue-plasma partition coefficient (Kp) in all tissues (except the heart and lungs) and the basal urine levels of ergothioneine. After intravenous administration, the pharmacokinetic profiles of ergothioneine were consistent with the basal levels of endogenous ergothioneine, with an increase in AUC∞ by 85%, a significant decrease in total clearance by 49%, and a decrease in Vss by 32% in 1,25(OH)2D3-treated rats. The Kp value and urinary recovery of ergothioneine also decreased in the 1,25(OH)2D3-treated group.ConclusionThis study showed the effects of 1,25(OH)2D3 on the expression and function of rOCTN1 by investigating the interaction between 1,25(OH)2D3 and ergothioneine. Dose adjustment and possible changes in bioavailability should be considered before the co-administration of vitamin D or its active forms and OCTN1 substrates.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40005-022-00563-1.

L-tetrahydropalmatine reduces oxaliplatin accumulation in the dorsal root ganglion and mitochondria through selectively inhibiting the transporter-mediated uptake thereby attenuates peripheral neurotoxicity

Oxaliplatin (OXA) is a third-generation platinum drug; however, its application is greatly limited due to the severe peripheral neurotoxicity. This study aims to confirm the transport mechanism of OXA and to explore whether L-tetrahydropalmatine (L-THP) would alleviate OXA-induced peripheral neurotoxicity by selectively inhibiting these uptake transporters in vitro and in vivo. Our results revealed that organic cation transporter 2 (OCT2), organic cation/carnitine transporter 1 (OCTN1) and organic cation/carnitine transporter 2 (OCTN2) were involved in the uptake of OXA in dorsal root ganglion (DRG) neurons and mitochondria, respectively. L-THP (1−100 μM) reduced OXA (40 μM) induced cytotoxicity in MDCK-hOCT2 (Madin–Darby canine kidney, MDCK), MDCK-hOCTN1, MDCK-hOCTN2, and rat primary DRG cells, and decreased the accumulation of OXA in above cells and rat DRG mitochondria, but did not affect its efflux from MDCK-hMRP2 cells. Furthermore, Co-administration of L-THP (5–20 mg/kg for mice, 10–40 mg/kg for rats; twice a week, iv or ig) attenuated OXA (8 mg/kg for mice, 4 mg/kg for rats; twice a week, iv) induced peripheral neurotoxicity and reduced the platinum concentration in the DRG. Whereas, L-THP (1−100 μM for cells; 10–20 mg/kg for mice) did not impair the antitumour efficacy of OXA (40 μM for cells; 8 mg/kg for mice) in HT29 tumour-bearing nude mice nor in tumour cells (HT29 and SW620 cells). In conclusion, OCT2, OCTN1 and OCTN2 contribute to OXA uptake in the DRG and mitochondria. L-THP attenuates OXA-induced peripheral neurotoxicity via inhibiting OXA uptake but without impairing the antitumour efficacy of OXA. L-THP is a potential candidate drug to attenuate OXA-induced peripheral neurotoxicity.

RIP3-deficience attenuates potassium oxonate-induced hyperuricemia and kidney injury

Recent preclinical and clinical evidence suggests that hyperuricemia (HU) is an independent risk factor for metabolic syndrome, hypertension, cardiovascular disease and chronic kidney disease. Receptor-interacting protein 3 (RIP3) is an important contributor in inducing programmed necrosis, representing a newly identified mechanism of cell death combining features of both apoptosis and necrosis. In our study, RIP3 was strongly expressed in mice with hyperuricemia. RIP3 deficiency attenuated hyperuricemia in mice, evidenced by reduced serum uric acid and creatinine and enhanced urinary uric acid and creatinine, as well as the improved histological alterations in renal sections. Additionally, RIP3-deletion reduced malondialdehyde (MDA), H2O2 and O2−, whereas enhanced superoxide dismutase (SOD), GSH and GSH-Px levels in potassium oxonate-induced mice. Potassium oxonate-treated mice showed significantly high mRNA levels of ATP-binding cassette, subfamily G, membrane 2 (ABCG2), organic anion transporter 1 (OAT1), OAT3, organic cation transporter 1 (OCT1) and organic cation/carnitine transporter 1 (OCTN1) in renal tissue samples, which were reversed by RIP3-deficiency. Meanwhile, down-regulation of circulating and kidney pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) were observed in RIP3-knockout mice with hyperuricemia, associated with inactivation of toll-like receptor 4 (TLR4), inhibitor of NF-κB alpha (IκBα) and nuclear factor kappa B (NF-κB). NLR family, pyrin domain-containing 3 (NLRP3) inflammasome was also suppressed by RIP3 knockout in potassium oxonate-treated mice. Importantly, RIP3-knockout mice exhibited the decrease of FAS-associated protein with a death domain (FADD), cleaved Caspase-8/-3 and Poly (ADP-ribose) polymerase (PARP) in renal samples, along with TUNEL reduction in mice with hyperuricemia. Similar results were observed in uric acid-incubated cells with RIP3 knockdown. Thus, we suggested that RIP3 played an important role in mice with hyperuricemia, which might be a novel signal pathway targeting for therapeutic strategies in future.

Anti-hyperuricemic and anti-inflammatory actions of vaticaffinol isolated from Dipterocarpus alatus in hyperuricemic mice

The present study was designed to examine the anti-hyperuricemic and anti-inflammatory effects and possible mechanisms of vaticaffinol, a resveratrol tetramer isolated from ethanol extracts of Dipterocarpus alatus, in oxonate-induced hyperuricemic mice. At 1 h after 250 mg·kg−1 potassium oxonate was given, vaticaffinol at 20, 40, and 60 mg·kg−1 was intragastrically administered to hyperuricemic mice once daily for seven consecutive days. Vaticaffinol significantly decreased serum uric acid levels and improved kidney function in hyperuricemic mice. It inhibited hepatic activity of xanthine dehydrogenase (XDH) and xanthine oxidase (XOD), regulated renal mRNA and protein levels of urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), organic anion transporter 1 (OAT1), organic cation transporter 1 (OCT1), OCT2, organic cation/carnitine transporter 1 (OCTN1), and OCTN2 in hyperuricemic mice. Moreover, vaticaffinol markedly down-regulated renal protein levels of NOD-like receptor 3 (NLRP3), apoptosis-associated speck-like (ASC), and Caspase-1, resulting in the reduction of interleukin (IL)-1β, IL-18, IL-6 and tumor necrosis factor-α (TNF-α) levels in this animal model. Additionally, HPLC and LC-MS analyses clearly testified the presence of vaticaffinol in the crude extract. These results suggest that vaticaffinol may be useful for the prevention and treatment of hyperuricemia with kidney inflammation.

Association study between OCTN1 functional haplotypes and Crohn's disease in a Korean population.

Crohn's disease (CD) is a chronic inflammatory bowel disease with multifactorial causes including environmental and genetic factors. Several studies have demonstrated that the organic cation/carnitine transporter 1 (OCTN1) non-synonymous variant L503F is associated with susceptibility to CD. However, it was reported that L503F is absent in Asian populations. Previously, we identified and functionally characterized genetic variants of the OCTN1 promoter region in Koreans. In that study, four variants demonstrated significant changes in promoter activity. In the present study, we determined whether four functional variants of the OCTN1 promoter play a role in the susceptibility to or clinical course of CD in Koreans. To examine it, the frequencies of the four variants of the OCTN1 promoter were determined by genotyping using DNA samples from 194 patients with CD and 287 healthy controls. Then, associations between genetic variants and the susceptibility to CD or clinical course of CD were evaluated. We found that susceptibility to CD was not associated with OCTN1 functional promoter variants or haplotypes showing altered promoter activities in in vitro assays. However, OCTN1 functional promoter haplotypes showing decreased promoter activities were significantly associated with a penetrating behavior in CD patients (HR=2.428, p=0.009). Our results suggest that the OCTN1 functional promoter haplotypes can influence the CD phenotype, although these might not be associated with susceptibility to this disease.

Sustained enhancement of OCTN1 transporter expression in association with hydroxyurea induced γ-globin expression in erythroid progenitors.

The clinical benefits of hydroxyurea (HU) treatment in patients with sickle cell disease (SCD) are due largely to increased γ-globinexpression. However, mechanisms that control γ-globin expression by HU in erythroid progenitors are incompletely understood. Here, we investigated the role of two HU transporters, urea transporter B (UTB) and organic cation/carnitine transporter 1 (OCTN1), in this process. Endogenous expression of both transporters peaked toward the end of erythroid differentiation. However, unlike UTB, HU-induced OCTN1 expression correlated positively with γ-globin level and was sustained throughout the period of induction. These results highlight a potential major role for OCTN1 in promoting the efficacy of HU.

Siwu decoction attenuates oxonate-induced hyperuricemia and kidney inflammation in mice

The aim of the study was to investigate the effects of Siwu decoction on hyperuricemia, kidney inflammation, and dysfunction in hyperuricemic mice. Siwu decoction at 363.8, 727.5, and 1 455 mg·kg−1 was orally administered to potassium oxonate-induced hyperuricemic mice for 7 days. Serum urate, creatinine, and blood urea nitrogen levels and hepatic xanthine oxidase (XOD) activity were measured. The protein levels of hepatic XOD and renal urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), organic anion transporters 1 (OAT1), ATP-binding cassette subfamily G member 2 (ABCG2), organic cation transporter 1 (OCT1), OCT2, organic cation/carnitine transporter 1 (OCTN1), OCNT2, Nod-like receptor family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), Caspase-1, and interleukin-1β (IL-1β) were determined by Western blotting. Renal histopathology change was obtained following hematoxylin-eosin staining. Our results indicated that Siwu decoction significantly reduced serum urate, creatinine and blood urea nitrogen levels and increased fractional excretion of uric acid in hyperuricemic mice. It effectively reduced hepatic XOD activity and protein levels in this animal model. Furthermore, Siwu decoction down-regulated URAT1 and GLUT9 protein levels, and up-regulated the protein levels of OAT1, ABCG2, OCT1, OCT2, OCTN1, and OCTN2 in the kidney of the hyperuricemic mice. Additionally, Siwu decoction remarkably reduced renal protein levels of NLRP3, ASC, Caspase-1, and IL-1β in the hyperuricemic mice. These results suggested that Siwu decoction exhibited anti-hyperuricemic and anti-inflammatory effects by inhibiting hepatic XOD activity, regulating renal organic ion transporter expression, and suppressing renal NLRP3 inflammasome activation, providing the evidence for its use in the treatment of hyperuricemia and associated kidney inflammation.

Correlation of Organic Cation/Carnitine Transporter 1 and Multidrug Resistance-Associated Protein 1 Transport Activities With Protein Expression Levels in Primary Cultured Human Tracheal, Bronchial, and Alveolar Epithelial Cells

Understanding how transporters contribute to the distribution of inhaled drugs in the lung is important for the discovery and development of such drugs. Protein expression levels may be useful to predict and understand drug distribution. As previously reported, organic cation/carnitine transporter 1 (OCTN1) and multidrug resistance-associated protein 1 (MRP1) have higher levels of protein expression among transporters in primary cultured human lung cells. Nevertheless, it is unclear to what extent transport activity correlates with transporter protein expression. The purpose is to evaluate whether differences in OCTN1 and MRP1 protein expression govern the respective transport activity in primary cultured human lung cells. The model substrates of 4-[4-(dimethylamino) styryl]-N-methylpyridinium iodide (ASP+) and carboxy-dichlorofluorescein (CDF) for OCTN1 and MRP1, respectively, were used in the lung cells from five donors. Significant correlation was found between the kinetic parameter Vmax for ASP+ and OCTN1 protein expression in plasma membrane of tracheal, bronchial, and alveolar cells (r2 = 0.965, 0.834, and 0.877, respectively), and between the efflux of CDF and MRP1 protein expression in plasma membrane of tracheal, bronchial, and alveolar cells (r2 = 0.800, 0.904, and 0.790, respectively). These findings suggest that OCTN1 and MRP1 protein concentrations are determinants for drug distribution in the lung.

Dissection of the Expression Patterns of Hydroxyurea Transporters during Erythropoiesis and Myelopoiesis

Background: Hydroxyurea (HU) is the only FDA approved drug for the treatment of moderate to severe sickle cell disease (SCD). It is becoming a standard of care for all SCD patients; clinical efficacy is attributed largely to enhanced expression of the γ-globin gene in erythroid progenitors. Dose-limiting toxicity of the drug in myeloid cells is believed to hinder its maximum effectiveness and utilization. Hitherto, the precise mechanisms that influence the on-target effects of HU in eyrthroid cells and the off-target cytotoxic effects in myeloid cells have not been defined. Cell surface transporters regulating the intracellular accumulation of compounds exert a major influence on drug action. For instance, differential expression of transporters in specific cell types elicits variable responses. We have previously shown that the organic cation/carnitine transporter 1 (OCTN1) and urea transporter B (UTB) modulate intracellular accumulation of HU. Both transporters are highly expressed in the bone marrow however their role in the on- and off-target effects of HU has never been examined. In this study, we reasoned that variation in the expression of UTB and OCTN1 on hematopoietic progenitor cells (HPCs) may influence how individual lineages of HPCs respond to the drug. Objective: In order to begin dissecting the role of OCTN1 and UTB transporters in modulating HU activity, the objective of the present study was to define lineage-specific expression patterns of UTB and OCTN1 in erythroid and myeloid progenitor cells during hematopoiesis. Methods: Human CD34+ HPCs isolated from peripheral blood were cultured in media containing erythropoietin or granulocyte-colony stimulating factor to induce erythropoiesis or myelopoiesis, respectively. Erythroid and myeloid differentiation was verified by flow cytometry and morphology. Specific populations of cells were isolated at defined time-points during differentiation, and gene expression for OCTN1 and UTB(encoded by SLC22A4 and SLC14A1 respectively) was determined by quantitative RT-PCR. Results: OCTN1 expression pattern was similar in erythroid and myeloid lineages but was variable during the course of differentiation. As cells differentiated from the common HPCs, OCTN1 expression was greatest in basophilic and polychromatic erythroblasts and promyelocytes. Expression increased 1.7- and 1.5-fold in erythroid and myeloid cells, respectively, compared to the common HPC. OCTN1 expression in late-stage precursor cells dropped to levels similar to those of common HPCs. Interestingly, UTB expression was 20-fold greater in othrochromatic erythroblasts and reticulocytes compared to common HPCs. However, the expression of this transporter decreased progressively in differentiating myeloid cells declining to a mean value 10-fold lower in late-stage myeloid band cells compared to the common HPCs. To examine whether HU treatment influences transporter expression, erythroblasts were treated with 100μM HU when OCTN1 and UTB expression was increased 10- and 20-fold, respectively (p<0.001). After 72 hours of HU exposure, γ-globin expression increased 2.5-fold (p<0.001) while OCTN1 expression increased 2-fold (p=0.01) compared to PBS-treated controls. UTB gene expression was significantly up regulated 1.5-fold (p<0.02) at 24 and 48 hours after HU treatment. Taken together these data demonstrate differential expression patterns of OCTN1 and UTB in HPCs that are altered by HU. Conclusions: These studies are the first to document HU transporter expression during erythroid and myeloid differentiation. We have shown that HU is a substrate for both OCTN1 and UTB, and we now demonstrate that their expression patterns are consistent with experimental evidence of HU response. Optimal γ-globin induction is observed when early- to mid-stage progenitor cells are treated with HU and this coincides with significant increases in transporter expression. Preferential expression of UTB in erythroid progenitors suggests it may be a primary modulator of HU-mediated γ-globin induction with a lesser role in myeloid cytotoxicity. A detailed understanding of the expression patterns of OCTN1 and UTB in HPCs may help to refine the use of HU to improve its overall efficacy and utilization in SCD. Disclosures No relevant conflicts of interest to declare.

Drug Transporter Protein Quantification of Immortalized Human Lung Cell Lines Derived from Tracheobronchial Epithelial Cells (Calu-3 and BEAS2-B), Bronchiolar–Alveolar Cells (NCI-H292 and NCI-H441), and Alveolar Type II-like Cells (A549) by Liquid Chromatography–Tandem Mass Spectrometry

Understanding the mechanisms of drug transport in the human lung is an important issue in pulmonary drug discovery and development. For this purpose, there is an increasing interest in immortalized lung cell lines as alternatives to primary cultured lung cells. We recently reported the protein expression in human lung tissues and pulmonary epithelial cells in primary culture, (Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) whereas comprehensive quantification of protein expressions in immortalized lung cell lines is sparse. Therefore, the aim of the present study was to clarify the drug transporter protein expression of five commercially available immortalized lung cell lines derived from tracheobronchial cells (Calu-3 and BEAS2-B), bronchiolar-alveolar cells (NCI-H292 and NCI-H441), and alveolar type II cells (A549), by liquid chromatography-tandem mass spectrometry-based approaches. Among transporters detected, breast cancer-resistance protein in Calu-3, NCI-H292, NCI-H441, and A549 and OCTN2 in BEAS2-B showed the highest protein expression. Compared with data from our previous study,(Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) NCI-H441 was the most similar with primary lung cells from all regions in terms of protein expression of organic cation/carnitine transporter 1 (OCTN1). In conclusion, the protein expression profiles of transporters in five immortalized lung cell lines were determined, and these findings may contribute to a better understanding of drug transport in immortalized lung cell lines.

Identification and Functional Characterization of Novel Genetic Variations in the OCTN1 Promoter.

Human organic cation/carnitine transporter 1 (OCTN1) plays an important role in the transport of drugs and endogenous substances. It is known that a missense variant of OCTN1 is significantly associated with Crohn's disease susceptibility. This study was performed to identify genetic variants of the OCTN1 promoter in Korean individuals and to determine their functional effects. First, the promoter region of OCTN1 was directly sequenced using genomic DNA samples from 48 healthy Koreans. OCTN1 promoter activity was then measured using a luciferase reporter assay in HCT-116 cells. Seven variants of the OCTN1 promoter were identified, two of which were novel. There were also four major OCTN1 promoter haplotypes. Three haplotypes (H1, H3, and H4) showed decreased transcriptional activity, which was reduced by 22.9%, 23.0%, and 44.6%, respectively (p<0.001), compared with the reference haplotype (H2). Transcription factor binding site analyses and gel shift assays revealed that NF-Y could bind to the region containing g.-1875T>A, a variant present in H3, and that the binding affinity of NF-Y was higher for the g.-1875T allele than for the g.-1875A allele. NF-Y could also repress OCTN1 transcription. These data suggest that three OCTN1 promoter haplotypes could regulate OCTN1 transcription. To our knowledge, this is the first study to identify functional variants of the OCTN1 promoter.

Role of Organic Cation/Carnitine Transporter 1 in Uptake of Phenformin and Inhibitory Effect on Complex I Respiration in Mitochondria

Phenformin causes lactic acidosis in clinical situations due to inhibition of mitochondrial respiratory chain complex I. It is reportedly taken up by hepatocytes and exhibits mitochondrial toxicity in the liver. In this study, uptake of phenformin and [(14)C]tetraethylammonium (TEA) and complex I inhibition by phenformin were examined in isolated liver and heart mitochondria. Uptake of phenformin into isolated rat liver mitochondria was higher than that into heart mitochondria. It was inhibited by several cat ionic compounds, which suggests the involvement of multispecific transport system(s). Similar characteristics were also observed for uptake of TEA; however, uptake of phenformin into mitochondria of organic cation/carnitine transporter 1 (OCTN1) knockout mice was lower than that in wild-type mice, whereas uptake of TEA was comparable between the two strains, suggesting the involvement of distinct transport mechanisms for these two cations in mitochondria. Inhibition by phenformin of oxygen consumption via complex I respiration in isolated rat liver mitochondria was greater than that in heart mitochondria, whereas inhibitory effect of phenformin on complex I respiration was similar in inside-out structured submitochondrial particles prepared from rat livers and hearts. Lactic acidosis provoked by iv infusion of phenformin was weaker in octn1(-/-) mice than that in wild-type mice. These observations suggest that uptake of phenformin into liver mitochondria is at least partly mediated by OCTN1 and functionally relevant to its inhibition potential of complex I respiration. This study was, thus, the first to demonstrate OCTN1-mediated mitochondrial transport and toxicity of biguanide in vivo in rodents.

Regulatory Role of Testosterone in Organic Cation Transport: in Vivo and in Vitro Studies

The renal proximal tubule (RPT) plays a crucial role in organic cation (OC) secretion and has a major impact on pharmacokinetics of OC drugs. Secretory transport is vectorial. Thus, it involves transporters located at both basolateral and apical membranes. Although sex hormones have been shown to regulate OC transport, there is little data on the effect of testosterone on OC secretion in a whole animal. Therefore, we determined the clearance of tetraethylammonium (TEA), a model OC substrate, in intact and castrated male mice. Castration significantly decreased renal TEA secretion by 30%, and testosterone supplementation returned TEA secretion to control levels in castrated mice. The mechanism of this effect was further examined in isolated mouse renal proximal tubules (mRPT). TEA uptake in isolated mRPT from castrated mice was reduced by 36%. This effect was reversed in tubules from castrated mice supplemented with testosterone. Kinetic analysis of [(3)H]-TEA uptake in isolated mRPT showed a decreased V(max) with no change in K(m), implying that the decrease in transport rate was caused by lowering in the number of transporters in castrated mice rather than a change in transporter affinity. Quantitative real time polymerase chain reaction (real time PCR) revealed that organic cation transporter (OCT)2 is the major TEA transporter in male mice. Moreover, OCT2 mRNA level was significantly reduced after castration. Castrated mice also showed a modest increase in organic cation/carnitine transporter 1 (OCTN1) mRNA level, indicating that testosterone may also regulate apical OCTN1 expression. These data suggest that testosterone regulates transepithelial transport of OC through modulation of OCT2 expression in male mice.

Functional Characterization of Ergothioneine Transport by Rat Organic Cation/Carnitine Transporter Octn1 (slc22a4)

It has been reported that organic cation/carnitine transporter 1 (OCTN1) is associated with rheumatoid arthritis and Crohn's disease. Additionally, we reported that OCTN1 is expressed in hematopoietic cells, and is associated with proliferation and differentiation of erythroid cells. However, physiological role of OCTN1 is still unclear. Ergothioneine, an anti-oxidant, was recently reported to be a good substrate of human OCTN1. However, the transport characteristics of ergothioneine in rat remains to be clarified. The present study, is to further investigate the role of rat Octn1 on transport of ergothioneine in rat Octn1 transfected cells and natively expressing cell line PC12 derived from rat adrenal pheochromocytoma. [(3)H]Ergothioneine uptake by rat Octn1 stably transfected HEK293 cells was saturable, sodium dependent with 1 : 1 stoichiometry of ergothioneine, and pH dependent. Since ergothioneine was reported to presumably play a protective role against oxidative stress-induced apoptosis in PC12 cells, its transport in this cell line was investigated. The expression of rat Octn1 and a saturable and Na(+)-dependent transport of ergothioneine were observed in PC12 cells, suggesting that ergothioneine transport in this cell line may be mediated by rat Octn1. These findings suggested that rat Octn1 may act as a survival factor by taking up ergothioneine to suppress oxidative stress in this cell line. In conclusion, functional characteristics of ergothioneine transport by rat Octn1 is similar to that of human OCTN1 and it is suggested that rat Octn1 is important by transporting anti-oxidant ergothioneine in PC12 cells, though its role in vivo is to be investigated.