Solute Transporter OCTN1/Slc22a4 Affects Disease Severity and Response to Infliximab in Experimental Colitis: Role of Gut Microbiota and Immune Modulation.

BACKGROUND: Inflammatory bowel diseases are chronic disabling conditions with a complex and multifactorial etiology, still incompletely understood. OCTN1, an organic cation transporter, could have a role in modulating the inflammatory response, and some genetic polymorphisms of this molecule have been associated with increased risk of inflammatory bowel diseases. Until now, limited information exists on its potential in predicting/modulating patient's response to therapies. The aim of this study was to evaluate the role of OCTN1 in modifying gut microbiota and mucosal immunity in response to infliximab therapy in murine colitis. METHODS: A dextran sodium sulphate model of colitis was used to assess the clinical efficacy of infliximab administered intravenously in ocnt1 gene knockout mice and their C57BL/6 controls. Stool, colon, and mesenteric lymph node samples were collected to evaluate differences in gut microbiota composition, histology, and T cell populations, respectively. RESULTS: Octn1 -/- influences the microbiota profile and is associated with a worse dysbiosis in mice with colitis. Infliximab treatment attenuates colitis-associated dysbiosis, with an increase of bacterial richness and evenness in both strains. In comparison with wild type, octn1-/- mice have milder disease and a higher baseline percentage of Treg, Tmemory, Th2 and Th17 cells. CONCLUSIONS: Our data support the murine model to study OCTN1 genetic contribution to inflammatory bowel diseases. This could be the first step towards the recognition of this membrane transporter as a biomarker in inflammatory conditions and a predictor of response to therapies.

In this article, we evaluated the role of OCTN1, an organic cation transporter, in modifying gut microbiota and immune T cell populations, as well as its effects on experimental colitis and the response to infliximab treatment.

Depression-like Behavior Induced by Repeated Administration of Dexamethasone to Lipopolysaccharide-inflamed Mice.

BACKGROUND: Over the years, animal models of depression have been developed by loading chronic stress, inducing neuroinflammation, or administering drugs that induce depression; however, these results have poor reproducibility. Therefore, it is necessary to develop animal models that exhibit definitive symptoms of depression for studies on potential therapeutics. OBJECTIVE: This study was aimed at investigating depression-like symptoms and their pathogenesis in lipopolysaccharide (LPS)-inflamed mice treated with dexamethasone (DEX). METHODS: Male ICR mice were injected with LPS, followed by injection with DEX a day later and each day for 6 consecutive days. Depression-like behavior, expression of the glial markers glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba1), and the number of the immature neuronal marker doublecortin (DCX)-positive cells were assessed using tail-suspension test (TST), forced swim test (FST), western blot analysis, and immunohistochemical analysis. RESULTS: Mice in the LPS+DEX group had significantly longer immobility time in the TST and FST than did those in the LPS- or DEX-only and control groups on day 7 post-LPS administration. GFAP and Iba1 expression was significantly elevated in the hippocampus of mice in the LPS group than in those of mice in the control group. Moreover, a significantly lower number of DCX-positive cells was observed in the hippocampal dentate gyrus of mice in the LPS+DEX group compared with that in mice in the LPS- or DEX-only and control groups on day 7 after LPS administration. CONCLUSION: Repeated DEX administration to LPS-inflamed mice may induce definitive depression-like symptoms by decreasing the number of immature neurons in the hippocampal dentate gyrus. This novel mouse model of depression was produced by repeated administration of steroids to inflamed mice.

Ergothioneine and central nervous system diseases.

Ergothioneine (ERGO) is a thiol contained in the food that exhibits an excellent antioxidant effect similar to that of glutathione. Although mammals lack a biosynthetic pathway for ERGO, the carnitine/organic cation transporter OCTN1/SLC22A4, which transports ERGO in vivo, is expressed throughout the body, and ERGO is distributed to various organs after oral intake. ERGO is a stable compound that remains in the body for a long time after ingestion. OCTN1 is also expressed in brain parenchymal cells, including neurons, and ERGO in the blood permeates the blood-brain barrier and is distributed to the brain, exhibiting a neuroprotective effect. Recently, the association between central nervous system (CNS) diseases and ERGO has become a research focus. ERGO concentrations in the blood components are lower in patients with cognitive impairment, Parkinson's disease, and frailty than in healthy subjects. ERGO exerts a protective effect against various neurotoxins and improves the symptoms of cognitive impairment, depression, and epilepsy in animal models. The promotion of neurogenesis and induction of neurotrophic factors, in addition to the antioxidant and anti-inflammatory effects, may be involved in the neuroprotective effect of ERGO. This review shows the association between ERGO and CNS diseases, discusses the possible biomarkers of peripheral ERGO in CNS diseases, and the possible preventive and improvement effects of ERGO on CNS diseases.

Oral Administration of the Food-Derived Hydrophilic Antioxidant Ergothioneine Enhances Object Recognition Memory in Mice.

BACKGROUND: The enhancement of learning and memory through food-derived ingredients is of great interest to healthy individuals as well as those with diseases. Ergothioneine (ERGO) is a hydrophilic antioxidant highly contained in edible golden oyster mushrooms (Pleurotus cornucopiae var. citrinopileatus), and systemically absorbed by its specific transporter, carnitine/organic cation transporter OCTN1/SLC22A4. OBJECTIVE: This study aims to examine the possible enhancement of object recognition memory by oral administration of ERGO in normal mice. METHODS: Novel object recognition test, spatial recognition test, LC-MS/MS, Golgi staining, neuronal culture, western blotting, immunocytochemistry, and quantitative RT-PCR were utilized. RESULT: After oral administration of ERGO (at a dose of 1-50 mg/kg) three times per week for two weeks in ICR mice, the novel object recognition test revealed a longer exploration time for the novel object than for the familiar object. After oral administration of ERGO, the spatial recognition test also revealed a longer exploration time for the spatially moved object than the unmoved one in mice fed ERGO-free diet. The discrimination index was significantly higher in the ERGO-treated group than the control in both behavioral tests. ERGO administration led to an increase in its concentration in the plasma and hippocampus. The systemic concentration reached was relevant to those found in humans after oral ERGO administration. Golgi staining revealed that ERGO administration increased the number of matured spines in the hippocampus. Exposure of cultured hippocampal neurons to ERGO elevated the expression of the synapse formation marker, synapsin I. This elevation of synapsin I was inhibited by the tropomyosin receptor kinase inhibitor, K252a. Treatment with ERGO also increased the expression of neurotrophin-3 and -5, and phosphorylated mammalian target of rapamycin in hippocampal neurons. CONCLUSION: Oral intake of ERGO may enhance object recognition memory at its plasma concentration achievable in humans, and this enhancement effect could occur, at least in part, through the promotion of neuronal maturation in the hippocampus.

Homostachydrine is a Xenobiotic Substrate of OCTN1/SLC22A4 and Potentially Sensitizes Pentylenetetrazole-Induced Seizures in Mice.

Understanding of the underlying mechanism of epilepsy is desired since some patients fail to control their seizures. The carnitine/organic cation transporter OCTN1/SLC22A4 is expressed in brain neurons and transports food-derived antioxidant ergothioneine (ERGO), L-carnitine, and spermine, all of which may be associated with epilepsy. This study aimed to clarify the possible association of this transporter with epileptic seizures. In both pentylenetetrazole (PTZ)-induced acute seizure and kindling models, ocnt1 gene knockout mice (octn1-/-) showed lower seizure scores compared with wild-type mice. Up-regulation of the epilepsy-related genes, c-fos and Arc, and the neurotrophic factor BDNF following PTZ administration was observed in the hippocampus of wild-type, but not octn1-/- mice. To find the OCTN1 substrate associated with the seizure, untargeted metabolomics analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry was conducted on extracts from the hippocampus, frontal cortex, and plasma of both strains, leading to the identification of a plant alkaloid homostachydrine as a compound present in a lower concentration in octn1-/- mice. OCTN1-mediated uptake of deuterium-labeled homostachydrine was confirmed in OCTN1-transfected HEK293 cells, suggesting that this compound is a substrate of OCTN1. Homostachydrine administration increased PTZ-induced acute seizure scores and the expression of Arc in the hippocampus and that of Arc, Egr1, and BDNF in the frontal cortex. Conversely, administration of the OCTN1 substrate/inhibitor ERGO inhibited PTZ-induced kindling and reduced the plasma homostachydrine concentration. Thus, these results suggest that OCTN1 is at least partially associated with PTZ-induced seizures, which is potentially deteriorated by treatment with homostachydrine, a newly identified food-derived OCTN1 substrate.

High-Speed SICM for the Visualization of Nanoscale Dynamic Structural Changes in Hippocampal Neurons.

Dynamic reassembly of the cytoskeleton and structural changes represented by dendritic spines, cargo transport, and synapse formation are closely related to memory. However, the visualization of the nanoscale topography is challenging because of the diffraction limit of optical microscopy. Scanning ion conductance microscopy (SICM) is an effective tool for visualizing the nanoscale topography changes of the cell surface without labeling. The temporal resolution of SICM is a critical issue of live-cell time-lapse imaging. Here, we developed a new scanning method, automation region of interest (AR)-mode SICM, to select the next imaging region by predicting the location of a cell, thus improving the scanning speed of time-lapse imaging. The newly developed algorithm reduced the scanning time by half. The time-lapse images provided not only novel information about nanoscale structural changes but also quantitative information on the dendritic spine and synaptic bouton volume changes and formation process of the neural network that are closely related to memory. Furthermore, translocation of plasmalemmal precursor vesicles (ppvs), for which fluorescent labeling has not been established, were also visualized along with the rearrangement of the cytoskeleton at the growth cone.

Metabolome Analysis Reveals Dermal Histamine Accumulation in Murine Dermatitis Provoked by Genetic Deletion of P-Glycoprotein and Breast Cancer Resistance Protein.

PURPOSE: P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are xenobiotic transporters which pump out variety types of compounds, but information on their interaction with endogenous substrates in the skin is limited. The purpose of the present study was to clarify possible association of these transporters in dermal accumulation of inflammatory mediators. METHODS: Dermatitis model was constructed by repeated topical application of oxazolone in wild-type, and P-gp and BCRP gene triple knockout (Mdr1a/1b/Bcrp-/-) mice to observe difference in phenotype. Target metabolome analysis of 583 metabolites was performed using skin and plasma. RESULTS: Dermatitis and scratching behavior in dermatitis model of Mdr1a/1b/Bcrp-/- mice were more severe than wild-type mice, suggesting protective roles of these transporters. This hypothesis was supported by the metabolome analysis which revealed that concentration of histamine and other dermatitis-associated metabolites like urate and serotonin in the dermatitis skin, but not normal skin, of Mdr1a/1b/Bcrp-/- mice was higher than that of wild-type mice. Gene expression of P-gp and BCRP was reduced in oxazolone-treated skin and the skin of patients with atopic dermatitis or psoriasis. CONCLUSIONS: These results suggest possible association of these efflux transporters with dermal inflammatory mediators, and such association could be observed in the dermatitis skin.

Maturational Characterization of Mouse Cortical Neurons Three-Dimensionally Cultured in Functional Polymer FP001-Containing Medium.

The aim of the present study is to construct and characterize a novel three-dimensional culture system for mouse neurons using the functional polymer, FP001. Stereoscopically extended neurites were found in primary mouse cortical neurons cultured in the FP001-containing medium. Neurons cultured with FP001 were distributed throughout the medium of the observation range whereas neurons cultured without FP001 were distributed only on the bottom of the dish. These results demonstrated that neurons can be three-dimensionally cultured using the FP001-containing medium. The mRNA expression of the glutamatergic neuronal marker vesicular glutamate transporter 1 in neurons cultured in the FP001-containing medium were higher than that in neurons cultured in the FP001-free medium. Expression of the matured neuronal marker, microtubule-associated protein 2 (MAP2) a,b, and the synapse formation marker, Synapsin I, in neurons cultured with FP001 was also higher than that in neurons cultured without FP001. The expression pattern of MAP2a,b in neurons cultured with FP001, but not that in neurons cultured without FP001, was similar to that in the embryonic cerebral cortex. Exposure to glutamate significantly increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity in neurons cultured with FP001 compared to that in neurons cultured without FP001. These results suggested that glutamatergic neurotransmission in neurons three-dimensionally cultured in the FP001-containing medium may be upregulated compared to neurons two-dimensionally cultured in the FP001-free medium. Thus, neurons with the properties close to those in the embryonic brain could be obtained by three-dimensionally culturing neurons using FP001, compared to two-dimensional culture with a conventional adhesion method.

[Possible Treatment of Neuropsychiatric Disorders by Promotion of Neuronal Differentiation through Organic Cation Transporters].

Neurons differentiated from neural stem cells mature to form a neuronal network. Neuronal maturation enables neurotransmission that regulates brain function. Therefore, abnormal neuronal differentiation causes dysfunction in neurotransmission, and is involved in the onset of various neuropsychiatric disorders. Most of the drugs currently available for the treatment of neuropsychiatric disorders act on membrane receptors and reuptake transporters of neurotransmitters, and control neurotransmission. These membrane proteins have a high affinity for a specific neurotransmitter, and are highly expressed in synapses. By contrast, xenobiotic transporters have a relatively lower affinity for neurotransmitters, but widely recognize various organic compounds, and are also expressed in brain neural cells. It has remained largely unknown why such xenobiotic transporters are expressed in neural cells that play a key role in neurotransmission. We have therefore attempted to clarify the physiological roles of organic cation transporters (OCTs) in neural stem cells in order to obtain new insight into the treatment of neuropsychiatric disorders. The carnitine/organic cation transporter OCTN1/SLC22A4 is expressed at much higher levels in neural stem cells compared with other OCTs, and promotes their differentiation into neurons through the uptake of the food-derived hydrophilic antioxidant ergothioneine after oral administration. In this review, we introduce current topics on the physiological/pathophysiological roles of OCTs in neural stem cells, and discuss their possible application to the treatment of neuropsychiatric disorders.

Bile Duct Obstruction Leads to Increased Intestinal Expression of Breast Cancer Resistance Protein With Reduced Gastrointestinal Absorption of Imatinib.

Liver dysfunction reduces systemic clearance of drugs that are primarily eliminated by the liver. However, liver dysfunction can cause a reduction in the plasma concentration profiles of certain drugs, including several tyrosine kinase inhibitors, after oral administration. The aim of the present study was to clarify the reduction in oral absorption of a tyrosine kinase inhibitor, imatinib, and the mechanisms of action involved under conditions of hepatic dysfunction, focusing on intestinal transporters. The maximum plasma concentration of imatinib after oral administration in mice subjected to bile duct ligation (BDL) was lower than that in sham-operated mice, whereas the plasma concentration profile after intravenous administration was essentially unaffected by BDL. The change in maximum plasma concentration was compatible with a reduction in small intestinal permeability of imatinib observed in the in situ closed loop. Gene expression of abcg2 was increased in BDL mice compared with that in sham-operated mice. Expression of breast cancer resistance protein and P-glycoprotein in the small intestinal brush border membrane fraction from BDL mice was also increased compared with that in sham-operated mice. In summary, the intestinal absorption and permeability of imatinib was decreased in BDL mice, and this may be attributed to the up-regulation of the efflux transporter(s).

Hydrolyzed Salmon Milt Extract Enhances Object Recognition and Location Memory Through an Increase in Hippocampal Cytidine Nucleoside Levels in Normal Mice.

Salmon milt extract contains high levels of nucleic acids and has antioxidant potential. Although salmon milt extract is known to improve impaired brain function in animal models with brain disease, its effects on learning and memory ability in healthy subjects is unknown. The purpose of the present study was to clarify the effect of hydrolyzed salmon milt extract (HSME) on object recognition and object location memory under normal conditions. A diet containing 2.5% HSME induced normal mice to devote more time to exploring novel and moved objects than in exploring familiar and unmoved objects, as observed during novel object recognition and spatial recognition tests, respectively. A diet containing 2.5% nucleic acid fraction purified from HSME also induced similar effects, as measured by the same behavioral tests. This suggests that the nucleic acids may be a functional component contributing to the effects of HSME on brain function. Quantitative polymerase chain reaction analysis revealed that gene expression of the markers for brain parenchymal cells, including neural stem cells, astrocytes, oligodendrocytes, and microglia, in the hippocampi of mice on an HSME diet was higher than that in mice on a control diet. Oral administration of HSME increased concentrations of cytosine, cytidine, and deoxycytidine in the hippocampus. Overall, ingestion of HSME may enhance object recognition and object location memory under normal conditions in mice, at least, in part, via the activation of brain parenchymal cells. Our results thus indicate that dietary intake of this easily ingestible food might enhance brain function in healthy individuals.

Influx and Efflux Transporters Contribute to the Increased Dermal Exposure to Active Metabolite of Regorafenib After Repeated Oral Administration in Mice.

The multikinase inhibitor regorafenib, which is a standard treatment for certain cancer patients after disease progression following other approved therapies, exhibits delayed-onset dermal toxicity. Here, we aimed to clarify the mechanisms that contribute to the increased dermal exposure to active metabolite M-5 of regorafenib after repeated oral administration. The dermal concentration of M-5 at 24 h after the last 5 oral administrations of regorafenib in mdr1a/1b/bcrp-/- mice was more than 190 times that in wild-type mice. The skin-to-plasma concentration ratio of M-5 in mdr1a/1b/bcrp-/- was also higher than in wild-type mice, suggesting possible involvement of P-glycoprotein and breast cancer resistance protein in regulating the dermal distribution. The area under the plasma concentration-time curve values of M-5 and its precursor M-2 in plasma of mdr1a/1b/bcrp-/- were at most 26 and 3 times those in wild-type mice, respectively. Interestingly, repeated administration of regorafenib markedly increased the area under the plasma concentration-time curve of M-5 in plasma, but not liver, compared with a single dose. Intravenous administration of M-5 dose-dependently reduced the liver-to-plasma concentration ratio. Our results indicate that hepatic uptake of M-5 may partially explain the accumulation of M-5 in the systemic circulation, but multiple factors, including influx and efflux transporters, are involved in determining dermal exposure to M-5.

Ergothioneine-induced neuronal differentiation is mediated through activation of S6K1 and neurotrophin 4/5-TrkB signaling in murine neural stem cells.

The promotion of neurogenesis is considered to be an effective therapeutic strategy for neuropsychiatric disorders because impairment of neurogenesis is associated with the onset and progression of these disorders. We have previously demonstrated that orally ingested ergothioneine (ERGO), a naturally occurring antioxidant and hydrophilic amino acid, promotes neurogenesis in the hippocampal dentate gyrus (DG) with its abundant neural stem cells (NSCs) and exerts antidepressant-like effects in mice. Independent of its antioxidant activities, ERGO induces in cultured NSCs this differentiation through induction of the basic helix-loop-helix transcription factor Math1. However, the upstream signaling of Math1 in the mechanisms underlying ERGO-induced neuronal differentiation remains unclear. The purpose of the present study was to elucidate the upstream signaling with the aim of discovering novel targets for the treatment of neuropsychiatric disorders. We focused on neurotrophic factor signaling, as it is important for the promotion of neurogenesis and the induction of antidepressant effects. We also focused on the signaling of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a known amino acid sensor, and the members of this signaling pathway, mTOR and p70 ribosomal protein S6 kinase 1 (S6K1). Exposure of cultured NSCs to ERGO significantly increased the expression of phosphorylated S6K1 (p-S6K1) at Thr389 in only 1 h, of phosphorylated mTOR (p-mTOR) in 6 h, and of the gene product of neurotrophin 4/5 (NT5) which activates tropomyosin receptor kinase B (TrkB) in 24 h. ERGO increased the population of ßIII-tubulin-positive neurons, and this effect was suppressed by the inhibitors of S6K1 (PF4708671), mTORC1 (rapamycin), and TrkB (GNF5837). Oral administration of ERGO to mice significantly increased in the DG the expression of p-S6K1 at Thr389, the gene product of NT5, and phosphorylated TrkB but not that of p-mTOR. Thus, neuronal differentiation of NSCs induced by ERGO is mediated, at least in part, through phosphorylation of S6K1 at Thr389 and subsequent activation of TrkB signaling through the induction of NT5. Thus, S6K1 and NT5 might be promising target molecules for the treatment of neuropsychiatric disorders.

Combination Metabolomics Approach for Identifying Endogenous Substrates of Carnitine/Organic Cation Transporter OCTN1.

PURPOSE: Solute carrier SLC22A4 encodes the carnitine/organic cation transporter OCTN1 and is associated with inflammatory bowel disease, although little is known about how this gene is linked to pathogenesis. The aim of the present study was to identify endogenous substrates that are associated with gastrointestinal inflammation. METHODS: HEK293/OCTN1 and mock cells were incubated with colon extracts isolated from dextran sodium sulfate-induced colitis mice; the subsequent cell lysates were mixed with the amino group selective reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), to selectively label OCTN1 substrates. Precursor ion scanning against the fragment ion of APDS was then used to identify candidate OCTN1 substrates. RESULTS: Over 10,000 peaks were detected by precursor ion scanning; m/z 342 had a higher signal in HEK293/OCTN1 compared to mock cells. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. Spermine concentration in peripheral blood mononuclear cells from octn1 gene knockout mice (octn1-/-) was significantly lower than in wild-type mice. Lipopolysaccharide-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1-/- mice was lower than in wild-type mice. CONCLUSIONS: The combination metabolomics approach can provide a novel tool to identify endogenous substrates of OCTN1.

Organic Cation Transporter 1 Is Responsible for Hepatocellular Uptake of the Tyrosine Kinase Inhibitor Pazopanib.

Pazopanib is an orally active tyrosine kinase inhibitor that exhibits hepatotoxicity in some patients. Despite the clinical importance of its hepatic distribution, the transporter(s) responsible for hepatic uptake of pazopanib in humans remain undetermined. To characterize its hepatic uptake mechanism, we screened the effects of several transporter inhibitors, including tetrapentylammonium (TPeA) for organic cation transporters (OCTs) and cyclosporin A (CsA) for organic anion-transporting polypeptides (OATPs), on both plasma disappearance and hepatic distribution of pazopanib in mice after its i.v. administration. Among the inhibitors, TPeA largely reduced hepatic distribution and plasma clearance of pazopanib, whereas CsA showed only partial reduction. Pazopanib uptake by isolated mouse hepatocytes was similarly reduced by these inhibitors, suggesting that OCTs play a major role in the overall hepatic uptake of pazopanib in mice. In human embryonic kidney cell line HEK293 cells stably transfected with human OCT1, pazopanib uptake was significantly higher than that in vector-transfected cells. Moreover, pazopanib uptake by OCT1 became saturated and was inhibited by TPeA, but not by CsA, confirming that pazopanib is also a substrate of human OCT1. Importantly, OCT1-mediated uptake of a typical OCT1 substrate metformin was inhibited by pazopanib with an IC50 value of 0.253 µM, indicating that pazopanib has the potential for clinically relevant inhibition of human OCT1. Finally, pazopanib was taken up by cryopreserved human pooled hepatocytes in a time-dependent manner, and this uptake was largely reduced by TPeA but only partially reduced by CsA. Thus, the present findings suggest that OCT1 is responsible for hepatocellular uptake of pazopanib.

Carnitine/Organic Cation Transporter OCTN1 Negatively Regulates Activation in Murine Cultured Microglial Cells.

Brain immune cells, i.e., microglia, play an important role in the maintenance of brain homeostasis, whereas chronic overactivation of microglia is involved in the development of various neurodegenerative disorders. Therefore, the regulation of microglial activation may contribute to their treatment. The aim of the present study was to clarify the functional expression of carnitine/organic cation transporter OCTN1/SLC22A4, which recognizes the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo, in microglia and its role in regulation of microglial activation. Primary cultured microglia derived from wild-type mice (WT-microglia) and mouse microglial cell line BV2 exhibited time-dependent uptake of [3H]- or d9-labeled ERGO. The uptake was markedly decreased in cultured microglia from octn1 gene knockout mice (octn1 -/--microglia) and BV2 cells transfected with small interfering RNA targeting the mouse octn1 gene (siOCTN1). These results demonstrate that OCTN1 is functionally expressed in murine microglial cells. Exposure of WT-microglia to ERGO led to a significant decrease in cellular hypertrophy by LPS-stimulation with concomitant attenuation of intracellular reactive oxygen species (ROS), suggesting that OCTN1-mediated ERGO uptake may suppress cellular hypertrophy via the inhibition of ROS production with microglial activation. The expression of mRNA for interleukin-1ß (IL-1ß) after LPS-treatment was significantly increased in octn1 -/--microglia and siOCTN1-treated BV2 cells compared to the control cells. Meanwhile, treatment of ERGO minimally affected the induction of IL-1ß mRNA by LPS-stimulation in cultured microglia and BV2 cells. Thus, OCTN1 negatively regulated the induction of inflammatory cytokine IL-1ß, at least in part, via the transport of unidentified substrates other than ERGO in microglial cells.

Usefulness of kidney slices for functional analysis of apical reabsorptive transporters.

Kidney plays a key role in the elimination and reabsorption of drugs and nutrients, however in vitro methods to evaluate renal disposition are limited. In the present study, we investigated usefulness of isolated kidney slice, which had been used for transport only at basolateral membrane of tubular epithelial cells, for evaluation of apical membrane transporters. As transporters that are easy to discriminate between apical and basolateral transports, apical membrane specific and sodium-dependent transporters (SGLTs and OCTNs) and pH-dependent transporters (PEPTs) are selected. Uptake of ergothioneine, carnitine and methyl-α-D-glucopyranoside, which are substrates of apical Octn1, Octn2, and Sglt1/2, respectively, by mice kidney slices showed clear Na+ dependence and reduction by selective inhibitors. In addition, sodium dependence of ergothioneine uptake was negligible in the kidney slice from Octn1-gene deficient mice. Moreover, uptake of PepT1/2 substrate glycyl-sarcosine, was higher than that in the presence of glycyl-leucine, a non-specific Pept inhibitor. The K m and IC 50 values for substrates and inhibitors of each transporter were mostly comparable to those obtained in transporter-transfected cells. In conclusion, it was demonstrated that kidney slices are promising tool to study transporters expressed at the apical membranes as well as basolateral membranes of kidney tubular epithelial cells.

Physiological Roles of Carnitine/Organic Cation Transporter OCTN1/SLC22A4 in Neural Cells.

Dysfunction in neurotransmission mediated by neurotransmitters causes various neurological disorders. Therefore, receptors and reuptake transporters of neurotransmitters have been focused on as a therapeutic target in neurological disorders. These membrane proteins have high affinity for a specific neurotransmitter and are highly expressed on synaptic membranes. In contrast, xenobiotic transporters have relatively lower affinity for neurotransmitters but widely recognize various organic cations and/or anions and are also expressed in brain neurons. However, it has been largely unknown why such xenobiotic transporters are expressed in neurons that play a key role in signal transduction. We have therefore attempted to clarify the physiological roles of one such xenobiotic organic cation transporter (OCT) in neural cells with the aim of obtaining new insight into the treatment of neurological disorders. Carnitine/organic cation transporter OCTN1/SLC22A4 is functionally expressed in neurons and neural stem cells. In particular, OCTN1 is expressed at much higher levels compared with other OCTs in neural stem cells and positively regulates their differentiation into neurons. OCTN1 accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a good in vivo substrate. Because ERGO is highly distributed into the brain after oral ingestion, OCTN1 may contribute to the alleviation of oxidative stress and promotion of neuronal differentiation via the uptake of ERGO in the brain, perhaps abating symptoms of neurological disorders. In this review, we introduce current topics on the physiological roles of OCTs with a focus on OCTN1 in neural cells and discuss its possible application to the treatment of neurological disorders.

Involvement of the Transporters P-Glycoprotein and Breast Cancer Resistance Protein in Dermal Distribution of the Multikinase Inhibitor Regorafenib and Its Active Metabolites.

Regorafenib is a multikinase inhibitor orally administered to colorectal cancer patients, and is known to often exhibit dermal toxicity. The purpose of this study is to clarify possible involvement of P-glycoprotein and breast cancer resistance protein (BCRP) in the dermal accumulation of regorafenib and its active metabolites M-2 and M-5. Following intravenous administration in triple knockout (Abcb1a/1b/bcrp-/-; TKO) and wild-type (WT) mice, delayed plasma clearance of M-2 and M-5, but not regorafenib, was observed in TKO mice compared to WT mice. Elacridar, an inhibitor of both transporters, also caused delayed clearance of M-2 and M-5, suggesting that these transporters are involved in their elimination. Skin-to-plasma concentration ratios of regorafenib, M-2, and M-5 were significantly higher in TKO mice than in WT mice. Elacridar increased skin-to-plasma and epidermis-to-plasma concentration ratios of regorafenib. Basal-to-apical transport of M-2 and M-5 was observed in LLC-PK1-Pgp and MDCKII/BCRP/PDZK1 cells, which was inhibited by elacridar and the BCRP inhibitor Ko143, respectively. The present findings thus indicate that P-glycoprotein and BCRP are involved in the accumulation of regorafenib and its active metabolites in the skin, by affecting either their systemic exposure or their plasma distribution in the circulating blood.