ABSTRACT
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.
Subject(s)
Central Nervous System Diseases , Ergothioneine , Neuroprotective Agents , Symporters , Animals , Antioxidants/metabolism , Antioxidants/pharmacology , Central Nervous System Diseases/drug therapy , Ergothioneine/pharmacology , Humans , Mammals/metabolism , Membrane Proteins/metabolism , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Organic Cation Transport Proteins/metabolismABSTRACT
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.
Subject(s)
Hippocampus/chemistry , Microscopy, Electrochemical, Scanning/methods , Nanoparticles/metabolism , Neurons/chemistry , Algorithms , Animals , Female , Hippocampus/cytology , Hippocampus/metabolism , Mice , Mice, Inbred ICR , Nanoparticles/analysis , Neurons/cytology , Neurons/metabolism , PregnancyABSTRACT
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.
Subject(s)
Epilepsy/metabolism , Organic Cation Transport Proteins/metabolism , Piperidines/pharmacology , Seizures/metabolism , Symporters/metabolism , Xenobiotics/pharmacology , Animals , Antioxidants/pharmacology , Brain-Derived Neurotrophic Factor/metabolism , Cytoskeletal Proteins/metabolism , Epilepsy/chemically induced , Ergothioneine/pharmacology , Gene Knockout Techniques , HEK293 Cells , Humans , Male , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Organic Cation Transport Proteins/genetics , Pentylenetetrazole , Piperidines/metabolism , Piperidines/urine , Seizures/chemically induced , Symporters/genetics , Xenobiotics/metabolism , Xenobiotics/urineABSTRACT
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.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , Dermatitis/metabolism , Histamine/metabolism , Metabolome/drug effects , Neoplasm Proteins/genetics , Skin/metabolism , Animals , Gene Deletion , Humans , Male , Mice , Mice, KnockoutABSTRACT
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.
Subject(s)
Cell Culture Techniques/methods , Cerebral Cortex/cytology , Culture Media/chemistry , Neurons/cytology , Polysaccharides, Bacterial/chemistry , Animals , Cerebral Cortex/embryology , Mice, Inbred C57BL , Mice, Inbred ICR , Microtubule-Associated Proteins/metabolism , Neurites/metabolism , Neurons/metabolism , Synapsins/metabolismABSTRACT
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.
Subject(s)
Liver/metabolism , Octamer Transcription Factor-1/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/pharmacokinetics , Sulfonamides/pharmacokinetics , Administration, Intravenous , Animals , Cyclosporine/pharmacology , HEK293 Cells , Hepatocytes/metabolism , Humans , Indazoles , Inhibitory Concentration 50 , Liver/cytology , Male , Metformin/pharmacokinetics , Mice , Mice, Inbred ICR , Octamer Transcription Factor-1/antagonists & inhibitors , Protein Kinase Inhibitors/administration & dosage , Pyrimidines/administration & dosage , Quaternary Ammonium Compounds/pharmacology , Sulfonamides/administration & dosage , Tissue Distribution , TransfectionABSTRACT
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.
Subject(s)
Biological Transport/drug effects , Carrier Proteins/metabolism , Ergothioneine/pharmacology , Membrane Proteins/metabolism , Microglia/drug effects , Neurons/drug effects , Animals , Antioxidants/metabolism , Antioxidants/pharmacology , Carnitine , Carrier Proteins/genetics , Cells, Cultured , Interleukin-1beta/metabolism , Lipopolysaccharides/pharmacology , Membrane Proteins/genetics , Mice , Microglia/metabolism , Neurons/metabolism , Organic Cation Transport Proteins/metabolism , SymportersABSTRACT
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.
Subject(s)
Organic Cation Transport Proteins/metabolism , Animals , Colitis/chemically induced , Colitis/metabolism , Colon/chemistry , Colon/metabolism , Cytokines/metabolism , HEK293 Cells , Humans , Inflammatory Bowel Diseases/metabolism , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Metabolomics , Mice , Mice, Inbred C57BL , Mice, Knockout , Organic Cation Transport Proteins/genetics , Spermine/metabolism , Symporters , Tissue Extracts/pharmacologyABSTRACT
PURPOSE: Monoamine oxidases (MAOs) are non-CYP enzymes that contribute to systemic elimination of therapeutic agents, and localized on mitochondrial membranes. The aim of the present study was to validate quantitative estimation of metabolic clearance of MAO substrate drugs using human liver microsomes (HLMs). METHODS: Three MAO substrate drugs, sumatriptan, rizatriptan and phenylephrine, as well as four CYP substrates were selected, and their disappearance during incubation with HLMs or mitochondria (HLMt) was measured. Metabolic clearance (CL) was then calculated from the disappearance curve. RESULTS: CL obtained in HLMs for sumatriptan and a typical MAO substrate serotonin was correlated with that obtained in HLMt among ten human individual livers. Hepatic intrinsic clearance (CLint,vitro) estimated from CL in HLMs was 14-20 and 2-5 times lower than in vivo hepatic intrinsic clearance (CLint,vivo) obtained from literature for MAO and CYP substrates, respectively. Utilization of HLMs for quantitatively assessing metabolic clearance of MAO substrates was further validated by proteomics approach which has revealed that numerous proteins localized on inner and outer membranes of mitochondria were detected in both HLMs and HLMt. CONCLUSION: CLint,vitro values of MAO substrate drugs can be quantitatively estimated with HLMs and could be used for semi-quantitative prediction of CLint,vivo values.
Subject(s)
Microsomes, Liver/metabolism , Monoamine Oxidase/metabolism , Phenylephrine/metabolism , Sumatriptan/metabolism , Triazoles/metabolism , Tryptamines/metabolism , Humans , Kinetics , Metabolic Clearance Rate , Phenylephrine/pharmacology , Sumatriptan/pharmacology , Triazoles/pharmacology , Tryptamines/pharmacologyABSTRACT
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.
Subject(s)
Neural Stem Cells/metabolism , Neurons/metabolism , Organic Cation Transport Proteins/metabolism , Animals , Carnitine/metabolism , Humans , Nervous System Diseases/metabolismABSTRACT
The high prevalence/incidence of hearing loss (HL) in humans makes it the most common sensory defect. The majority of the cases are of genetic origin. Non-syndromic hereditary HL is extremely heterogeneous. Genetic approaches have been instrumental in deciphering genes that are crucial for auditory function. In this study, we first used NADf chip to exclude the implication of known North-African mutations in HL in a large consanguineous Tunisian family (FT13) affected by autosomal recessive non-syndromic HL (ARNSHL). We then performed genome-wide linkage analysis and assigned the deafness gene locus to ch:5q23.2-31.1, corresponding to the DFNB60 ARNSHL locus. Moreover, we performed whole exome sequencing on FT13 patient DNA and uncovered amino acid substitution p.Cys113Tyr in SLC22A4, a transporter of organic cations, cosegregating with HL in FT13 and therefore the cause of ARNSHL DFNB60. We also screened a cohort of small Tunisian HL families and uncovered an additional deaf proband of consanguineous parents that is homozygous for p.Cys113Tyr carried by the same microsatellite marker haplotype as in FT13, indicating that this mutation is ancestral. Using immunofluorescence, we found that Slc22a4 is expressed in stria vascularis (SV) endothelial cells of rodent cochlea and targets their apical plasma membrane. We also found Slc22a4 transcripts in our RNA-seq library from purified primary culture of mouse SV endothelial cells. Interestingly, p.Cys113Tyr mutation affects the trafficking of the transporter and severely alters ergothioneine uptake. We conclude that SLC22A4 is an organic cation transporter of the SV endothelium that is essential for hearing, and its mutation causes DFNB60 form of HL.
Subject(s)
Cochlea/pathology , Consanguinity , Endothelium/pathology , Genes, Recessive/genetics , Hearing Loss/genetics , Mutation/genetics , Organic Cation Transport Proteins/genetics , Amino Acid Sequence , Animals , Cells, Cultured , Cochlea/metabolism , Endothelium/metabolism , Exome/genetics , Female , HEK293 Cells , Hearing Loss/pathology , High-Throughput Nucleotide Sequencing , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Pedigree , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Rats , Rats, Sprague-Dawley , Sequence Homology, Amino Acid , SymportersABSTRACT
We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100 µM in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.
Subject(s)
Amino Acid Transport System A/genetics , Cell Differentiation/genetics , Glutamates/pharmacology , Neural Stem Cells/drug effects , Up-Regulation , Animals , Cell Proliferation/genetics , Cells, Cultured , Mice , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , RNA, Messenger/genetics , Rats , Rats, WistarABSTRACT
The aim of the present study is to discover multidrug resistance-associated protein (MRP) inhibitors with neuroblastoma-selective cytotoxicity by means of fluorescence assay with a membrane-permeable fluorescent dye, Fluo-8 AM, based on our observation that gene expression of Mrp3 in neuroblastoma Neuro2a cells was remarkably higher than that in primary cultured cortical neurons, as determined by real-time PCR. Neuro2a cells showed minimal fluorescence upon incubation with Fluo-8 AM. However, blocking of Mrp3 efflux function by small interfering RNA (siRNA) transfection or inhibition with probenecid resulted in significant dye accumulation, observed as an increase of fluorescence. Interestingly, Mrp3 siRNA or probenecid treatment also resulted in increased cytotoxicity, as evidenced by decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-reducing activity of Neuro2a, with a concomitant increase in release of lactate dehydrogenase. On the other hand, primary cultured neurons exhibited higher fluorescence intensity after incubation with Fluo-8 AM regardless of addition of probenecid. Also, probenecid only minimally affected MTT-reducing activity. Thus, probenecid showed selective cytotoxicity towards Neuro2a cells. Based on these findings, we screened a series of established therapeutic agents for ability to induce Fluo-8 accumulation in Neuro2a cells. Several uricosuric and nonsteroidal anti-inflammatory drugs were identified, and these drugs were confirmed to decrease MTT-reducing activity selectively in Neuro2a. There was a negative linear correlation between Fluo-8 accumulation and cytotoxicity of these agents. Although the compounds identified here are insufficiently potent for practical application, further screening to discover higher-affinity MRP3 inhibitors using larger chemical libraries may uncover drug candidates with potent neuroblastoma-selective cytotoxicity.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Neuroblastoma/drug therapy , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Female , Mice , Mice, Inbred ICR , Neuroblastoma/metabolism , Neurons/drug effects , Neurons/metabolism , Pregnancy , Uricosuric Agents/pharmacologyABSTRACT
Posttraumatic stress disorder is a long-lasting psychiatric disease with the consequence of hippocampal atrophy in humans exposed to severe fatal stress. We demonstrated a positive correlation between the transient decline of 5-bromo-2'-deoxyuridine (BrdU) incorporation in the hippocampal dentate gyrus (DG) and long-lasting behavioral abnormalities in mice with traumatic stress. Here, we investigated pharmacological properties of theanine on the declined BrdU incorporation and abnormal behaviors in mice with traumatic stress. Prior daily oral administration of theanine at 50-500 mg/kg for 5 days significantly prevented the decline of BrdU incorporation, while theanine significantly prevented the decline in the DG even when administered for 5 days after stress. Consecutive daily administration of theanine significantly inhibited the prolonged immobility in mice with stress in forced swimming test seen 14 days later. Although traumatic stress significantly increased spontaneous locomotor activity over 30 min even when determined 14 days later, the increased total locomotion was significantly ameliorated following the administration of theanine at 50 mg/kg for 14 days after stress. These results suggest that theanine alleviates behavioral abnormalities together with prevention of the transient decline of BrdU incorporation in the hippocampal DG in adult mice with severe traumatic stress.
Subject(s)
Behavior, Animal/drug effects , Bromodeoxyuridine/metabolism , Dentate Gyrus/metabolism , Glutamates/administration & dosage , Glutamates/pharmacology , Mental Disorders/drug therapy , Mental Disorders/etiology , Stress Disorders, Post-Traumatic/drug therapy , Stress Disorders, Post-Traumatic/psychology , Administration, Oral , Animals , Disease Models, Animal , Locomotion/drug effects , Male , Mice, Inbred Strains , Motor Activity/drug effects , Severity of Illness Index , Stress Disorders, Post-Traumatic/complicationsABSTRACT
5-Aminosalicylic acid (5-ASA) is an orally administered therapeutic agent for inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease. We hypothesized that the absorption of 5-ASA is mediated by the polyspecific carnitine/organic cation transporter (OCTN1/SLC22A4), based on the similarity of chemical structure between 5-ASA and other OCTN1 substrates. Therefore, we examined the involvement of this transporter in the disposition of 5-ASA in vivo by using octn1 gene knockout (octn1(-/-)) mice. After oral administration of 5-ASA, the plasma concentrations of 5-ASA and its primary metabolite, N-acetyl-5-aminosalicylate (Ac-5-ASA), in octn1(-/-) mice were much lower than those in wild-type mice. The time required to reach maximum plasma concentration was also delayed in octn1(-/-) mice. On the other hand, the plasma concentration profiles of both 5-ASA and Ac-5-ASA after intravenous administration of 5-ASA (bolus or infusion) were similar in the two strains. Uptake of 5-ASA from the apical to the basal side of isolated small-intestinal tissues of octn1(-/-) mice, determined in an Ussing-type chamber, was lower than that in wild-type mice. Further, uptake of 5-ASA in HEK293 cells stably transfected with the OCTN1 gene, assessed as the sum of cell-associated 5-ASA and Ac-5-ASA, was higher than that in HEK293 cells transfected with the vector alone. Overall, these results indicate that OCTN1 is involved, at least in part, in the gastrointestinal absorption of 5-ASA.
Subject(s)
Carnitine/metabolism , Inflammatory Bowel Diseases/drug therapy , Intestinal Absorption , Intestine, Small/metabolism , Mesalamine/pharmacokinetics , Organic Cation Transporter 1/metabolism , Animals , Biological Availability , Biological Transport, Active , HEK293 Cells , Humans , Male , Membrane Proteins , Mesalamine/blood , Mesalamine/therapeutic use , Mice , Mice, Knockout , Organic Cation Transporter 1/genetics , Sequence Deletion , TransfectionABSTRACT
Eltrombopag (ELT), an orally available thrombopoietin receptor agonist, is a substrate of organic anion transporting polypeptide 1B1 (OATP1B1), and coadministration of ELT increases the plasma concentration of rosuvastatin in humans. Since the pharmacokinetic mechanism(s) of the interaction is unknown, the present study aimed to clarify the drug interaction potential of ELT at transporters. The OATP1B1-mediated uptake of ELT was inhibited by several therapeutic agents used to treat lifestyle diseases. Among them, rosuvastatin was a potent inhibitor with an IC(50) of 0.05 µM, which corresponds to one-seventh of the calculated maximum unbound rosuvastatin concentration at the inlet to the liver. Nevertheless, a simulation study using a physiologically based pharmacokinetic model predicted that the effect of rosuvastatin on the pharmacokinetic profile of ELT in vivo would be minimal. On the other hand, ELT potently inhibited uptake of rosuvastatin by OATP1B1 and human hepatocytes, with an IC(50) of 0.1 µM. However, the results of the simulation study indicated that inhibition of OATP1B1 by ELT can only partially explain the clinically observed interaction with rosuvastatin. ELT also inhibited transcellular transport of rosuvastatin in MDCKII cells stably expressing breast cancer resistance protein (BCRP), and was found to be a substrate of BCRP. The interaction of ELT with rosuvastatin can be almost quantitatively explained on the assumption that intestinal secretion of rosuvastatin is essentially completely inhibited by ELT. These results suggest that BCRP in small intestine may be the major target for interaction between ELT and rosuvastatin in humans.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Benzoates/pharmacokinetics , Blood Platelets/drug effects , Fluorobenzenes/pharmacokinetics , Hydrazines/pharmacokinetics , Intestine, Small/metabolism , Neoplasm Proteins/antagonists & inhibitors , Organic Anion Transporters/antagonists & inhibitors , Pyrazoles/pharmacokinetics , Pyrimidines/pharmacokinetics , Sulfonamides/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Adult , Animals , Benzoates/pharmacology , Biological Transport , Blood Platelets/cytology , Computer Simulation , Dogs , Drug Interactions , Fluorobenzenes/blood , HEK293 Cells , Hepatocytes/metabolism , Humans , Hydrazines/pharmacology , LLC-PK1 Cells , Liver-Specific Organic Anion Transporter 1 , Madin Darby Canine Kidney Cells , Male , Mice , Models, Biological , Neoplasm Proteins/genetics , Organic Anion Transporters/genetics , Pyrazoles/pharmacology , Pyrimidines/blood , Receptors, Thrombopoietin/agonists , Rosuvastatin Calcium , Sulfonamides/blood , SwineABSTRACT
PURPOSE: Clinical study has previously revealed that plasma concentration of 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, was higher in patients with end-stage renal failure than those with normal kidney function although SN-38 is mainly eliminated in the liver. Here, we focused on inhibition by uremic toxins of hepatic SN-38 uptake and down-regulation of uptake transporter(s) by uremic plasma in humans. METHODS: We evaluated SN-38 uptake and its inhibition by uremic toxins, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoxyl sulfate (Indox), hippuric acid (HA) and indole acetate (IA), with cryopreserved human hepatocytes and HEK293 cells stably expressing hepatic uptake transporters, organic anion transporting polypeptides (OATPs). We also collected plasma samples from patients with severe renal failure to examine their effects on mRNA level of OATPs in primary cultured human hepatocytes. RESULTS: SN-38 was taken up by hepatocytes, which showed biphasic saturation patterns. The SN-38 uptake by hepatocytes was significantly inhibited by a uremic toxin mixture including clinically relevant concentrations of CMPF, Indox, HA and IA. Kinetic analyses for OATP-mediated transport revealed that the uptake of SN-38 by OATP1B1 was the highest, followed by OATP1B3. Among the uremic toxins, CMPF exhibited most potent inhibition of OATP1B1-mediated SN-38 uptake and directly inhibited the uptake of SN-38 also in hepatocytes. In addition, gene expression of OATP1B1 and OATP1B3 in hepatocytes was significantly down-regulated by the treatment with the uremic plasma. CONCLUSIONS: OATP1B1-mediated hepatic uptake of SN-38 was inhibited by uremic toxins, and gene expression of OATP1B1 was down-regulated by uremic plasma.
Subject(s)
Biological Transport/drug effects , Camptothecin/analogs & derivatives , Down-Regulation/drug effects , Liver/drug effects , Adult , Camptothecin/metabolism , Camptothecin/pharmacology , Cell Line , Female , HEK293 Cells , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Irinotecan , Liver/metabolism , Male , Middle Aged , Organic Anion Transporters/metabolismABSTRACT
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.
ABSTRACT
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.
ABSTRACT
The underlying mechanisms are still unclear for the neuroprotective properties of nicotine to date, whereas we have shown functional expression of nicotinic acetylcholine receptors (nAChRs) responsible for the influx of extracellular Ca(2+) in cultured rat cortical astrocytes. In this study, we investigated the possible involvement of astrocytic nAChRs in the neuroprotection by this agonist. Exposure to nicotine predominantly induced mRNA expression of glial cell line-derived neurotrophic factor (GDNF) among the different neurotrophic factors examined in cultured astrocytes, in a manner sensitive to nAChR antagonists, nifedipine, and aCa(2+) chelator. Nicotine significantly increased GDNF in a concentration-dependent manner in cultured astrocytes but not in neurons or neural progenitors even at the highest concentration used. In cultured astrocytes, a transient increase was seen in the expression of mRNA and corresponding protein for GDNF during sustained exposure to nicotine for 24 hr. Cytotoxicity mediated by oxidative, calcium, mitochondrial, or endoplasmic reticulum stress was invariably protected against in cortical neurons cultured with conditioned medium from astrocytes previously exposed to nicotine, and preincubation with the anti-GDNF antibody reduced the neuroprotection by conditioned medium from astrocytes exposed to nicotine. Intraperitoneal administration of nicotine transiently increased the number of cells immunoreactive for both GDNF and glial fibrillary acidic protein in rat cerebral cortex. These results suggest that astrocytic nAChRs play a role in the neuroprotection against different cytotoxins after predominant upregulation of GDNF expression through a mechanism relevant to the acceleration of extracellular Ca(2+) influx in rat brain in a particular situation.