ABSTRACT
Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min-1 mg protein-1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.
Subject(s)
Renal Insufficiency, Chronic , Toxins, Biological , Humans , Uremic Toxins , Indican/metabolism , Chromatography, Liquid , HEK293 Cells , Tandem Mass Spectrometry , Renal Insufficiency, Chronic/metabolism , Cresols/metabolism , Toxins, Biological/metabolism , Cell Membrane/metabolism , Organic Anion Transporters, Sodium-IndependentABSTRACT
The ergothioneine transporter ETT (formerly OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). Recently, Sparreboom et al. reported that the ETT would transport nucleosides and nucleoside analogues such as cytarabine and gemcitabine with the highest efficiency. In our assay system, we could not detect any such transport. Subsequently, Sparreboom suggested that the intracellular metabolization of the nucleosides occurs so fast that the original compounds cannot be detected by LC-MS/MS after inward transport. Our current experiments with 293 cells disprove this hypothesis. Uptake of gemcitabine was easily detected by LC-MS/MS measurements when we expressed the Na+/nucleoside cotransporter CNT3 (SLC28A3). Inward transport was 1280 times faster than the intracellular production of gemcitabine triphosphate. The deoxycytidine kinase inhibitor 2-thio-2'-deoxycytidine markedly blocked the production of gemcitabine triphosphate. There was no concomitant surge in intracellular gemcitabine, however. This does not fit the rapid phosphorylation of gemcitabine. Uptake of cytarabine was very slow, but detection by MS was still possible. When the ETT was expressed and incubated with gemcitabine, there was no increase in intracellular gemcitabine triphosphate. We conclude that the ETT does not transport nucleosides.
Subject(s)
Ergothioneine , Chromatography, Liquid , Cytarabine , Deoxycytidine/analogs & derivatives , Humans , Organic Cation Transport Proteins/metabolism , Tandem Mass Spectrometry , GemcitabineABSTRACT
The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC-MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious. We wanted to normalize active transporters by the activity of a second transporter. A transporter tandem, generated by joining two transporter cDNAs into a single open reading frame, should guarantee a 1 : 1 stoichiometry. Here we created a series of tandems with different linkers between the human ergothioneine (ET) transporter ETT (gene symbol SLC22A4) and organic cation transporter OCT2 (SLC22A2). The linker sequence strongly affected the expression strength. The stoichiometry was validated by absolute peptide quantification and untargeted peptide analysis. Compared with wild-type ETT, the normalized ET clearance of the natural variant L503F was higher (f = 1.34); G462E was completely inactive. The general usefulness of the tandem strategy was demonstrated by linking several transporters with ETT; every construct was active in both parts. Transporter tandems can be used - without membrane isolation or protein quantification - as precise tools for transporter number normalization, to identify, for example, relevant transporters for a drug. It is necessary, however, to find suitable linkers, to check the order of transporters, and to verify the absence of functional interference by saturation kinetics.
Subject(s)
Cell Membrane/metabolism , Biological Transport/physiology , DNA Mutational Analysis/methods , DNA, Complementary/genetics , DNA, Complementary/metabolism , Ergothioneine/metabolism , Humans , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Organic Cation Transporter 2/metabolismABSTRACT
The N-α-trimethyl 2-selenohistidine selenoneine is the selenium isolog of the natural antioxidant ergothioneine. Sulfur-to-selenium substitutions are known to endow proteins and nucleic acids with special activities. In contrast, secondary metabolites that exploit selenium-specific chemistry are rare. Selenoneine therefore provides a unique opportunity to study how natural organoselenides interact with cellular processes. In this report we describe the chemical synthesis of selenoneine and other 2-selenoimidazoles. With synthetic selenoneine at hand we discovered a set of reactivities that distinguish selenoneine from ergothioneine, showing that the two compounds can fill distinct functional niches. Synthetic access to 2-selenoimidazoles should pave the way to explore the pharmaceutical potential and physiological function of this heretofore inaccessible class of compounds.
Subject(s)
Antioxidants/chemical synthesis , Antioxidants/pharmacology , Ergothioneine/pharmacology , Histidine/analogs & derivatives , Organoselenium Compounds/chemical synthesis , Organoselenium Compounds/pharmacology , Antioxidants/chemistry , HEK293 Cells , Histidine/chemical synthesis , Histidine/chemistry , Histidine/pharmacology , Humans , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Organoselenium Compounds/chemistry , Oxidation-ReductionABSTRACT
The candidate vitamin ergothioneine (ET) is a unique antioxidant. Expression of the ET transporter (ETT) (gene symbol SLC22A4) in distinct cells is thought to signal intracellular ET activity, since we have previously shown that the ETT is highly selective for ET. Unfortunately, some continue to hold the ETT as a relevant drug transporter, using the misleading functional name OCTN1, novel organic cation transporter. The present study was provoked by two recent reports in which new ETT substrates were declared. Astonishingly, the transport efficiencies (TEs) of ETT for saracatinib and some nucleoside drugs were as high as the TE for ET. Here we examined, based on regulated expression of ETT from human and rat in 293 cells and liquid chromatography-mass spectrometry quantification, the transport of several drugs. With the nucleosides cytarabine, gemcitabine, 2'-deoxycytidine, and 2'-deoxyadenosine, and the drugs saracatinib, ipratropium, metformin, and oxaliplatin, the uptake into cells expressing ETT was not increased over control cells. ETT-mediated uptake of gabapentin was detectable, but the TE was approximately 100-fold lower than the TE for ergothioneine (50-200 µl/min per milligram of protein). In conclusion, the ETT remains highly specific for its physiologic substrate ergothioneine. Our results contradict several reports on additional substrates. The ETT does not provide multiple substrate specificities, and it is not a transporter of cationic drugs. Only compounds that are related to ET in substructure-for example, gabapentin, carnitine, and TEA-can be transported, but with very low efficiency. Thus, ETT persists as a specific molecular indicator of ET activity.
Subject(s)
Biological Transport/physiology , Ergothioneine/metabolism , Animals , Antioxidants/metabolism , Cell Line , HEK293 Cells , Humans , Organic Cation Transport Proteins/metabolism , Pharmaceutical Preparations/metabolism , Rats , Substrate SpecificityABSTRACT
l-Ergothioneine (ET) is a sulfur-containing derivative of the amino acid histidine that offers unique antioxidant properties. The enzyme independent redox-chemistry of ET relies on the availability of the thiol tautomer to allow oxidative formation of disulfide bridges, i.e., the tautomeric equilibrium. To study the intrinsic properties of ET the tautomeric equilibrium is studied in the gas-phase by infrared multiphoton dissociation (IRMPD) spectroscopy. The IR ion spectra of isolated molecular ions of ET and of the biosynthetic precursors of ET, i.e., hercynine and Nε-methyl-hercynine are acquired. The analyte structures are independently investigated by density functional theory (DFT) and computed linear IR-spectra of tautomer ion structures are compared with the gas-phase spectra for identification. For the molecular ion of ET the simulated IR spectra of thione and thiol structures match the recorded IRMPD spectrum and that prevents an individual structure assignment. On the other hand, theory suggests that ET adopts a thione tautomer in MeOH solution which could be carried over from the condensed phase to gas phase and could be kinetically trapped after effective electrospray phase transfer and desolvation. Such a non-thermal behavior is also found for the molecular ions of protonated hercynine and Nε-methyl-hercynine. Contrary to that, the sodium complex ions of ET, hercynine and Nε-methyl-hercynine adopt the respective ground structures predicted by theory, which are reliably identified spectroscopically. For ET the thione tautomer is by far the most stable isomer in the sodium complex molecular ion.
ABSTRACT
In vertebrates, SLC22A13 is an evolutionarily conserved transport protein of the plasma membrane. In humans and rat, it is principally expressed in the kidney. The precise localization and physiological function are unknown. In the present study, immunohistochemistry revealed that expression of SLC22A13 is confined to the basolateral membrane of type A intercalated cells in rat kidney. Double-staining confirmed that SLC22A13 co-localizes with anion exchanger 1. LC-MS difference shading showed that heterologous expression of human and rat SLC22A13 in HEK (human embryonic kidney)-293 cells stimulates efflux of guanidinosuccinate, aspartate, glutamate and taurine. Time courses of uptake of [3H]aspartate and [3H]glutamate revealed that SLC22A13 counteracted endogenous uptake. By contrast, OAT2 (organic anion transporter 2), a bidirectional glutamate transporter, increased accumulation of [3H]glutamate. Thus SLC22A13 catalyses unidirectional efflux. Velocity of efflux of standard amino acids was measured by LC-MS/MS. Expression of SLC22A13 strongly stimulated efflux of aspartate, taurine and glutamate. When the intracellular concentrations of aspartate and taurine were increased by pre-incubation, velocities of efflux increased linearly. We propose that in type A intercalated cells, SLC22A13 compensates luminal exit of protons by mediating the basolateral expulsion of the anions aspartate and glutamate. In this context, unidirectional efflux is essential to avoid anion re-entering. Loss of SLC22A13 function could cause distal tubular acidosis.
Subject(s)
Aspartic Acid/metabolism , Epithelial Cells/metabolism , Glutamic Acid/metabolism , Kidney Tubules, Collecting/metabolism , Organic Anion Transporters/biosynthesis , Animals , Catalysis , Gene Expression Regulation , HEK293 Cells , Humans , Organic Anion Transporters/genetics , Protein Transport/physiology , RatsABSTRACT
The liver is the principal source of glutamate in blood plasma. Recently we have discovered that efflux of glutamate from hepatocytes is catalyzed by the transporter OAT2 (human gene symbol SLC22A7). Organic anion transporter 2 (OAT2) is an integral membrane protein of the sinusoidal membrane domain; it is primarily expressed in liver and much less in kidney, both in rats and humans. Many years ago, Häussinger and coworkers have demonstrated in isolated perfused rat liver that benzoic acid or specific 2-oxo acid analogs of amino acids like e.g. 2-oxo-4-methyl-pentanoate ('2-oxo-leucine') strongly stimulate release of glutamate (up to 7-fold); '2-oxo-valine' and the corresponding amino acids were without effect. The molecular mechanism of efflux stimulation has remained unclear. In the present study, OAT2 from human and rat were heterologously expressed in 293 cells. Addition of 1 mmol/l benzoic acid to the external medium increased OAT2-specific efflux of glutamate up to 20-fold; '2-oxo-leucine' was also effective, but not '2-oxo-valine'. Similar effects were seen for efflux of radiolabeled orotic acid. Expression of OAT2 did not increase uptake of benzoic acid; thus, benzoic acid is no substrate, and trans-stimulation can be excluded. Instead, further experiments suggest that increased efflux of glutamate is caused by direct interaction of benzoic acid and specific 2-oxo acids with OAT2. We propose that stimulators bind to a distinct extracellular site and thereby accelerate relocation of the empty substrate binding site to the intracellular face. Increased glutamate efflux at OAT2 could be the main benefit of benzoate treatment in patients with urea cycle defects.
Subject(s)
Acids/chemistry , Benzoic Acid/pharmacology , Glutamic Acid/metabolism , Liver/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , Animals , Benzoic Acid/chemistry , Binding Sites , Biological Transport , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Kidney/metabolism , Ligands , Mice , Models, Biological , Rats , Substrate Specificity , Time Factors , Valine/chemistryABSTRACT
BACKGROUND: The limited regenerative capacity of injured axons hinders functional recovery after nerve injury. Although no drugs are currently available in the clinic to accelerate axon regeneration, recent studies show the potential of vasohibin inhibition by parthenolide, produced in Tanacetum parthenium, to accelerate axon regeneration. However, due to its poor oral bioavailability, parthenolide is limited to parenteral administration. PURPOSE: This study investigates another sesquiterpene lactone, cnicin, produced in Cnicus benedictus for promoting axon regeneration. RESULTS: Cnicin is equally potent and effective in facilitating nerve regeneration as parthenolide. In culture, cnicin promotes axon growth of sensory and CNS neurons from various species, including humans. Neuronal overexpression of vasohibin increases the effective concentrations comparable to parthenolide, suggesting an interaction between cnicin and vasohibin. Remarkably, intravenous administration of cnicin significantly accelerates functional recovery after severe nerve injury in various species, including the anastomosis of severed nerves. Pharmacokinetic analysis of intravenously applied cnicin shows a blood half-life of 12.7 min and an oral bioavailability of 84.7 % in rats. Oral drug administration promotes axon regeneration and recovery after nerve injury in mice. CONCLUSION: These results highlight the potential of cnicin as a promising drug to treat axonal insults and improve recovery.
Subject(s)
Nerve Regeneration , Sesquiterpenes , Animals , Humans , Male , Mice , Rats , Axons/drug effects , Axons/physiology , Biological Availability , Cell Cycle Proteins/metabolism , Lactones/pharmacology , Nerve Regeneration/drug effects , Rats, Sprague-Dawley , Sesquiterpenes/pharmacologyABSTRACT
According to the 2012 WHO global tuberculosis (TB) report (http://apps.who.int/iris/bitstream/10665/75938/1/9789241564502_eng.pdf), the death rate for tuberculosis was over 1.4 million patients in 2011, with â¼9 million new cases diagnosed. Moreover, the frequency of comorbidity with human immunodeficiency virus (HIV) and with diabetes is on the rise, increasing the risk of these patients for experiencing drug-drug interactions (DDIs) due to polypharmacy. Ethambutol is considered a first-line antituberculosis drug. Ethambutol is an organic cation at physiological pH, and its major metabolite, 2,2'-(ethylenediimino)dibutyric acid (EDA), is zwitterionic. Therefore, we assessed the effects of ethambutol and EDA on the function of human organic cation transporter 1 (hOCT1), hOCT2, and hOCT3 and that of EDA on organic anion transporter 1 (hOAT1) and hOAT3. Potent inhibition of hOCT1- and hOCT2-mediated transport by ethambutol (50% inhibitory concentration [IC50] = 92.6 ± 10.9 and 253.8 ± 90.8 µM, respectively) was observed. Ethambutol exhibited much weaker inhibition of hOCT3 (IC50 = 4.1 ± 1.6 mM); however, significant inhibition (>80%) was observed at physiologically relevant concentrations in the gastrointestinal (GI) tract after oral dosing. EDA failed to exhibit any inhibitory effects that warranted further investigation. DDI analysis indicated a strong potential for ethambutol interaction on hOCT1 expressed in enterocytes and hepatocytes and on hOCT3 in enterocytes, which would alter absorption, distribution, and excretion of coadministered cationic drugs, suggesting that in vivo pharmacokinetic studies are necessary to confirm drug safety and efficacy. In particular, TB patients with coexisting HIV or diabetes might experience significant DDIs in situations of coadministration of ethambutol and clinical therapeutics known to be hOCT1/hOCT3 substrates (e.g., lamivudine or metformin).
Subject(s)
Antitubercular Agents/pharmacology , Ethambutol/pharmacology , Organic Cation Transport Proteins/metabolism , Organic Cation Transporter 1/metabolism , Animals , Biological Transport/drug effects , CHO Cells , Cricetulus , Drug Interactions , Enterocytes/drug effects , Hepatocytes/drug effects , Humans , Lamivudine/pharmacology , Metformin/pharmacologyABSTRACT
Fluoroquinolones (FQs) are important antimicrobials that exhibit activity against a wide range of bacterial pathogens and excellent tissue permeation. They exist as charged molecules in biological fluids, and thus, their disposition depends heavily on active transport and facilitative diffusion. A recent review of the clinical literature indicated that tubular secretion and reabsorption are major determinants of their half-life in plasma, efficacy, and drug-drug interactions. In particular, reported in vivo interactions between FQs and cationic drugs affecting renal clearance implicated organic cation transporters (OCTs). In this study, 13 FQs, ciprofloxacin, enoxacin, fleroxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, pefloxacin, prulifloxacin, rufloxacin, and sparfloxacin, were screened for their ability to inhibit transport activity of human OCT1 (hOCT1) (SLC22A1), hOCT2 (SLC22A2), and hOCT3 (SLC22A3). All, with the exception of enoxacin, significantly inhibited hOCT1-mediated uptake under initial test conditions. None of the FQs inhibited hOCT2, and only moxifloxacin inhibited hOCT3 (~30%), even at a 1,000-fold excess. Gatifloxacin, moxifloxacin, prulifloxacin, and sparfloxacin were determined to be competitive inhibitors of hOCT1. Inhibition constants (K(i)) were estimated to be 250 ± 18 µM, 161 ± 19 µM, 136 ± 33 µM, and 94 ± 8 µM, respectively. Moxifloxacin competitively inhibited hOCT3-mediated uptake, with a K(i) value of 1,598 ± 146 µM. Despite expression in enterocytes (luminal), hepatocytes (sinusoidal), and proximal tubule cells (basolateral), hOCT3 does not appear to contribute significantly to FQ disposition. However, hOCT1 in the sinusoidal membrane of hepatocytes, and potentially the basolateral membrane of proximal tubule cells, is likely to play a role in the disposition of these antimicrobial agents.
Subject(s)
Anti-Infective Agents/metabolism , Fluoroquinolones/metabolism , Organic Cation Transport Proteins/metabolism , Organic Cation Transporter 1/metabolism , Biological Transport, Active , Drug Interactions , Enterocytes/metabolism , HEK293 Cells , Hepatocytes/metabolism , Humans , Kidney Tubules, Proximal/metabolism , Organic Cation Transporter 2ABSTRACT
Recently we have uncovered a non-enzymatic multi-step cycle for the regeneration of ergothioneine (ET), after reaction with noxious singlet oxygen (1O2), by glutathione (GSH). When living cells were loaded with ET labeled with deuterium and N-15 atoms (D5-ET) and exposed to light in the presence of a photosensitizer, no loss of deuterium at position 5 of the imidazole ring was observed, in contradiction to our previous mechanistic proposal. Therefore, it was necessary to reexamine the in vitro products of ET and 1O2 by liquid chromatography coupled to high resolution mass spectrometry. Pure 1O2 was generated by thermolysis at 37 °C of the endoperoxide DHPNO2. The use of D5-ET enabled us to revise and extend the reaction scheme. On the main pathway, 1O2 attacks the imidazole ring, and the hydroperoxide intermediates are reduced rapidly by ET or GSH via different mechanisms. The intramolecular water elimination from the 5-hydroperoxide described previously is slower and not a part of the cycle. On another side path, 1O2 attacks the sulfur of ET to form a sulfine (S-oxide). The reduction of the sulfine also allows for the complete regeneration of ET. Experiments with methanol instead of water as solvent revealed that, in the absence of GSH, ET was attacked 6 times more frequently at the ring than at the sulfur. In the presence of 1 mM GSH or higher, both side paths were abandoned. ET efficiently captures 1O2 with its ring and can then be regenerated to a large extent by GSH, without enzyme involvement.
Subject(s)
Ergothioneine , Ergothioneine/chemistry , Singlet Oxygen/chemistry , Hydrogen Peroxide/chemistry , Deuterium , Glutathione/metabolism , Imidazoles , Water , OxygenABSTRACT
PURPOSE: Cocktails of transporter probe drugs are used in vivo to assess transporter activity and respective drug-drug interactions. An inhibitory effect of components on transporter activities should be ruled out. Here, for a clinically tested cocktail consisting of adefovir, digoxin, metformin, sitagliptin, and pitavastatin, inhibition of major transporters by individual probe substrates was investigated in vitro. METHODS: Transporter transfected HEK293 cells were used in all evaluations. Cell-based assays were applied for uptake by human organic cation transporters 1/2 (hOCT1/2), organic anion transporters 1/3 (hOAT1/3), multidrug and toxin extrusion proteins 1/2K (hMATE1/2K), and organic anion transporter polypeptide 1B1/3 (hOATP1B1/3). For P-glycoprotein (hMDR1) a cell-based efflux assay was used whereas an inside-out vesicle-based assay was used for the bile salt export pump (hBSEP). All assays used standard substrates and established inhibitors (as positive controls). Inhibition experiments using clinically achievable concentrations of potential perpetrators at the relevant transporter expression site were carried out initially. If there was a significant effect, the inhibition potency (Ki) was studied in detail. RESULTS: In the inhibition tests, only sitagliptin had an effect and reduced hOCT1- and hOCT2- mediated metformin uptake and hMATE2K mediated MPP+ uptake by more than 70%, 80%, and 30%, respectively. The ratios of unbound Cmax (observed clinically) to Ki of sitagliptin were low with 0.009, 0.03, and 0.001 for hOCT1, hOCT2, and hMATE2K, respectively. CONCLUSION: The inhibition of hOCT2 in vitro by sitagliptin is in agreement with the borderline inhibition of renal metformin elimination observed clinically, supporting a dose reduction of sitagliptin in the cocktail.
Subject(s)
Metformin , Organic Cation Transport Proteins , Humans , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolism , HEK293 Cells , Biological Transport , Sitagliptin Phosphate/pharmacology , Metformin/metabolism , Organic Cation Transporter 2/metabolism , Drug InteractionsABSTRACT
BACKGROUND: Organic cation transporters (OCT) are responsible for the uptake and intracellular inactivation of a broad spectrum of endogenous substrates and detoxification of xenobiotics and chemotherapeutics. The transporters became pharmaceutically interesting, because OCTs are determinants of the cytotoxicity of platin derivates and the transport activity has been shown to correlate with the sensitivity of tumors towards tyrosine kinase inhibitors. No data exist about the relevance of OCTs in hepatocellular carcinoma (HCC). METHODS: OCT1 (SLC22A1) and OCT3 (SLC22A3) mRNA expression was measured in primary human HCC and corresponding non neoplastic tumor surrounding tissue (TST) by real time PCR (n = 53). Protein expression was determined by western blot analysis and immunofluorescence. Data were correlated with the clinicopathological parameters of HCCs. RESULTS: Real time PCR showed a downregulation of SLC22A1 and SLC22A3 in HCC compared to TST (p ≤ 0.001). A low SLC22A1 expression was associated with a worse patient survival (p < 0.05). Downregulation was significantly associated with advanced HCC stages, indicated by a higher number of T3 tumors (p = 0.025) with a larger tumor diameter (p = 0.035), a worse differentiation (p = 0.001) and higher AFP-levels (p = 0.019). In accordance, SLC22A1 was less frequently downregulated in tumors with lower stages who underwent transarterial chemoembolization (p < 0.001) and liver transplantation (p = 0.001). Tumors with a low SLC22A1 expression (< median) showed a higher SLC22A3 expression compared to HCC with high SLC22A1 expression (p < 0.001). However, there was no significant difference in tumor characteristics according to the level of the SLC22A3 expression.In the western blot analysis we found a different protein expression pattern in tumor samples with a more diffuse staining in the immunofluorescence suggesting that especially OCT1 is not functional in advanced HCC. CONCLUSION: The downregulation of OCT1 is associated with tumor progression and a worse patient survival.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Neoplasm Proteins/metabolism , Organic Cation Transport Proteins/metabolism , Organic Cation Transporter 1/metabolism , Adult , Aged , Aged, 80 and over , Blotting, Western , Down-Regulation , Female , Humans , Male , Middle Aged , Organic Cation Transport Proteins/genetics , Organic Cation Transporter 1/genetics , Prognosis , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Survival AnalysisABSTRACT
Organic cation transporter 3 (OCT3, SLC22A3) is a low-affinity, high-capacity transporter widely expressed in the central nervous system (CNS) and other major organs in both humans and rodents. It is postulated that OCT3 has a role in the overall regulation of neurotransmission and maintenance of homeostasis within the CNS. It is generally believed that all antidepressant drugs in current clinical use exert their primary therapeutic effects through inhibition of one or more of the high-affinity neuronal plasma membrane monoamine transporters, such as the norepinephrine transporter and the serotonin transporter. In the present study, we investigated the inhibitory effects of selected antidepressants on OCT3 activity in OCT3-transfected cells to evaluate whether OCT3 inhibition may at least in part contribute to the pharmacological effects of tested antidepressants. The studies demonstrated that all examined antidepressants inhibited OCT3-mediated uptake of the established OCT3 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) in a concentration-dependent manner. The IC(50) values were determined to be 4.7 µM, 7.4 µM, 12.0 µM, 18.6 µM, 11.2 µM, and 21.9 µM for desipramine, sertraline, paroxetine, amitriptyline, imipramine, and fluoxetine, respectively. Additionally, desipramine had an IC(50) value of 0.7 µM for the uptake of NE by OCT3, while the IC(50) value of sertraline was 2.3 µM for 5-HT uptake. Both desipramine and sertraline appeared to inhibit OCT3 activity via a non-competitive mechanism. In vivo studies are warranted to determine whether such effects on OCT3 inhibition are of sufficient magnitude to contribute to the overall therapeutic effects of antidepressants.
Subject(s)
Antidepressive Agents/pharmacology , Organic Cation Transport Proteins/antagonists & inhibitors , Cell Survival/drug effects , HEK293 Cells , Humans , Methylamines/metabolism , Norepinephrine/metabolism , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolism , Pyridinium Compounds/metabolism , Serotonin/metabolism , TransfectionABSTRACT
Ergothioneine (ET) is a natural compound which humans and other vertebrates cannot synthesize themselves; it must be absorbed from food in which it is distributed very unevenly. In general, ET is considered an intracellular antioxidant. However, the precise physiological purpose of ET and the consequences of ET deficiency are still unclear. The ergothioneine transporter ETT (old name OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ET. Cells lacking ETT do not accumulate ET, since the plasma membrane is virtually impermeable for this hydrophilic zwitterion compound. The existence of an evolutionary conserved ergothioneine transporter implies a beneficial role for ET. ETT is the first and so far only biomarker of ET activity. Only cells with strong expression of ETT can accumulate ET to high levels. In the human body, the ability to absorb, distribute, and retain ET depends entirely on this transporter. Blockade or inactivation of ETT in animal models may be essential to at last understand the function of ET. In this review of ETT, the focus is on substrate specificity, subcellular localization, human expression profile and expression profiles across species.
Subject(s)
Antioxidants/metabolism , Ergothioneine/metabolism , Organic Cation Transport Proteins/metabolism , Animals , Humans , Models, Animal , SymportersABSTRACT
OAT (organic anion transporter) 2 [human gene symbol SLC22A7 (SLC is solute carrier)] is a member of the SLC22 family of transport proteins. In the rat, the principal site of expression of OAT2 is the sinusoidal membrane domain of hepatocytes. The particular physiological function of OAT2 in liver has been unresolved so far. In the present paper, we have used the strategy of LC (liquid chromatography)-MS difference shading to search for specific and cross-species substrates of OAT2. Heterologous expression of human and rat OAT2 in HEK (human embryonic kidney)-293 cells stimulated accumulation of the zwitterion trigonelline; subsequently, orotic acid was identified as an excellent and specific substrate of OAT2 from the rat (clearance=106 µl·min⻹·mg of protein⻹) and human (46 µl·min⻹·mg of protein⻹). The force driving uptake of orotic acid was identified as glutamate antiport. Efficient transport of glutamate by OAT2 was directly demonstrated by uptake of [³H]glutamate. However, because of high intracellular glutamate, OAT2 operates as glutamate efflux transporter. Thus expression of OAT2 markedly increased the release of glutamate (measured by LC-MS) from cells, even without extracellular exchange substrate. Orotic acid strongly trans-stimulated efflux of glutamate. We thus propose that OAT2 physiologically functions as glutamate efflux transporter. OAT2 mRNA was detected, after laser capture microdissection of rat liver slices, equally in periportal and pericentral regions; previous reports of hepatic release of glutamate into blood can now be explained by OAT2 activity. A specific OAT2 inhibitor could, by lowering plasma glutamate and thus promoting brain-to-blood efflux of glutamate, alleviate glutamate exotoxicity in acute brain conditions.
Subject(s)
Glutamic Acid/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , Orotic Acid/metabolism , Alkaloids/metabolism , Animals , Biological Transport, Active/genetics , Catalytic Domain/genetics , Cell Line, Transformed , HEK293 Cells , Humans , Organic Anion Transporters, Sodium-Independent/genetics , Rats , Substrate Specificity/geneticsABSTRACT
In all vertebrates including mammals, the ergothioneine transporter ETT (obsolete name OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). ETT is not expressed ubiquitously and only cells with high ETT cell-surface levels can accumulate ET to high concentration. Without ETT, there is no uptake because the plasma membrane is essentially impermeable to this hydrophilic zwitterion. Here, we review the substrate specificity and localization of ETT, which is prominently expressed in neutrophils, monocytes/macrophages, and developing erythrocytes. Most sites of strong expression are conserved across species, but there are also major differences. In particular, we critically analyze the evidence for the expression of ETT in the brain as well as recent data suggesting that the transporter SLC22A15 may also transport ET. We conclude that, to date, ETT remains the only well-defined biomarker for intracellular ET activity. In humans, the ability to take up, distribute, and retain ET depends principally on this transporter.
Subject(s)
Ergothioneine , Organic Cation Transport Proteins/physiology , Symporters/physiology , Animals , Antioxidants/metabolism , Biological Transport , Ergothioneine/metabolism , Humans , Mammals , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolism , Substrate Specificity , Symporters/genetics , Symporters/metabolismABSTRACT
PURPOSE: Quaternary ammonium salts have demonstrated marked accumulation in the left ventricular (LV) myocardium of rodents and swine. To investigate the mechanism underlying this uptake, the present study examined the interaction of [18F]fluoroethylquinolinium ([18F]FEtQ) with the family of organic cation transporters (OCTs). PROCEDURES: The cellular uptake of [18F]FEtQ into HEK293 cells, expressing human OCT1, -2, or -3 (HEK293-hOCT), and its inhibition by corticosterone was evaluated in vitro. The inhibitory effect of decynium 22 (D 22) in vivo was also studied, using PET/CT of HEK293-hOCT tumor-bearing mice. Furthermore, the distribution kinetics of [18F]FEtQ were determined in rats, with and without pre-administration of corticosterone, and following administration to a non-human primate (NHP). RESULTS: The accumulation of [18F]FEtQ in HEK293-hOCT cells was 15-20-fold higher than in control cells and could be inhibited by corticosterone. in vivo, the uptake of [18F]FEtQ in the LV myocardium of corticosterone-treated rats was significantly reduced compared to that of untreated animals. Similarly, following administration of D 22 to HEK293-hOCT tumor-bearing mice, the peak tumor uptake of [18F]FEtQ was reduced by 40-45 % compared to baseline. Contrary to the distinct accumulation of [18F]FEtQ in the LV myocardium of rats, no cardiac uptake was observed following its administration to a NHP. CONCLUSIONS: The quinolinium salt derivative [18F]FEtQ interacts with the family of OCTs, and this interaction could account, at least in part, for the increased uptake in the LV myocardium of rodents. Nonetheless, its low affinity for hOCT3 and the results of PET/CT imaging in a NHP indicate a limited clinical applicability as a radiopharmaceutical for cardiac and/or OCT imaging.
Subject(s)
Organic Cation Transport Proteins , Salts , Humans , Animals , Rats , Mice , Swine , HEK293 Cells , Rodentia , Radiopharmaceuticals , Corticosterone , Positron Emission Tomography Computed Tomography , Quaternary Ammonium Compounds , Myocardium , CationsABSTRACT
With the gaining prevalence of obesity, related risks during pregnancy are rising. Inflammation and oxidative stress are considered key mechanisms arising in white adipose tissue (WAT) sparking obesity-associated complications and diseases. The established anti-diabetic drug metformin reduces both on a systemic level, but only little is known about such effects on WAT. Because inhibiting these mechanisms in WAT might prevent obesity-related adverse effects, we investigated metformin treatment during pregnancy using a mouse model of diet-induced maternal obesity. After mating, obese mice were randomised to metformin administration. On gestational day G15.5, phenotypic data were collected and perigonadal WAT (pgWAT) morphology and proteome were examined. Metformin treatment reduced weight gain and visceral fat accumulation. We detected downregulation of perilipin-1 as a correlate and observed indications of recovering respiratory capacity and adipocyte metabolism under metformin treatment. By regulating four newly discovered potential adipokines (alpha-1 antitrypsin, Apoa4, Lrg1 and Selenbp1), metformin could mediate anti-diabetic, anti-inflammatory and oxidative stress-modulating effects on local and systemic levels. Our study provides an insight into obesity-specific proteome alterations and shows novel modulating effects of metformin in pgWAT of obese dams. Accordingly, metformin therapy appears suitable to prevent some of obesity's key mechanisms in WAT.