PROBING THE SUBSTRATE SPECIFICITY OF THE ERGOTHIONEINE TRANSPORTER WITH METIMAZOLE, HERYCINE, AND ORGANIC CATIONS
Probing the substrate speci.city of the ergothioneine transporter with methimazole, hercynine, and organic cations
Silke Grigat a, Stephanie Harl.nger a, Sonia Pal a, Ralph Striebinger a, Stefan Golz b, Andreas Geerts b,Andreas Lazar a,Edgar Schomiga,c, DirkGrundemanna,c,*
a Department of Pharmacology, University of Cologne, Gleueler Strabe 24, 50931 Cologne, Germany b Pharma Research—Target Research, Bayer Healthcare AG, 42096 Wuppertal, Germany c Center for Molecular Medicine, University of Cologne (CMMC), Germany
article info abstract
Article history:
Received 16 February 2007 Accepted 16 April 2007
Keywords: SLC22A4
Chronic in.ammatory disease Ergothioneine Ergothioneine transporter Methimazole Organic cations Recently, we have identi.ed the ergothioneine (ET) transporter ETT (gene symbol SLC22A4). Much interest in human ETT has been generated by case-control studies that suggest an association of polymorphisms in the SLC22A4 gene with susceptibility to chronic in.ammatory diseases. ETT was originally designated a multispeci.c novel organic cation transporter (OCTN1). Here we reinvestigated, based on stably transfected 293 cells and with ET as reference substrate, uptake of quinidine, verapamil, and pyrilamine. ETT from human robustly catalyzed transport of ET (68 ml/(min mg protein)), but no transport of organic cations was discernible. With ET as substrate, ETT was relatively resistant to inhibition by selected drugs; the most potent inhibitor was verapamil (Ki =11 mmol/l). The natural compound hercynine and antithyroid drug methimazole are related in structure to ET. However, ef.ciency of ETT-mediated transport of methimazole (Ki = 7.5 mmol/l) was 130fold lower, and transport of hercynine (Ki = 1.4 mmol/l) was 25-fold lower than transport of ET. ETT from mouse, upon expression in 293 cells, catalyzed high af.nity, sodium-driven uptake of ET very similar to ETT from human. Additional real-time PCR experiments based on 16 human tissues revealed ETT mRNA levels considerably lower than in bone marrow. Our experiments establish that ETT is highly speci.c for its physiological substrate ergothioneine. ETT is not a cationic drug transporter, and it does not have high af.nity for organic cation inhibitors. Detection of ETT mRNA or protein can therefore be utilized as a speci.c molecular marker of intracellular ET activity.
# 2007 Elsevier Inc. All rights reserved.
1. Introduction food in which it is distributed very unevenly; a distinguished
source of ET are mushrooms (0.1-1 mg/g dried material). Ergothioneine (ET) is a natural antioxidant which is ET is rapidly cleared from the circulation and then biosynthesized solely by fungi and mycobacteria [1]. avidly retained with minimal metabolism. The content of Humans like other mammals absorb it exclusively from ET varies greatly among human tissues [2]. High ET levels
* Corresponding author. Tel.: +49 221 478 7455; fax: +49 221 478 5022. E-mail address: dirk.gruendemann@uni-koeln.de (D. Gru ndemann). Abbreviations: ETT, ergothioneine transporter; LC, liquid chromatography; MPP+, 1-methyl-4-phenylpyridinium; MS, mass spectro
metry; TEA, tetraethylammonium 0006-2952/$ - see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2007.04.015
have been found in erythrocytes, bone marrow, seminal .uid and eye.
Chemically, ET is the betaine of histidine with a sulfur atom attached to position 2 of the imidazole ring (Fig. 1). It should not be considered a thiol compound, but rather a thione, a derivative of thiourea. As a consequence of the prevailing thione tautomer, ET is a very stable antioxidant with unique properties; e.g. it does not auto-oxidize at physiological pH and does not promote the generation of hydroxyl radical from H2O2 and Fe2+ ions (=Fenton reaction) [2]. The precise physiological role of ET is still unclear. Most authors consider it an intracellular antioxidant.
Recently, we have discovered an ET transporter (ETT; gene symbol SLC22A4) [3]. ETT from human (ETTh) has high af.nity for ET (Km =21 mmol/l) and catalyzes cotransport of ET with Na+. Cells lacking ETT do not accumulate ET, since their plasma membrane is virtually impermeable for this compound. By contrast, cells with expression of ETT accumulate ET to high levels. Based on the expression pro.le, we judge ETT to be necessary for the supply of ET primarily to erythrocyte progenitor cells and to monocytes.
Much interest in ETT has been generated by case-control studies that suggest an association of polymorphisms in the SLC22A4 gene with susceptibility to chronic in.ammatory diseases. The association with Crohn’s disease [4] has been largely con.rmed [5-8]. Moreover, associations with ulcerative colitis [9] and Type I diabetes [10] have been reported. There was association with rheumatoid arthritis in a Japanese cohort [11], but this could not be replicated in British [12] and Spanish cohorts [13]. Clearly, in order to elucidate the role of ETT in the genesis of chronic in.ammatory diseases, it will be necessary to fully understand substrate speci.city and localization of the carrier.
Our previous results suggest that expression of ETT in speci.c cells indicates intracellular ergothioneine activity. However, the gene product of SLC22A4 was originally designated a multispeci.c novel organic cation transporter (OCTN1), because it was reported to transport tetraethylammonium [14], and quinidine, verapamil, and pyrilamine [15].If ETT also functions as an organic cation transporter, then it cannot serve as a speci.c molecular marker of ET activity. Thus, it was one of our aims here to clarify, with ET as reference substrate, whether ETT transports organic cations. In addition, we tested organic cations as inhibitiors to see if ETT displays high af.nity towards organic cations with ET as substrate.
The widely used antithyroid drug methimazole (=1methyl-imidazole-2-thione) and the side chain of ET have, except for the methyl moiety, identical structures (cf. Fig. 1). In view of the possible involvement of ETT in chronic in.ammatory diseases, it is very interesting that methimazole has immunosuppressive [16-18] and powerful anti-in.ammatory
[19] activity. In order to evaluate whether ETT could provide a speci.c route of entry of methimazole into those cells that express this carrier, we tested methimazole as a substrate. The substrate speci.city of ETT was probed further with the natural precursor of ET, hercynine, which lacks the sulfur atom but is otherwise identical to ET (Fig. 1).
Finally, we expressed and analyzed ETT from mouse in 293 cells to see if key functional properties are conserved over species.
2. Materials and methods
2.1. Plasmid constructs
The construction of pEBTetD/ETTh has been described
previously [20]. The cDNA of ETTm was inserted into the polylinker of a plasmid related to pEBTet but without the cassette for expression of the Tet repressor; the latter was supplied by a second plasmid [20]. The cDNA sequence of ETTm corresponds to GenBank entry AB016257 except for a single base deviation at position 66 downstream of the stop codon (C > T). The 50-interface between cDNA and vector is GTTTAAACTT AAGCTT CGCGCCGAAT (polylinker in bold, cDNA underlined); the 30-interface is TCAAAAGCCT GGATCC ACTA. The construct was assembled by standard cloning methods; the whole insert was veri.ed by DNA sequencing.
2.2. Cell culture
293 cells (ATCC CRL-1573), a transformed cell line derived from human embryonic kidney, were grown at 37 8C in a humidi.ed atmosphere (5% CO2) in plastic culture .asks (Falcon 3112, Becton Dickinson, Heidelberg, Germany). The growth medium was Dulbecco’s Modi.ed Eagle Medium (Life Technologies 31885-023, Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (PAA Laboratories, Colbe, Germany). Medium was changed every 2-3 days and the culture was split every 5 days.
Stably transfected cell lines were generated as reported previously for the pEBTetD vector [20]; cell culture medium always contained 3 mg/ml puromycin (PAA Laboratories) to ascertain plasmid maintenance. To turn on protein expression, cells were cultivated for at least 20 h in regular growth medium supplemented with 1 mg/ml doxycycline (195044, MP Biomedicals, Eschwege, Germany).
2.3. Transport assays
For measurement of solute uptake by LC-ESI-MS/MS, cells were grown in surface culture on 60 mm polystyrol dishes (Nunclon 150288, Nunc, Roskilde, Denmark) precoated with
0.1 g/l poly-L-ornithine in 0.15 M boric acid-NaOH, pH 8.4. Cells were used for uptake experiments at a con.uence of at least 70%. Uptake was measured at 37 8C. Uptake buffer contains 125 mmol/l NaCl, 25 mmol/l HEPES-NaOH pH 7.4, 5.6 mmol/l (+)glucose, 4.8 mmol/l KCl, 1.2 mmol/l KH2PO4, 1.2 mmol/l CaCl2, and 1.2 mmol/l MgSO4. With inhibitor concentrations >1 mmol/l, the NaCl concentration was isoosmotically reduced. After preincubation for at least 20 min in 4 ml of uptake buffer, the buffer was replaced with 2 ml of substrate in uptake buffer. Incubation was stopped after 1 min by rinsing the cells four times with each 4 ml ice-cold uptake buffer. Subsequently, the cells were solubilized with 4 mmol/l HClO4 and stored at .20 8C. After centrifugation (1 min, 16,000 .g, 20 8C) of the thawed lysates, 100 ml of the supernatant was mixed with 10 ml unlabelled MPP+ iodide (5.0 ng/ml) which served as internal standard. Of this mixture, 20 ml samples were analyzed by LC-MS/MS on a triple quadrupole mass spectrometer (TSQ Quantum, Thermo Electron, Dreieich, Germany). Atmospheric pressure ionization with positive electrospray was used. The LC system consisted of Surveyor LC-pump, autosampler, and Waters Atlantis HILIC silica column (length 100 mm, diameter 3 mm, particle size 5 mm). The solvent for isocratic chromatography (.ow rate 250 ml/ min) was made of methanol (70%) and 0.1% formic acid (30%). For quanti.cation by SRM (selected reaction monitoring; scan time 0.3 s), at .rst the optimal collision energy (CE) for argon-induced fragmentation in the second quadrupole was determined for each analyte. From the product ion spectra, the following fragmentations were selected for SRM (m/z parent, m/z fragment, CE)—ergothioneine: 230, 127, 24 V; hercynine: 198, 95, 20 V; methimazole: 115, 57, 24 V; MPP+: 170, 128, 25 V; pyrilamine: 286, 121, 17 V; quinidine: 325, 184, 32 V; verapamil: 455, 165, 26 V. For each analyte, the area of the intensity versus time peak was integrated and divided by the area of the MPP+ peak to yield the analyte response ratio. Linear calibration curves (R2 > 0.99) were constructed from at least six standards which were prepared using control cell lysates as solvent. Sample analyte content was calculated from the analyte response ratio and the slope of the calibration curve, obtained by weighted linear regression.
Protein was measured by the BCA assay with bovine serum albumin as standard. The protein content of MS samples was estimated from the response ratio for proline, which was calibrated against the BCA assay (four to six matched cell dishes) for each MS session.
2.4. Synthesis of hercynine
Since hercynine is not available commercially, it was synthesized from a-N,N-dimethyl-L-histidine (F3625, Bachem, Bubendorf, Switzerland) and methyl iodide as described [21]. The raw product was puri.ed by preparative thin layer chromatography on a glass backed silica gel plate (cat. no. 113792, Merck, Darmstadt, Germany) with methanol/H2O/25% (v/v) NH3 (8:2:1) as developing solvent. Hercynine was eluted with water from the silica gel fraction, dried, and weighed. Our preparation was 91% pure as judged by LC-MS total ion current (positive mode).
2.5. Calculations and statistics
The clearance is directly proportional to kcat/Km(kcat: turnover number) and thus a valid measure of ef.ciency of transport (provided that the substrate concentration is much smaller than the respective Km) [22]. The clearance equals initial rate of speci.c uptake (=uptake mediated by expressed carrier) divided by substrate concentration. Speci.c uptake equals total uptake minus uptake into control cells (=non-speci.c uptake).
Analysis of saturation curves and calculation of Ki-values have been reported previously [23].Fitted parameters such as Km-and Ki-values are given as geometric mean with 95% con.dence interval. Arithmetic means are given with S.E.M. Velocity of uptake of ET as a function of Na+ concentration (Fig. 7) was described with a modi.ed Hill function: v ¼ V0 þ
Vlim =ð1 þKc.hcNaþ
NaþÞ with h = Hill coef.cient and = sodium concentration. In the absence of any sign of inhibition, a Kivaluewas estimatedasthe highest used inhibitor concentration multiplied by 5. The unpaired t-test was used to test for signi.cance; two-tailed P values are given.
2.6. Drugs
5-Aminosalicylic acid (819019, Merck, Darmstadt, Germany), L-carnitine (C-0283, Sigma-Aldrich, Munich, Germany), L-(+)ergothioneine (F-3455, Bachem, Bubendorf, Switzerland), methimazole (M-8506, Sigma-Aldrich), 1-methyl-4-phenylpyridinium iodide (D-048, Sigma-Aldrich), pyrilamine (P5514, Sigma-Aldrich), quinidine (Q-0875, Sigma-Aldrich), thioperamide (T-123, RBI, Natick, MA, USA), verapamil-HCl (Knoll AG (Abbott Laboratories), Liestal, Switzerland). Disprocynium24 (1,10-diisopropyl-2,4-cyanine iodide) was synthesized as described previously [24]. All other chemicals were at least of analytical grade.
3. Results
3.1. Transport of organic cations
Uptake mediated by ETT from human (ETTh) of 1 mmol/l quinidine, verapamil, pyrilamine, and ET was investigated with 293 cells stably transfected with pEBTetD/ETTh. pEBTetD is an Epstein-Barr plasmid vector for doxycycline-inducible protein expression in human cell lines based on the simple tetracycline repressor [20]. Expression is turned on by addition of 1 mg/ml doxycycline to the culture medium for about 20 h. This system provides a high rate of ETTh-mediated transport in the on-state (=100%) and a low rate (4%) in the off-state [20]. The results (Fig. 2) indicate high levels of quinidine, verapamil, and pyrilamine in off-state and on-state cell lysates. However, expression of the transporter did not increase lysate contents (P = 0.38, 0.76, and 0.50, respectively; n = 5). By contrast, the ET
Fig. 2 - Determination by LC-MS/MS of uptake of ET and organic cations into 293 cells with or without expression of ETTh. Cells grown in dishes were incubated for 1 min with 1.0 mmol/l substrate in uptake buffer, washed, and lysed with methanol. The substrate content of cell lysates was determined by LC-MS/MS. Endogenous ET, corresponding to 30 W 1 pmol/(min mg protein) for off-state cells and 77 W 4 pmol/(min mg protein) for on-state cells, was subtracted to yield the uptake rates shown. The clearance for ET thus amounts to 62 W 8 ml/ (min mg protein).
content was much higher in on-state compared to off-state cell lysates (P < 0.0001; n = 3), indicating robust expression of functional transporter. Note that for both cell states, ET already present in the cells at the beginning of the uptake period due to uptake from the cell culture medium was subtracted; it was determined with matching dishes incubated in uptake buffer devoid of substrate. For the xenobiotics, no such correction is necessary. Our data suggests that ETT does not transport quinidine, verapamil or pyrilamine.
3.2. Inhibition by organic cations of ET transport
293 cells expressing ETTh were analyzed for inhibition of ET uptake by selected drugs (Fig. 3, Table 1). None of the drugs displayed high af.nity (Ki < 1 mmol/l) towards ETTh. Verapamil (Ki =11 mM) was the most potent inhibitor, followed by disprocynium24, pyrilamine, and thioperamide. Lidocaine up to a concentration of 64 mM failed to inhibit ETTh noticeably. ETTh was also fully resistant to inhibition by carnitine and 5aminosalicylic acid, each up to 640 mM.
3.3. Methimazole
Uptake of methimazole and ET was determined in parallel with 293 cells stably transfected with pEBTetD/ETTh as above. Cell lysates were analyzed by LC-MS/MS. Initial rates of speci.c uptake were calculated as the difference in substrate content of on-state-cells and off-state-cells, divided by uptake time (=1 min). Endogenous ET was determined and subtracted as above. With 100 mmol/l methimazole, a clearance of
0.3 .0.2 ml/(min mg protein) was calculated (n = 3). With 10 mmol/l ET, measured with paired dishes, the clearance was 39 .2 ml/(min mg protein). Thus, transport of methima-
Fig. 3 - Inhibition of ETTh-mediated uptake of ET by selected drugs. An uptake period of 1 min was chosen to approximate initial rates of transport. Shown is mean W S.E.M. (n = 3) of the specific uptake of ET (10 mmol/ l) in the presence of inhibitor relative to control. Specific uptake was calculated as total content minus endogenous content of control cells divided by uptake time. A Hill coefficient of 1 was used for non-linear regression.
zole by ETTh is negligible. The af.nity of ETTh for methimazole was determined by inhibition of uptake of ET (Fig. 4). A Ki of 7.5 mmol/l was determined from the data (Table 1).
3.4. Hercynine
Hercynine was synthesized, puri.ed, and analyzed for purity as described in Section 2. Uptake of hercynine and ET was determined in parallel with 293 cells stably transfected with pEBTetD/ETTh as above. Cell lysates were analyzed by LC-MS/ MS. Endogenous substrate was determined and subtracted as above. With 100 mmol/l hercynine, a clearance of 1.1 .0.2 ml/ (min mg(protein)) was calculated (n = 4). With 10 mmol/l ET, measured with paired dishes, the clearance was 28 .3 ml/
Fig. 4 - Inhibition of ETTh-mediated uptake of ET by methimazole and hercynine. See legend to Fig.
3
for experimental conditions.
(min mg(protein)). It follows that ETT transports hercynine, albeit at low transport ef.ciency. The af.nity of ETTh for hercynine was determined by inhibition of uptake of ET (Fig. 4). A Ki of 1.4 mmol/l was extrapolated from the data (Table 1).
3.5. Functional characterization of ETT from mouse
ETT from mouse (ETTm) was expressed in 293 cells and analyzed for uptake of ET. In saturation analysis, a Km of 50 mmol/l (95% con.dence interval, 33-76) was determined
Fig. 5 - Saturation of uptake of ET mediated by ETT from mouse. An uptake period of 1 min was chosen to approximate initial rates of transport. Shown is mean W S.E.M. (n = 3). Expressed uptake equals total content minus endogenous content divided by uptake time minus non-specific uptake. Non-specific uptake increased linearly with ET concentration, slope = 0.20 ml/ (min mg protein). Vmax = 4.7 W 0.3 nmol/(min mg protein). Inset: Eadie-Scatchard transformation.
Fig. 6 - Sodium dependence of uptake of ET mediated by ETTm. Cells were rapidly washed with modified uptake buffers in which N-methyl-D-glucosamine isoosmotically substituted Na+ as indicated and then assayed for uptake in the same buffer. Control uptake buffer was completely free of Na+. Shown is mean W S.E.M. (n = 3) of specific initial rates of uptake relative to control (uptake period = 1 min). A Hill coefficient of 1.1 W 0.4 resulted from curve fitting.
(Fig. 5). Replacement of Na+ in the uptake buffer by N-methylD-glucosamine demonstrated that uptake of ET by ETTm is strongly stimulated by extracellular sodium ions (Fig. 6).
3.6. Analysis of ETTh mRNA levels
The expression of ETT was investigated by real-time PCR as described previously [3] in several additional human tissues (Fig. 7). mRNA levels in all tested tissues were much lower than in bone marrow, with the highest levels still in mammary gland, prostate and ovary (>0.2 relative to bone marrow). Note that there is virtually no ETTh mRNA in thyroid and salivary gland.
4. Discussion
There is an enduring notion that ETT (OCTN1) functions as a proton/organic cation antiporter or organic cation/organic cation antiporter [25,26]. For the most part, this notion is probably based on inhibition experiments [15,27], but it must be stressed that it is impossible to tell whether an inhibitor is actually a substrate [28]. In fact, there is only a single report that actually states transport of organic cations other than TEA, i.e. quinidine, verapamil, and pyrilamine [15]. All other functional assays were based on transport of radiolabeled TEA or carnitine. Recently, we have demonstrated that ET is transported >100 times more ef.ciently than TEA and carnitine [3]. With the discovery of ET as a high performance substrate, it has become possible to test the relevance of transport of organic cations. 293 cells rapidly accumulate the highly lipophilic organic cations, but expression of ETT does not increase accumulation (Fig. 2). Functional expression of ETTh was veri.ed by robust uptake of ET. Thus, ETT does not discernibly transport quinidine, verapamil, and pyrilamine. By contrast, Yabuuchi et al. recorded signi.cantly higher radioactivity (by a factor of 2.3, 1.6, and 2.2, respectively) with transporter-expressing versus water-injected oocytes from Xenopus laevis (see Fig. 3 in the cited work). One possible explanation would be, rather than transport, mere binding of radiolabel to the carrier [24]; this binding may not be apparent in a mammalian cell line since transporter overexpression in the plasma membrane of the oocyte appears to be exceptionally high [29].
We have measured inhibition of ET transport by selected drugs to see if ETT displays, in conjunction with its physiological substrate, high af.nity towards organic cations. In a previous study with TEA as substrate [4], the most potent inhibitor of the wild-type carrier (=OCTN1-Leu503) was reported as the antiarrhythmic and local anaesthetic lidocaine (Ki = 0.83 mmol/l), followed by calcium channel antagonist verapamil (8.4 mmol/l) and carnitine (24 mmol/l). By contrast, we can extrapolate from our experiments - also with the wild-type carrier, but with ET as substrate - Ki-values of .320 mmol/l for lidocaine and .3200 mmol/l for carnitine. To explain the discrepancies one could assume that ET and TEA bind to different transport sites and thus interact differently with the tested inihibitors. TEA, for example, could bind to the sodium-binding site. However, our Ki for verapamil (11 mmol/l) agrees with the previous report. Verapamil represents the most potent inhibitor in our assays, but we rate its af.nity only as moderate. Disprocynium24 displayed similar af.nity (Ki =15 mmol/l); note that it is by three orders more potent on the organic cation transporter type 2 (OCT2, gene symbol SLC22A2) and on the extra-neuronal monoamine transporter (EMT, gene symbol SLC22A3) [30,31]. Low af.nity was observed for pyrilamine (Ki = 180 mmol/l), a histamine H1 receptor antagonist also known as mepyramine, and thioperamide (Ki = 250 mmol/l), a histamine H3 receptor antagonist that contains, similar to ET, both thiourea and imidazole structures. The anti-in.ammatory drug 5-aminosalicylic acid, also known as mesalazine or mesalamine, is considered the active moiety of sulfasalazine. Since it shows predominant actions in the gut, it is used to treat Crohn’s disease and mild to moderate ulcerative colitis. Its mechanism of action is not entirely clear. With an extrapolated Ki of .3200 mmol/l, our data suggest that ETT is not a target of 5-aminosalicylic acid. Altogether, it appears from our data that ETT is largely resistant to inhibition by organic cations, at least when assayed with its physiological substrate. In other words, currently no potent inhibitor is available for ETT.
The substrate speci.city of ETT was probed further with hercynine. Hercynine is a precursor in the biosynthesis of ET [1]. It lacks the sulfur atom, but is otherwise identical in structure to ET. Our results indicate that hercynine is transported, albeit at low ef.ciency; ET is transported 25-fold better than hercynine. Concordantly, hercynine inhibited uptake of ET with low af.nity (Ki = 1.4 mmol/l).
The glycine betaine moiety of ET may be regarded a hydrophilic handle that minimizes membrane permeability. The imidazole-2-thione moiety likely is responsible for the physiological activity of ET. Hence, the widely used antithyroid drug methimazole (Fig. 1) with its highly similiar structure might supply important clues to the function of ET. In the human body, methimazole is concentrated by cells in the thyroid, salivary glands, and polymorphonuclear leucocytes [32]. It is well documented that methimazole has immunosuppressive activity. This was traced back to monocytes and macrophages, the tissue-resident derivatives of monocytes [32]. In monocytes, methimazole inhibits the respiratory burst,
i.e. the production of oxygen radicals and H2O2 [16,17]. This was interpreted as inhibition of peroxidase or scavenging of free oxygen radicals. Interestingly, monocytes and macrophages, like neutrophils and thyroid cells, accumulate methimazole. These cells apparently express a speci.c transport protein, since lymphocytes and two cell lines did not accumulate methimazole [32]. It is clear from previous work that ETT is strongly expressed in monocytes [3,11].We therefore have tested whether ETT could provide a speci.c route of entry of methimazole into monocytes. Our results indicate that methimazole is virtually no substrate of ETT. Consistently, with a Ki of 7.5 mmol/l (Fig. 4, Table 1), ETTh has very low af.nity for methimazole. It follows that methimazole must use a carrier other than ETT to enter cells. We expect that this putative methimazole transporter will not transport ET, but this cannot be tested at present since no such carrier has been molecularly identi.ed. Interestingly, our real-time PCR data suggest that methimazole is accumulated in cells that negligibly express ETT, if at all, i.e. salivary glands and thyroid. This makes good sense, since lack of ETT protects the thyroid from import of ET, a potential inhibitor of thyroid peroxidase (see below). It is likely therefore that ET and methimazole in general distribute to different cells. Still, in monocytes, because of concurrent expression of transporters, methimazole could indeed mimic a bene.cial effect of ET. Altogether, previous reports on the in vivo effects of methimazole cannot be used directly to understand the function of ET. However, in vitro, without any membrane barrier, methimazole can be considered a valid surrogate of ET.
The existence of a speci.c transporter suggests that ergothioneine is advantageous for our long-term health. The key question to understand the purpose of ET is—what is the unique intracellular bene.t from ET in the presence of 10-fold higher concentrations of the ubiquitous hydrophilic antioxidants glutathione(GSH)and ascorbate? Thereare manyreports that imidazole-2-thiones like ET and methimazole are potent antioxidants in vitro [33,34]; however, thereis nocomprehensive evidence that imidazole-2-thiones are much more potent than GSH, ascorbate, or e.g. trolox, the water-soluble analog of vitamin E [35]. Thus, ET clearly has properties of a general antioxidant or radical scavenger, but other intracellular antioxidants could probably replace this function.
Our present results with ETT from mouse con.rm that ETT catalyzes high af.nity, sodium-driven uptake of ET (Figs. 5 and 6). Our real-time PCR data emphasize strong expression of ETT in bone marrow [36], since all other tissues in Fig. 7 had considerably lower mRNA levels. More precisely, ETT is strongly expressed in CD71+ (= transferrin receptor) cells [3]. These .ndings suggest that ETT charges developing erythrocytes with available ET. We entertain the hypothesis that what really distinguishes ET from other antioxidants is its interaction with protein-bound heme [3]. As detailed previously, we expect ET not to affect native hemoglobin (HbFeII), but only to bind to or react with ferryl hemoglobin (HbFeIV
O). The HbFeIV
O species is a highly reactive intermediate in the autocatalytic oxidation, caused by many xenobiotics, of HbFeIIO2 to methemoglobin (HbFeIII) and is also considered a starting point for detrimental radical reactions including heme degradation [37]. Thus, the primary function of ET can be considered as protecting erythrocytes against damage related to HbFeIV
O [38]. Monocytes do not express hemoglobin, so there must be another target for ET. A critical clue comes from the fact that hemoglobin has peroxidase activity, analogous to that of horseradish peroxidase [39]. Peroxidases like myeloperoxidase, eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase (TPO) are closely related in structure and function. The second important clue comes from a detailed cell-free analysis of the reaction of methimazole, the in vitro substitute of ET, with its pharmacological target TPO. Methimazole does not react with reduced heme. However, it is highly interesting that methimazole binds to and covalently inactivates the heme group of TPO only if the heme is in an oxidized state (=compound II) [40]. Compound II corresponds directly to ferryl hemoglobin [39]. Most notably, GSH was inactive in this assay. Thus, af.nity towards oxidized peroxidase may be the key feature of imidazole-2-thione compounds. Indeed, it has been shown that potent inhibition of the prostaglandin H synthase complex by methimazole is not caused by inhibition of the cyclooxygenase but rather by inhibition of the hydroperoxidase component [19]. From these pieces of evidence we infer that ET, by analogy with methimazole, may provide protection for monocytes by speci.c interaction with peroxidase(s). A lack of ET may thus represent a precipitating factor in the genesis of chronic in.ammatory diseases.
In conclusion, our experiments with hercynine and methimazole establish that ETT is highly speci.c for its physiological substrate ergothioneine. ETT is not a ‘‘multispeci.c’’ cationic drug transporter, it does not show broad substrate speci.city, and it does not even have high af.nity for organic cation inhibitors. Detection of ETT mRNA or protein in cells can therefore be utilized as an accurate and speci.c molecular marker of ET activity. Methimazole, in view of its strong antithyroid activity, cannot be employed as an anti-in.ammatory agent. However, since methimazole and ET rely on disparate uptake mechanisms for cellular uptake, and since ETT is not expressed in the thyroid (Fig. 7), supplementation of ET to correct a dietary de.cit could provide a new therapeutic strategy for chronic in.ammatory diseases. Finally, our results provide a basis for creating a knock-out mouse model to further investigate the role of ETT in chronic in.ammation.
Acknowledgments
Supported by Deutsche Forschungsgemeinschaft (GR 1681/21). We thank Beatrix Steinru cken, Simone Kalis, and Regina Baucks for skillful technical assistance, Bjorn Reinartz and Reinhard Berkels for providing the cDNA of ETTm, and Olaf Utermohlen for a critical discussion of the manuscript.
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