Probing the substrate specificity of the ergothioneine transporter with methimazole, hercynine, and organic cations

Article abstract of DOI:10.1016/j.bcp.2007.04.015

Recently, we have identified 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 inflammatory diseases. ETT was originally designated a multispecific 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 μl/(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 (K_i = 11 μmol/l). The natural compound hercynine and antithyroid drug methimazole are related in structure to ET. However, efficiency of ETT-mediated transport of methimazole (K_i = 7.5 mmol/l) was 130-fold lower, and transport of hercynine (K_i = 1.4 mmol/l) was 25-fold lower than transport of ET. ETT from mouse, upon expression in 293 cells, catalyzed high affinity, 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 specific for its physiological substrate ergothioneine. ETT is not a cationic drug transporter, and it does not have high affinity for organic cation inhibitors. Detection of ETT mRNA or protein can therefore be utilized as a specific molecular marker of intracellular ET activity.

Full text of DOI:10.1016/j.bcp.2007.04.015

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Probing the substrate specificity of the ergothioneine
transporter with methimazole, hercynine,
and organic cations
Silke Grigat a, Stephanie Harlfinger a, Sonia Pal a, Ralph Striebinger a, Stefan Golz b,
Andreas Geerts b, Andreas Lazar a, Edgar Scho¨mig a,c, Dirk Grundemann a,c,
*
¨
a Department of Pharmacology, University of Cologne, Gleueler Straße 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, we have identified 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 inflam-
matory diseases. ETT was originally designated a multispecific novel organic cation trans-
porter (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, efficiency of ETT-mediated transport of methimazole (Ki = 7.5 mmol/l) was 130-
fold 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 affinity, 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 specific for its physiological substrate ergothio-
neine. ETT is not a cationic drug transporter, and it does not have high affinity for organic
cation inhibitors. Detection of ETT mRNA or protein can therefore be utilized as a specific
molecular marker of intracellular ET activity.
Received 16 February 2007
Accepted 16 April 2007
Keywords:
SLC22A4
Chronic inflammatory disease
Ergothioneine
Ergothioneine transporter
Methimazole
Organic cations
# 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
avidly retained with minimal metabolism. The content of
ET varies greatly among human tissues [2]. High ET levels
biosynthesized solely by fungi and mycobacteria [1].
Humans like other mammals absorb it exclusively from
* Corresponding author. Tel.: +49 221 478 7455; fax: +49 221 478 5022.
E-mail address: dirk.gruendemann@uni-koeln.de (D. Grundemann).
¨
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

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b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 3 0 9 – 3 1 6
designated a multispecific novel organic cation transporter
(OCTN1), because it was reported to transport tetraethylam-
monium [14], and quinidine, verapamil, and pyrilamine [15]. If
ETT also functions as an organic cation transporter, then it
cannot serve as a specific 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 affinity towards organic cations with ET as
substrate.
The widely used antithyroid drug methimazole (=1-
methyl-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 inflam-
matory diseases, it is very interesting that methimazole has
immunosuppressive [16–18] and powerful anti-inflammatory
[19] activity. In order to evaluate whether ETT could provide a
specific route of entry of methimazole into those cells that
express this carrier, we tested methimazole as a substrate. The
substrate specificity 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).
Fig. 1 – Structures of ET, hercynine, and methimazole.
Finally, we expressed and analyzed ETT from mouse in 293
cells to see if key functional properties are conserved over
species.
have been found in erythrocytes, bone marrow, seminal
fluid 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.
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 verified by DNA sequencing.
Recently, we have discovered an ET transporter (ETT; gene
symbol SLC22A4) [3]. ETT from human (ETTh) has high affinity
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 com-
pound. By contrast, cells with expression of ETT accumulate
ET to high levels. Based on the expression profile, we judge ETT
to be necessary for the supply of ET primarily to erythrocyte
progenitor cells and to monocytes.
2.2.
Cell culture
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 inflammatory
diseases. The association with Crohn’s disease [4] has been
largely confirmed [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 inflammatory diseases, it will be necessary
to fully understand substrate specificity and localization of the
carrier.
293 cells (ATCC CRL-1573), a transformed cell line derived from
human embryonic kidney, were grown at 37 8C in a humidified
atmosphere (5% CO2) in plastic culture flasks (Falcon 3112,
Becton Dickinson, Heidelberg, Germany). The growth medium
was Dulbecco’s Modified 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 expres-
sion, cells were cultivated for at least 20 h in regular growth
Our previous results suggest that expression of ETT in
specific cells indicates intracellular ergothioneine activity.
However, the gene product of SLC22A4 was originally

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311
medium supplemented with 1 mg/ml doxycycline (195044, MP
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).
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 confluence 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 (flow rate 250 ml/
min) was made of methanol (70%) and 0.1% formic acid (30%).
For quantification by SRM (selected reaction monitoring; scan
time 0.3 s), at first the optimal collision energy (CE) for argon-
induced fragmentation in the second quadrupole was deter-
mined 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.
2.5.
Calculations and statistics
The clearance is directly proportional to kcat/Km (kcat: turnover
number) and thus a valid measure of efficiency of transport
(provided that the substrate concentration is much smaller
than the respective Km) [22]. The clearance equals initial rate of
specific uptake (=uptake mediated by expressed carrier) divided
by substrate concentration. Specific uptake equals total uptake
minus uptake into control cells (=non-specific 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%
confidence 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 modified Hill function: v ¼ V0 þ
V
lim=ð1 þ KcꢀNhaþ Þ with h = Hill coefficient and cNaþ = sodium
concentration. In the absence of any sign of inhibition, a Ki-
valuewasestimatedasthehighestusedinhibitorconcentration
multiplied by 5. The unpaired t-test was used to test for
significance; 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-phenyl-
pyridinium iodide (D-048, Sigma–Aldrich), pyrilamine (P-
5514, Sigma–Aldrich), quinidine (Q-0875, Sigma–Aldrich),
thioperamide (T-123, RBI, Natick, MA, USA), verapamil-HCl
(Knoll AG (Abbott Laboratories), Liestal, Switzerland). Dis-
procynium24 (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
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 purified by preparative thin layer

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b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 3 0 9 – 3 1 6
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/
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.
(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.
zole by ETTh is negligible. The affinity of ETTh for methima-
zole 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, purified, 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 ꢂ 0.2 ml/
(min mg(protein)) was calculated (n = 4). With 10 mmol/l ET,
measured with paired dishes, the clearance was 28 ꢂ 3 ml/
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 affinity (Ki < 1 mmol/l) towards ETTh. Verapa-
mil (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 5-
aminosalicylic acid, each up to 640 mM.
Table 1 – Inhibition of ETTh-mediated uptake of er-
gothioneine by selected drugs and compounds
3.3.
Methimazole
Drug
Ki (mmol/l)
95% CI
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
specific 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
Verapamil
10.8
14.6
182
9.1, 12.8
11.2, 19.1
134, 247
209, 311
700, 2990
5090, 9940
Disprocynium24
Pyrilamine
Thioperamide
Hercynine
254
1450
7520
Methimazole
as above. With 100 mmol/l methimazole,
a clearance of
Uptake (1 min) of 10 mmol/l ET was assayed in stably transfected 293
cells (n = 12 for each drug). Given is the Ki (assuming a Hill coefficient
of 1) with the corresponding 95% confidence interval (CI).
0.3 ꢂ 0.2 ml/(min mg protein) was calculated (n = 3). With
10 mmol/l ET, measured with paired dishes, the clearance
was 39 ꢂ 2 ml/(min mg protein). Thus, transport of methima-

b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 3 0 9 – 3 1 6
313
Fig. 4 – Inhibition of ETTh-mediated uptake of ET by
Fig. 6 – Sodium dependence of uptake of ET mediated by
methimazole and hercynine. See legend to Fig. 3 for
experimental conditions.
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.
(min mg(protein)). It follows that ETT transports hercynine,
albeit at low transport efficiency. The affinity 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% confidence interval, 33–76) was determined
(Fig. 5). Replacement of Na+ in the uptake buffer by N-methyl-
D-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 efficiently than TEA and
carnitine [3]. With the discovery of ET as a high performance
substrate, it has become possible to test the relevance of
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.

314
b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 3 0 9 – 3 1 6
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 affinity only as
moderate. Disprocynium24 displayed similar affinity
(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 affinity 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 struc-
tures. The anti-inflammatory 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 ꢃ3200 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 physiolo-
gical substrate. In other words, currently no potent inhibitor is
available for ETT.
The substrate specificity 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 efficiency; ET is transported 25-fold
better than hercynine. Concordantly, hercynine inhibited
uptake of ET with low affinity (Ki = 1.4 mmol/l).
Fig. 7 – Tissue distribution of ETTh analyzed by real-time
PCR. Results are given relative to the mRNA level of bone
marrow. NS: no signal.
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 antithyr-
oid 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 immuno-
suppressive 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 macro-
phages, like neutrophils and thyroid cells, accumulate
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 verified by robust uptake of ET. Thus, ETT does not
discernibly transport quinidine, verapamil, and pyrilamine. By
contrast, Yabuuchi et al. recorded significantly higher radio-
activity (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 exception-
ally high [29].
We have measured inhibition of ET transport by selected
drugs to see if ETT displays, in conjunction with its
physiological substrate, high affinity 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 ꢃ320 mmol/l
for lidocaine and ꢃ3200 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
methimazole. These cells apparently express a specific
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 specific
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 affinity 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 identified. Interestingly, our real-time PCR

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315
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, methima-
zole could indeed mimic a beneficial 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.
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
specific interaction with peroxidase(s). A lack of ET may thus
represent a precipitating factor in the genesis of chronic
inflammatory diseases.
In conclusion, our experiments with hercynine and methi-
mazole establish that ETT is highly specific for its physiological
substrate ergothioneine. ETT is not a ‘‘multispecific’’ cationic
drug transporter, it does not show broad substrate specificity,
and it does not even have high affinity for organic cation
inhibitors. Detection of ETT mRNA or protein in cells can
therefore be utilized as an accurate and specific molecular
marker of ET activity. Methimazole, in view of its strong
antithyroid activity, cannot be employed as an anti-inflamma-
tory 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 deficit could provide a new therapeutic
strategy for chronic inflammatory diseases. Finally, our results
provide a basis for creating a knock-out mouse model to further
investigate the role of ETT in chronic inflammation.
The existence of
a specific 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 benefit from ET in the presence of 10-fold
higher concentrations of the ubiquitous hydrophilic antiox-
idantsglutathione(GSH)andascorbate?Therearemanyreports
that imidazole-2-thiones like ET and methimazole are potent
antioxidantsinvitro [33,34]; however, thereisnocomprehensive
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 anti-
oxidants could probably replace this function.
Our present results with ETT from mouse confirm that ETT
catalyzes high affinity, 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 findings suggest that ETT charges developing erythro-
cytes with available ET. We entertain the hypothesis that what
really distinguishes ET from other antioxidants is its interac-
tion 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 hemoglo-
bin, 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, affinity towards oxidized
peroxidase may be the key feature of imidazole-2-thione
compounds. Indeed, it has been shown that potent inhibition
Acknowledgments
Supported by Deutsche Forschungsgemeinschaft (GR 1681/2-
1). We thank Beatrix Steinrucken, 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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