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Departments of Internal Medicine (E.C.H.F., R.D., E.P.C.M.M., E.P.K., T.J.V.) and Nuclear Medicine (E.P.K., G.H.), Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands; and Institute of Physiology, University of Zürich (F.V.), CH-8057 Zürich, Switzerland
Address all correspondence and requests for reprints to: Theo J. Visser, Ph.D., Department of Internal Medicine, Erasmus University Medical Center, Room Bd234, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: visser{at}inw3.azr.nl
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Abstract |
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T3 >
T4 (10 nM), which in all cases was
Na+ independent. Saturation analysis provided apparent
Michaelis constant (Km) values of 7.9 µM for
T4, 0.8 µM for T3, 12.5
µM for rT3, 7.9 µM for
3,3'-diiodothyronine, 46 µM for leucine, and 19
µM for tryptophan. Uptake of leucine, tyrosine, and
tryptophan (10 µM) was inhibited by the different
iodothyronines (10 µM), in particular T3.
Vice versa, uptake of 0.1 µM
T3 was almost completely blocked by coincubation with 100
µM leucine, tryptophan, tyrosine, or phenylalanine. Our results demonstrate stereospecific Na+-independent transport of iodothyronines by the human heterodimeric system L amino acid transporter.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although T3 is the receptor-active form of thyroid hormone, its precursor T4 is the predominant product secreted by the follicular cells of the thyroid gland (5, 6). Although some T3 is also secreted, most T3 is produced by enzymatic outer ring deiodination of T4 in peripheral tissues (5, 6). Both T4 and T3 are inactivated by inner ring deiodination to the metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2), respectively (5, 6). The three deiodinases (D1–D3) involved in these conversions are homologous selenoproteins with different catalytic profiles, tissue distributions, and physiological functions (7, 8). D1 in liver and kidney appears important for systemic T3 production, D2 in tissues such as brain and pituitary for local T3 production, and D3 in brain and other tissues for T4 and T3 degradation. All three deiodinases are transmembrane proteins with their active site exposed to the cytoplasm (5, 6, 7, 8).
The metabolism and action of thyroid hormone are intracellular events requiring the uptake of extracellular hormone through the plasma membrane. Although iodothyronines are lipid-soluble compounds, they cannot readily cross the lipid bilayer of the cell membrane by simple diffusion. This is because the polar zwitter-ionic alanine side-chain prevents passage of the iodothyronine molecule through the hydrophobic inner part of the cell membrane constituted of the aliphatic fatty acid chains. Evidence accumulated over the last two decades indicates that uptake of thyroid hormone in different tissues is mediated by transporters (6, 9, 10, 11, 12). Work in our laboratory has demonstrated the presence of multiple iodothyronine transporters in rat and human liver cells (6, 11, 12). Two energy- and Na+-dependent transporters appear of particular importance for hepatic uptake of T4 and rT3, and of T3, respectively, showing nanomolar affinities for their ligands, but different dependencies on cellular ATP levels (6, 11, 12). However, iodothyronine transporters in other tissues show different characteristics. Isolated rat pituitary cells also show carrier-mediated uptake of different iodothyronines, but this appears to be mediated by a single transporter (13). In contrast, cultured neonatal rat cardiomyocytes show specific uptake of T3, but not of T4 (14). A variety of cells, including rat pituitary cells (13), GH-producing tumor cells (15), erythrocytes (16, 17), cardiomyocytes (14), and astrocytes (18); mouse neuroblastoma (19) and thymocytes (20); and human choriocarcinoma cells (21, 22), show competition between uptake of iodothyronines and neutral amino acids such as leucine (Leu) and tryptophan (Trp). This may not be surprising, because iodothyronines are iodinated amino acid derivatives built from two tyrosine (Tyr) molecules. Based on the above observations it has been suggested that thyroid hormone may be taken up in different tissues at least in part through system L or system T amino acid transporters (15, 16, 17, 18, 19).
In particular through the pioneering work of Christensen (23, 24), different classes of amino acid transporters have been distinguished on the basis of their preference for certain types of amino acids (e.g. neutral, acidic, or basic), their specificity for natural or artificial prototypic ligands, as well as their mechanism of transport (e.g. Na+ dependent or Na+ independent). A rapidly increasing number of amino acid transporters has been characterized in recent years, including the 4F2-related heterodimeric transporters (for reviews, see Refs. 25, 26, 27). The 4F2 or CD98 cell surface antigen has been known for some time to be expressed in many tissues, especially on activated lymphocytes and tumor cells, but only recently has it been identified as a family of amino acid transporters (25, 26, 27, 28). These heterodimeric transporters each consist of a common 4F2 heavy chain (4F2hc) and a member of a family of homologous light chains, 7 of which have now been cloned (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). 4F2hc is a glycosylated protein with a single transmembrane domain, whereas the light chains are not glycosylated and have 12 putative transmembrane domains; they are linked through a disulfide bond (25, 26, 27, 28). One of the 7 light chains mentioned above appears to dimerize preferentially with another heavy chain, termed rBAT (for related to basic amino acid transport), which is homologous to 4F2hc, suggesting the existence of a superfamily of heterodimeric amino acid transporters consisting of multiple heavy and light chains (25, 26, 27, 28, 38, 39, 40).
In combination with 4F2hc, two 4F2 light chains mediate the Na+-independent transport of large neutral (branched chain and aromatic) amino acids such as Leu, Tyr, Trp, and phenylalanine (Phe). This is typical for the system L amino acid transporter, hence the names LAT1 and LAT2 for these light chains (25, 26, 27, 29, 30, 31, 32, 33). Two other light chains forming heterodimers with 4F2hc mediate the Na+-dependent uptake of neutral amino acids such as Leu as well as the Na+-independent uptake of basic amino acids such as arginine (Arg). This is characteristic of the system y+L amino acid transporter, which is why these light chains are named y+LAT1 and y+LAT2 (25, 26, 27, 34, 35, 36). We have tested the possible involvement of these heterodimeric 4F2 transporters in the transport of thyroid hormone by studying the uptake of the iodothyronines T4, T3, rT3, and 3,3'-T2 by Xenopus laevis oocytes injected with cRNA coding for human 4F2hc (h4F2hc) alone or in combination with cRNA coding for human LAT1 (hLAT1), mouse LAT2 (mLAT2), hy+LAT1, or hy+LAT2. Whereas the system y+L transporters did not mediate the uptake of iodothyronines, effective thyroid hormone transport was observed with the system L transporters, in particular the h4F2hc/hLAT1 heterodimer, which is the subject of this report.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA preparation
The plasmids containing cDNA coding for h4F2hc and
hLAT1, pSPORT1-h4F2hc (43) and
pcDNA1-E16 (29), were linearized with
HindIII and EcoRV (Roche,
Mannheim, Germany), respectively, and transcribed using the Ampliscribe
High Yield T7 RNA transcription kit (Epicentre, Madison, WI). The
cRNAs were capped with the m7G (5')ppp (5')G cap analog
(Epicentre) and stored in sterile water at -80 C.
Oocyte isolation and cRNA injection
Oocytes were prepared as described previously (44).
After isolation, oocytes were sorted on morphological criteria and
defolliculated manually. Healthy-looking stage V–VI oocytes were kept
at 18 C in modified Barth’s solution containing 20 IU/ml penicillin
and 20 µg/ml streptomycin (44). The next day, oocytes
were injected with 2.3 ng h4F2hc cRNA and/or 2.3 ng
hLAT1 cRNA in 23 nl water using the Nanoject system
(Drummond Scientific, Broomall, PA). Uninjected oocytes were used as
controls, as similar results were obtained using water-injected
oocytes. Injected and uninjected oocytes were kept for 2 d at 18 C
in modified Barth’s solution.
Uptake
Uptake assays were performed as reported previously
(44). Groups of 8–10 oocytes were incubated for 2–60 min
at 25 C with 0.01–10 µM
[125I]T4,
[125I]T3,
[125I]rT3, or
[125I]3,3'-T2, or with
10–100 µM [3H]Arg,
[3H]Leu, [3H]Phe,
[3H]Tyr, or [3H]Trp in
0.1 ml incubation medium [100 mM NaCl or choline chloride
(ChCl), 2 mM KCl, 1 mM
CaCl2, 1 mM
MgCl2, 10 mM HEPES, and 10
mM Tris, pH 7.5]. After incubation, oocytes were washed
four times with 2.5 ml ice-cold Na+-containing
incubation medium containing 0.1% BSA. Oocytes were transferred to new
tubes and counted individually.
Efflux
Oocytes injected with cRNAs coding for h4F2hc and
hLAT1 were incubated in groups of 8–10 for 30 min at 25 C
with 10 µM [3H]Leu or
0.1 µM
[125I]T3 or
[125I]T2 in 0.1 ml
Ch+-containing incubation medium. One group of
oocytes was processed to determine the total uptake of each ligand as
described above. Efflux of internalized ligand from other groups of
oocytes was analyzed as follows. After removal of the medium, oocytes
were rapidly washed with 0.5 ml Ch+-containing
incubation medium at 25 C and incubated for successive 2-min periods at
25 C with 0.5 ml of the same medium without or with 10
mM unlabeled Leu. After each interval, medium was
rapidly replaced by fresh medium and counted for radioactivity.
Radioactivity still associated with the oocytes at the end of the
20-min total efflux period was counted as well. Efflux was quantified
by expressing the cumulative release of radioactivity as a percentage
of that present in the oocytes at the start of the efflux period.
Statistics
Data are presented as the mean ± SEM.
Differences were tested for statistical significance by t
test. Kinetic parameters were determined by fitting the plot of uptake
rate (v) vs. ligand concentration (S) to the
Michaelis-Menten equation: v = Vmax/(1 +
Km/S), where Vmax is the
maximum uptake rate, and Km is the Michaelis
constant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the effects of injection of 2.3
ng h4F2hc cRNA alone or together with 2.3 ng
hLAT1 cRNA on the uptake of Leu, Phe, Tyr, Trp, and Arg.
Incubations were performed 2 d after cRNA injection at a ligand
concentration of 50 µM using incubation medium
containing Na+ or choline
(Ch+). Injection of oocytes with cRNA coding for
hLAT1 alone did not stimulate transport of the different
amino acids (data not shown). Injection of h4F2hc cRNA alone
did not effect transport of Phe, Tyr, or Trp, but induced
Na+dependent transport of Leu and
Na+-independent transport of Arg. This is
characteristic for the induction of a y+L-type
transporter, which has been documented in different studies and is
explained by the dimerization of exogenous h4F2hc with an
endogenous y+LAT-type light chain expressed in
native oocytes (25, 26, 27, 28, 29, 30). This assumption is supported by
observations that injection of cRNA coding for
hy+LAT1 or
hy+LAT2 in addition to
h4F2hc cRNA further markedly increased
Na+-dependent transport of Leu and
Na+-independent transport of Arg compared with
those in oocytes injected with h4F2hc cRNA alone (data not
shown). Coinjection of oocytes with h4F2hc cRNA and
hLAT1 cRNA did not increase Arg transport above that
observed after injection of h4F2hc cRNA alone. Compared with
oocytes injected with h4F2hc cRNA alone, oocytes injected in
addition with hLAT1 cRNA showed a further marked increase in
Leu uptake, which, however, became almost completely
Na+ independent. Coinjection of cRNA coding for
h4F2hc and hLAT1 also resulted in a large
induction of the transport of Phe and Tyr and a much smaller increase
in the uptake of Trp, which in all cases was Na+
independent. This is in agreement with previous reports, and
characteristic for the induction of an L-type amino acid transporter.
Marked stimulation of the Na+-independent uptake
of 100 µM Leu was also observed after
coinjection of h4F2hc and mLAT2 cRNA (data not
shown).
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shows the uptake of 10 µM Leu, Tyr, and Trp in
Ch+-containing medium by oocytes coinjected with
h4F2hc and hLAT1 cRNA as a function of incubation
time. Data were corrected for transport in uninjected oocytes, which
was significant only for Trp because of the relatively small induction
of Trp transport by h4F2hc plus hLAT1 cRNA
injection. Trp uptake was linear with time of incubation for at least
60 min. Transport of Leu and Tyr was much faster than Trp uptake and
was linear with time for only approximately 15 min.
Figure 2A shows the uptake of
T4, T3,
rT3, or 3,3'-T2 by
uninjected oocytes and oocytes injected with cRNA coding for
h4F2hc and hLAT1 after incubation for 1 h
with 10 nM iodothyronine in medium with or
without Na+. As shown previously, significant
uptake of iodothyronines was observed in uninjected oocytes, which is a
major drawback of this expression system for the cloning of thyroid
hormone transporters. Iodothyronine uptake by native oocytes decreased
in the order 3,3'-T2 T3
> T4 > rT3 and was
somewhat lower in incubation medium containing
Ch+ instead of Na+.
Injection of oocytes with h4F2hc cRNA alone or with
hLAT1 cRNA alone did not increase the uptake of any
iodothyronine (data not shown). The lack of effect of injection with
h4F2hc cRNA alone suggests that the
y+L-type transporter generated by dimerization of
the exogenous heavy chain with an endogenous light chain does not
mediate transport of iodothyronines. This is supported by findings that
coinjection of h4F2hc cRNA and cRNA coding for
hy+LAT1 or
hy+LAT2 did not stimulate
iodothyronine transport in oocytes (data not shown). However, injection
of oocytes with both cRNA coding for h4F2hc and
hLAT1 resulted in significant increases in net iodothyronine
uptake, which decreased in the order 3,3'-T2 >
rT3 T3 >
T4, and in all cases was
Na+ independent. Smaller increments in
iodothyronine uptake were noted after coinjection of h4F2hc
cRNA and mLAT2 cRNA (data not shown).
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shows the uptake of 0.1 µM
T3 and 3,3'-T2 induced by
injection of oocytes with both h4F2hc and hLAT1
cRNA as a function of time of incubation in
Ch+-containing medium. Transport of
T3 was linear with time for at least 60 min.
Transport of 3,3'-T2 was much faster than
T3 uptake and was linear with time for about 45
min.
The saturation kinetics of iodothyronine uptake by the heterodimeric
h4F2hc/hLAT1 transporter were studied by
incubation of oocytes injected with cRNA for both subunits during
1 h with 0.1–10 µM ligand in
Ch+-containing medium. Iodothyronine uptake
through the oocytes’ endogenous transporter(s) was determined in
parallel incubations with uninjected oocytes. The results are presented
in Fig. 3, showing that iodothyronine
uptake was saturable in both uninjected and cRNA-injected oocytes.
Michaelis-Menten analysis of the results obtained with uninjected
oocytes provided apparent Km values of 2–14
µM for the different iodothyronines.
Iodothyronine transport mediated by the
h4F2hc/hLAT1 transporter was determined by
subtraction of the uptake rates in uninjected oocytes from those
observed in oocytes injected with the cRNAs for both subunits.
Michaelis-Menten analysis of the corrected data provided apparent
Km values of 7.9 µM for
T4, 0.8 µM for
T3, 12.5 µM for
rT3, and 7.9 µM for
3,3'-T2. Vmax values were
2.6, 1.1, 11.3, and 28 pmol/oocyte·h for T4,
T3, rT3, and
3,3'-T2, respectively. The fold stimulation of
iodothyronine uptake induced by injection of oocytes with cRNA for
h4F2hc and hLAT1 varied with increasing ligand
concentration (0.1–10 µM) from 2.1–2.7 for
T4, from 2.2–1.9 for T3,
from 4.4–7.6 for rT3, and from 3.2–13.7 for
3,3'-T2. The kinetics of transport of Leu and Trp
by the h4F2hc/hLAT1 transporter were analyzed
similarly using ligand concentrations of 1–100
µM, yielding Km values of
46 µM for Leu and 19 µM
for Trp (data not shown). These data are in good agreement with
previous reports (29, 30, 31, 32).
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demonstrates that uptake of 10
µM Leu, Tyr, and Trp by
h4F2hc/hLAT1 was inhibited by the different
iodothyronines. The degree of competition was greatest with
T3, in agreement with its low apparent
Km value. Vice versa, uptake of 0.1
µM T3 by the
h4F2hc/hLAT1 transporter was almost completely
inhibited by 100 µM Leu, Trp, Tyr, or Phe,
whereas iodothyronine uptake by the endogenous transporter(s) was not
affected (Fig. 4B).
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). Uptake was studied in the
absence of Na+ using 0.1
µM
[125I]T3 or 10
µM [3H]Leu as ligands
and oocytes injected with cRNA for h4F2hc and
hLAT1 and was corrected for uptake in oocytes injected with
h4F2hc cRNA only. In general, Leu uptake was somewhat less
sensitive to the different competitors than T3
uptake, perhaps because Leu uptake was tested at a relatively high
ligand concentration. Neither
[125I]T3 nor
[3H]Leu uptake was significantly inhibited by
10 µM
D-T3 or Triac, in contrast
to the potent inhibition by 10 µM
L-T3. Tested at 100
µM, BCH produced at least the same marked
inhibition of [3H]Leu and
[125I]T3 uptake as Leu
itself. The prototypic organic anion transporter ligand BSP and the
bile acid TC had little effect on uptake of either ligand (Fig. 5).
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). In contrast, 10
mM extracellular Leu only slightly stimulated the
efflux of 3,3'-T2 internalized by
h4F2hc/hLAT1-expressing oocytes (Fig. 6B),
whereas it did not affect the efflux of T3 taken
up by such oocytes (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Injection of oocytes with hLAT1 cRNA alone does not induce
the transport of the various amino acids and iodothyronines, indicating
that expression of a 4F2-like heavy chain by native oocytes is
negligible. However, if oocytes are coinjected with cRNA for both
h4F2hc and hLAT1, uptake of the large, neutral
amino acids Leu, Phe, Tyr, and Trp, but not that of the basic amino
acid Arg, is markedly stimulated above that seen after injection of
h4F2hc cRNA alone. The hLAT1-induced increment in
amino acid transport is completely independent of
Na+, which confirms the characteristics of
the L-type amino acid transporter (23, 24, 25, 26, 27). Also,
transport of the different iodothyronines is markedly stimulated by
coexpression of h4F2hc and hLAT1, and for all
iodothyronines the induced transport is completely
Na+ independent. Tested at low ligand
concentrations, the rate of iodothyronine uptake by the
h4F2hc/hLAT1 transporter decreases in the order
3,3'-T2 > rT3
T3 > T4. This does not
appear to be a simple reflection of the affinity of the different
iodothyronines for the h4F2hc/hLAT1 transporter,
as the apparent Km value is much lower for
T3 than for T4,
rT3, and 3,3'-T2. The
apparent Km of 0.8 µM for
T3 is the lowest value reported for a ligand of
the h4F2hc/hLAT1 transporter (25, 26, 27, 29, 30). Among the different iodothyronines, by far the highest
Vmax value is observed for
3,3'-T2.
As iodothyronines and large neutral amino acids are all ligands for the
h4F2hc/hLAT1 transporter, it is not surprising
that they inhibit each other’s transport. Leu uptake is more strongly
inhibited by L-T3 than by
L-T4,
L-rT3,
3,3'-L-T2,
D-T3, and Triac, in keeping
with the low Km value for
T3 and the stereospecificity of this L-type amino
acid transporter (24, 25, 26, 27, 28, 29, 30). This is reminiscent of the
competition between iodothyronine and amino acid uptake in different
cell systems reported previously (13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Thus, uptake of
T4 and T3 by NB41A3 mouse
neuroblastoma cells is stereospecific, saturable
(Km: T3, 3
nM; T4, 6
nM), and inhibited by high concentrations of Leu
and Phe, but not by 
-aminoisobutyric acid, a system A
transporter-specific ligand (19). Similar characteristics
of iodothyronine uptake were observed in the Hs683 human glioma cell
line (47). Somewhat different results were reported for
T3 uptake by cultured rat astrocytes, which
express both high affinity (L1) and low affinity (L2) system L
transporters (18). Apparent Km
values for uptake of Leu and Trp by the L1 transporter amount to 8–9
µM. T3 uptake by these
cells is Na+ independent and saturable, with an
apparent Km of 2–3 µM.
L1-mediated uptake of Leu and Trp is competitively inhibited by
T3, and T3 uptake is
competitively inhibited by Trp, with corresponding
Km and Ki values. However,
T3 uptake is not inhibited by up to 30
mM Leu (18). It is also interesting
to mention the characterization of saturable and stereospecific
iodothyronine transport in GH4C1 rat pituitary tumor cells
(15), showing high affinity for T3
(Km, 0.4 µM) and
T4, low affinity for rT3
and thyronine, and strong inhibition by Leu, Phe, Tyr, Trp, and the
L-type transporter-specific ligand BCH. GH4C1 cells also show high
affinity transport of Leu (Km, 17
µM), which is potently inhibited by
T3 (IC50, 2
µM), further supporting the involvement of an
L-type transporter in T3 (and
T4) uptake (15). Also in cultured
rat anterior pituitary cells, uptake of T4 and
T3 is mediated by a common transporter and
inhibited by the aromatic amino acids Phe, Tyr, and Trp
(13).
Blondeau and co-workers (16, 17) demonstrated that T3 transport by rat erythrocytes is Na+ independent, saturable (Km, 0.14 µM), and specific (L-T3 >> D-T3 > T4 > rT3 > thyronine). T3 uptake is competitively inhibited by the aromatic amino acids Trp, Phe, and Tyr, but not by D-Trp or Leu. They also showed low affinity uptake of Trp by rat erythrocytes, with an apparent Km of 558 µM. Trp uptake is competitively inhibited by Phe, Tyr, and iodothyronine analogs, with Ki values identical to those for inhibition of T3 transport. These results suggest the involvement of a T-type, aromatic amino acid-specific transporter in the uptake of both Trp and T3 (16, 17). Interestingly, T3 uptake is markedly trans-stimulated by intracellular Trp, although Trp uptake is trans-inhibited by intracellular T3 (16, 17). Both T3 and Trp uptake are inhibited by the thiol-blocking reagent N-ethylmaleimide. A 45-kDa protein was identified by photoaffinity labeling with [125I]T3 that may be a subunit of the T-type transporter, but this was not further characterized (48).
Competition between iodothyronine and aromatic amino acid (e.g. Trp) transport has also been demonstrated in other cells, e.g. JAR human choriocarcinoma cells (21, 22) and neonatal rat cardiomyocytes (14), but it has not been established whether iodothyronine uptake in these cells is indeed mediated by amino acid transporters. Of special interest are observations of countertransport of Tyr derivatives by the system h transporter located in thyroidal lysosomal membranes (49, 50). Loading of lysosomes with Tyr or 3-(mono)iodotyrosine (MIT) greatly stimulates the influx of Tyr, MIT, 3,5-diiodotyrosine, Phe, and Leu. Potent competition by T4 and T3 suggests that iodothyronines are also countertransported against Tyr derivatives. The apparent Km value for MIT is 1.5 µM, and Tyr, 3,5-diiodotyrosine, T4, and T3 show similar high affinities. This exchange mechanism probably plays an important role in thyroid hormone biosynthesis, as the iodotyrosines released by lysosomal hydrolysis of Tg must be transported to the cytoplasm for deiodination, and the iodothyronines to the cell membrane for secretion (6).
The apparent Km values of T4, T3, Leu, and Trp in the 10-6–10-5 M range for the h4F2hc/hLAT1 transporter expressed in oocytes most closely resemble those reported for their uptake by rat GH4C1 pituitary tumor cells (15), supporting the involvement of an L-type transporter. They are also in reasonable agreement with the apparent Km values for T4, T3, Leu, and Trp uptake by cultured rat astrocytes (18) and rat erythrocytes (16, 17), but the complete lack of effect of >10-2 M Leu on iodothyronine uptake by these cells suggests that an aromatic amino acid-specific (T-type) transporter is involved. Also, the much lower Km values reported for T4 and T3 uptake by the NB41A3 mouse neuroblastoma cells appear to implicate another (sub)type of amino acid transporter than 4F2hc/LAT1.
The L-type amino acid transporter mediates not only influx, but also efflux, of amino acids. Our results show that the release of intracellular Leu is stimulated by exchange with extracellular Leu in agreement with previous reports (29, 30). Extracellular Leu only induces a small increase in the release of 3,3-T2 from the oocytes and no release of internalized T3, which may be explained by strong binding of iodothyronines to intracellular sites in oocytes.
The above-mentioned properties of the thyroidal lysosomal system h
transporter, mediating the exchange of amino acids such as Leu, Tyr,
iodotyrosines, and iodothyronines, suggest that it may actually be an
L-type amino acid transporter. This is also supported by evidence that
the high uptake of radioiodine-labeled MIT and
3-iodo--methyltyrosine by different tumors is mediated by an L-type
transporter (51, 52, 53). This principle is used in nuclear
medicine for the scintigraphic visualization of such tumors. If the
h4F2hc/hLAT1 and/or
h4F2hc/hLAT2 transporters are indeed responsible
for tumor uptake of the radioactive Tyr derivatives, then the
availability of cell systems overexpressing these transporters would
greatly facilitate the development of improved tumor-seeking
radiopharmaceuticals.
Obviously, all cells require amino acid transporters, but, in contrast to the ubiquitous expression of the 4F2 heavy chain, the LAT1 and, in particular, LAT2 light chains show restricted tissue distributions; neither of them is expressed in liver (25, 26, 27, 28, 29, 30, 31, 32, 33). This suggests the existence of other light chains as yet to be identified, which are involved in the uptake of aromatic amino acids in tissues that do not express LAT1 or LAT2. Presumably, one of these constitutes with 4F2hc a T-type transporter specific for aromatic amino acids, including iodothyronines (16, 17, 18). Perhaps, additional light chains exist that associate specifically with the homologous rBAT heavy chain (28), generating transporters that also accept iodothyronines. However, cellular uptake of iodothyronines is not only mediated by amino acid transporters. We and others have demonstrated recently that iodothyronines are also transported into liver by Na+-dependent and Na+independent organic anion transporters, although various members of the Na+-independent organic anion transporter family are also expressed in other tissues, in particular kidney and brain (46, 54, 55, 56, 57, 58). Typical ligands for these organic anion transporters, BSP and TC, have no effect on iodothyronine uptake by the h4F2hc/hLAT1 transporter, in contrast to the potent inhibition by the L-type ligand BCH. However, the major, Na+-dependent hepatic transporters for T4 and T3 remain to be identified. Iodothyronine uptake by uninjected oocytes is saturable (apparent Km, 2–14 µM), suggesting that it is carrier mediated. The lack of effect of high concentrations of different amino acids on iodothyronine uptake by native oocytes argues against the involvement of an amino acid transporter. The type of endogenous iodothyronine transporter(s) in Xenopus oocytes remains to be determined.
In summary, we have demonstrated that h4F2hc/hLAT1 and, albeit less effectively, also h4F2hc/mLAT2 are capable of transporting iodothyronines, in agreement with previous suggestions that thyroid hormone is taken up in different tissues via L-type amino acid transporters. Our findings are in agreement with a recent report published after completion of our study, showing iodothyronine transport by the heterodimeric transporter composed of h4F2hc and the IU12 light chain from Xenopus, which is homologous to hLAT1 (59). One of the questions that remains to be clarified is the extent to which cellular uptake of iodothyronines through 4F2-related transporters is stimulated by countertransport of different intracellular amino acids. Of course, these transporters may also mediate cellular efflux of iodothyronines.
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Footnotes |
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Received February 26, 2001.
Accepted for publication June 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  E. K. Wirth, S. Roth, C. Blechschmidt, S. M. Holter, L. Becker, I. Racz, A. Zimmer, T. Klopstock, V. Gailus-Durner, H. Fuchs, et al. Neuronal 3',3,5-Triiodothyronine (T3) Uptake and Behavioral Phenotype of Mice Deficient in Mct8, the Neuronal T3 Transporter Mutated in Allan-Herndon-Dudley Syndrome J. Neurosci., July 29, 2009; 29(30): 9439 - 9449. [Abstract] [Full Text] [PDF]
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 W. E. Visser, E. C. H. Friesema, and T. J. Visser Transport of Thyroxine and 3,3',5-Triiodothyronine in Human Umbilical Vein Endothelial Cells Endocrinology, March 1, 2009; 150(3): 1552 - 1557. [Abstract] [Full Text] [PDF]
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L. M. Roberts, K. Woodford, M. Zhou, D. S. Black, J. E. Haggerty, E. H. Tate, K. K. Grindstaff, W. Mengesha, C. Raman, and N. Zerangue Expression of the Thyroid Hormone Transporters Monocarboxylate Transporter-8 (SLC16A2) and Organic Ion Transporter-14 (SLCO1C1) at the Blood-Brain Barrier Endocrinology, December 1, 2008; 149(12): 6251 - 6261. [Abstract] [Full Text] [PDF]
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 E. C. H. Friesema, G. G. J. M. Kuiper, J. Jansen, T. J. Visser, and M. H. A. Kester Thyroid Hormone Transport by the Human Monocarboxylate Transporter 8 and Its Rate-Limiting Role in Intracellular Metabolism Mol. Endocrinol., November 1, 2006; 20(11): 2761 - 2772. [Abstract] [Full Text] [PDF]
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 N Shimada and K Yamauchi Characteristics of 3,5,3'-triiodothyronine (T3)-uptake system of tadpole red blood cells: effect of endocrine-disrupting chemicals on cellular T3 response J. Endocrinol., December 1, 2004; 183(3): 627 - 637. [Abstract] [Full Text] [PDF]
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K. Tohyama, H. Kusuhara, and Y. Sugiyama Involvement of Multispecific Organic Anion Transporter, Oatp14 (Slc21a14), in the Transport of Thyroxine across the Blood-Brain Barrier Endocrinology, September 1, 2004; 145(9): 4384 - 4391. [Abstract] [Full Text] [PDF]
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 T. Mikkaichi, T. Suzuki, T. Onogawa, M. Tanemoto, H. Mizutamari, M. Okada, T. Chaki, S. Masuda, T. Tokui, N. Eto, et al. Isolation and characterization of a digoxin transporter and its rat homologue expressed in the kidney PNAS, March 9, 2004; 101(10): 3569 - 3574. [Abstract] [Full Text] [PDF]
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 E. C. H. Friesema, S. Ganguly, A. Abdalla, J. E. M. Fox, A. P. Halestrap, and T. J. Visser Identification of Monocarboxylate Transporter 8 as a Specific Thyroid Hormone Transporter J. Biol. Chem., October 10, 2003; 278(41): 40128 - 40135. [Abstract] [Full Text] [PDF]
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H. H. A. G. M. van der Putten, E. C. H. Friesema, N. A. Abumrad, M. E. Everts, and T. J. Visser Thyroid Hormone Transport by the Rat Fatty Acid Translocase Endocrinology, April 1, 2003; 144(4): 1315 - 1323. [Abstract] [Full Text] [PDF]
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J. W. A. Ritchie, Y.-B. Shi, Y. Hayashi, F. E. Baird, R. W. Muchekehu, G. R. Christie, and P. M. Taylor A Role for Thyroid Hormone Transporters in Transcriptional Regulation by Thyroid Hormone Receptors Mol. Endocrinol., April 1, 2003; 17(4): 653 - 661. [Abstract] [Full Text] [PDF]
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), or Trp (
) in Ch+-containing medium.
Data were corrected for minor uptake observed in uninjected oocytes.
Data are the mean ± SEM of 8–10 oocytes. *,
P < 0.001 vs. corresponding oocytes
injected with h4F2hc cRNA alone.
) or
oocytes injected with cRNAs coding for h4F2hc and
hLAT1 (
). Oocytes were incubated for 60 min with
0.1–10 µM 125I-labeled T4,
T3, rT3, or 3,3'-T2 in
Ch+-containing medium. Uptake induced by expression of
h4F2hc/hLAT1 was calculated by
subtraction of uptake in uninjected oocytes from that observed in
injected oocytes (
