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Nuclear Medicine Research Laboratory, Veterans Administration Medical Center (R.R.C.), San Francisco, California; the Department of Pharmaceutical Sciences, University of Brasilia (L.A.S., R.C.J.R.), Brasilia, DF Brazil; University of California (S.W.P.), Davis, California 95817; and the Metabolic Research Unit (J.D.B.), Gastroenterology Division (B.F.S., N.L.), and Liver Center (R.R.C., B.F.S., N.L.), Department of Medicine, University of California, San Francisco, California 94143-0538
Address all correspondence and requests for reprints to: Dr. Noureddine Lomri, Gastroenterology Division, University of California, 513 Parnassus Avenue, S-357, P.O. Box 0538, San Francisco, California 94143. E-mail: nlomri{at}itsa.ucsf.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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170 kDa)
suggest that a novel protein is involved in the transport of
T3 out of cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to what is known about uptake, the cellular export of thyroid hormones is poorly understood. Thyroid hormone export has been described in rat hepatocytes (7, 20, 21), fibroblasts and cardiocytes (7), rat and human red blood cells (22, 23), rat pituitary GC (24) and GH4C1 (10) cells, human choriocarcinoma (JAR) cells (25, 26), and human hepatocytes and fibroblasts (8). In most of these publications (20, 21, 22, 23, 24, 25) hormone uptake was the major focus of study, with observations on efflux included as incidental findings. T3 export was shown to be saturable in some studies (7, 20, 23, 26), but not in another study (21). To date no cellular protein linked to thyroid hormone export has been identified.
In a prior study we obtained evidence that cellular export plays a role in regulating cellular content of thyroid hormone (7). We found that there is accelerated thyroid hormone export (7) in rat hepatoma (HTC) cells that were selected for resistance (HTC-R) to a glycocholic acid (27). We further found that HTC-R cells are resistant to thyroid hormone action compared with parental HTC cells and that the resistance was due not to decreased uptake or increased metabolic disposal but, rather, to enhanced efflux of the hormone (7). The HTC-R cells overexpress several novel multidrug resistance (mdr)-like P-glycoproteins (27), members of the ATP-binding cassette (ABC) superfamily of plasma membrane transporters (28). Various mdr/P-glycoproteins transport corticosteroids and a wide range of anticancer drugs out of cells, thereby conferring multidrug resistance (9, 10, 11, 12, 13, 14, 29). Verapamil, which inhibits mdr/P-glycoproteins and reverses multidrug resistance (29), slowed T3 efflux from HTC-R cells, but not HTC cells, suggesting that efflux is mediated by these proteins (7).
These observations led to the question of what type of protein mediates the efflux. A candidate protein ideally should bind T3 specifically, and the binding should be blocked by verapamil. The presence of the protein should also correlate with T3 export activity. In the current work, we have expanded these studies to other cell types, and we show that rat FRTL-5 thyroid cells and mouse NIH-3T3 cells display verapamil-inhibitable T3 efflux. Further, in these cells T3 specifically photoaffinity labels a protein(s) different from mdr or mrp proteins. This labeling is reduced by verapamil and is not observed in rat hepatoma (HTC) cells that do not exhibit verapamil-sensitive T3 export activity. We also found that verapamil-inhibitable T3 efflux activity in several cell lines does not correlate with the expression of two members of the ABC superfamily: MDR1, and the multidrug resistance-associated protein, mrp1, which mediates the cellular export of a spectrum of chemotherapeutic drugs different from those transported by mdr1b. These results, taken together, suggest that a novel protein mediates T3 export.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cellular uptake of T3
Transport studies were conducted as described previously (7).
Briefly, cells grown in monolayer to 80–100% confluence in six-well
plates were washed twice with PBS prewarmed to 37 C and incubated with
the appropriate growth medium containing 0.01% NBS and 1–2
nM L-T3 labeled with
125I in the 3'-position (540 Ci/mmol;
NEN Life Science Products, Boston, MA). After incubation for varying
periods (usually 5 min), uptake was stopped by aspirating the medium
containing [125I]T3 and washing the cells six
times with ice-cold PBS to remove superficially adherent tracer on the
cell surface. Cells were detached with 1 ml/well prewarmed
calcium-/magnesium-free PBS with 1 mM EDTA (EDTA buffer).
Cellular radioactivity and protein were determined. The effect of
verapamil on the uptake of T3 was studied by adding the
drug (final concentration, 100 µM) to the appropriate
medium containing 0.01% NBS 20 min before addition of the
[125I]T3.
Efflux of T3
Cells were first preloaded with
[125I]T3 in preincubation medium containing
0.01% NBS and 1–2 nM [125I]T3
at 37 C for 90–180 min, after which cells were washed six times with
ice-cold PBS (as in the uptake studies described above). Efflux was
initiated by adding to each well 1 ml medium containing 10% NBS
without tracer, prewarmed to 37 C. Incubation of the monolayers was
continued at 37 C, and the medium was collected and replaced by fresh
prewarmed efflux medium at 5, 10, 30, and 60 min. The purpose of the
10% NBS is to decrease the proportion of medium
L-T3 in the free form, thus minimizing reuptake
by cells during efflux.2 At
the end of the efflux period, cells were removed from the plates by
adding 1 ml/well EDTA buffer. Percentages of the tracer remaining in
the cells at each time were computed, and efflux curves were
constructed using a curve-fitting algorithm as described previously
(7). To determine the effect of verapamil on efflux, two protocols were
followed. In one, the drug was added to the cold PBS washes and efflux
medium, but not to the preincubation media. In the other protocol,
verapamil was present during preincubation as well as during
efflux.
The effects of unlabeled L-T3 and D-T3 (Sigma Chemical Co.) on T3 efflux were determined by adding these substances only to the preincubation medium. The net accumulation of radiolabeled T3 during the 90- to 180-min preloading period was decreased, but not abolished, by excess L-T3 in agreement with the observations of others that only a portion of cellular T3 uptake is saturable (1, 2, 3, 4, 5, 6). In FRTL-5 cells, accumulation of radiolabeled T3 during preincubation was reduced by 52% in 100 µM L-T3 and was decreased by 26% in 100 µM D-T3. Thus, even at the highest concentration of added T3, there was sufficient loading with tracer to permit studies of T3 efflux.
Assay of mdr function
The efflux rate of rhodamine-123 (R123; Molecular Probes, Inc., Eugene, OR) was used to assess mdr function. Briefly,
after 1-h preincubation of MDR1/NIH-3T3 and control NIH-3T3 cells at 37
C in DMEM-H21 medium containing 10% NBS and R123 at 10 nM,
cells were washed six times with ice-cold PBS. Efflux was begun by
adding 1 ml/well prewarmed DMEM-H21 medium containing 10% NBS. This
efflux medium was collected and replaced by fresh efflux medium at 1,
5, 10, and 20 min. Fluorescence was measured using the Fluorolog 2
spectrofluorometer (Spex Industries, Edison, NJ).
Column chromatography
The 125I-labeled compounds in efflux medium were
analyzed by column chromatography according to the method described by
Docter et al. (31). Briefly, Pasteur pipettes containing
Sephadex LH-20 (0.5 x 4 cm) and plugged with cotton wool were
equilibrated with 0.1 M HCl. One milliliter of efflux
medium was acidified to pH 1.0 with HCl and applied to the column.
Iodide was eluted with 3 x 1 ml 0.1 M HCl.
Subsequently, conjugates of T3 were eluted with 7 x 1
ml H2O, and finally, T3 was eluted with
3x 1 ml 0.1 M NaOH-ethanol (1:1).
Plasma membrane isolation
Cells were grown to confluence, washed, and scraped with PBS.
They were then harvested by centrifugation (180 x g, 2
min, 4 C) and resuspended in hypotonic buffer (1 mM
Tris-Cl, pH 7.0). Eight hundred units of recombinant Serratia
marcescens nuclease (benzonase) was added, and the mixture was
stirred on ice for 2 h. The cell lysate was centrifuged at
100,000 x g for 30 min at 4 C, and the resulting crude
membrane pellet was resuspended in buffer (50 mM Tris and
50 mM mannitol, pH 7.0). All solutions were supplemented
with the proteinase inhibitors leupeptin (4 µM),
pepstatin (2 µM), EGTA (2 mM), and
phenylmethylsulfonylfluoride (0.5 mM; Sigma Chemical Co.). Aliquots were removed for determination of protein
concentration.
Electrophoresis and immunoblotting procedures
Total and plasma membrane proteins (40 µg) isolated from
resistant (HTC-R), nonresistant (HTC), MDR1/NIH-3T3, NIH-3T3, and
FRTL-5 cells were each fractionated in duplicate by electrophoresis on
7.5% SDS-PAGE (32). The first gel was used for Coomassie blue
staining, and the second gel was transferred to nitrocellulose membrane
by electroblotting (33). The nitrocellulose membrane was incubated
with a 1:500 dilution of mouse monoclonal antibody (C219) against the
nucleotide-binding domain of human P-glycoprotein (34) or a 1:1000
dilution of rat monoclonal antibody (MRPr1) against the human MRP1
protein (Signet). Immune complexes were detected using antimouse and
antirat antibodies coupled to alkaline phosphatase, respectively.
Ribonuclease (RNase) protection assay
Total RNA (40 µg) isolated from resistant (HTC-R) and
nonresistant (HTC) FRTL-5 cells and primary rat hepatocytes was
coprecipitated with 105 cpm of a 32P-labeled
complementary RNA (cRNA) probe for rat mdr1b (nucleotides 1317–1706)
or with a cRNA probe for rat glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). The pellet was rinsed with 100% ethanol and thoroughly
resuspended in 20 µl hybridization buffer. The RNA species were
denatured (3 min, 90 C) and rapidly transferred to the appropriate
hybridization temperature for 16 h, after which 200 µl ice-cold
ribonuclease digestion buffer containing a 1:200 dilution of RNase A
(250 U/ml) and RNase T1 (10,000 U/ml) was added, and the mixture was
incubated for 60 min at 37 C. Precipitation solution was added, and the
suspension was chilled at -80 C for 10 min and centrifuged for 20 min
at 14,000 rpm. The pellets were redissolved in 8 µl RNA loading
buffer, denatured for 3 min at 90 C, chilled on ice, and loading onto a
6% denaturing polyacrylamide gel. After electrophoresis, the gel was
exposed to x-ray film.
Photoaffinity labeling
Cells grown in monolayers to near confluence in 10-cm petri
dishes were washed with warm PBS and incubated at 37 C in medium
containing 0.01% NBS and
[125I]L-T3 (4 nM; 2
µCi/ml) for 120–180 min. The medium was removed by aspiration, and
the cells were washed with 6 ml/dish ice-cold PBS three times. The
final wash was left on the dish. The effect of cold
L-T3, D-T3, and
verapamil on photolabeling of the cells was studied by adding these
substances (all at 100 µM, final concentration) to the
[125I]L-T3-containing medium
during the final 15 min of incubation and to the PBS washes, so that
the agent was present during photolabeling. All of the following steps
were carried out at 4 C. Photolabeling was carried out by exposing the
dish (uncovered) to a UV source (wavelength, 354 nm) for 30 min. The
PBS overlying the cells was removed by aspiration, and the cells were
collected by adding 6 ml ice-cold EDTA buffer using a rubber policeman.
After centrifugation of the cell suspension at 1500 x
g for 10 min, the supernatant was removed, and the cell
pellet was snap-frozen in a dry ice-ethanol mixture.
PAGE
The cell pellets were dissolved in sample electrophoresis buffer
[2% (wt/vol) SDS, 5% (vol/vol) mercaptoethanol, 10% (wt/vol)
glycerol, and 60 mM Tris-HCl, pH 6.8] and boiled for 5
min, as described by Samson et al. (35). The samples were
electrophoresed in 7.5% SDS-polyacrylamide slab gels (1.5 mm thick) by
the method of Laemmli (32), with a 5% polyacrylamide stacking gel. The
Mr of the radiolabeled bands was estimated using
Mr standards (BRL, Rockville, MD). Gels were
fixed, stained, destained, and dried, and autoradiography was performed
at room temperature for 1–3 weeks.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, cellular uptake
at 5 min, expressed as picomoles of T3 accumulated per mg
cellular protein, was highest in FRTL-5 cells, lowest in HTC and HTC-R
cells, and intermediate in MDR1-transduced and control NIH-3T3 cells.
Verapamil (final concentration, 100 µM), added to the
incubation medium 20 min before [125I]T3,
diminished T3 uptake by 72% in FRTL-5 cells and by 79%
and 88%, respectively, in MDR1/NIH-3T3 and control NIH-3T3 cells. In
contrast, the drug had no significant effect on T3 uptake
by HTC and HTC-R cells, confirming our previous findings (7).
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, FRTL-5 cells
exported T3 rapidly. After 60 min, only 8.8% of initial
cellular T3 remained in the cells. The curve depicting
cellular [125I]T3 vs. time was
best described by a two-component exponential function; the exit rate
constant (minutes-1) of the major component averaged
0.104 ± 0.018 (SD), and that of the slower, minor
component was 0.030 ± 0.004. The efflux of
125I-labeled T3 was inhibited by adding excess
unlabeled L-T3 during preincubation with tracer
(Fig. 1). At 1 µM L-T3 the rate
of [125I]T3 efflux was significantly reduced;
the efflux curve fit a single exponential with a rate constant
(0.036 ± 0.006) similar to that of the slower component of the
control efflux curve. At 10 µM
L-T3 the rate constant of the single
exponential was decreased to 0.018 ± 0.002 (Fig. 1), and at 100
µM L-T3 it was decreased to
0.014 ± 0.002 (not shown). Thus, T3 efflux is
saturable. Furthermore, D-T3 (100
µM) had no effect, indicating that the efflux process is
stereospecific.
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and Table 1). In the presence of verapamil the efflux
curve fit a single exponential function with an exit rate constant
(minutes-1) of 0.017 ± 0.004 (P <
0.005 vs. cells without verapamil). Thus, the drug virtually
eliminated the initial rapid component of the efflux curve and
inhibited the second component that was seen in the control
experiments. The effect of verapamil was concentration dependent.
T3 efflux from FRTL-5 cells was measured at three
concentrations of the drug in the efflux medium. The percentages of
cellular T3 remaining after 60 min were 13.0 ±
3.5, 19.4 ± 4.6, and 35.5 ± 4.1 at 1, 10, and 100
µM verapamil, respectively, whereas the mean value in the
controls was 10.7 ± 1.7. The efflux kinetics can be interpreted
as indicating that T3 exits from two compartments in FRTL-5
cells and that both are verapamil sensitive and saturable. The cellular accumulation of [125I]T3 during prolonged incubation is the net result of uptake (influx) and efflux. The accumulation of [125I]T3 by FRTL-5 cells during a 2-h preincubation was reduced by 21% when verapamil was present in the medium. Therefore, verapamil-induced inhibition of T3 uptake slightly exceeded its inhibition of efflux. Most studies of the verapamil effect on T3 efflux were performed by adding the drug only to the cold PBS washes and to the efflux medium, not to the preincubation medium with [125I]T3. However, virtually identical T3 efflux rates were obtained in experiments in which verapamil was also present during preloading of cells with [125I]T3 as well as during efflux (data not shown).
Chromatographic analysis of the 125I-labeled compounds in the efflux medium from FRTL-5 cells at 30 and 60 min showed that less than 4.0% of the total 125I was in the form of iodide, and more than 92% was in the form of T3 at both times. Furthermore, the proportion of iodide in the medium did not increase from 30–60 min. These results indicate that the T3 in the cells or medium did not undergo 3'-deiodination to a significant extent during the period of efflux.
In contrast to FRTL-5 cells, HTC cells exhibited very slow
T3 efflux, which was not affected by verapamil (Table 1).
However, HTC cells that had been selected for glycocholic acid
resistance (HTC-R cells) showed rapid T3 efflux that was
inhibited by verapamil (100 µM).
T3 export in cells expressing the multidrug
resistance P-glycoprotein, MDR1
To test the hypothesis that the multidrug resistance
P-glycoprotein is involved in T3 export, we studied
T3 efflux in NIH-3T3 cells that had been transduced with
MDR1, the human analog of the rodent mdr1b gene, and in NIH-3T3 cells
transduced with the retroviral vector without MDR1 (controls).
Nontransduced NIH-3T3 cells express little or no mdr1b (data not
shown). To confirm that the product of MDR1 in the MDR1-transduced
NIH-3T3 cells was functional, the efflux of R123 in these cells was
49 ± 4% more rapid than that in control cells and was verapamil
inhibitable. As shown in Table 1 and Fig. 2A, T3 efflux in both
MDR1-transduced and control NIH-3T3 cells was equally rapid and equally
inhibited by verapamil. In contrast to FRTL-5 cells, NIH-3T3 cells
(without verapamil) showed T3 efflux kinetics that fit a
single exponential curve. The exit rate constant
(minutes-1) in control cells was 0.033 ± 0.005, and
that in MDR1-transduced cells was 0.029 ± 0.005. These were
reduced by verapamil to 0.006 ± 0.001 and 0.007 ± 0.001,
respectively. The similarity in T3 efflux rates between
MDR1-transduced and control NIH-3T3 cells suggests that the multidrug
resistance P-glycoprotein MDR1 does not mediate T3
export.
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, T3 efflux in control NIH-3T3 cells was
inhibited by excess L-T3 in a
concentration-dependent manner, indicating that the process is
saturable, but was not affected by D-T3,
indicating that it is stereospecific. Control cells and nontransduced
(parent) NIH-3T3 cells exhibited similar degrees of
verapamil-inhibitable, saturable, and stereospecific T3
efflux activity as MDR1/NIH-3T3 cells (data not shown).
Absence of correlation between T3 export
and expression of mdr1b and mrp1
RNase protection assay analysis of the RNA extracted from primary
hepatocytes, FRTL-5, HTC, and HTC-R cells showed prominent bands
representing mdr1b RNA-protected fragments (313 nucleotides) in
hepatocytes and HTC-R cells and a weak band in HTC cells, but no
detectable protected fragments in FRTL-5 cells. There was no difference
in the number or density of RNA fragments from the various cell types
with the GAPDH probe (Fig. 3).
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). These results closely
parallel the findings of the RNase protection assay. The cell lines
that showed little or no detectable mdr expression were those that
exhibited as much T3 efflux activity as the cells that
expressed high levels of mdr. Immunoblots using specific antibody
against mrp1 protein showed expression of mrp1 in all of the cell lines
tested: HTC, HTC-R, FRTL-5, and NIH-3T3 cells (Fig. 5). All cell lines expressed mrp1 in
nearly equal degrees, but they differed greatly in T3
efflux activity. Thus, there was no correlation between the presence of
verapamil-inhibitable T3 efflux activity and the expression
of either mdr1b or mrp1.
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, A–C). A discrete band in the range of 90–100 kDa was present in all
cells except HTC cells, which do not exhibit verapamil-inhibitable
T3 export. In fact, in contrast to the other cells studied,
including HTC-R cells, very little T3 labeling of any
cellular proteins was detectable in HTC cells. In FRTL-5 cells, the
labeling of the 90- to 100-kDa band was reduced when either verapamil
or L-T3 (both 100 µM) was present
in the cell medium during exposure to UV light.
D-T3 (100 µM) did not inhibit
labeling of this protein (Fig. 6B). Although there were other, lower
Mr proteins in the FRTL-5 cells, the labeling of which was
partially inhibited by verapamil and L-T3, the
90- to 100-kDa protein was the only one common to FRTL-5, MDR/NIH-3T3,
and HTC-R cells. Thus, the photolabeling properties of the 90- to
100-kDa protein correlate well with the characteristics of cellular
T3 export in that both photolabeling and T3
efflux are saturable, stereospecific, and verapamil sensitive.
Nondenaturing PAGE of proteins from cells photoaffinity labeled with
[125I]T3 showed nearly all the radioactivity
at the origin and no detectable signal in the region of 90–100 kDa,
indicating that this is a membrane-associated protein (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Second, we provide evidence that neither mdr1b nor mrp1 gene products
are involved in T3 export. This contradicts our previous
suggestion that T3 efflux is mediated by an ABC protein of
the mdr type, based on the observations that HTC-R cells acquire
accelerated, verapamil-sensitive T3 efflux and overexpress
several novel mdr-like P-glycoproteins. Although verapamil is known to
be a potent reversal agent of the function of mdr/P-glycoproteins (29)
and of mrp (36), our data argue strongly against the hypothesis that an
ABC protein of the mdr or mrp type is involved in T3
efflux. This contention is based on the lack of correlation between
T3 efflux activity and MDR1 or mrp1 expression among the
various cell lines tested and the absence of
[125I]T3-labeled proteins (170 kDa)
compatible with either mdr/P-glyco-proteins (37) or mrp proteins
(38). In fact, we detected in these cells by photoaffinity labeling a
90- to 100-kDa protein band with verapamil- and T3-binding
properties consistent with the putative transporter.
Nelson and Hinkel showed that pituitary tumor cells with acquired multidrug resistance exhibit increased export of hydrocortisone, but not of thyroid hormones (10). They showed that various multidrug resistance-reversing agents, including verapamil (but not other calcium channel blockers), increased the cellular content of hydrocortisone, but did not alter the T3 content of the cells. Our finding of similar T3 efflux rates in MDR1-transduced and control NIH-3T3 cells confirms their data pertaining to thyroid hormone. We found that verapamil inhibited thyroid hormone uptake by FRTL-5 and NIH-3T3 cells, which is consistent with the inhibition by various calcium channel blockers, including verapamil, on T3 uptake by rat H4 hepatoma cells, L6 myoblast cells (6), and human JAR cells (26). In contrast, verapamil did not inhibit T3 uptake in rat HTC or HTC-R cells, suggesting that there may be differences among various cell lines with regard to thyroid hormone uptake.
Cellular proteins in the range of 45–55 kDa that bind T3
and are possibly involved in hormone uptake have been identified in rat
liver using immunoprecipitation (39) and in rat erythrocytes using
photoaffinity labeling (35). Recent reports indicate that one or more
subtypes of the family of organic anion transporter polypeptides, oatp
(16), and Na+/taurocholate cotransporting polypeptide,
Ntcp, may mediate the cellular uptake (influx) of thyroid hormone and
various derivatives, e.g. iodothyronine sulfates (17). One
of the forms of oatp with putative thyroid hormone transport activity
consists of 670 amino acids (Mr, 75 kDa) (16).
Ntcp has an apparent Mr of 51 kDa (40). There are many
cellular proteins associated with thyroid hormones, and in only a few
cases has the function been defined (18, 19). No transport protein
mediating thyroid hormone efflux has yet been identified. Although we
cannot exclude the possibility that the photolabeled band represents a
proteolytic fragment of a larger protein, our findings suggest that a
novel 90- to 100-kDa protein, different from mdr or mrp, may be linked
to T3 efflux. However, the protein must be isolated and
functionally characterized to determine whether it is indeed involved
in the transport of T3 out of the cell.
The steady state intracellular concentration of T3 is determined by several factors, including the rates of influx and efflux, the metabolic disposal of T3, and the de novo production of T3 by 5'-deiodination of T4. The latter pathway appears to be particularly important in tissues of the central nervous system and the anterior pituitary (18). The results of the present study and those previously reported (7) indicate that carrier-mediated T3 efflux is present in many mammalian tissues. It is reasonable to postulate that this represents an additional physiological mechanism for regulating the intracellular concentration of active thyroid hormone. The present study has not addressed all possible mechanisms by which thyroid hormones exit cells. As we pointed out in our previous publication (7), in addition to the T3 export mechanism we have described, there appears to be a basal hormone efflux mechanism that is not saturable, is not inhibited by verapamil, and probably involves passive diffusion. Many questions remain to be explored. For example, is verapamil-sensitive efflux an active, i.e. energy-requiring, process? Are uptake and efflux of thyroid hormones under different regulatory controls?
The present demonstration of a stereospecific, saturable, T3 export mechanism in a thyroid-derived cell raises a number of other questions, such as, is the mechanism influenced by TSH? It is tempting to speculate that in the thyroid gland, this process of T3 export may provide a means of regulating hormone secretion. These studies also provide new insights into thyroid hormone transport and form the basis for further investigations needed to elucidate the molecular mechanisms that govern the biology of thyroid hormone transport.
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Footnotes |
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2 Separate experiments were performed to estimate
reuptake of the T3 released from cells during the efflux
period. In medium containing 10% NBS, only 0.2% of the T3
in each well was taken up by cells in 5 min. Therefore, during the
longest interval between efflux medium changes (30 min), 1.2% of
T3 in the medium would have reentered cells. The reuptake
from medium containing verapamil would have been even lower (0.6%).
Correction of the data for this amount of reuptake resulted in an
increase of less than 0.4% in the slopes of the efflux curves for both
control and verapamil groups. Therefore, we made no correction for
reuptake.
Received March 11, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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