Characterization of the Thyroid Hormone Transport …THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No 6 , Issue of Fehruary 25. pp. 2685-2692.1986 Printed in U.S.A.

Characterization of the Thyroid Hormone Transport System of Isolated Hepatocytes*

(Received for publication, September 22, 1987)

Jean-Paul BlondeauS, Jeanine Osty, and Jacques Francon From the Unite de Recherche sur la Glande Thyroide et la Regulation Hormonule (Unite 96) de l’lnstitut National de la Sante et de la Recherche Medicale, 78 rue du Geniral Leclerc, 94275 Le Kremlin-Bicitre Ceder, France

Transport of 3,5-[3’-’2511triodo-~-thyronine ([12’1] T3) was studied in isolated rat liver hepatocytes. T3 transport was temperature-sensitive, the initial velocity of uptake, at low substrate concentration, was 60 times higher at 25 “C than at 0 “C. The activation energy of cellular uptake (26 kcal/mol) was different from that of binding to cytosolic proteins (6 kcal/mol), indicating that the latter was not the rate-limiting step. Uptake obeyed simple Michaelis-Menten kinetics, with an apparent K , of 0.34 pM and a V,,, of 0.15 fmol/ min/cell at 25 “C. No simple diffusion occurred. Unla- beled T3, L-thyroxine (T4), 3,5,3’-triiodo-~-thyronine, and triiodothyroacetic acid inhibited T3 uptake with Kt values of 0.32, 1.4, 4.1, and 5.4 p ~ , respectively, indicating specificity of uptake which was different from specificity of intracellular binding sites. [ 12’1]T4 was also taken up by a saturable process (K,,, = 0.65 pM and Vmax= 0.16 fmol/min/cell at 25 “C). T3 was a better competitor than T4 for the uptake of [12‘I]T4, indicating that both hormones were taken up by the same carrier system. Metabolic inhibitors had either no effect on T3 uptake or inhibitory effects unrelated to cellular ATP depletion. Uptake was not affected by modification of the membrane potential or the sodium ion gradient. T3 and T4 uptake was pH-dependent. It is suggested that the un-ionized 4’-OH form of the hormones was preferentially taken up. Inhibition of uptake by various compounds was compared to inhibition of thyroid hormone binding to transthyretin, nuclear receptor, and cytosolic-binding proteins. We conclude that, in freshly isolated hepatocytes, thyroid hormones are taken up by a saturable, stereospecific, Na+-independent carrier system.

The chromatin-associated receptor is thought to be the main intracellular target of thyroid hormones, where 3,5,3‘- triiodo-L-thyronine (T,)’ influences the expression of specific genes (1). The T3 bound to the nuclear receptor of liver cells is of plasma origin (2) and must therefore cross the plasma membrane. The entry of T,, into the hepatocyte is also of biological interest since the liver actively deiodinates T, and contributes significantly to the peripheral production of Ta.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. The abbreviations used are: T,, 3,5,3’-triiodo-~-thyronine; D-T,,

3,5,3’-triiodo-~-thyronine; T,, L-thyroxine; To, DL-thyronine; ANS, 8-anilino-1-naphthalene sulfonic acid; Hepes, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; pHMB, p-hy- droxymercuribenzoate.

The translocation of TB and T, from plasma to the intracellular compartment was initially thought to take place by passive diffusion (3). But specific binding sites for these molecules have been detected in purified rat liver plasma membrane (4, 5), and it has been suggested that they are implicated in the entry of thyroid hormones into cells. There is experimental evidence for the existence of limited capacity systems that mediate the entry of thyroid hormones through the membrane of various cells (6-10).

The entry of T3 into cultured fibroblasts and GH3 cells by receptor-mediated endocytosis was described using drugs known to interfere with this route (11,12). Recently, a mono- clonal antibody raised against a membrane protein was shown to inhibit the cellular uptake and subsequent deiodination of thyroid hormones by cultured rat hepatocytes (13).

Despite this substantial body of evidence, several conflict- ing points remain. Recent investigations did not show T, saturation uptake kinetics, characteristic of mediated uptake, in freshly isolated hepatocytes (14) or perfused liver (15). The entry of T, into target cells is reported to occur by simple diffusion (10, 16) or to be mediated by a saturable process distinct from that which is involved in T, uptake (17). There are also discrepancies as to the energetic requirements for the uptake of T,. Carriers can work either by facilitated diffusion, in which a solute flows down its electrochemical gradient, or by secondary active transport, with the electrochemical gradient of an ion (generally Na’) driving another solute against its electrochemical gradient (18). Energy-independent facilitated uptake of T, was reported for human erythrocytes (9) and frog, rat, and human red blood cells and rat thymus cells (lo), whereas active energy-dependent uptake has been described in cultured hepatocytes (7, 17) and fibroblasts (12). Whether these active processes are carrier-mediated uptake of free hormone or receptor-mediated endocytosis is still an open question (19).

This study concerns the transmembrane movement of thyroid hormones in freshly isolated rat hepatocytes. These cells, prepared by enzymatic dispersion of the liver, have been widely used to study a variety of facilitated and active transport systems and to investigate receptor-mediated endocytosis.

We have previously reported the conditions for the measurement of saturable association of Ta with the isolated hepatocytes (20). In this work, we show that Ts is taken up exclusively by a saturable, stereospecific carrier system whose transport characteristics are described.

MATERIALS AND METHODS

Chemi~als-[3’-’*~I]T~ (specific activity = 3 mCi/rg) and [3’,5’-

sham Corp. T,, D-T3, T4, triiodothyroaeetic acid, To, ANS, bovine serum albumin (type V), bromosulfophthalein, chloroquine, choline

125 IlT, (specific activity = 1.5 mCi/pg) were purchased from Amer-

2685

2686 Thyroid Hormone Transport in Hepatocytes

chloride, collagenase (type I), dansyl chloride, 5,5-diphenylhydantoin, 2,4-dinitrophenol, Mes, monensine, monodansylcadaverine, N-ethylmaleimide, oligomycin (0-4876), ouabain, ovalbumin, phenylmethylsulfonyl fluoride, phloretin, pHMB, soybean trypsin inhibitor, and valinomycin were from Sigma. Antimycin A and Hepes were from Boehringer Mannheim. Eagle's basal medium was purchased from GIBCO, Percoll from Pharmacia LKB Biotechnology Inc., and Dowex AG 1-X8 from Bio-Rad. Iopanoic acid was a generous gift from Laboratoires Winthrop (Dijon, France). Plastic tubes and pipette tips were siliconized (Sigmacote, Sigma).

Preparation of Isolated Hepatocytes-Suspensions of isolated hepatocytes were prepared from the livers of male Wistar rats (200 g of body weight) which had been thyroidectomized 4-10 weeks earlier and fed ad libitum. The cells were isolated by the collagenase perfusion method of Seglen (21) as modified by Oldberg et al. (22), incubated in an oxygenated glucose-containing medium, and purified by Percoll centrifugation as described previously (20). Briefly, the liver was perfused in situ with oxygenated calcium-free buffer A (142 mM NaCl, 6.7 mM KC1, 10 mM Hepes, pH 7.4) at 37 "C, transferred to an in uitro system, and perfused for 18 min at 37 "C with buffer A, pH 7.6, containing colleganase (85 units/ml), bovine serum albumin (1.5%), soybean trypsin inhibitor (0.2 mg/ml), and CaC1, (1 mM). The hepatocytes were released into the collagenase perfusion medium and slowly diluted with chilled buffer B (137 mM NaC1, 47 mM KC1, 0.65 mM MgSO,, 1.2 mM CaC12, 8.5 mM Hepes, pH 7.4) containing 1.5% bovine serum albumin. The cell suspension was filtered through a 45- pm nylon gauze, centrifuged (70 X g, 2 rnin), and washed twice by resuspension/centrifugation with cold buffer B without albumin. The cells were resuspended, in oxygenated medium C (Eagle's basal medium supplemented with 2.6 g/liter glucose and 10 mM Hepes, pH 7.4) and incubated for 30 min at 37 "C in order to recover from acute stress resulting from collagenase treatment and to restore transmembrane gradients (23,24). After centrifugation (70 X g, 2 rnin), the cell pellet was resuspended in isotonic Percoll diluted with medium C (d = 1.09) and centrifuged for 5 min at 200 X g. The damaged cells remained floating at the surface; the pellet contained the viable hepatocytes which were resuspended in medium C and kept on ice. The viability of the cell suspension, estimated by the trypan blue exclusion method, was 97% or over and did not drop for at least 4 h at 0 "C or 30 min at 37 "C. After 24 h at 0 "C, viability was still 80- 85%. Cell counts were done in a Malassez hemocytometer.

Measurement of Uptake by Hepatocytes-Unless otherwise stated, the experiments were carried out in oxygenated medium C at the indicated temperature. The cells were routinely suspended in the incubation medium and equilibrated for 2-3 min at the choosen temperature. The transport experiments were started by addition of hepatocytes to pre-equilibrated medium containing labeled thyroid hormones (25-50 nCi/ml) and other additives in order to obtain a final suspension containing 4 X IO5 hepatocytes/ml. Under these conditions, the intracellular aqueous volume represented about 0.1% of the volume of the incubation mixture (25). 1-ml aliquots of suspension were removed at timed intervals, transferred to siliconized (Sig- macote, Sigma) polystyrene tubes containing medium C and unlabeled T3 to give a final T3 concentration of M, and placed in an ice bath. The cells were centrifuged (200 X g, 2 rnin), and the pellet was washed once with 1.5 ml of ice-cold glycine buffer (50 mM glycine, 150 mM NaCl, 3% (w/v) bovine serum albumin, pH 10.5) containing

M unlabeled T3 and twice with 2 ml of ice-cold buffer B. AS described previously (20), cell viability was not affected by the alkaline washing procedure provided the cells were processed at a temperature close to 0 "C. Efflux experiments conducted with [I2'I]T3- loaded cells indicated that efflux of intracellular ['251]T3 during the washing procedure was negligible (<2%). The radioactivity was measured on the pellets with an efficiency of 80%.

Following incubation with ["'II]T,, the hepatocytes (2 X 10' cells) Measurement of T3 Binding to Nuclei of Intact Hepatocytes-

were centrifuged and treated with 1 ml of cold 0.32 M sucrose, 1 mM MgCl,, 2 mM CaCl,, 0.1 mM dithiothreitol, 0.5% (v/v) Triton X-100, 20 mM Tris-HC1, pH 7.4. The nuclei were pelleted (1000 X g, 10 min) and washed with 1 ml of the same buffer as described (12). The bound radioactivity was measured on the final nuclear pellet.

Binding of T3 to Isolated Liver Nuclei-Triton X-100-treated nuclei were prepared from rat liver as described by Spindler et al. (26). The nuclei, suspended in 0.25 M sucrose, 50 mM NaCl, 1 mM MgCl,, 0.1 mM dithiothreitol, 2 mM EDTA, 5% (v/v) glycerol, 20 mM Tris-HCl, pH 7.6 (0.15 mg of DNA/ml), were incubated with 50 pM [12sI]T3 for the times and at the temperatures indicated in the figure legends. The incubations were stopped by the addition of 10 p M unlabeled TS

to 0.5-ml aliquots and cooling at 0 "C. The nuclei were centrifuged (1000 X g, 10 min), and the pellets were washed twice by resuspension centrifugation with 1 ml of cold 0.32 M sucrose, 1 mM MgCl,, 2 mM CaCl,, 0.1 mM dithiothreitol, 0.5% (v/v) Triton X-100, 20 mM Tris- HC1, pH 7.4, as described (12). The bound radioactivity was measured on the pellet.

Binding of T:j to Liuer Cytosol-Cytosol was prepared from the livers of male Wistar rats as described by Lennon et al. (27). The buffer used was 0.32 M sucrose, 1 mM MgCl,, 1 mM dithiothreitol, 5% (v/v) glycerol, 2 pg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM Hepes, pH 7.6. The preparations were incubated with ["'I]T3 under the conditions described in the appropriate figure legends, and bound and unbound hormones were separated by Dowex treatment. An equal volume of 20% (w/v) Dowex AG 1-X8 suspension was added to the cytosol, and the mixture was allowed to stand 1 min at 0 "C and was centrifuged for 30 s at 2500 X g. The bound radioactivity was measured on the supernatant. The nonsaturable binding, measured in the presence of 5 X M unlabeled T3, was subtracted.

Binding of T3 to Purified Transthyretin-Rat transthyretin was purified according to the method of Bleiberg et al. (28) using phenol precipitation of the serum followed by a single preparative polyacryl- amide gel electrophoresis and was about 95% pure. The transthyretin preparation (2.5 pg of protein/ml of 150 mM NaC1,lO mM Hepes, pH 7.5) was incubated with 4.3 X 10"' M ["'I]T, for 30 min at 25 'C in the presence of 0.4 mg/ml ovalbumin. Bound and unbound hormones were separated by Dowex treatment as described above. Ovalbumin was added to prevent transthyretin adsorption to the Dowex resin. Nonsaturable binding was determined in the presence of M unlabeled T, and was subtracted from the total binding.

Statistical Treatment of Data-Standard deviations of the regression coefficients, correlation coefficients ( r ) , and standard deviations were calculated as described by Snedecor and Cochran (29).

RESULTS

Validation of the Washing Procedure-In previous work (ZO), we showed that alkaline washing of the hepatocytes (glycine buffer, pH 10.5) following incubation with ['*'1]T3 may be used to distinguish between a saturable, alkaline- resistant compartment of cell-associated T3 and an unsaturable, alkaline-suppressible compartment. In this previous work, the binding of ['*'1]T3 to isolated hepatocytes was studied using 2 x lo6 cells/ml and a 4-h incubation a t 0 "C. Raising the pH of the washing buffer from 7.5 to 10.5 caused a 20% decrease in the binding measured at low (0.1 nM) [1251] T3 concentration (total binding) and a 85% decrease in that measured in the presence of excess (10 p ~ ) unlabeled TJ (unsaturable binding). As a consequence, the unsaturable binding varied from 65 to 13% of the total binding when the pH of the washing buffer was raised from 7.4 to 10.5. In this work, we have used a lower cell concentration (4 X lo5 cells/ ml). Under this condition, the alkaline washing caused simi- larly a 20% decrease in total binding and a 80% decrease in nonsaturable binding (not shown). The amounts of nonsaturable binding were lower, being 25% of total binding when the pH of the washing buffer was 7.4 and 5% when the pH was 10.5.

The removal of the unsaturable compartment by the alkaline buffer was instantaneous, with no further loss using longer contact times (up to 5 min) or by a second alkaline wash. Conversly, the saturable compartment was little affected by the duration of the washing procedure, and dissociation was very slow at 0 "C and pH 7.4 in the presence of excess unlabeled T:L (t),> > 15 h). This suggests that alkaline washing removes membrane-bound, but not internalized, ligand. A substantial unsaturable binding of aromatic amino acids to the cell surface of isolated hepatocytes, irrelevant to the transport of the amino acids themselves, has been reported by others (30).

We investigated the possibility that both compartments (alkaline-resistant and -suppressible) could be a source of hormone for nuclear receptor, The time course of in situ

Thyroid Hormone Transport in Hepatocytes 2687

nuclear transfer of [1251]T3 (0.05 nM) at 25 "C (control condition) is shown in Fig. la. Maximum nuclear binding was obtained after a 2-h incubation. A similar time course was obtained with cells preincubated for 4 h at 0 "C with ['"I]T3 and washed with Hepes buffer, pH 7.4. An identical time course was obtained with preincubated hepatocytes submitted to the alkaline washing procedure (which reduced the unsaturable compartment from 25% of total binding to 5%). The reduction of the equilibrium level of nuclear binding to 60% of the control value could be related to the fact that the amount of TS associated with the cells, after 4 h at 0 "C, was about 65% of the equilibrium level of cellular uptake (see Fig. 2). The in situ saturation of the nuclear receptor was studied under control conditions or using the preincubation/alkaline washing procedure (Fig. lb). The saturation curves were very similar since 50% displacement of ['251]T3 by unlabeled Ts was obtained at 0.24 and 0.32 nM, respectively. These values, which approximate the apparent Kd of the nuclear receptor, are close to the value of 0.2 nM reported by Mooradian et al. (14) for the in situ nuclear binding of T3 in isolated hepatocytes.

These experiments indicate that the saturable, alkaline- resistant T3 compartment is biologically active since it con- stitutes the reservoir for nuclear transfer of the hormone, whereas the unsaturable, alkaline-suppressible compartment is not necessary for this purpose. Therefore, the alkaline washing procedure was used routinely for the characterization of saturable T3 uptake.

Time Course of Association of T3 with Cells as a Function of Temperature-The time course of uptake of T3 (0.2 nM) by cells is shown in Fig. 2. The initial rates of uptake increased over the temperature range 0-25 "C. Equilibration of T3 between cells and medium converged to a level which was independent of the temperature, suggesting that T3 also en- tered the cells at 0 "C. At equilibrium, 45% of the added T3 was cell-associated. Trapped isotope at time 0 was less than

A

0.1 i TIME (hours) UNLABELLED T3 (nM)

FIG. 1. Effect of the washing procedure on the T, nuclear translocation. a, isolated hepatocytes (4 x lo5 cells/ml) were preincubated with labeled T, (50 PM) for 4 h at 0 "C in medium C and washed by resuspension/centrifgation either twice with buffer B (A) or once with glycine buffer, pH 10.5, and once with buffer B (0). They were resuspended in oxygenated medium C. The control hepatocytes (0) were not preincubated with labeled T,, but were directly suspended in oxygenated medium C containing labeled T, (50 PM). Nuclear transfer was initiated by heating the cells at 25 "C and was stopped by cooling at 0 "C and addition of 10 p~ unlabeled T,. Duplicate aliquots (2 X lo6 cells) were taken up at timed intervals. Nuclear bound T, was determined as described under "Materials and Methods." b, the preincubation conditions were the same as described above except that the medium contained 50 pM labeled T, and various concentrations of unlabeled T3 (0.05-5 nM). Cells were washed once with glycine buffer, pH 10.5, and once with buffer B (0). The control hepatocytes (0) were directly suspended in medium C containing 50 p~ labeled T3 and the same concentrations of unlabeled T, as described above. Nuclear transfer was for 2 h at 25 "C. The results are expressed as the percentage of ["'1]T3 bound in the absence of unlabeled T, and are the mean of duplicates.

i 2'.4 8 TIME (hours)

FIG. 2. Time course of T3 uptake. Isolated hepatocytes (4 x lo6 cells/ml) were incubated in medium C in the presence of 0.2 nM [lZ5I] T, (4.5 X IO' cpm/ml) at the following temperatures: 25 'C (O), 20 "C (B), 15 "C (*), 10 "C (A), 5 "C (+), and 0 "C (V), or in the presence of the same amount of [1251]T3 and M unlabeled T, at 25 "C (0) and 0 "C (V). 1-ml aliquots were taken up in duplicate at timed intervals and added to 0.5 ml of cold stop solution (medium C containing 3 X M unlabeled T3). Cells were centrifuged and washed once with glycine buffer, pH 10.5, and twice with buffer B as described under "Materials and Methods." Results are the mean of duplicates.

TEMPERATURE (X) 20 10 0

I 1

3.4 35 3.6 103/T (OK")

FIG. 3. Arrhenius plot: effect of temperature on T3 uptake by isolated hepatocytes and on T3 binding by liver nuclei and cytosol. The initial rates of [**'I]T3 uptake (0.2 nM, 4.5 X lo4 cpm/ ml) by isolated hepatocytes were determined as described in the legend to Fig. 1, with incubation times ranging from 30 s at 25 'C to 8 min at 0 "C. Incubations were carried out in the absence (0) or presence (W) of M unlabeled T,. ['z51]T3 binding (50 PM) to rat liver nuclei was measured at timed intervals as described under "Materials and Methods." The linear phase of binding, which ranged from 4 min at 25 "C to 30 min at 0 "C, was used to calculate initial rates of nuclear binding (A). ['251]T3 binding (20 PM) to rat liver cytosol (0.1 mg of protein/ml) was measured at timed intervals as described under "Materials and Methods." The initial rates of binding (+) were determined by regression on the linear initial portion of the progress curves (from 50 s at 15 "C to 2 min at 0 "C). The results are normalized as the percentage of initial rate of uptake or binding at 25 "C. Each point is the mean of duplicate determinations. The Arrhenius activation energy was calculated by linear regression.

2% of the equilibrium level and was subtracted. The initial velocity of uptake was routinely measured as

the amount of Tu accumulated by cells between 20 and 40 s at 25 "C or 15 and 30 min at 0 "C following the initiation of the transport process since the process was linear during this initial phase and less than 10% of the added T, was taken up during this time.

The initial velocity of uptake was about 60 times higher at 25 "C than at 0 "C, corresponding to half-times of equilibration of 2 min at 25 "C and 2 h at 0 "C. The temperature dependence of the initial velocity of uptake was analyzed by an Arrhenius plot (Fig. 3). The plot was linear ( r = -0.998), showing no evidence of abrupt breaks attributable to discon-


tinuous behavior of the uptake process between 0 and 25 "C. The activation energy (E,) calculated by linear regression was 26 & 1 kcal/mol, in the range generally observed for transport systems which carry out facilitated diffusion or active transport (25,31,32). The E,, of the association of T3 to the nuclear receptor in isolated rat liver nuclei was 17 f 1 kcal/mol and that for association to the rat liver cytosol binding proteins was 6.0 k 0.3 kcal/mol (Fig. 3). This suggested that binding to intracellular proteins was not the rate-limiting step in the uptake of T3 by cells.

The time course of cellular uptake of a tracer amount of [1251]T3 in the presence of a large amount ( M ) of unlabeled T3 was also studied a t temperatures from 0 to 25 "C. For clarity, only the curves obtained at 0 and 25 "C are shown in Fig. 2. Uptake was saturable at all temperatures studied since the initial velocity was reduced 60-fold at 25 "C and 250-fold at 0 "C in the presence of excess T3. In contrast, long-term incubation at 25 "C showed that equilibrium uptake was not saturable. The temperature dependence of the initial velocity of uptake in the presence of M T3 is illustrated in Fig. 3. The E, of the process was calculated to be 34 f 1 kcal/mol.

Initial Uptake as a Function of the Concentration of T3 in the Medium: Saturability and Effect of Temperature-The effect of TR concentration in the medium on the initial velocity of uptake a t 0 and 25 "C was studied over the range 1.25 X lo-'' to M, keeping the concentration of ['2sII]T3 constant at 1.25 X lo-" M. Inhibition of initial velocity of ['"1]T3 uptake by increasing concentrations of unlabeled T3 became significant at 10 nM a t 0 "c and at 50 nM at 25 "c and was practically complete at M (Fig. 4a). Eadie-Hofstee plots of the data (Fig. 4, b and c ) gave straight lines ( r = -0.992 at 0 "C and -0.994 at 25 "C), indicating that saturable T3 transport was compatible with simple Michaelis-Menten kinetics and that virtually no unsaturable, simple diffusion occurred, even at high concentrations of T3. In the experiment shown in Fig. 4, the apparent K,, calculated by linear regression,

UNLABELLED Ts(VM) 0.01 01 1 10

V,/S (rnin")(.1o3) V, /S (rnin")(.lO)

FIG. 4. Saturability of T, uptake at 0 and 25 "C. Isolated hepatocytes (4 X lo5 cells/ml) were incubated in medium C with [1251] TI (12.5 PM, 4.5 X lo4 cpm/ml) and varying concentrations of unlabeled T,. 1-ml aliquots were removed at timed intervals and treated as described in the legend to Fig. 2. The initial velocities of uptake were calculated as the amount of T, accumulated by the cells between 20 and 40 s at 25 "C (W) and between 15 and 30 min at 0 "C (0). Each point represents the mean value of two separate experiments. a, the initial velocity of ["'I]T, uptake is plotted against the logarithm of the concentration of unlabeled T,. The initial velocity in the absence of unlabeled T, (8.1 X IO3 cpm/min/4 X lo5 cells a t 25 "C and 1.7 X 10' cpm/min/4 X lo5 cells a t 0 "C) was taken as 100%. b and c, Eadie- Hofstee plots of the T, uptake data. The slopes and the intercepts were calculated by linear regression.

was 74 f 4 nM a t 0 "c and 0.38 & 0.01 pM at 25 "c. The mean values (fS.D.) from three independent experiments performed at 25 "C with three separate hepatocyte preparations were K,,, = 0.34 f 0.04 p~ and V,,, = 0.15 f 0.02 fmol/min/ cell. The mean values from two independent experiments performed at 0 "c were K,,, = 90 nM (range = 74-106) and V,,, = 0.83 amol/min/cell (range = 0.66-1.00).

It was calculated that K,,, increased 4-fold and V,,, increased 200-fold when the temperature was raised from 0 to 25 "C, corresponding to Arrhenius activation energies of 8.7 kcal/mol for K , and 34 kcal/mol for V,,, (Table I). This meant that the temperature dependence of K, was not dom- inated by that of the rate-limiting steps contributing to VmaX. Table I summarizes the thermodynamic data. Results obtained from initial velocity measurements at low and high concentrations of substrate are in agreement with those obtained from the constants derived from Eadie-Hofstee plots. 1) The E, of Vmax is identical to the E. of initial velocity measured at high substrate concentration (10 PM). Therefore, the latter primarily reflected the V,,, of transport rather than nonsaturable uptake. 2) The initial velocity measured at low substrate concentration (i.e. << K,) is proportional to the first-order rate constant of uptake, which is equal to the ratio VmaX/K,,,. This is confirmed by the similarity of the E, values measured under these two experimental conditions.

Effect of Analogues on the Uptake of T3-The specificity of the T3 transport system was examined by inhibition of ['"I] TB uptake at 25 "C by increasing concentrations of unlabeled analogues. Initial velocities of T3 transport were plotted against inhibitor concentrations (Dixon plot) as shown in Fig. 5. The respective inhibition constants (K,) for unlabeled TB, T,, D-T~, and triiodothyroacetic acid were 0.32, 1.4, 4.1, and 5.4 p~ as calculated by linear regression. Thus, T4, D-T,, and triiodothyroacetic acid were 4-, 13-, and 17-fold less potent competitors than T3, respectively, indicating a high degree of specificity. To did not compete even at 10 pM.

The fact that unlabeled T, inhibited T, uptake did not prove that T, was also taken up by the TI, transport system: T, might inhibit T3 binding without being itself transported by this transport system. The uptake of ['TIT4 and its inhibition by unlabeled T, and T, were therefore examined.

TABLE I Temperature dependence of the kinetic parameters for T3 uptake in

isohted rat hepatocytes Kinetic constants for uptake (K, and VmaJ were determined on

the same preparation of hepatocytes a t 25 and 0 "C. They were calculated by linear regression from Eadie-Hofstee plots of the initial rates of uptake at concentrations between 0.1 nM and 50 p M as described in the legend to Fig. 4. The ratio of the kinetic constants measured at 25 "C to those measured at 0 "C and the values for the activation energies (E, ) were calculated from two independent experiments conducted with two distinct preparations of hepatocytes. The mean values are given, followed in parentheses by the range of the two experiments. The ratio of initial velocities (VJ, measured at high concentration (50 p ~ ) , and the corresponding E. were calculated from the data of Fig. 3. V, values a t low T, concentrations (0.1-0.2 nM) were measured between 20 and 40 s of uptake by the procedure described in the legend to Fig. 2. The results are the mean f S.D. of four independent experiments performed with distinct hepatocyte preparations.

Kinetic parameter Ratio 25/0 'C E, kcallrnol

K, 4.0 (2.9-5.1) 8.7 (6.9-10.5) Vm*X 200 (154-247) 34 (32.6-35.6) Vm.,/Km 50 (48-53) 25 (25.1-25.7) V.

High T, conc 190 34 Low T, conc 57.6 (f5.7) 26 (f l)


' L

ANALOGUE CONCENTRATION (pM)

FIG. 5. Inhibition of ['261]T3 uptake by unlabeled analogues. Isolated hepatocytes (4 X lo5 cells/ml) were incubated in medium C at 25 ' C with 25 PM [1Z51]T3 (9 X 10' cpm/ml) and varying concentrations of unlabeled T, (O), T, (a), D-T, (*), triiodothyroacetic acid (A), and To (e). The uptake was measured after 20 and 40 s of incubation on I-ml aliquots as described in the legend to Fig. 2, and initial uptake velocities were obtained by taking the difference between these two values. The reciprocal of the initial velocity is plotted uersus the analogue concentration (Dixon plot). The points are the average of two experiments. The line 1/Vmex is superposed on the abscissa so that the x intercepts of the regression lines give direct estimates of the inhibition constants.

ANALOGUE CONCENTRATION (,,MI

FIG. 6. Dixon plot for the inhibition of ['2SI]T4 uptake. Ex- perimental conditions were identical to those described in the legend to Fig. 5, except that cells were incubated with 0.1 nM ['251]T, (1.8 X lo6 cpm/ml) and varying concentrations of unlabeled T3 (0) or T4 (a). Inset, Eadie-Hofstee plot of the data obtained in the presence of unlabeled T, (0). Uptake in the presence of IO-' M T4, which was 15% of total uptake, was subtracted to correct for nonsaturable association of [12sI]T, with the cells.

A Dixon plot of the initial velocities of [1z51]T4 uptake is shown in Fig. 6. The k, values were calculated to be 0.68 p~ for T4 and 0.35 p~ for TJ, showing that unlabeled Ta was a better competitor than unlabeled T4, as expected if both analogues were transported by the same carrier. The Eadie- Hofstee plot of the uptake data for T4 (Fig. 6, inset), corrected for nonsaturable association, was linear ( r = -0.995) and indicated a K , of 0.65 k 0.03 p~ and a V,,,, of 0.16 fmol/ min/cell. The similarity of the Vmax values for TB and T4 reinforced the idea that both hormones were transported, at least in part, by the same carrier.

Effect of Metabolic Inhibitors and Sodium Substitution- The dependence of transport on intracellular energy supplies was tested with hepatocytes that had been preincubated with drugs (30 min at 25 "C), conditions that have been shown to deplete cellular ATP (7, 25, 33). Preincubation with 4 mM KCN was ineffective, and 0.5 mM ouabain (an inhibitor of the sodium/potassium pump) had only a very small effect. Preincubation with 2 mM dinitrophenol, 50 p~ oligomycin,

and 20 WM antimycin inhibited T3 transport by 30, 60, and 80%, respectively, when measured a t 0 "C and by 30, 80, and 85%, respectively, when measured a t 25 "C (not shown). How- ever, control experiments, in which the drugs were added at the same time as [1Z51]T3, indicated that these compounds inhibited transport at 0 and 25 "C independently of a preincubation step. Inhibition was observed as early as 20 s at 25 "C and 15 min a t 0 "C. Parallel time course experiments, conducted in the presence of 50 p~ unlabeled TJ, indicated that the inhibitors did not promote nonsaturable uptake or binding whether the cells were preincubated or not with the drugs.

The dependence of the transport system on the presence of extracellular Na+ was investigated by isosmotic substitution of NaCl in the external medium by KCl, choline C1, Tris-C1, or sucrose. Cells were rinsed twice with the appropriate buffer prior to the addition of [lZ5I]T3. Substitution of Na' by choline had a moderate effect on initial velocity uptake (12% inhibition), whereas KC1, Tris, and sucrose buffers gave values not significantly different from the control (not shown), pointing to a lack of dependence on external Na'. Addition of CaC12 and MgSO, to the incubation buffer (at concentrations equal to that of buffer B) or use of medium C did not significantly alter the rate of uptake relative to that observed in NaCl buffer. The effect of the sodium ionophore monensin (10 pg/ ml) was tested on initial uptake of TJ in the presence or absence of external Na'. In the presence of Na+, monensin should dissipate the transmembrane Na' gradient (outside > inside) and should lead to an inhibition of Na'-driven uptake. Any effect of monensin not linked to the perturbation of the Na' gradient should also be observed in the absence of external Na' (replaced by K+). Monensin had no significant effect in either case, reinforcing the idea that the transmembrane Na' gradient was not essential as the driving force for the uptake of T,.

Effect of pH on Thyroid Hormone Influx-The initial velocity of T, uptake was dependent on the pH of the external medium (Fig. 7). Uptake was maximum at pH 6.7-7.4 and rapidly decreased with increasing pH. At pH 9.1, uptake was one-third of the value measured at pH 6.7. This decrease might be due to alterations of the membrane proteins involved

. ; I \ '* I

pH

FIG. 7. Effect of pH on TS and T4 uptake. Isolated hepatocytes (4 X lo5 cells/ml) were preincubated for 2 min at 25 "C in buffer (140 mM NaCl, 10 mM Hepes, 10 mM Mes, and 5 mM Tris) adjusted to the desired pH. Uptake was measured after 20 and 40 s of incubation at 25 "C, in the presence of ['z51]T, or ['251]T, (0.2 nM), on 1-ml aliquots as described in the legend to Fig. 2. The initial velocities of T, (0) and T, (A) uptake were obtained from the difference between these two values and were plotted uersus the pH of the buffer. The T, uptake was corrected for nonsaturable association with the cells (15% of total uptake). Inset, the uptake was plotted versus the percentage of T, (0) or T4 (A) of the undissociated phenolic form, calculated as a function of TB or T4 pK, and buffer pH. The points are the mean of two uptake experiments.


in transport and/or the influence of pH on the dissociation of the 4'-hydroxyl group of T, whose pKa is 8.45 (34). When initial velocity of uptake was plotted against the calculated percent of Ts in the uncharged phenolic form (Fig. 7, inset), a linear relation was found (r = 0.996), suggesting that this electroneutral form was preferentially taken up.

Uptake of T, was also pH-dependent with an optimum at pH 5.9-6.4, where uptake of T4 was nearly equivalent to uptake of T, at pH 6.7-7.4 (Fig. 7). Increasing the pH caused an inhibition of T, uptake which, at pH 7.7, decreased to the level of T, uptake observed at pH 9.1. The relation to the ionization state of the phenolic group of T4, whose pK, is 6.7 (34), is less clear than in the case of TS since the relation between uptake and the calculated percent of T, in the uncharged phenolic form is linear only in the pH range 7.7-6.4 (r = 0.955). This may be due to pH-induced alterations of the macromolecules involved in uptake of T4, and this is further suggested by the fact that T4 uptake was inhibited below pH 5.9.

Uptake in the Presence of Various Inhibitors-Various compounds which are reported to interfere with the binding of thyroid hormones to some of their binding proteins (in serum and intracellular compartment) or with the uptake of various substrates taken up by cells were tested. The inhibitory effect on cellular uptake of T3 measured at 25 "C was compared with the effect on binding of T3 to hepatic cytosol binding proteins and nuclear receptor and on binding of T, to purified rat serum transthyretin (also named thyroxine-binding prealbu- min). The results are given in Table 11. 1 mM ANS, 0.1 mM dansyl chloride, 2 mM dinitrophenol, 0.3 mM iopanoic acid, and 0.3 mM bromosulfophthalein all strongly inhibited the binding of Tq to transthyretin and, to a large extent, the

cellular uptake of T,. Of these, iopanoic acid appeared to be the best inhibitor of cellular uptake relative to transthyretin binding. A study of the dose-dependent inhibition of T, uptake by iopanoic acid (not shown) gave an estimated K, of about 30 p ~ . ANS and bromosulfophthalein were also strong inhibitors of cytosol binding proteins and nuclear receptor, whereas dansyl chloride had little effect.

Of the two SH reagents used in this study, pHMB was the most efficient; 5 X M pHMB completely inhibited the saturable uptake of T3, whereas the same concentration of N - ethylmaleimide was ineffective. pHMB inhibited T, binding to cytosol binding proteins and to nuclear receptor to a smaller extent.

Chloroquine (1 mM) and monodansylcadaverine (1 mM), frequently used as inhibitors of receptor-mediated endocytosis, inhibited T, cellular uptake. Dose-dependent inhibition studies (not shown) gave an estimated Ki of 1 mM for chloroquine and 450 ~ L M for monodansylcadaverine. The inhibitory properties of these compounds were similar whether the uptake experiments were conducted a t 25 or 0 "C (not shown). They did not inhibit nuclear receptor binding, but monodansylcadaverine had a significant inhibitory effect on cytosol binding proteins and on transthyretin binding.

Diphenylhydantoin (0.3 mM) was found to be a specific inhibitor of cellular uptake since the binding proteins studied were hardly affected. Phloretin (0.1 mM), a widely used inhibitor of mediated transport, markedly reduced T, uptake, had little effect on nuclear and cytosol binding proteins, and was a very potent inhibitor of T4 binding to transthyretin.

As stated above, 20 p M antimycin and 10 p M oligomycin inhibited uptake when added simultaneously with T3. The K, of oligomycin was estimated to be about 4 pM from dose-

TABLE I1 Effect of inhibitors on T3 uptake by isolated hepatocytes, T3 binding to liver cytosol and nuclei, and T4 binding to

serum tramthyretin Uptake was measured on isolated hepatocytes suspended in medium C (4 X lo6 cells/ml) in the presence of 0.2

nM ['"1]T3 (9 X lo4 cpm/ml). Drugs were added at the same time as labeled T,, and initial velocity of uptake was measured between 20 and 40 s of incubation at 25 "C by the procedure described in the legend to Fig. 2. Results are the mean of two uptake experiments and are expressed as the percent of inhibition relative to the control value (32.1 fmol/min/4 X 10' cells). Binding to liver cytosol proteins (1 mg/ml) was determined after a 30-min incubation at 0 "C in the presence of 20 PM [Iz5I]T3. Bound and unbound T1 were separated as described under "Materials and Methods." Non-saturable binding was obtained from parallel incubations performed in the presence of 5 p M unlabeled T, and was subtracted from total binding. Results are the mean of triplicates. Liver nuclei were incubated for 30 min at 25 "C with the drugs and labeled T3 (50 PM), and T3 binding was determined as described under "Materials and Methods." Results are the mean of duplicates. The procedures for purification of serum transthyretin and measurement of saturable [1251]T4 binding are described under "Materials and Methods." The values are the mean of triplicates. The results of the binding experiments are given as the percent of inhibition relative to control values. Since some drugs were dissolved in ethanol, the final ethanol concentration in uptake and binding experiments, including the controls, was 1% (v/v).

Inhibition of initial velocity of:

DNg Conc Cellular Cytosol Nuclear Transthyretin uptake binding binding binding

M %

Control ANS Dansyl chloride Dinitrophenol Iopanoic acid Bromosulfophthalein

pHMB N-Ethylmaleimide

Chloroquine Monodansylcadaverine Diphenylhydantoin Phloretin Antimycin Oligomycin

1 0 - ~ 10"

3 X 10-~ 3 X 10-~ 5 X 10-~ 5 X 10-~

3 X 10-~ 10"

2 x

2 x lo-' lo-'

0 64.6 67.5 28.6 94.4 72.0 1.8

52.3 74.0 59.0 82.8 82.6 75.1

100

0 81.1 7.6 7.7

55.1 79.0 0

64.8 2.5

21.5 5.0 9.8 0 2.4

0 82.6 0

30.5 40.1

29.2 43.1 0 0 6.0

17.0 10.0 0

100

0 98.8 97.5 96.7 87.2 96.0 0

15.6 0

50.8 0

95.5 8.1 0


dependent studies (not shown). Antimycin was slightly inhibitory toward binding to nuclear receptor and transthyretin, whereas oligomycin was without effect.

DISCUSSION

There is still controversy as to whether thyroid hormones cross the plasma membrane of target cells by simple diffusion through the membrane lipids or by facilitated or active carrier- mediated transport. In a recent study with isolated hepatocytes (14), cellular accumulation of TS, studied with an oil centrifugation technique, was reported to be unsaturable, even at 2 X M. This is the concentration range for which we observed half-saturation of uptake. In our experiments, saturation became significant at 1-5 X IO-' M (Fig. 4a). As reported in a previous work (ZO), we were also unable to detect any saturable uptake using an oil centrifugation technique. Saturable uptake could be detected only after repeated washing of the hepatocytes with buffer. Furthermore, we have reported than an alkaline washing step facilitated the characterization of the saturable uptake.

An alternative model to mediated uptake could be an instantaneous simple diffusion through the cell membrane followed by binding to intracellular sequestration system(s) which would determine the net uptake of T, (35). However, we have made several observations which are not compatible with this model. 1) The initial rate of uptake of [1z51]T3 was saturable, whereas the equilibrium value was not (Fig. 2). A diffusion/sequestration system would give just the opposite result (36). 2) Since the Arrhenius plot measures the temperature dependence of the rate-limiting step for the process, the large difference between activation energies for uptake and binding to cytosol proteins suggests that the latter was not the rate-limiting step of uptake and that the initial velocity of uptake primarily reflects the passage of T, through the cell membrane. The activation energy of cellular uptake reported here is within the range normally found for mediated transport processes (25, 31, 32). 3) Competition experiments indicated that T, uptake by hepatocytes was specific. The fact that T3 was a more than 10-fold better competitor than D-T, indicates a high degree of specificity which is not compatible with lipid-mediated simple diffusion. Intracellular binding by previously described binding sites in cytosol and nuclei cannot account for the reported specificity (37-39). 4) Various drugs differentially affected the cellular uptake and the binding of T3 to intracellular binding proteins.

Kinetic analyses indicate that T, was transported by an apparently homogeneous carrier system. Negligible amounts of T3, if any, were taken up by unsaturable processes, partic- ularly a t hormone concentrations <lo-' M. These results are not compatible with the hypothesis of free diffusion, lipid- mediated uptake of T3 and substantiate the carrier-mediated pathway reported by others for hepatocytes (6, 7) and other cell types (8-10).

The K, value that we report for Tn agrees approximately with those already reported in hepatocytes and other cell types. Holm et al. (8) reported a K , of 1 p~ at 37 "C for cultured hepatocytes. Extrapolation of our thermodynamic data gives 0.6 p M as an estimation of the K,,, of our isolated hepatocytes at 37 "C, assuming homogeneous temperature dependence between 0 and 37 "C. Rao et al. (6), working on isolated hepatocytes, reported the presence of two saturable uptake systems for T3 (K, = 50 nM and 1.5 p~ at 23 "C). However, the same group (40) subsequently reported consid- erably lower K, values (90 and 700 p ~ ) . Krenning et al. (7) reported a K, of 20 nM at 21 "C in cultured hepatocytes in the presence of 1% albumin. Our experiments were conducted

in the absence of albumin (which extensively binds thyroid hormones). With other cell types, such as human lymphocytes (8), human and rat erythrocytes (9), rat thymus cells (lo), and rat muscle (19), uptake data are compatible with only one saturable system whose K, ranged from 50 to 400 nM.

The K, that we report for T3 ( e 1 pM) is substantially lower than those reported for other substances taken up by facilitated or active processes, which usually lie between 1 p~ and 1 mM (la), but is several orders of magnitude higher than the free plasma hormone level. I t appears that, under physiolog- ical conditions, the fractional entrance of T3 and T4 into the liver cell is essentially independent of the ambient concentration of free hormones. This means that the saturation of the uptake process is not a limiting step for access of hormones to the intracellular targets since the carrier system works far below saturation.

The analogue specificity of uptake is similar to those re- portedpreviously by us for association (uptake) to hepatocytes at 0 "C (20). This is also in the range of potency reported by Holm et al. (8) in human lymphocytes and rat hepatocytes and by Cheng (12) in cultured fibroblasts. D - T ~ was also reported to have a poor affinity for the transport system of Ta in red blood cells and thymus cells (10) and in skeletal muscle (19). This could explain why triiodothyroacetic acid and D-T, have lower biological potencies than T, (41) despite their high affinity for nuclear receptors. Mooradian et at. (14) reported preferential uptake of [1251]D-T3 into isolated hepatocytes and preferential transport from cytosol to nucleus of the L-isomer. Whether this nonphysiological isomer enters the cell by simple diffusion or by a specialized transport system remains to be established. In our work, uptake of T, also shows saturation kinetics. Cross-inhibition experiments are consistent with the idea that T4 is taken up, at least in part, by the same uptake system as T,.

The effect of pH on TB uptake was coherent with the idea that the undissociated 4"OH form was preferentially recog- nized and taken up. Our results suggest that the difference in apparent K, between T3 and T, may be due, in part, to the different ionization states of the two molecules at pH 7.4. Similar observations were reported concerning the differential effect of pH on the binding of T, and Tq to the nuclear receptor (34, 42). The 2-fold difference between the K, for labeled T, uptake and the K, for T, inhibition of labeled T, uptake is not clear. This may be due to the fact that, at pH 7.4, for the same total concentration of T3 and T4, the concentration of the active 4"OH form is smaller for T, than for TS.

Assuming that T3 is distributed in an intracellular water space of about 3 pl/hepatocyte (26, 43), T, would be concen- trated about 700 times at equilibrium and 70 times after 20 s of uptake, if it was not bound intracellularly. The question is therefore raised whether T, accumulation inside the hepatocyte results only from intracellular trapping or if the uptake process is an active one which results in a concentration gradient of free hormone.

Carrier-mediated uptake of T, into various cells has been reported either to be energy-independent (9, 10) or to be inhibited by preincubation with metabolic inhibitors (7, 8, 17). However, the use of inhibitors to deduce information about metabolic processes in intact cellular systems is less than satisfactory. Among the factors which must be consid- ered are the multiple modes of action that these compounds can exert (441, including direct inhibition of the transport system. Our results show that preincubation with KCN was without effect and that several compounds were equally active in the absence of a preincubation step, suggesting that their


inhibitory properties were unrelated to their ability to deplete cellular ATP. Oligomycin has been reported to strongly inhibit Ts uptake in the human erythrocytes by a mechanism independent of ATP depletion (9). On the other hand, the transmembrane Na+ gradient did not appear to be the driving force for uptake since replacement of Na+ by other monova- lent cations or sucrose, use of the ionophore monensin, and inhibition of the Na’ pump with ouabain were without effect. Our data are compatible with facilitated transport of TS into isolated hepatocytes, although the role of some oxidative phosphorylation intermediate in the uptake process cannot be excluded.

The T3 uptake system appears to be inhibited by a variety of structurally unrelated drugs. Some of these drugs may have multiple modes of action. Several have already been reported to inhibit thyroid hormone uptake, e.g. iopanoic acid (45), diphenylhydantoin (46), monodansylcadaverine (11,12), chloroquine (47), and oligomycin (7, 17). Others, such as ANS, dansyl chloride, dinitrophenol, iopanoic acid, diphenylhydantoin, and bromosulfophthalein have been reported to inhibit binding of thyroid hormones to some of their binding proteins or deiodinase activities. The SH reagent pHMB and the mitochondrial inhibitor dinitrophenol could act as phenol analogues, whereas the lysosomotropic agent chloroquine could also act as a membrane-active agent. The effect of the endocytotic inhibitor monodansylcadaverine may be related to the presence of the dansyl group which has been reported to be a ligand for transthyretin (48). Phloretin, a well-known inhibitor of a variety of carrier-mediated transport systems (49), may act at the recognition site of the T3 uptake system, as suggested by its effects on iodothyronine deiodinase (50) and T4 binding to transthyretin (this work).

The drugs can be roughly classified into two categories, those which interact with both the cellular uptake of T3 and the binding of T4 to transthyretin and those which inhibit uptake more specifically. This suggests that transthyretin binding could serve as a model for direct interaction of various compounds with the uptake system. Therefore, drugs which do not inhibit transthyretin may act by more indirect mech- anisms. We have checked that transthyretin is not directly involved in the uptake mechanism since T4 uptake is inhibited by about 85% in the presence of 5 pg/ml transthyretin (the serum concentration) (not shown).

In conclusion, we have shown that T, enters the isolated hepatocyte exclusively by a carrier-mediated process, which has characteristic saturation kinetics and stereospecificity. This process may act primarily as a “filter,” allowing selective entry of biologically active iodothyronines.

Acknowledgments-The secretarial assistance of A. Lefevre and M. Bahloul is acknowledged.

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