J. Endocrinol. Invest. 4: 177, 1981

Thyroid hormone binding to plasma membrane preparations: studies in different thyroid states and tissues J. Gharbi-Chihi, and J. Torresani Laboratoire de Biochimie Medicale et groupe U 38lnserm, Faculte de Mececlne, 13385 Marseille Cedex 4, France

ABSTRACT. Two orders of high-affinity saturable binding sites for L-T 4 and L-T 3 were evidenced in purified plasma membrane preparations from rat liver (apparent equilibrium dissociation constant K o forT 4 ~ 0.6 and 23 nM, forT 3 ~ 9 and 237 nM) and kidney (K o forT 4 ~ 4 and 127 nM; for T 3 ~ 15 and 270 nM). Differences of statistical significance were only found for the higher affinity T 4 binding site. In contrast, no saturable T 4 or T 3 binding could be detected in spleen plasma membranes. Testis plasma membranes exhibited 2 sets of T 4 binding sites but with a lower affinity than in liver and kidney (K; ~ 28 and 286 nM), and only one set ot T, binding sites (Ko~ 266 nM). A good correlation was found between the plasmalemma T 4 and T 3 binding properties of a tissue and its ability to respond to and/or metabolize thyroid hormones. T 4 and T 3 binding was also examined in liver plasma membranes of rats under various thyroid status; no difference could be detected in either K o or total capacity for both sets of T 4 and T 3 binding sites when comparing normal with hyper- or hypothyroid rats. The distribution of plasmalemma high-affinity specific T 4 and T 3 binding sites in different tissues suggests that these sites are involved in hormone action, or in the transport of these hormones within the cell. T 4 binding sites, at least those of the highest affinity (K o ~ 0.4 nM), are true plasmalemma constituents and are likely to be different from the highest affinity T3 binding sites (K o ~ 6 nM); we also detailed the structural requirements of iodothyronines for membrane T 4 binding sites (12). The biological relevance of such specific high-affinity T 4 and T3binding in plasma membranes is unknown. In order to investigate this point, T 4 and T 3 binding were studied in liver plasma membranes of rats under different thyroid states. T 4 and T3 binding were also examined in 2 types of tissues: (i) tissues considered to be responsive to thyroid hormones, i.e. liver and kidney, based on conventional criteria (increased oxygen consumption (13) and increased levels of mitochondrial alphaglycerophosphate dehydrogenase activity (14) under the action of thyroactive substances); (ii) tissues held to be unresponsive, i.e. spleen and testis. Thyroid hormone binding data for these different tissues correlate with their ability to respond to thyroid hormones.

INTRODUCTION Thyroid hormones, L-thyroxine (T 4) and 3,5,3'-triiodoL-thyronine (T 3) exert multiple effects on numerous tissues of vertebrates (1). Their mechanism of action is currently unknown. Thyroid hormones enter the cells of their target tissues and it has been evidenced that a large part of cellular T 3 is derived from intracellular deiodination of T 4 (2). In recent years, several intracellular saturable binding components for T3and T 4 have been described. A small number of saturable highaffinity T 3 binding sites were first detected in nuclei from various target tissues (3). These nuclear T3binding sites, which have been partly purified and characterized (4, 5) and are mainly localized in the perichromatin region when complexed to radioiodinated T 3 (6), could represent a receptor mediating T 3 effects at a transcriptional level (3). More recently, high-affinity sites for T 3 have been identified in a fraction solubilized from inner mitochondrial membranes (7, 8). T 3 and T 4 also bind, but with a lower affinity, to cytosol proteins (9, 10). Furthermore, rat liver plasma membranes were recently shown to possess high-affinity, low capacity sites for T3 (11) and T 4 (12). We demonstrated that the

MATERIALS AND METHODS Male Sprague Dawley rats (250-300 g) were used in all experiments. Normal rats were fed ad libitum with standard diet. Hypothyroid rats were thyroidectomized at 80 g bw, placed on a low iodine diet with 0.5% CaCI 2 in drinking water, and 1 week later injected ip with 80 J.1Ci 1311Nal. The animals were sacrificed 6 to 8 weeks later. Hyperthyroid rats were normal rats given a standard diet with T 4added in drinking water (3 mg/I in 0.05%

Key-words: Thyroxine, triiodothyronine, plasma membranes, hyperthyroid, hypothyroid, liver, kidney, spleen, testis. Correspondence: Gharbi-Chihi Jouda, Laboratoire de Biochimie Medicale, Faculte de Medecine, 13385 Marseille Cedex 5, France.

Received September 1,1980: accepted December 4,1980.

177

J. Gharbi-Chihi, and J. Torresani bovine serum albumin) during 8 days prior to sacrifice. Animals were fasted overnight, stunned and killed by exsanguination. Tissues were quickly removed and chilled in hypotonic medium (1 mM NaHC0 3 + 0.5 mM CaCI 2). Liver, kidney and spleen plasma membranes were prepared from the 1000 x g pellet of tissue homogenate in hypotonic medium and collected at d := 1.16/1.18 sucrose interface after isopycnic.centrifugation as described by Ray (15). Membrane yields were about 1.5, 1.0 and 0.6 mg protein per g liver, kidney and spleen respectively. Purified testis plasma membranes were kindly given by Dr. Roullier and prepared according to Abou-Issa and Reichert (16). Purified liver plasma membranes were enriched in glucagon-stimulated adenylate cyclase and had reduced mitochondrial and microsomal enzymatic activities as previously described (12). The purification of other plasmalemma preparations was assessed by their enrichment in ouabain-sensitive activities of sodium, potassium-dependent adenosine triphosphatase (Na ", K+-ATPase) and potassium-dependent p-nitrophenylphosphatase (K+-pNPPase), determined according to Jorgensen (17) and Wolff and Jones (18) respectively. When compared to homogenate, liver, kidney, sp1een and testis membrane preparations showed a 16.1 ± 1.0 (n == 7), 15.2 ± 1.1, (n == 4),9.8 ± 0.1 (n == 3) and 5.3 ± 0.2 (n == 3) - fold increase in Na '. K+-ATPase specific activity respectively, and a 7.3 ± 0.7,10.4 ± 1.1,7.1 ± 0.3 and 4.2 ± 0.5 - fold increase in K+-pNPPase specific activity, respectively. DNA determinations showed negligible contamination with nuclei (DNA < 5 IJ.g 1mg protein in liver, kidney .md spleen, 17 IJ.g 1mg protein in testes membranes); similarly, cytosol contarni.iation was very low as assessed by lactate dehydrogenase activity determinations /10) (0.15,0.05 and 0.33 IU/mg proteins in liver, kidney and testes plasma .rnernbranes respectively as compared to > 3 IU/mg protein in homogenates). 1251-T4 (T 4*) and 1251-T3 (T 3*) were radioiodinated by the chloramine T method to a specific radioactivity of about 4700 mCi/mg for T 4* and about 2800 mCi/mg for T3* and showed no detectable contamination with other labelled compounds by thin-layer chrornatoqraphy as previously described (12). Plasma membrane preparations (usually 50 IJ.g protein) were incubated with trace concentrations of T/ or T 3* (0.1 nM) in 0.1 ml of 20 mM Tris-CI, 2mM EDTA, 1 mM MgCI 2, 10 mM NaCI, 200 mM sucrose, 1 mM dithiothreitol pH 7.6. After 2 h at 0 C, 1 ml of cold buffer was added and the incubates were immediately centrifuged (5 min, 10,000 x g, 2C). The membrane pellets, containing bound hormone, were counted in a gamma spectrometer. Non-specific binding was determined in parallel incubations with an excess of unlabelled T4or T3(13IJ.M) and substracted from total binding to obtain specific binding. All incubations were perforrred in duplicate or triplicate. Binding parameters (apparent equilibrium dissociation con-

stant K o and maximum binding capacity MBC) were obtained in saturation experiments with hormone concentrations ranging from 0.1 nM to 131J.M, and analyzed according to Scatchard (21 ) considering specifically bound T4 or T3' Proteins were estimated by the method of Lowry et al (22) using bovine serum albumin as reference. T 4 and T3 concentrations in serum were determined by radioimmunoassays using Lepetit kits for T 3and Traven01 kits for T4' Results are expressed as mean ± SE. Data were statistically evaluated by one way analysis of variance. The significance of differences between control and experimental groups was assessed by the Student's test for unpaired data. T4'T3'ATP (tris), p-nitrophenylphosphate, ouabain, bovine serum albumin and DNA were purchased from Sigma (St. Louis, Mo, USA); 1251-Nal was from the Radiochemical Center (Amersham, England).

RESULTS i) Thyroid hormone binding to liver plasma membranes from rats in different thyroid states Binding studies were carried out with liver plasma membranes prepared from control rats (N), rats rendered hypothyroid by surgical- and radiothyroidectomy (H) and normal rats given T 4in drinking water for 8 days prior to use (N+). All comparative studies were performed with rats of the same age. After T4 treatment, circulating levels of TSH were significantly decreased from 450 to 100 ng/ml, as described by Roques and Tirard in parallel studies (23). T 4and T3concentrations in serum were enhanced .~ 3.5 fold (51 ± 0 to 170 ± 7 nM for T 4; <0.5 to 1.4 ± 0.1 nM for T 3 in 3 rats). The hypothyroid state was assessed by growth failure and a liver weight never exceeding 4 g (Table 1). Furthermore, in agreement with results reported by Lo and Edelman (24), the specific activity of Na", K+-ATPase in liver plasma membrane preparations from H rats was significantly decreased; values, in urnot. Pi 1min 1mg protein were 0.923 ± 0.17,0.757 ± 0.023 and 0.991 ± 0.096 respectively, in 5 series of N, Hand N+ rats. It should be noted that membrane yields, in mg protein 1g liver, were equivalent in the 3 groups studied. The T 4 binding parameters, K o and MBC, derived from saturation experiments, are summarized in Table 1. Two sets of saturable T4binding sites were consistently found in liver plasma membranes from normal rats as previously described (12) and from H or N+ rats. No statistically significant difference (p > 0.05) could be detected in K o or in MBC values for both sets of T 4 binding sites when comparing experimental and control groups of rats. Two orders of saturable binding sites for T 3 were also evidenced as previously described, but K o values were about one order of magnitude higher than those found for T4' Furthermore, saturable T3 binding always represented less than 20% of total binding. In one series of 178

T4 and T3 binding to plasma membranes Table 1 - Influenced of thyroid status on T4 binding to rat liver plasma membranes.

Second site

Liver weight (g)

Ko (nM)

Capacity (pmol/mg protein)

Ko

(nM)

Capacity (pmol/mg protein)

337.5 ± 9.52 110.0 ± 4.0 358.5 ± 5.5

15.75 ± 0.5 3.85 ± 0.1 17.25 ± 0.5

0.76 ± 0.17 0.73 ± 0.13 1.03 ± 0.08

0.72 ± 0.14 0.55 ± 0.1 0.58 ± 0.2

12.9± 2.7 25.9 ± 7.2 34.8 ± 6.0

4.56 ± 0.3 5.6 ± 0.46 8.95 ± 2.8

Animal status Normal (N)1 Hypothyroid (H) Hyperthyroid (N+)

T4 binding

First site

Body weight (g)

1 5 series of N, Hand N+ rats of the same age were used.

2 All data are given as mean

± SE.

Table 2 - T4 binding to purified plasma membranes from different tissues. First site

Second site

N1

Tissue

17 7

Liver Kidney Testis Spleen

4 4

Ko

(nM)

Ko

Capacity (pmol/mg protein)

(nM)

0.57 ± 0.05 2 4.54 ± 0.78 27.77 ± 3.05

0.50 ± 0.06 0.42 ± 0.04 0.65 ± 0.13

23.84 ± 3.86 127.0 ± 29.0 285.7 ± 59.4

o

o

Capacity (pmol/mg protein)

6.40 ± 0.82 12.9 ± 2.5 22.0 ± 5.0

o

o

1 Number of experiments.

2 Values are expressed as the mean

± SE.

Table 3 - T3 binding to purified plasma membranes from different tissues. First site Tissue

Ko (nM) Liver Kidney Testis Spleen 1

Second site

N1

9 4

3 4

9.7 ± 2.42 15.8± 4.3 266.0 ± 48.0 0

Capacity (pmol/mg protein)

Ko (nM)

Capacity (pmoi/mg protein)

1.3 ± 0.47 0.97 ± 0.15 24.0 ± 8.0 0

237 ± 3.2 270 ± 35

25.5 ± 4.7 18.5 ± 5.7

0

0

Number of experiments.

2 Values are expressed as the mean

± SE.

assays, N, Hand N+ rat liver plasma membranes were similar in K o and MBC for both T 3 binding sites. K o values in N, Hand N+ rats were 6.9, 11.9 and 7.2 nM respectively for the higher affinity site and 288,199 and 206 nM respectively for the lower affinity site.

richment in plasmalemma components. Plasma membrane preparations from the other tissues always bound T 4 and T 3 to saturable sites and with saturation characteristics compatible with the existence of 2 sets of binding sites, except for T 3 binding to testis membranes where only one low affinity site was detected. Nevertheless, differences between the tissues under study were found when evaluating the binding parameters. Regarding the highest affinity T 4 binding sites, similar MBC were found in liver, kidney and testis (no significant differences, p > 0.05) with values at about 0.5 pmol T 4 per mg membrane protein (Table 2). However, differences of marked statistical significance were observed in K o values: K o values in liver (range

ii) Thyroid hormone binding to purified plasma membrane from different tissues T 4 and T 3 binding were studied in plasma membrane preparations from liver, kidney, testis and spleen. Results are summarized in Tables 2 and 3. No saturable T 4 or T 3 binding could be detected in 4 preparations of spleen plasma membranes, although the increase ih Na ", K+-ATPase specific activity suggested an en179

J. Gharbi-Chihi, and J. Torresani

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Fig.1 - Inhibition of specific 1251_T4 binding to liver and kidney plasma membranes by increasing concentrations of non radioactive T 3 and T 4 added to incubation medium: L- T4 (0-0),' L- T3 (0---0). Incubations, in duplicate, were performed under standard conditiotis. Results from 3 separate experiments were pooled. Each point represents the mean value ± SF Maximum and non.speciiic binding of 1251_T 4amounted in liver to ~ 35% and 7% of total T 4 respectively and in kidney to ~ 14% and 8% respectively.

purified from rat liver (12). In a large series of liver plasmalemma preparations, T 4 and T 3 binding always displayed characteristics compatible with the existence of 2 orders of binding sites. T 4 was bound with the highest affinity, with mean Ko values of about 0.6 and 23 nM. T3 binding studies were more difficult to interpret due not only to an about 10-fold lower affinity for both sites (mean Ko of about 9 and 237 nM), but mainly to a high level of non saturable binding. Our results also indicate that plasma membranes purified from kidney, another tissue sensitive to thyroid hormones and actively metabolizing them, also bind T 4 and T 3 to 2 sets of high-affinity sites. Similar results were obtained with kidney plasma membranes prepared either under hypotonic conditions as described above, or under isotonic conditions according to Fitzpatrick (26) (not shown). When comparing binding to liver and kidney membranes, similar characteristics were found for T3' whereas T 4 was always bound in the kidney with an about tenfold lower affinity (K o about 4 and 127 nM), the concentration of binding sites being of the same order of magnitude. Competition experiments with T 3 for the T 4 binding entities also suggest that the highest affinity T 4 binding sites may be different in liver and kidney. Of interest is the finding that in purified plasma membranes from spleen, a tissue which is held unresponsive to thyroid hormones and almost completely devoid of nuclear T 3 receptors (27), no saturable binding of T 4 or T3 could be detected. Testis is another tissue poorly sensitive to thyroid hormone action and poor in nuclear receptors (27); in our study, testis plasma membrane preparations exhibited very·low levels of saturable T 4 and T3 binding; T 4 was bound to 2 sets of sites, but the first set was found with a low capacity and an affinity lower than that found in liver and kidney (KD~28 nM), while the second one showed characteristics close to those found for the single set of low affinity T 3 binding sites (K o ~ 266 nM). T 4 and T 3 are known to be bound in several compartments of the cell, and a contamination of our plasma membrane preparations by other subcellular fractions cannot be completely excluded, although marker enzymes from other membrane fractions or from cytosol were poorly represented. Nevertheless, nuclear bind-

0.24 to 0.95 nM) were smaller than in kidney (range 1.25 to 7.20 nM; p < 0.001) and in testis (range 12 to 35 nM; p < 0.001 when cornpared to liver; p < 0.01 when compared to kidney). Differences were also evidenced in the second set of T 4 binding sites with smaller Ko values in liver than in kidney (p <0.001) and testis (p < 0.001); likewise, Ko values measured in kidney were significantly lower than in testis (p < 0.05). MBC values for the second site were higher in kidney and testis than in liver (p < 0.01 ). T3 was constantly bound with a markedly lower affinity than that observed for T 4 (Table 3). The single class of sites evidenced in testis membranes had Ko and MBC values of the same order of magnitude as those observed for the second class of T3 binding sites in liver and kidney. No statistically significant difference (p > 0.05) was found between liver and kidney when comparing Ko or MBC values for both sets of T 3 binding sites (Table 3). In order to further examine differences between T 4 binding characteristics in liver and kidney plasma membranes, competition experiments with T3 were performed (Fig. 1). As previously described in liver plasmalemma, T 3 competes with T 4 for the T 4 binding sites with less efficiency; i.e. when compared to T 4' about 200-fold higher concentrations of T3 were needed to obtain 500/ 0 inhibition of T/ binding. This result and the observation that T 4 competed less efficiently than T 3 for the T 3 binding sites suggested that T 4 and T 3 binding sites of the highest affinity were different (12,25). In kidney membranes, T 3 was also less effective than T 4 in inhibiting T/ binding but the competition curves were oitterent, particularly in the range of low hormone concentrations. Although such experiments do not allow a clear discrimination between different sets of binding sites, this observation supports the idea that the highest affinity T 4 binding site may be different in liver and kidney.

DISCUSSION The present study confirms our previous findings which showed the presence of saturable high-affinity T4 and T3 binding sites in a plasma membrane fraction 180

T 4 and T 3 binding to plasma membranes

ing sites cannot be involved in our study since they bind T 3 more strongly than T 4 in liver and kidney (3), while their concentration is very low in spleen and testis (27). Cytosol and microsomal sites bind T4 and T3 with a lower affinity (12, 25). Lower Ko was found for T 3 than for T 4 in liver cytosol (9,25); by contrast we found in rat kidney cytosol a strongest binding of T 4 as compared to T 3 (2 sets of sites with Ko 120 and 5000 nM respectively' for T 4; 1 set of sites of Ko 2000 nM for T 3)' in rough 'agreement with Davis et al. results in dog kidney (1 O).lt is thus likely that high affinity binding sites for T 4 and T 3 in liver and kidney plasma membrane preparations are true plasmalemma constituents. The plasmalemma origin of lower affinity sites, although probable, cannot be definitely ascertained. The physiological relevance of such specific highaffinity T 4 and T3 binding sites in plasma membrane preparations is currently unknown. A preferential distribution of these sites in tissues which actively metabolize and / or are sensitive to T 4 and T3 suggests that these sites could playa biological role. In an attempt to investigate this role, studies were performed with liver plasma membranes prepared from rats in different thyroid states; no detectable correlation was found between thyroid status and subsequently determined Ko and capacities for T4 and T 3' This finding suggests that under our conditions thyroid hormones do not by themselves regulate their own plasmalemma binding sites. Such an absence of variation has also been reported for nuclear T3 binding sites (28, 29). Thyroid hormones may alter the activity of some plasmalemma enzymes. Modified activities of adenylate cyclase (30) or phosphodiesterases (31,32), and modified quantities of Na+, ,K+-ATPase (24) have been described in hyper or hypothyroid states; but data are scarce as to the existence of a direct effect of thyroid hormones upon membrane enzymes (33, 34, 35). Working with isolated liver plasma membranes, under conditions where a significant specific T4 binding was maintained, we were unable to dectect any effect of several concentrations of T 4 or T 3 upon either Na+, K+ -ATPase activity or adenylate cyclase activity whether the latter was measured under basal conditions or after stimulation by glucagon or isoproterenol (25). Thyroid hormones may also regulate the entry of some low molecular weight compounds within the cell. At close to physiological concentrations, T 3 stimulates the uptake of several neutral aminoacids in thyrnocytes (36) and the uptake of 2-deoxy-D-glucose in embryonic cardiac cells (37), but T 4 was shown to be about 1O-fold tess efficient than T3 on these transport systems. It is also possible that plasma membrane binding sites for T 4 and T3 are involved in cellular uptake and metabolism of these hormones. In this respect, stepwise deiodination is a major metabolic pathway for T 4 and T3; it is particularly active in liver and kidney and recently a 5' -deiodinase activity, which forms T 3 from T 4' has been described in a subcellular fraction en-

riched in plasma membranes (38). Furthermore recent reports indicate that the transport of T 3 within the cells is not passive and involves protein components of the membrane. High affinity transport systems have been described for T 3 in hepatocytes (39, 40) and for T 3 and T 4 in adipocytes (41). A transport of T 3 has also been described within erythrocyte ghosts (42). Furthermore, very recently, the use of rhodamine- T3 allowed visualization of a clustering and internalization phenomenon in cultured fibroblasts, suggesting a receptor-mediated uptake of T3 (43). Plasma membrane binding sites for T 4 and T 3 may represent the protein entities involved in the transport system delivering thyroid hormone into the cell.

ACKNOWLEDGMENTS The authors are grateful to Dr. E. Castanas for advice in statistical analysis, Prof. S. Lissitzky for helpful discussions, Dr. E. Goldstein for revising the manuscript and E. Giraud for typing. This work was supported in part by CNRS LA 178.

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