Eur. J. Biochem. 80, 25-33 (1977)
Adsorption Equilibria of Thyroid Hormones in the Liver Cell Richard WAHL, Dietrich GEISELER, and Ekkehard KALLEE Medizinische Klinik, Isotopenabteilung, Universitat Tiibingen (Received April 4/June 15, 1977)
The adsorptive distribution of L-thyroxine and L-triiodothyronine at 0.04- 25 nM concentrations was determined in suspensions of mitochondria in soluble cytoplasmic proteins from rat livers, at protein concentrations of 0.5 - 4.7 mg total protein/ml suspension. In the concentration ranges measured no saturation of both mitochondria and soluble proteins with thyroid hormones could be observed. The distribution of the hormones between the liquid phase of cytoplasmic proteins and the solid phase of the mitochondria depended mainly on the ratio of the amounts of proteins in both phases. Certain drugs such as chlorpromazine and phenobarbital in concentrations used therapeutically can interfere with the processes of adsorptive transport in vitro. Chlorpromazine, in the presence of desorptively functioning cytoplasmic proteins or serum proteins, promotes the adsorption of thyroxine and triiodothyronine onto the mitochondria. In contrast, phenobarbital weakens the binding of the hormones to the mitochondria. The processes of adsorptive binding of thyroid hormones to cell proteins in vitro are suggested as possible links in a transport chain between different compartments in vivo. The new model may also explain the effect of drugs on the distribution of thyroid hormones within the cell. The distribution of thyroid hormones in the body comprises an essential part of a biocybernetical feedback system. Thyroxine-binding globulin, thyroxinebinding prealbumin and albumin are known to transport thryoid hormones in the blood stream to the organs [1,2]. On an intracellular level the thyroid hormones are adsorbed to proteins of mitochondria, microsomes and soluble cytoplasm according to principles similar to those governing the binding of serum proteins [3]. Triiodothyronine and thyroxine binding proteins were previously found in the liver, kidneys, heart, skeletal muscular system, and adenohypophysis [3 - 111. Mitochondria1 proteins comprise one of the largest constituents of all proteins in the liver [12]. Furthermore, it appears that mitochondria are one of the major metabolic targets of thyroid hormones. The binding processes to be described here are diphasic, the cytoplasmic proteins forming the liquid phase, the mitochondria the solid phase. The distribution of triiodothyronine and thyroxine between the two phases depends on the association-dissociation equilibrium between the hormone-binding proteins [13]. These protein phases are neither uniform chemically nor univalent with regard to the adsorbed hormones indicating that the distributions are mostly non-specific reactions. If the mitochondria1 triiodothyronine and thyroxine binding receptors were specific only for triiodothyronine and thyroxine, then no
other substances, for example certain drugs, could be effective competitors [14]. The adsorption-desorption processes can be rendered more readily comprehensible utilizing a kinetic model. The level of thyroid hormones in the serum is generally believed to constitute a measure of their biological effectiveness. However, the actual site of hormone action is intracellular. The associationdissociation equilibria can be shifted within the cell itself as well as in the blood stream by drugs displacing the hormones from their binding sites. This can result either in increased hormone adsorption onto cellular organelles or desorption out of them [14,15].
MATERIALS AND METHODS Female Sprague Dawley/SIV-50 rats (Ivanovas, Kisslegg) weighing 200 - 300 g, were bled by cardiac puncture under ether anesthesia. The liver was perfused via the portal vein with an electrolyte solution containing 136mmol Na+, 5 mmol K + , 2mmol Ca2+, 1 mmol Mg2+, 95 mmol C1- and 48 mmol lactate at 37 "C until it appeared bloodless. Subsequently, it was perfused with a sucrose solution of 15 g/dl at body temperature until the surface became glassy. After the surplus sugar solution had run off,
26
the liver was minced and homogenized on ice with sucrose solution at pH 6.2 in a Dounce glass homogenizer (Braun, Melsungen) [16]. In order to adjust the pH value to about 6.2,4 ml sucrose solution and 0.03 0.04 ml of a fresh 0.1 M citric acid solution per gram liver were added. The homogenate was diluted with sucrose solution to twice its former volume under refrigeration and centrifuged at 675 x g for 20 min at 4 "C to sediment nuclei and intact cells. Mitochondria and microsomes were then separated from the supernatnat by centrifugation at 13000 x g (10 min) and 25 000 - 30000 x g (30 min) respectively. The sediment containing the mitochondria was washed once with 10 ml sucrose solution and 6 ml sucrose solution were added to the mitochondria obtained from 1 g liver. The mitochondria were rehomogenized and mixed with 1.9 ng ['251]triiodothyronine and 2.1 ng [13'1]thyroxine per ml suspension for the experiments with drugs, the concentration of radioactivity hence varying from 76.4 to 68.0 nCi/ml mitochondrial suspension. 5-ml aliquots of this stock solution were then pipetted into 25-ml flasks. The final concentrations of thyroxine and triiodothyronine were thus 0.54 nM and 0.58 nM respectively. The drugs were added in varying concentrations and according to the individual experiment mixed with 0.9-1.0 ml rat serum or 10 ml of cytoplasm corresponding to 1 g liver and diluted with sucrose solution. In some series of experiments the microsomes were still present in the cytoplasm; in others the microsomes had been removed from the cytoplasm previously. Each measuring flask with 5 ml of the mitochondria stock solution was then made up to 25 ml with cold sucrose solution. 3 ml of each suspension were centrifuged at 13000 x g, the mitochondria were washed with sucrose solution once and the radioactivities of the sediments were compared with those of the suspensions. All tests were run in duplicate. The drug concentrations given apply to the 25-ml suspensions. In choosing the initial drug concentration the basic assumption was made that the entire daily dose of a drug is contained in a volume of 3 1 plasma after administering the drug intravenously. Radioactive triiodothyronine and thyroxine with specific activity values of approximately 20 - 60 Ci/g were obtained from Amersham/Buchler (Braunschweig) and Abbott (Ingelheim). Chromatograms were made for purposes of purity control through random samples. Cold thyroid hormones were a gift from Henning Co. (Berlin). Protein determinations were carried out according to Lowry as modified by Bensadoun and Weinstein [17] with bovine albumin serving as standard. Since the mitochondrial content of different suspensions cannot be kept constant, the ratio of sedimented to soluble proteins is subject to variations
Adsorptive Transport Chain for Thyroid Hormones
from animal to animal and comparing the absolute values in the drug experiments is therefore not possible. The preparation method was electron-microscopically controlled, for which we thank Prof. Dr W. Schlote (Institute for Submicroscopic Pathology and Neurophathology, University of Tuebingen). As far as an accurate estimate from the photographs is possible the mitochondrial preparations contained no more than 10- 20 non-mitochondria1 matter, e.g. microsomes and unidentifiable debris from nuclei, membranes and other tissue constituents. RESULTS Adsorptive Distribution of Thyroid Hormones between Cytoplasmic Proteins and Mitochondria
Triiodothyronine and thyroxine, labelled with or '"1, were added to mitochondria from rat livers. The hormone radioactivity was determined in the mitochondria pellets after centrifugation and washing. The association-dissociation equilibrium of the hormones on the proteins appeared to establish itself at once in all trials. At 0 "C no change in the equilibrium could be detected over a 24-h period, nor did the - SH group stabilizer dithiothreitol show any effect on the binding over this period. The percentage binding of the thyroid hormones to the mitochondria was independent of the hormone concentration within the range measured, i.e. between 37 pg and 20000 pg of hormone per ml suspension medium containing 0.04-2.16 mg protein at association-dissociation equilibrium. For various concentrations of soluble cytoplasmic proteins and constant concentrations of mitochondrial proteins a whole set of parallels was obtained (Fig.1 for thyroxine). Fig. 2 shows the dependence of the amount of actually bound thyroxine on the concentration of hormone added at a constant concentration of mitochondria. The linear shape of the curves shows that a saturation of the mitochondrial proteins cannot be reached in the measured range of hormone concentration. For triiodothyronine the situation was very similar. Neither in the absence nor in the presence of serum or soluble cytoplasmic proteins could any triiodothyronine (0.3 ng/ml medium) be displaced by increasing thyroxine concentrations (0.35 - 200 ng thyroxine/ml medium). Cold triiodothyronine and 'reverse triiodothyronine' (3,3',5'-~-triiodothyronine)also had no influence on the binding of radioactive triiodothyronine to mitochondria. Fig. 3a and b show the desorption of thyroxine and triiodothyronine from a constant concentration of mitochondrial proteins as increasing concentrations of soluble cytoplasmic proteins were added. The slopes of the curves show only slight quantitative differences. When the ratio of mitochondrial proteins I3'I
21
R. Wahl, D. Geiseler, and E. Kallee
r
.
.
* -a 0
.-•
o
m
m
L
X
X
X
X
X
+
+-+ 0
0 ' 0
37.5
75
150
200
: >
300
Thyroxine added [Altota, (pg/rn[ suspension) Fig. 1. Adsorption of thyroxine to mitochondria oj rut 1ivrr.s ut ~ r i r i o u sconce~1trutio11.c of soluble cy/c~plusmicproteins. The percentage of hormone radioactivity bound to mitochondria is plotted against the total hormone concentration used. Only soluble mitochondrial proteins (0.04 mg/ml suspension) are contained in the supernatant in the uppermost curve (0).The course of the curves remains unchanged up to 20000 pg/ml (25.6 nmol/l). Mitochondria1 protein, [B1], was 0.66 mg/ml suspension. Soluble cytoplasmic proteins added, [B2], were, in mg/ml suspension: (A) 0.133; (0) 0.265; (H) 0.398; ( x ) 0.530; (+) 1.060; (0)2.120
. m
E 200 m a
-m-a =
100
0
n c .-m x
ex + r
o
0
57
114
Thyroxine added,
228
[AItotal
303
455
(pg/mg mitochondrial protein)
Fig. 2. Adsorption curves,for the binding of thyroxine to mitochondriu ut d(fjeiwi1 c~oncenlrcttionsof ,so/u/J/ec~~/op/usntic~ prorc4n.T. The concentrations shown are converted to pg/mg mitochondrial protein. The uppermost curve ( 0 )contains only soluble mitochondrial proteins (0.04 mg/ ml suspension). The linear slope of the curves continues to 30300 pg thyroxine per mg mitochondrial protein (not shown here). Mitochondria1 protein, [BI], was 0.66 mgjml suspension. Soluble cytoplasmic proteins added, [Bz]were, in mgjml suspension: (A) 0.133; (0) 0.265; (H) 0.398; (x) 0.530; (+) 1.060; (0)2.120
to soluble proteins was 1:1, similar percentages of triiodothyronine and thyroxine were adsorbed to the mitochondria. The thyroid hormones, however, can be desorbed to a greater extent using serum proteins instead of cytoplasmic proteins (Table 1). In the former case only approximately lj5 of the protein concentration is needed to attain desorption of a given hormone activity using serum proteins [3,5,14]. Microsomes in suspension exhibited similar desorption properties as soluble cytoplasm. If they were
allowed to remain in the cytoplasm the desorption curves were merely displaced downwards 5 - 10 % on a parallel as a result (not being shown here). In Fig.4a and b the extent of the percentage binding of thyroid hormones (200 pg triiodothyronine and 230 pg thyroxine per ml medium) to increasing concentrations of mitochondria, at constant amounts of cytoplasmic proteins, is represented. The binding approaches a saturation value corresponding to adsorption of the entire amount of hormones to the mitochondria and thus desorbing all thyroid hormones
28
Adsorptive Transport Chain for Thyroid Hormones
0
> 0
250
500
750
loo0
1250
1500
1750
2000
2250
Soluble protein, [Bz] (NQirnl supernatant)
c
: P
U
LL
0 0
250
500
750
Soluble protein,
1000
1250
1500
[Bz](wglrnl supernatant)
Fig. 3. Desorption Of thyroxine und triiodorhyronine out of mitochondria at increusing concmtrations of cytoplasmic proteins. For each determination three different hormone concentrations were used. 1 = 300 pg/ml suspension; 2 = 150 pg/ml suspension; 3 = 37.5 pg/ml suspension. (a) Thyroxine: the controls (0)to which no cytoplasmic protein had been added contained 0.04 mg soluble mitochondrial proteins per ml suspension medium, otherwise mitochondrial protein [BI] was 0.66 mg/ml suspension. Cytoplasmic proteins [Bz] added were, in mg/ml suspension: (A) 0.133; (0)0.265; (W) 0.398; ( x ) 0.530; (+) 1.060; (0)2.120. (b) Triiodothyronine: mitochondrial proteins, [BI] were 0.45 mg/ ml suspension. The controls (0)contained 0.04 mg soluble mitochondrial proteins per ml suspension. Cytoplasmic proteins, [B2] added were, in mg/ml suspension: (A) 0.073; (0)0.15; (W) 0.23; ( x ) 0.29; (+) 0.59; (0)1.18
from the soluble proteins. The final point of saturation was not reached experimentally, however. One problem encountered during this experimental series was the release of a certain percentage of soluble structural proteins into the surrounding medium by washed mitochondria which under most unfavourable conditions accounted for as much as one tenth of the mitochondrial proteins. This has a slight adverse effect on the invariance of the cytoplasmic proteins during the increase in the concentration of mitochondria. To minimize this error, a relatively high
protein concentration of the cytoplasmic proteins was selected compared to the concentration of the mitochondria. Secondly, the measured radioactivity ( A ) was linked to the mean values of the cytoplasmic protein concentration (c) by the following corrective quantities, I .Y I:
where A l , A2 = measured radioactivity values for two adjacent points plotted for equal concentrations
29
R. Wahl, D. Geiseler, and E. Kallee
'I a
80
0.84
x-x/x
60 --
1.51
++-+-
0 d . .
40 .x
2?
-
c
m
0
U
E
> 0
1000
500
1500
2000
Mitochondrial proteins, [ B,] ( p g l m l suspension)
Mitochondrial poteins, [B,] (Lgiml SUSpenSiOn)
Fig. 4. Adsorption of thyroxine and triiodothyronim to increasirzg ~ ~ O I ~ ~ ~ ~ , ~ of ~ / mitochondriul ~ ~ I / ~ ( I I I . S proteins at three different concentrations of soluble cy.ytoplasmicproteins. The extrapolated parts of the curves were drawn as dashed lines. (a) Thyroxine: the suspensions contained 230 pg ['311]thyroxine/ml. (b) Triiodothyronine : the suspensions contained 200 pg ['2sI]triiodothyronine/ml. [BZ] = soluble proteins in the supernatant
of mitochondria; el, c2 = respective cytoplasmic protein concentrations measured; C = mean value of the cytoplasmic protein concentration within an experimental series for increasing concentrations of mitochondria.
Effects of Drugs on the Mechanism of Adsorptive Transport of Thyroid Hormones Drugs can exhibit dissimilar effects on the association-dissociation equilibrium. On the one hand, drugs can facilitate the adsorption of thyroid hormones to the mitochondria. This may occur when a certain
drug is administered in a sufficiently high concentration comparable to those used therapeutically, even in the presence of either serum proteins or cytoplasmic proteins which otherwise act in a desorbing manner. An example of this is the phenothiazine derivative, chlorpromazine. On the other hand, with drugs such as phenobarbital, desorption of thyroid hormones out of the mitochondria can occur even without adding soluble proteins. Cytoplasmic and serum proteins weaken this effect, however (Table 1). These patterns could be demonstrated on both liver and heart muscle mitochondria as well [14,15]. Besides these opposite effects on the association-dissociation equilibrium there exists a multitude of intermediate forms.
30
Adsorptive Transport Chain for Thyroid Hormones
Table 1. Shifting eflects ojdrugs on distribution of triiodothyronine and thyroxine Influence of chlorpromazine and phenobarbital on the binding of [131 Ilthyroxine and ['251]triiodothyronine to rat liver mitochondria in vitro in association-dissociation equilibria with rat serum and fluid cytoplasm. Values indicate percentage of applied hormone radioactivity bound to mitochondria. The suspensions contained both 2.1 ng ['311]thyroxine(T4) and 1.9 ng ['251]triiodothyronine (T3) per ml Drug
Concentration
Amount of hormone bound to mitochondria without adding proteins
with serum
55.8 59.6 56.9 64.2 64.6 64.5 61.3
22.7
with cytoplasmic proteins
mmolil Chlorpromazine
-
0.009 0.018 0.035 0.071 0.140 0.280 0.560 Phenobarbital
~
63.1 60.7 53.2 52.9 46.0 42.6 39.8 39.4
-
0.027 0.054 0.108 0.216 0.432 0.865 1.730
Development of a Kinetic Model of the Association-Dissociation Equilibrium The adsorptive distribution of a thyroid hormone (concentration = [A]) in two protein phases (concentration of solid [mitochondrial] proteins = [B1], concentration of soluble [cytoplasmic] proteins = [Bz]) results from the different affinities of the proteins for the hormone as well as on the ratio in which the two proteins are present. The concentrations [BI] and [Bz] react with [A] yielding [ABI] and [ABz] (concentrations of thyroid hormones bound to proteins) until equilibrium is reached (Fig. 5). At equilibrium the total concentration of added hormone is distributed as follows : [Altotat = [A] [ABi] [A&].
+
60.1 63.3 59.4 65.6 64.8 65.4 61.4 -
24.5 26.7 29.4 30.6 39.9 46.2
9.4 10.3 13.1 16.2 28.7 45.4
20.8 19.8 19.6 22.2 26.0 36.7 51.6 77.0
68.4 63.0 55.2 53.8 42.2 34.7 21.6 21.8
35.2 35.2 37.0 34.2 33.2 35.5 31.1 29.7
14.8 12.8 13.1 12.4 10.9 14.2 13.3 12.5
29.9 31.1 28.8 27.4 25.8 24.2 24.2 24.2
-
7.1 -
20.8 20.4 19.6 23.0 25.9 35.9 50.6 73.1 31.3 31.8 30.0 29.0 27.9 25.6 23.3 20.1
the area represented in Fig. 2, implying that the number of unbound protein binding sites lies far above the number of binding sites already occupied, thus in good approximation [Biltotal = [BI] and [B~ltotal= [B21. Consequently one obtains the following differential equations for the transport rates of [ABl] and [ABz] using the pattern of Fig. 5 :
+
Analogously, the total concentrations of the proteins are : [Blltota~= [BI] [Bzltota~= [Bz]
+ [ABlI + [AB2].
Experimental results now allow the assumption that [ABl] and [AB2] are negligibly small compared with [B1] and [Bz]. As Fig.2 shows for thyroxine, the hormone concentration bound to mitochondrial proteins increase in a linear fashion with increasing hormone concentrations. The results with cytoplasm correspond to these. Saturation of the proteins cannot be obtained at hormone concentrations of up to 20000 pg, i.e. concentrations which lie far outside
At equilibrium, d[ABl]/dt and d[ABz]/dt become equal to zero so that [ABl] and [ABz] can be determined from two algebraic equations. One obtains :
31
R. Wahl, D. Geiseler, and E. Kallee
[ 821 Fig. 5. Schematic representation 01 tire kinetics of the associationdisfissociation equilibrium for thyroid hormones in concentration [ A ] hetween two phases in protein concentrations [ B I ] and [&I. k l , k i = actual velocity constants for the association reactions. k2, k i = actual velocity constants for the dissociation reactions. [A] = concentration of hormone. [Bl] = concentration of mitochondrial proteins in the solid phase 1. [Bz] = concentration of cytoplasmic proteins in the liquid phase 2
A-
-
k2
association constants of the reaction
__ =
Ki
.[A] ~~
ki kl
+ [Bi]
[Ah],
- -~ =
K2
association constants of the reaction [A]
+ [Bz]
[A&].
In this way an association between the fraction of hormone bound to protein and the concentration ratio of the protein phases has been formulated. The actual mean association constants l/Kl and l/K2 can be determined experimentally. All measured curves can be easily explained using Eqn (3). Influence of Drugs on the Association-Dissociation Equilibrium It has been shown using two examples how drugs can promote as well as hinder the adsorption of thyroid hormones to mitochondria. This action can be easily explained using the model developed here. Because of the relative unspecificity of the adsorptive processes concerned, not only hormones but also numerous other substances such as dyes, drugs or fatty acids can be bound by the proteins [5,14,15,18,19, 23,241. While no saturation of the binding sites occurred with thyroid hormones in the concentration range 0.04 - 25 pmol/mg protein, indications of saturation have been found with drugs because of the approximately lo3- lo6 times higher concentrations used, i.e. 0.01 - 1.7 pmol/mg protein. Hence, a drug can
[CBd
Fig. 6. Kinetic representation of [he as.soc~iarion-dissociationequilibrium for thyroid hormones under the influence of u drug in concentration [ C ] . k3, kq, k;, k i = velocity constants of the competing reactions C + BI tCBI and C + Bz tCBZ.[A] = concentration of hormone; [BI] = concentration of mitochondrial proteins in the solid phase 1 ; [Bz] = concentration of cytoplasmic proteins in the liquid phase 2; [C] = concentration of drug added
compete with the hormone for binding sites on the proteins. Such a situation is schematically depicted in Fig.6. The proteins B1, Bz not only adsorb the hormone quantity [A], but also can bind the quantity [C] of a drug. The drug C then deprives A of free binding sites, changing the association-dissociation equilibrium for A between B1 and B2. According to the ratio of the reaction constants kj, kq and kj, k:, either B1 or BZcan be withdrawn from the association-dissociation equilibrium, so that the adsorption of hormone is reinforced either on cytoplasmic proteins or on the mitochondria. Adsorption or desorption of thyroid hormones to or from the mitochondria is, therefore, determined indirectly through the characteristics of the adsorption of the drug to mitochondrial and cytoplasmic proteins.
DISCUSSION Among the numerous proteins of cytoplasm and mitochondria [20] about two to five are capable of binding thyroid hormones [5,18,19,25]. Unlike nuclear non-histone proteins [26 - 281 which bind preferably and perhaps specifically triiodothyronine, the proteins of mitochondria, microsomes and cytoplasm also adsorb thyroxine, dyes, drugs, fatty acids and other substances. The question whether or not all hormone binding proteins inside the cell are ‘specific receptors’ has not been settled as yet. Sterling and Milch [29,30] have detected so-called specific binding sites for triiodothyronine on the inner mito-
32
chondrial membrane. The respective association constants of these binding sites are even higher than those of the nuclear triiodothyronine-binding proteins. This finding would be in agreement with the original concept of mitochondria being the major target of thyroid hormones. Physico-chemically the binding of thyroxine and triiodothyronine to mitochondria is predominantly of a strictly adsorptive nature showing little distinct specificity.The distribution depends on the equilibrium between the bound and unbound hormone. As shown in Fig. 3 there exists a certain component of unbound hormone radioactivity even in the absence of soluble cytoplasm. This unbound part may well be available for diffusion processes and could penetrate to the receptors on the inner mitochondrial membrane which are coupled to the hormone-specific mechanism. The transfer of unbound hormone into the mitochondria would be one link in the intracellular transport chain. As unbound hormone is lost through diffusion, binding sites on the outer mitochondrial membrane become available again resulting in withdrawal of bound hormone from the soluble cytoplasm corresponding to the association-dissociation equilibrium. A flow of hormone A into the mitochondria occurs until the equilibrium is reached which depends solely on the ratio of both protein phases B1 and Bz [cf Eqn (3)]. Intramitochondrial metabolism would then be coupled to the intracellular and extracellular hormone pools by means of the associationdissociation equilibrium. Certain drugs can interfere with the diphasic association-dissociation equilibrium inside the cell [14,31]. They compete for binding sites because of their high concentration compared to the physiological concentrations of thyroid hormones in the cell and in the serum. Gwinup and Rapp [32] found a drop in the thyroxine level of psychiatric patients under chlorpromazine treatment to an average of 2.9 pg thyroxine/dl serum (normal: 8.2 pg/dl), however, no myxoedema resulted. The metabolic processes occuring cannot be solely attributed to the level of thyroxine. Our findings, however, can explain such a situation. Chlorpromazine promotes the binding of triiodothyronine and thyroxine to mitochondria in the presence of soluble proteins. In turn, the increased concentration in the cell causes a decrease in the thyroid hormone level in the serum to subnormal. Using the serum level alone to assess a patient’s condition would thus produce results contradictory to those clinically observed. Phenobarbital has the opposite effect. It displaces thyroxine and triiodothyronine out of the mitochondria onto soluble proteins. On the one hand, microsomes catabolize the hormones but act like soluble cytoplasm as far as distribution is concerned. There-
Adsorptive Transport Chain for Thyroid Hormones
fore, the binding of hormones to microsomes may constitute one of the steps toward inactivation of thyroid hormones and their accelerated disappearance from blood after administration of phenobarbital [33]. Computing the binding capacity of mitochondria according to Scatchard [34] is impossible if subcellular particles are suspended in protein-containing solutions as is always the case in vivo and in vitro. This is evident from our model which simplifies the complex of biological interrelationships of the cellular transport processes of triiodothyronine and thyroxine. Its prime significance, however, lies in the fact that it provides a working hypothesis which can be expanded upon to cover further regulatory interactions as well. We wish to thank Miss H. Kaltenbach for her technical assistance. The support of the Deutsche For~chungsgc.meinschaftftis gratefully acknowledged.
REFERENCES 1. DeGroot, L. J. & Stanbury, J. B. (1975) The Thyroid and Its Diseases, 4th edn, Wiley Biochemical Division, New York. 2. Oppenheimer, J . H. (1968) N . Engl. J . Med. 278, 1153-1162. 3. Kallee, E. (1960) in Protides Biol. Fluids Proc. Colloq. Bruges, 7, 161 - 164. 4. Oppenheimer, J. H., Schwartz, H. L. & Surks, M. I. (1974) Endocrinology, 95, 897 - 903. 5. Kallee, E. (1966) Acta Isot. 6, Suppl. I , 1-95. 6. Freinkel, N., Ingbar, S. H. & Dowling, J. T. (1957) J . Clin. Invest. 36, 25 - 37. 7. Lissitzky, S. (1960) in Radioaktive Isotope in Klinik und Forschung, vol. 4 (Fellinger, H. & Hofer, R., eds) pp. 315-316, Urban & Schwarzenberg, Miinchen. 8. Tata, J. R. (1962) Recent Progr. Hormone Res. 18,221 -2268. 9. Toccafondi, R. S.&Sufi, S. B. (1973) Horm. Metah. Res. 5.62. 10. Oppenheimer, J. H. & Surks, M. I. (1974) in Handbook of Physiology, Sect. 7, vol. 3, Thyroid(Greep, R. 0.& Astwood, E. B., eds) pp. 197-214, American Physiological Society, Washington. 11. Dillmann, W., Surks, M. 1. & Oppenheimer, J. H. (1974) Endocrinology, 95,492 - 498. 12. Siebert, G. (1968) in Handbuch der Allgerneinen Pathologie. Stqjjiiwhsel und Feinstruktur der Zelle I, vol. 2 (Buchner, F., ed.) pp. 1-237, Springer, Berlin. 13. Kallee, E., Bohner, J., Wahl, R. & Geiseler, D. (1976) in Nuklearmedizin (Oeff, K., ed.) Medico-Informationsdienste, Berlin 1977, in the press. 14. Wahl, R. & Kallee, E. (1974) Z. Naturforsrh. 29c, 608-617. 15. Locher, M., Kaltenbach, 11.. Wahl, R. &Kallee, E. (1975) Verh. Dtsch. Ges. Inn. Med. 81, 1564- 1567. 16. Dounce, A. L., Witter, R. F., Monty, K . J., Pate, S. & Cottone, M. A. (1955) J . Biophys. Biochem. Cytol. I , 139- 153. 17. Bensadoun, A. & Weinstein, D. (1976) Anal. Biochem. 70,241 250. 18. Grimminger, H. (1962) Acta Isotop. 2, 21 -39. 19. Grimminger, H., Heni, F. & Kallee, E. (1962) Z. Naturjbrsclz. 17b, 769-772. 20. Baudry, M., Clot, J.-P. & Michel, R. (1975) Biochimie, 57, 7783. 21. Hamada, S. & Fukase, M. (1976) in ThyroidResearch (Robbins, J. & Bravermann, L. E., eds) pp. 338 - 341, Excerpta M e d i a , American Elsevier. 22 Spaulding, S. W. & Davids, P. J. (1971) Biochirn. Biophys. Acta, 229, 219 - 283.
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R. Wahl, D. Geiseler, and E. Kallee
23. Kallee, E. (1956) Arch. Biochem. 60, 262-263. 24. Kallee, E. & Oppermann, W. (1958) Z . Naturforsch. 13h, 532- 538. 25. Lichter, M. G., Fleischner, G., Kirsch, R., Levi, A. J., Kamisaka, K. & Arias, I. M. (1976) Am. J . Physiol. 230, 11131120. 26. Oppenheimer, J. H., Koerner, D., Schwartz, H. L. & Surks, M. I. (1972) J . Clin. Endocrinol. Metab. 35, 330-333. 21. Torresani, J., Wahl, R. & Anselmet, A. (1975) Ahstr. Commun. 10th Meet. Fed. EM:. Biochem. Soc. 1348. 28. Burns, A. H. & Reddy, W. J. (1976) Liff Sci. 18, 319-328.
29. Sterling, K. & Milch, P. 0. (1975) Proc. Nut1 Acad. Sci. U . S . A . 72,3225 - 3229. 30. Sterling, K. & Milch, P. 0. (1976) Acta Endocrinol. 84, Suppl. 204, Abstr. 96. 31. Wechsler, M. B. & Roizin, L. (1960) J . Ment. Sci. 106, 15021505. 32. Gwinup, G. & Rapp, N. (1975) Am. J . Clin.Pathol. 63,9497. 33. Oppenheimer, J. H., Bernstein, G . & Surks, M. I. (1 968) J . Clin. Invest. 47, 1399- 1406. 34. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672.
R. Wahl, D. Geiseler, and E. Kallee, Isotopenabteilung, Medizinische Klinik der Eberhard-Karls-Universitat Tubingen, Otfried-Muller-StraBe 10, D-7400 Tiibingen, Federal Republic of Germany
Adsorption Equilibria of Thyroid Hormones in the Liver Cell Richard WAHL, Dietrich GEISELER, and Ekkehard KALLEE Medizinische Klinik, Isotopenabteilung, Universitat Tiibingen (Received April 4/June 15, 1977)
The adsorptive distribution of L-thyroxine and L-triiodothyronine at 0.04- 25 nM concentrations was determined in suspensions of mitochondria in soluble cytoplasmic proteins from rat livers, at protein concentrations of 0.5 - 4.7 mg total protein/ml suspension. In the concentration ranges measured no saturation of both mitochondria and soluble proteins with thyroid hormones could be observed. The distribution of the hormones between the liquid phase of cytoplasmic proteins and the solid phase of the mitochondria depended mainly on the ratio of the amounts of proteins in both phases. Certain drugs such as chlorpromazine and phenobarbital in concentrations used therapeutically can interfere with the processes of adsorptive transport in vitro. Chlorpromazine, in the presence of desorptively functioning cytoplasmic proteins or serum proteins, promotes the adsorption of thyroxine and triiodothyronine onto the mitochondria. In contrast, phenobarbital weakens the binding of the hormones to the mitochondria. The processes of adsorptive binding of thyroid hormones to cell proteins in vitro are suggested as possible links in a transport chain between different compartments in vivo. The new model may also explain the effect of drugs on the distribution of thyroid hormones within the cell. The distribution of thyroid hormones in the body comprises an essential part of a biocybernetical feedback system. Thyroxine-binding globulin, thyroxinebinding prealbumin and albumin are known to transport thryoid hormones in the blood stream to the organs [1,2]. On an intracellular level the thyroid hormones are adsorbed to proteins of mitochondria, microsomes and soluble cytoplasm according to principles similar to those governing the binding of serum proteins [3]. Triiodothyronine and thyroxine binding proteins were previously found in the liver, kidneys, heart, skeletal muscular system, and adenohypophysis [3 - 111. Mitochondria1 proteins comprise one of the largest constituents of all proteins in the liver [12]. Furthermore, it appears that mitochondria are one of the major metabolic targets of thyroid hormones. The binding processes to be described here are diphasic, the cytoplasmic proteins forming the liquid phase, the mitochondria the solid phase. The distribution of triiodothyronine and thyroxine between the two phases depends on the association-dissociation equilibrium between the hormone-binding proteins [13]. These protein phases are neither uniform chemically nor univalent with regard to the adsorbed hormones indicating that the distributions are mostly non-specific reactions. If the mitochondria1 triiodothyronine and thyroxine binding receptors were specific only for triiodothyronine and thyroxine, then no
other substances, for example certain drugs, could be effective competitors [14]. The adsorption-desorption processes can be rendered more readily comprehensible utilizing a kinetic model. The level of thyroid hormones in the serum is generally believed to constitute a measure of their biological effectiveness. However, the actual site of hormone action is intracellular. The associationdissociation equilibria can be shifted within the cell itself as well as in the blood stream by drugs displacing the hormones from their binding sites. This can result either in increased hormone adsorption onto cellular organelles or desorption out of them [14,15].
MATERIALS AND METHODS Female Sprague Dawley/SIV-50 rats (Ivanovas, Kisslegg) weighing 200 - 300 g, were bled by cardiac puncture under ether anesthesia. The liver was perfused via the portal vein with an electrolyte solution containing 136mmol Na+, 5 mmol K + , 2mmol Ca2+, 1 mmol Mg2+, 95 mmol C1- and 48 mmol lactate at 37 "C until it appeared bloodless. Subsequently, it was perfused with a sucrose solution of 15 g/dl at body temperature until the surface became glassy. After the surplus sugar solution had run off,
26
the liver was minced and homogenized on ice with sucrose solution at pH 6.2 in a Dounce glass homogenizer (Braun, Melsungen) [16]. In order to adjust the pH value to about 6.2,4 ml sucrose solution and 0.03 0.04 ml of a fresh 0.1 M citric acid solution per gram liver were added. The homogenate was diluted with sucrose solution to twice its former volume under refrigeration and centrifuged at 675 x g for 20 min at 4 "C to sediment nuclei and intact cells. Mitochondria and microsomes were then separated from the supernatnat by centrifugation at 13000 x g (10 min) and 25 000 - 30000 x g (30 min) respectively. The sediment containing the mitochondria was washed once with 10 ml sucrose solution and 6 ml sucrose solution were added to the mitochondria obtained from 1 g liver. The mitochondria were rehomogenized and mixed with 1.9 ng ['251]triiodothyronine and 2.1 ng [13'1]thyroxine per ml suspension for the experiments with drugs, the concentration of radioactivity hence varying from 76.4 to 68.0 nCi/ml mitochondrial suspension. 5-ml aliquots of this stock solution were then pipetted into 25-ml flasks. The final concentrations of thyroxine and triiodothyronine were thus 0.54 nM and 0.58 nM respectively. The drugs were added in varying concentrations and according to the individual experiment mixed with 0.9-1.0 ml rat serum or 10 ml of cytoplasm corresponding to 1 g liver and diluted with sucrose solution. In some series of experiments the microsomes were still present in the cytoplasm; in others the microsomes had been removed from the cytoplasm previously. Each measuring flask with 5 ml of the mitochondria stock solution was then made up to 25 ml with cold sucrose solution. 3 ml of each suspension were centrifuged at 13000 x g, the mitochondria were washed with sucrose solution once and the radioactivities of the sediments were compared with those of the suspensions. All tests were run in duplicate. The drug concentrations given apply to the 25-ml suspensions. In choosing the initial drug concentration the basic assumption was made that the entire daily dose of a drug is contained in a volume of 3 1 plasma after administering the drug intravenously. Radioactive triiodothyronine and thyroxine with specific activity values of approximately 20 - 60 Ci/g were obtained from Amersham/Buchler (Braunschweig) and Abbott (Ingelheim). Chromatograms were made for purposes of purity control through random samples. Cold thyroid hormones were a gift from Henning Co. (Berlin). Protein determinations were carried out according to Lowry as modified by Bensadoun and Weinstein [17] with bovine albumin serving as standard. Since the mitochondrial content of different suspensions cannot be kept constant, the ratio of sedimented to soluble proteins is subject to variations
Adsorptive Transport Chain for Thyroid Hormones
from animal to animal and comparing the absolute values in the drug experiments is therefore not possible. The preparation method was electron-microscopically controlled, for which we thank Prof. Dr W. Schlote (Institute for Submicroscopic Pathology and Neurophathology, University of Tuebingen). As far as an accurate estimate from the photographs is possible the mitochondrial preparations contained no more than 10- 20 non-mitochondria1 matter, e.g. microsomes and unidentifiable debris from nuclei, membranes and other tissue constituents. RESULTS Adsorptive Distribution of Thyroid Hormones between Cytoplasmic Proteins and Mitochondria
Triiodothyronine and thyroxine, labelled with or '"1, were added to mitochondria from rat livers. The hormone radioactivity was determined in the mitochondria pellets after centrifugation and washing. The association-dissociation equilibrium of the hormones on the proteins appeared to establish itself at once in all trials. At 0 "C no change in the equilibrium could be detected over a 24-h period, nor did the - SH group stabilizer dithiothreitol show any effect on the binding over this period. The percentage binding of the thyroid hormones to the mitochondria was independent of the hormone concentration within the range measured, i.e. between 37 pg and 20000 pg of hormone per ml suspension medium containing 0.04-2.16 mg protein at association-dissociation equilibrium. For various concentrations of soluble cytoplasmic proteins and constant concentrations of mitochondrial proteins a whole set of parallels was obtained (Fig.1 for thyroxine). Fig. 2 shows the dependence of the amount of actually bound thyroxine on the concentration of hormone added at a constant concentration of mitochondria. The linear shape of the curves shows that a saturation of the mitochondrial proteins cannot be reached in the measured range of hormone concentration. For triiodothyronine the situation was very similar. Neither in the absence nor in the presence of serum or soluble cytoplasmic proteins could any triiodothyronine (0.3 ng/ml medium) be displaced by increasing thyroxine concentrations (0.35 - 200 ng thyroxine/ml medium). Cold triiodothyronine and 'reverse triiodothyronine' (3,3',5'-~-triiodothyronine)also had no influence on the binding of radioactive triiodothyronine to mitochondria. Fig. 3a and b show the desorption of thyroxine and triiodothyronine from a constant concentration of mitochondrial proteins as increasing concentrations of soluble cytoplasmic proteins were added. The slopes of the curves show only slight quantitative differences. When the ratio of mitochondrial proteins I3'I
21
R. Wahl, D. Geiseler, and E. Kallee
r
.
.
* -a 0
.-•
o
m
m
L
X
X
X
X
X
+
+-+ 0
0 ' 0
37.5
75
150
200
: >
300
Thyroxine added [Altota, (pg/rn[ suspension) Fig. 1. Adsorption of thyroxine to mitochondria oj rut 1ivrr.s ut ~ r i r i o u sconce~1trutio11.c of soluble cy/c~plusmicproteins. The percentage of hormone radioactivity bound to mitochondria is plotted against the total hormone concentration used. Only soluble mitochondrial proteins (0.04 mg/ml suspension) are contained in the supernatant in the uppermost curve (0).The course of the curves remains unchanged up to 20000 pg/ml (25.6 nmol/l). Mitochondria1 protein, [B1], was 0.66 mg/ml suspension. Soluble cytoplasmic proteins added, [B2], were, in mg/ml suspension: (A) 0.133; (0) 0.265; (H) 0.398; ( x ) 0.530; (+) 1.060; (0)2.120
. m
E 200 m a
-m-a =
100
0
n c .-m x
ex + r
o
0
57
114
Thyroxine added,
228
[AItotal
303
455
(pg/mg mitochondrial protein)
Fig. 2. Adsorption curves,for the binding of thyroxine to mitochondriu ut d(fjeiwi1 c~oncenlrcttionsof ,so/u/J/ec~~/op/usntic~ prorc4n.T. The concentrations shown are converted to pg/mg mitochondrial protein. The uppermost curve ( 0 )contains only soluble mitochondrial proteins (0.04 mg/ ml suspension). The linear slope of the curves continues to 30300 pg thyroxine per mg mitochondrial protein (not shown here). Mitochondria1 protein, [BI], was 0.66 mgjml suspension. Soluble cytoplasmic proteins added, [Bz]were, in mgjml suspension: (A) 0.133; (0) 0.265; (H) 0.398; (x) 0.530; (+) 1.060; (0)2.120
to soluble proteins was 1:1, similar percentages of triiodothyronine and thyroxine were adsorbed to the mitochondria. The thyroid hormones, however, can be desorbed to a greater extent using serum proteins instead of cytoplasmic proteins (Table 1). In the former case only approximately lj5 of the protein concentration is needed to attain desorption of a given hormone activity using serum proteins [3,5,14]. Microsomes in suspension exhibited similar desorption properties as soluble cytoplasm. If they were
allowed to remain in the cytoplasm the desorption curves were merely displaced downwards 5 - 10 % on a parallel as a result (not being shown here). In Fig.4a and b the extent of the percentage binding of thyroid hormones (200 pg triiodothyronine and 230 pg thyroxine per ml medium) to increasing concentrations of mitochondria, at constant amounts of cytoplasmic proteins, is represented. The binding approaches a saturation value corresponding to adsorption of the entire amount of hormones to the mitochondria and thus desorbing all thyroid hormones
28
Adsorptive Transport Chain for Thyroid Hormones
0
> 0
250
500
750
loo0
1250
1500
1750
2000
2250
Soluble protein, [Bz] (NQirnl supernatant)
c
: P
U
LL
0 0
250
500
750
Soluble protein,
1000
1250
1500
[Bz](wglrnl supernatant)
Fig. 3. Desorption Of thyroxine und triiodorhyronine out of mitochondria at increusing concmtrations of cytoplasmic proteins. For each determination three different hormone concentrations were used. 1 = 300 pg/ml suspension; 2 = 150 pg/ml suspension; 3 = 37.5 pg/ml suspension. (a) Thyroxine: the controls (0)to which no cytoplasmic protein had been added contained 0.04 mg soluble mitochondrial proteins per ml suspension medium, otherwise mitochondrial protein [BI] was 0.66 mg/ml suspension. Cytoplasmic proteins [Bz] added were, in mg/ml suspension: (A) 0.133; (0)0.265; (W) 0.398; ( x ) 0.530; (+) 1.060; (0)2.120. (b) Triiodothyronine: mitochondrial proteins, [BI] were 0.45 mg/ ml suspension. The controls (0)contained 0.04 mg soluble mitochondrial proteins per ml suspension. Cytoplasmic proteins, [B2] added were, in mg/ml suspension: (A) 0.073; (0)0.15; (W) 0.23; ( x ) 0.29; (+) 0.59; (0)1.18
from the soluble proteins. The final point of saturation was not reached experimentally, however. One problem encountered during this experimental series was the release of a certain percentage of soluble structural proteins into the surrounding medium by washed mitochondria which under most unfavourable conditions accounted for as much as one tenth of the mitochondrial proteins. This has a slight adverse effect on the invariance of the cytoplasmic proteins during the increase in the concentration of mitochondria. To minimize this error, a relatively high
protein concentration of the cytoplasmic proteins was selected compared to the concentration of the mitochondria. Secondly, the measured radioactivity ( A ) was linked to the mean values of the cytoplasmic protein concentration (c) by the following corrective quantities, I .Y I:
where A l , A2 = measured radioactivity values for two adjacent points plotted for equal concentrations
29
R. Wahl, D. Geiseler, and E. Kallee
'I a
80
0.84
x-x/x
60 --
1.51
++-+-
0 d . .
40 .x
2?
-
c
m
0
U
E
> 0
1000
500
1500
2000
Mitochondrial proteins, [ B,] ( p g l m l suspension)
Mitochondrial poteins, [B,] (Lgiml SUSpenSiOn)
Fig. 4. Adsorption of thyroxine and triiodothyronim to increasirzg ~ ~ O I ~ ~ ~ ~ , ~ of ~ / mitochondriul ~ ~ I / ~ ( I I I . S proteins at three different concentrations of soluble cy.ytoplasmicproteins. The extrapolated parts of the curves were drawn as dashed lines. (a) Thyroxine: the suspensions contained 230 pg ['311]thyroxine/ml. (b) Triiodothyronine : the suspensions contained 200 pg ['2sI]triiodothyronine/ml. [BZ] = soluble proteins in the supernatant
of mitochondria; el, c2 = respective cytoplasmic protein concentrations measured; C = mean value of the cytoplasmic protein concentration within an experimental series for increasing concentrations of mitochondria.
Effects of Drugs on the Mechanism of Adsorptive Transport of Thyroid Hormones Drugs can exhibit dissimilar effects on the association-dissociation equilibrium. On the one hand, drugs can facilitate the adsorption of thyroid hormones to the mitochondria. This may occur when a certain
drug is administered in a sufficiently high concentration comparable to those used therapeutically, even in the presence of either serum proteins or cytoplasmic proteins which otherwise act in a desorbing manner. An example of this is the phenothiazine derivative, chlorpromazine. On the other hand, with drugs such as phenobarbital, desorption of thyroid hormones out of the mitochondria can occur even without adding soluble proteins. Cytoplasmic and serum proteins weaken this effect, however (Table 1). These patterns could be demonstrated on both liver and heart muscle mitochondria as well [14,15]. Besides these opposite effects on the association-dissociation equilibrium there exists a multitude of intermediate forms.
30
Adsorptive Transport Chain for Thyroid Hormones
Table 1. Shifting eflects ojdrugs on distribution of triiodothyronine and thyroxine Influence of chlorpromazine and phenobarbital on the binding of [131 Ilthyroxine and ['251]triiodothyronine to rat liver mitochondria in vitro in association-dissociation equilibria with rat serum and fluid cytoplasm. Values indicate percentage of applied hormone radioactivity bound to mitochondria. The suspensions contained both 2.1 ng ['311]thyroxine(T4) and 1.9 ng ['251]triiodothyronine (T3) per ml Drug
Concentration
Amount of hormone bound to mitochondria without adding proteins
with serum
55.8 59.6 56.9 64.2 64.6 64.5 61.3
22.7
with cytoplasmic proteins
mmolil Chlorpromazine
-
0.009 0.018 0.035 0.071 0.140 0.280 0.560 Phenobarbital
~
63.1 60.7 53.2 52.9 46.0 42.6 39.8 39.4
-
0.027 0.054 0.108 0.216 0.432 0.865 1.730
Development of a Kinetic Model of the Association-Dissociation Equilibrium The adsorptive distribution of a thyroid hormone (concentration = [A]) in two protein phases (concentration of solid [mitochondrial] proteins = [B1], concentration of soluble [cytoplasmic] proteins = [Bz]) results from the different affinities of the proteins for the hormone as well as on the ratio in which the two proteins are present. The concentrations [BI] and [Bz] react with [A] yielding [ABI] and [ABz] (concentrations of thyroid hormones bound to proteins) until equilibrium is reached (Fig. 5). At equilibrium the total concentration of added hormone is distributed as follows : [Altotat = [A] [ABi] [A&].
+
60.1 63.3 59.4 65.6 64.8 65.4 61.4 -
24.5 26.7 29.4 30.6 39.9 46.2
9.4 10.3 13.1 16.2 28.7 45.4
20.8 19.8 19.6 22.2 26.0 36.7 51.6 77.0
68.4 63.0 55.2 53.8 42.2 34.7 21.6 21.8
35.2 35.2 37.0 34.2 33.2 35.5 31.1 29.7
14.8 12.8 13.1 12.4 10.9 14.2 13.3 12.5
29.9 31.1 28.8 27.4 25.8 24.2 24.2 24.2
-
7.1 -
20.8 20.4 19.6 23.0 25.9 35.9 50.6 73.1 31.3 31.8 30.0 29.0 27.9 25.6 23.3 20.1
the area represented in Fig. 2, implying that the number of unbound protein binding sites lies far above the number of binding sites already occupied, thus in good approximation [Biltotal = [BI] and [B~ltotal= [B21. Consequently one obtains the following differential equations for the transport rates of [ABl] and [ABz] using the pattern of Fig. 5 :
+
Analogously, the total concentrations of the proteins are : [Blltota~= [BI] [Bzltota~= [Bz]
+ [ABlI + [AB2].
Experimental results now allow the assumption that [ABl] and [AB2] are negligibly small compared with [B1] and [Bz]. As Fig.2 shows for thyroxine, the hormone concentration bound to mitochondrial proteins increase in a linear fashion with increasing hormone concentrations. The results with cytoplasm correspond to these. Saturation of the proteins cannot be obtained at hormone concentrations of up to 20000 pg, i.e. concentrations which lie far outside
At equilibrium, d[ABl]/dt and d[ABz]/dt become equal to zero so that [ABl] and [ABz] can be determined from two algebraic equations. One obtains :
31
R. Wahl, D. Geiseler, and E. Kallee
[ 821 Fig. 5. Schematic representation 01 tire kinetics of the associationdisfissociation equilibrium for thyroid hormones in concentration [ A ] hetween two phases in protein concentrations [ B I ] and [&I. k l , k i = actual velocity constants for the association reactions. k2, k i = actual velocity constants for the dissociation reactions. [A] = concentration of hormone. [Bl] = concentration of mitochondrial proteins in the solid phase 1. [Bz] = concentration of cytoplasmic proteins in the liquid phase 2
A-
-
k2
association constants of the reaction
__ =
Ki
.[A] ~~
ki kl
+ [Bi]
[Ah],
- -~ =
K2
association constants of the reaction [A]
+ [Bz]
[A&].
In this way an association between the fraction of hormone bound to protein and the concentration ratio of the protein phases has been formulated. The actual mean association constants l/Kl and l/K2 can be determined experimentally. All measured curves can be easily explained using Eqn (3). Influence of Drugs on the Association-Dissociation Equilibrium It has been shown using two examples how drugs can promote as well as hinder the adsorption of thyroid hormones to mitochondria. This action can be easily explained using the model developed here. Because of the relative unspecificity of the adsorptive processes concerned, not only hormones but also numerous other substances such as dyes, drugs or fatty acids can be bound by the proteins [5,14,15,18,19, 23,241. While no saturation of the binding sites occurred with thyroid hormones in the concentration range 0.04 - 25 pmol/mg protein, indications of saturation have been found with drugs because of the approximately lo3- lo6 times higher concentrations used, i.e. 0.01 - 1.7 pmol/mg protein. Hence, a drug can
[CBd
Fig. 6. Kinetic representation of [he as.soc~iarion-dissociationequilibrium for thyroid hormones under the influence of u drug in concentration [ C ] . k3, kq, k;, k i = velocity constants of the competing reactions C + BI tCBI and C + Bz tCBZ.[A] = concentration of hormone; [BI] = concentration of mitochondrial proteins in the solid phase 1 ; [Bz] = concentration of cytoplasmic proteins in the liquid phase 2; [C] = concentration of drug added
compete with the hormone for binding sites on the proteins. Such a situation is schematically depicted in Fig.6. The proteins B1, Bz not only adsorb the hormone quantity [A], but also can bind the quantity [C] of a drug. The drug C then deprives A of free binding sites, changing the association-dissociation equilibrium for A between B1 and B2. According to the ratio of the reaction constants kj, kq and kj, k:, either B1 or BZcan be withdrawn from the association-dissociation equilibrium, so that the adsorption of hormone is reinforced either on cytoplasmic proteins or on the mitochondria. Adsorption or desorption of thyroid hormones to or from the mitochondria is, therefore, determined indirectly through the characteristics of the adsorption of the drug to mitochondrial and cytoplasmic proteins.
DISCUSSION Among the numerous proteins of cytoplasm and mitochondria [20] about two to five are capable of binding thyroid hormones [5,18,19,25]. Unlike nuclear non-histone proteins [26 - 281 which bind preferably and perhaps specifically triiodothyronine, the proteins of mitochondria, microsomes and cytoplasm also adsorb thyroxine, dyes, drugs, fatty acids and other substances. The question whether or not all hormone binding proteins inside the cell are ‘specific receptors’ has not been settled as yet. Sterling and Milch [29,30] have detected so-called specific binding sites for triiodothyronine on the inner mito-
32
chondrial membrane. The respective association constants of these binding sites are even higher than those of the nuclear triiodothyronine-binding proteins. This finding would be in agreement with the original concept of mitochondria being the major target of thyroid hormones. Physico-chemically the binding of thyroxine and triiodothyronine to mitochondria is predominantly of a strictly adsorptive nature showing little distinct specificity.The distribution depends on the equilibrium between the bound and unbound hormone. As shown in Fig. 3 there exists a certain component of unbound hormone radioactivity even in the absence of soluble cytoplasm. This unbound part may well be available for diffusion processes and could penetrate to the receptors on the inner mitochondrial membrane which are coupled to the hormone-specific mechanism. The transfer of unbound hormone into the mitochondria would be one link in the intracellular transport chain. As unbound hormone is lost through diffusion, binding sites on the outer mitochondrial membrane become available again resulting in withdrawal of bound hormone from the soluble cytoplasm corresponding to the association-dissociation equilibrium. A flow of hormone A into the mitochondria occurs until the equilibrium is reached which depends solely on the ratio of both protein phases B1 and Bz [cf Eqn (3)]. Intramitochondrial metabolism would then be coupled to the intracellular and extracellular hormone pools by means of the associationdissociation equilibrium. Certain drugs can interfere with the diphasic association-dissociation equilibrium inside the cell [14,31]. They compete for binding sites because of their high concentration compared to the physiological concentrations of thyroid hormones in the cell and in the serum. Gwinup and Rapp [32] found a drop in the thyroxine level of psychiatric patients under chlorpromazine treatment to an average of 2.9 pg thyroxine/dl serum (normal: 8.2 pg/dl), however, no myxoedema resulted. The metabolic processes occuring cannot be solely attributed to the level of thyroxine. Our findings, however, can explain such a situation. Chlorpromazine promotes the binding of triiodothyronine and thyroxine to mitochondria in the presence of soluble proteins. In turn, the increased concentration in the cell causes a decrease in the thyroid hormone level in the serum to subnormal. Using the serum level alone to assess a patient’s condition would thus produce results contradictory to those clinically observed. Phenobarbital has the opposite effect. It displaces thyroxine and triiodothyronine out of the mitochondria onto soluble proteins. On the one hand, microsomes catabolize the hormones but act like soluble cytoplasm as far as distribution is concerned. There-
Adsorptive Transport Chain for Thyroid Hormones
fore, the binding of hormones to microsomes may constitute one of the steps toward inactivation of thyroid hormones and their accelerated disappearance from blood after administration of phenobarbital [33]. Computing the binding capacity of mitochondria according to Scatchard [34] is impossible if subcellular particles are suspended in protein-containing solutions as is always the case in vivo and in vitro. This is evident from our model which simplifies the complex of biological interrelationships of the cellular transport processes of triiodothyronine and thyroxine. Its prime significance, however, lies in the fact that it provides a working hypothesis which can be expanded upon to cover further regulatory interactions as well. We wish to thank Miss H. Kaltenbach for her technical assistance. The support of the Deutsche For~chungsgc.meinschaftftis gratefully acknowledged.
REFERENCES 1. DeGroot, L. J. & Stanbury, J. B. (1975) The Thyroid and Its Diseases, 4th edn, Wiley Biochemical Division, New York. 2. Oppenheimer, J . H. (1968) N . Engl. J . Med. 278, 1153-1162. 3. Kallee, E. (1960) in Protides Biol. Fluids Proc. Colloq. Bruges, 7, 161 - 164. 4. Oppenheimer, J. H., Schwartz, H. L. & Surks, M. I. (1974) Endocrinology, 95, 897 - 903. 5. Kallee, E. (1966) Acta Isot. 6, Suppl. I , 1-95. 6. Freinkel, N., Ingbar, S. H. & Dowling, J. T. (1957) J . Clin. Invest. 36, 25 - 37. 7. Lissitzky, S. (1960) in Radioaktive Isotope in Klinik und Forschung, vol. 4 (Fellinger, H. & Hofer, R., eds) pp. 315-316, Urban & Schwarzenberg, Miinchen. 8. Tata, J. R. (1962) Recent Progr. Hormone Res. 18,221 -2268. 9. Toccafondi, R. S.&Sufi, S. B. (1973) Horm. Metah. Res. 5.62. 10. Oppenheimer, J. H. & Surks, M. I. (1974) in Handbook of Physiology, Sect. 7, vol. 3, Thyroid(Greep, R. 0.& Astwood, E. B., eds) pp. 197-214, American Physiological Society, Washington. 11. Dillmann, W., Surks, M. 1. & Oppenheimer, J. H. (1974) Endocrinology, 95,492 - 498. 12. Siebert, G. (1968) in Handbuch der Allgerneinen Pathologie. Stqjjiiwhsel und Feinstruktur der Zelle I, vol. 2 (Buchner, F., ed.) pp. 1-237, Springer, Berlin. 13. Kallee, E., Bohner, J., Wahl, R. & Geiseler, D. (1976) in Nuklearmedizin (Oeff, K., ed.) Medico-Informationsdienste, Berlin 1977, in the press. 14. Wahl, R. & Kallee, E. (1974) Z. Naturforsrh. 29c, 608-617. 15. Locher, M., Kaltenbach, 11.. Wahl, R. &Kallee, E. (1975) Verh. Dtsch. Ges. Inn. Med. 81, 1564- 1567. 16. Dounce, A. L., Witter, R. F., Monty, K . J., Pate, S. & Cottone, M. A. (1955) J . Biophys. Biochem. Cytol. I , 139- 153. 17. Bensadoun, A. & Weinstein, D. (1976) Anal. Biochem. 70,241 250. 18. Grimminger, H. (1962) Acta Isotop. 2, 21 -39. 19. Grimminger, H., Heni, F. & Kallee, E. (1962) Z. Naturjbrsclz. 17b, 769-772. 20. Baudry, M., Clot, J.-P. & Michel, R. (1975) Biochimie, 57, 7783. 21. Hamada, S. & Fukase, M. (1976) in ThyroidResearch (Robbins, J. & Bravermann, L. E., eds) pp. 338 - 341, Excerpta M e d i a , American Elsevier. 22 Spaulding, S. W. & Davids, P. J. (1971) Biochirn. Biophys. Acta, 229, 219 - 283.
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R. Wahl, D. Geiseler, and E. Kallee
23. Kallee, E. (1956) Arch. Biochem. 60, 262-263. 24. Kallee, E. & Oppermann, W. (1958) Z . Naturforsch. 13h, 532- 538. 25. Lichter, M. G., Fleischner, G., Kirsch, R., Levi, A. J., Kamisaka, K. & Arias, I. M. (1976) Am. J . Physiol. 230, 11131120. 26. Oppenheimer, J. H., Koerner, D., Schwartz, H. L. & Surks, M. I. (1972) J . Clin. Endocrinol. Metab. 35, 330-333. 21. Torresani, J., Wahl, R. & Anselmet, A. (1975) Ahstr. Commun. 10th Meet. Fed. EM:. Biochem. Soc. 1348. 28. Burns, A. H. & Reddy, W. J. (1976) Liff Sci. 18, 319-328.
29. Sterling, K. & Milch, P. 0. (1975) Proc. Nut1 Acad. Sci. U . S . A . 72,3225 - 3229. 30. Sterling, K. & Milch, P. 0. (1976) Acta Endocrinol. 84, Suppl. 204, Abstr. 96. 31. Wechsler, M. B. & Roizin, L. (1960) J . Ment. Sci. 106, 15021505. 32. Gwinup, G. & Rapp, N. (1975) Am. J . Clin.Pathol. 63,9497. 33. Oppenheimer, J. H., Bernstein, G . & Surks, M. I. (1 968) J . Clin. Invest. 47, 1399- 1406. 34. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672.
R. Wahl, D. Geiseler, and E. Kallee, Isotopenabteilung, Medizinische Klinik der Eberhard-Karls-Universitat Tubingen, Otfried-Muller-StraBe 10, D-7400 Tiibingen, Federal Republic of Germany
