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The triiodothyronine carrier of rat erythrocytes
46
Biochimica et Biophysica Acta, 1051 (1990) 46 51
Elsevier BBAMCR 12613
The triiodothyronine carrier of rat erythrocytes: asymmetry and mechanisms of trans-inhibition Jeannine Osty, Ya Zhou, Francoise Chantoux, Jacques Francon and Jean-Paul Blondeau Unitd de Recherche sur la Glande Thyro~de et la Rdgulation Hormonale (U.96), lnstitut National de la Santd et de la Recherche M~dicale, Kremlin-Bic~tre (France)
(Received 19 June 1989)
Key words: Thyroid hormone; Triiodothyronine; Cell membrane; Transport kinetics; (Rat erythrocyte)
The kinetic properties of the carrier-mediated transport of 3,5,3'-triiodo-L-thyronine (T3) in washed rat erythrocytes were investigated (1) by studying the effects of trans unlahelled T3 on influx and efflux of labelled substrate and (2) by testing some predictions of the theory of Lieb and Stein ((1974) Biochim. Biophys. Acta 373, 165-177). The carrier was trans-inhibited by T 3 on both sides of the membrane. Under zero-trans conditions, the carrier displayed asymmetrical properties, the Michaelis constant and the maximal velocity being more than 6-times higher for influx than for efflux. Under equilibrium-exchange conditions, the Michaelis constant was lower than the zero-trans values, as expected when tram-inhibition occurs. This kinetic behaviour is consistent with a carrier which is accessible to T3 simultaneously from both sides of the erythrocyte membrane.
Introduction There is now a great deal of experimental evidence for the existence of specific, saturable systems transporting thyroid hormones across cell membranes [1-6]. These carriers are thought to work by facilitated diffusion, allowing the rapid entry of biologically active iodothyronines and restricting that of other iodothyronines [5], or by generating a concentration gradient of hormones by active, energy-dependent transport [6]. However, there have been no detailed studies of the kinetic behaviour of these carriers in terms of hypothetical transport mechanisms. We have recently shown [7] that 3,5,3'-triiodo-Lthyronine (T3) is transported across the plasma membrane of rat erythrocytes by a stereospecific, Na+-inde pendent, carrier-mediated process, and is accumulated by intracellular trapping. We suggested that, since erythrocytes are not considered to be target cells for thyroid hormones, the rat erythrocyte carrier might play a role in the blood transport of T3. This study also showed that excess unlabelled T 3 in the extracellular medium prevented the efflux of labelled T3 from preloaded cells, but not whether this effect was due to direct trans-inhibition by unlabelled T 3 or to cis-inhibi-
Abbreviation: T3, 3,5,3'-triiodo-L-thyronine. Correspondence: J.P. Blondeau, INSERM U.96, 78, rue du Gtntral Leclerc, 94275 Kremlin-BicStre, France.
tion following the rapid entry of unlabelled T 3 into the cells. The present study was designed to ascertain whether unlabelled substrate acted on the trans side of the membrane and to determine the kinetic characteristics of the T 3 carrier in terms of the formalism developed by Lieb and Stein [8,9]. The kinetic tests proposed by these authors can be used to distinguish between two transport mechanisms: one for which the access of substrate to the carrier occurs simultaneously from both sides of the membrane (simple pore model) and the other for which the access occurs alternately (simple carrier model). Only the second model can account for countertransport [9]. This difference is biologically important, since, in the case of countertransport, the driven substrate (i.e., T3) is coupled to the downhill flow of other intracellular substrates and would be pumped against its concentration gradient. Materials and Methods Chemicals. [3'-125I]T3 (SA 3 m C i / # g ) was obtained from Amersham Corp. (England) and diluted to the desired specific radioactivities with unlabeUed T 3 purchased from Sigma Chemicals (St. Louis, MO). Since T 3 binds avidly to various types of plasticware, all plastic tubes and pipette tips were siliconized (Sigmacote, Sigma). Rat erythrocytes. Male Wistar rats (250 g, Iffa Credo, Lyon, France) were decapitated. Blood was collected on
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
47 heparin (20 U / m l blood), filtered through gauze and centrifuged (2200 x g, 10 min). The plasma and buffy coat were discarded, and the erythrocytes were washed three times with 7 vol. buffer A (137 mM NaCI, 2.7 mM KC1, 8.1 mM N a 2 H P O 4, 1.5 mM K H 2 P O 4 (pH 7.5)), once with 7 vol. buffer B (125 mM NaC1, 20 mM KCI, 4 mM MgC12, 10 mM glucose, 4.05 mM N a 2 H P O 4, 0.95 mM N a H 2 P O 4 (pH 7.4)) and suspended in 1 vol. buffer B. Washed erythrocytes were kept on ice and used within 48 h of their preparation. They were equilibrated for 10-15 min at 2 5 ° C before experiments. Cell concentrations were determined in a Coulter counter. Measurement of T3 fluxes. All assays were performed in triplicate at 25 ° C and pH 7.4 in siliconized plastic tubes. Since the equilibrium level is highly temperaturedependent [7], a constant temperature was maintained throughout the experimental period until addition of the stop solution. Influx experiments were performed in a final volume of 1 ml containing approx. 105 cpm [125I]T3 and 108 erythrocytes. (a) Zero-trans influx: experiments were initiated by mixing erythrocytes with medium (preequilibrated at 25 ° C) containing labelled T 3 and various concentrations of unlabeUed T 3. (b) Inside trans-inlfibition and equilibrium-exchange influx: erythrocytes were preincubated at 2 5 ° C with various concentrations of unlabelled T 3 until equilibrium was reached (10 min). The cells were centrifuged at 250C and the supernatants were either discarded (trans-inhibition) or supplemented with 10 5 cpm [ 125i]Ts (equilibrium-exchange). Washing of the pellets was avoided because it would induce a rapid efflux of internalized T 3 even at 0 ° C [7]. T 3 adsorbed at the cell surface represented less than 4% of the equilibrium values. Uptake was initiated by resuspending the erythrocyte pellets in either 1 ml buffer B supplemented with 0.2 nM T 3 and 10 5 c p m / m l [125I]T3 (trans-inhibition) or in 1 ml of erythrocyte supernatant supplemented with labelled T 3 (equilibrium-exchange). Efflux experiments were performed as follows: 10 s erythrocytes were preincubated for 10 min at 25 ° C with a tracer amount of labelled T 3 (105 cpm) and either various concentrations of unlabelled T 3 (zero-trans procedure) or 0.2 nM unlabelled T 3 (outside trans-inhibition) (final volume 0.25 ml). The cells were centrifuged at 25 ° C, and the supernatants were saved for radioactivity measurements. Cells were used without further washing. T 3 exit was initiated by resuspending the erythrocyte pellets in 2 ml of buffer B (preequilibrated at 25°C) containing no unlabeUed T 3 (zero-trans) or containing various concentrations of unlabelled T 3
(trans-inhibition). Influx and efflux experiments were terminated by the rapid addition of 1 / 3 vol ice-cold stop solution (buffer B containing 3 . 1 0 -5 M unlabelled T3) and subsequent manipulations were done at 0 - 2 ° C . Assay times were
monitored audibly with a metronome set at 60 beats/min. Zero-time assays were performed by adding the stop solution to the cells immediately prior to adding the initiating solution. The cells were centrifuged (2600 x g, 2 min) and the pellets were washed two times with 1.5 ml ice-cold buffer A containing 10 -5 M unlabelled T 3. The radioactivity of the pellets was measured with an efficiency of 80%. Influx or efflux were measured over the first 5 s (initial velocity conditions). These values were either subtracted from the zero-time values (efflux experiments) or the zero-time values were subtracted from them (influx experiments). In the latter case, the zerotime assays contained approx. 10% of the counts present in the cell pellet after 5 s of uptake or exchange.
Determination of the equilibrium concentration of T3. We previously reported that T 3 uptake by rat erythrocytes was Na+-independent and that the accumulation of T 3 by the cells at 25 ° C was due to intracellular trapping [7]. Also, others reported evidence that T 3 uptake in human [10] and rat [11] erythrocytes was not dependent on metabolic energy. Therefore, intra- and extracellular concentrations of free T 3 were assumed to be equal at equilibrium. In the efflux experiments (zero-trans efflux and outside trans-inhibition), aliquots of the preincubation mixture and of the supernatant (after removal of the cells) were assayed for radioactivity, allowing the determination of the equilibrium T 3 concentration in the medium (and hence the free T 3 concentration in the cells). In influx experiments (inside trans-inhibition and equilibrium-exchange), this was done in parallel incubations performed under the same conditions as those of the preincubation step, except that [~25I]T3 tracer was present. The free T 3 concentration at equilibrium was 25-30% of the starting concentration. Test of the kinetic models. The outside of the erythrocytes was arbitrarily designated side 1 and the cytoplasmic side as side 2. Maximal velocities (V) and apparent Michaelis-Menten constant ( K ) were determined in the three transport modes: zero-tram influx (zt,12), zero-trans efflux (zt,21) and equilibrium-exchange (ee). R terms are the reciprocals of the maximal velocities in each of the three modes studied and thus represent 'resistance' factors. For the simple pore kinetic model [8] R ee = R ~ + R~tl and 1 / r ee = ( 1 / r ~ t g ) + ( 1 / K ~ )
whereas for the simple carrier model [9] the relations are:
R ee + R
oo
-
zt R I 2 + R~tl
and
( 1 / K ' * ) + ( 1 / K °°) = (1/K~t~) + ( 1 / K ~ I )
48 TABLE I
150~
Kinetic parameters of Ts transport in rat erythrocytes
The kinetic parameters K (Michaelis constant), V (maximal velocity) and /7 (first-order rate constant = V / K ) were obtained from EadieHofstee plots by linear regression as described in the legend to Fig. 2. Values are mean =t:S.D. of four independent experiments performed with four different batches of rat erythrocytes. The means were compared by the paired t-test. The values of the kinetic constants are statistically different (P <1%), from one experimental protocol to another, except for those marked with an asterisk (P > 5%). A is the asymmetry parameter in the zero-trans modes. Experimental protocol zt 21
'
lOO. I
~X~o-transinflux
A 50"
ee
zt 12
K (nM) 18.1+1.2 119+6 15.6+1.9 6.6+0.6 V (nM/min) 20.2+2.7 * 130+20 18.7+2.0 * 6.4+0.2 /7 (rain -1) 1.12+0.14 * 1.10+0.15 * 1.21+0.14 * -
~trans
efflux
ecluilil~um-exchange [ ~ = ' = ~ j ~ _ 0
,
0
~, ,'""Dxn'e
0.5
1.0 v / S (rnin-1)
where R °° is the resistance experienced b y the e m p t y carrier ( c o n f o r m a t i o n a l changes of the e m p t y carrier) a n d K °° is the simple carrier affinity parameter. S t a t i s t i c a l t r e a t m e n t o f data. S t a n d a r d deviations a n d paired t-test for c o m p a r i s o n s of m e a n s were calculated according to Snedecor a n d C o c h r a n [12].
Fig. 2. Zero-trans influx and efflux and equilibrium-exchangeof 1"3 in rat erythrocytes. Flux measurements were performed as described under Materials and Methods using the same population of cells. Initial velocity data (v, mean of triplicates) are plotted according to the Eadie-Hofstee linearization v vs. v/Is], where [s] is the initial (zero-trans influx) or equilibrium (zero-trans efflux and equilibrium exchange) concentration of T3. The slopes are equal to - K (Michaelis constants) and the y-intercepts to maximal velocities of transport. (o) zero-trans influx; (A) zero-trans efflux (o); equilibrium-exchange.
Results
m e d i u m . This i n h i b i t i o n was studied at various conc e n t r a t i o n s of t r a n s u n l a b e l l e d substrate, a n d the outside i n h i b i t i o n c o n s t a n t was calculated from a D i x o n plot (Fig. lb). T h e m e a n value from two i n d e p e n d e n t experiments was 125 n M (range: 1 1 8 - 1 3 0 nM). T h e Michaelis c o n s t a n t (K21) zt a n d the m a x i m u m velocity (V2~t) of efflux were d e t e r m i n e d by m e a s u r i n g the initial velocity of T 3 efflux i n t o b u f f e r from erythrocytes preloaded with increasing c o n c e n t r a t i o n s of
Zero-trans and infinite-trans efflux
Fig. l a shows the time-course of labelled T 3 efflux f r o m preloaded erythrocytes. Efflux i n t o buffer (zerot r a n s efflux) was linear for 5 s a n d reached a n e w e q u i l i b r i u m after 1 rain. T h e logarithmic t r a n s f o r m a t i o n of efflux d a t a (not shown) indicates that there was o n l y o n e c o m p o n e n t of efflux with a half-life of 11 s. Efflux of labelled T 3 was almost completely i n h i b i t e d b y the presence of 10 btM u n l a b e l l e d T 3 in the extracellular
100,AA~A~A"
'A
B
/e
c~
o/
t~J
~'
<
•
o~
•
•
O
o
i
i
TIME (rain)
~
o
~o
2oo
o
EXTRACELLUt.AR UNLABELLED T3 (nM)
Fig. 1. Trans-inhibifion of T3 efflux in rat erythrocytes. Washed erythrocytes were preincubated with [125I]T3,centrifuged and the supernatant was discarded. (A) the cells were re~uspended in buffer B (u) or in buffer B containing 10 ~M unlabelled T3 (A). Influx was stopped at the indicated times and the cell-associated radioactivity was determined (see Materials and Methods). (B) Dixon plot of trans-inhibition. The cells were resuspended in buffer B containing various concentrations of unlabelled T3. Initial velocities of efflux (mean of triplicates) were determined as described under Materials and Methods.
49 labelled T 3. Under these conditions, re-uptake of hormone was negligible. The kinetic parameters were calculated by linear regression from an Eadie-Hofstee plot of the data (Fig. 2) and the mean values of four independent experiments are given in Table I. Zero-trans and equilibrium-exchange influx In Fig. 2 are shown typical Eadie-Hofstee plots of uptake data obtained by the zero-trans and equilibrium-exchange procedures. The kinetic constants ( K ~t and V(~ for zero-trans uptake and K ~ and V ~ for equilibrium-exchange uptake) are given in Table I (mean of four separate experiments). T 3 transport displayed asymmetrical properties since the asymmetry factor (A = V12/V21 zt zt -_ "'12/"21, I," zt/r,- zt was approx. 6.5 K ~ was significantly lower than K ~t and K2~ ( P < 1%) and Vee was significantly lower than V~] ( P < 1%) but not statistically different from V2~t ( P > 5%). The limiting permeabilities ( H ) (pseudo-first-order rate constants) were not statistically different from each other. The mean /7 value was 1.14 + 0.14 rain -~ (n = 12). R ¢~ and 1 / K ~ were obtained from the data used to calculate the mean values of V and K in Table I and were tested for equality with, respectively, the sums 2:R zt = R~t~ + R ~ and ~ ( 1 / K zt) = 1/KI~ + 1/K2~. By the paired t-test (n = 4, P > 5%), R ~ (53.9 + 5.5 min/xM -1) is not significantly different from ~ R zt (58.0-48.0 m i n - # M -1) and 1 / K ~¢ (65.0+7.5 # M -~) is not significantly different from , ~ ( 1 / ( K zt) ( 6 4 . 0 + 4 . 0 # M - ~ ) . This means that the calculated values of R °°, the resistance of the empty simple cartier, and 1 / K °°, the reciprocal of the simple carrier affinity parameter, are not significantly different from zero.
Inside trans-inhibition The trans-inlfibition of labelled T 3 influx was studied with erythrocytes preloaded with increasing concentrations of unlabelled T 3 (Fig. 3a). The mean inside inhibition constant from two independent experiments was 21 nM (range: 19.2-22.2 nM). The apparent K zt and V zt were determined at several intracellular concentrations of unlabelled T 3. Fig. 3b shows that the inside trans-inhibition was competitive, since increasing intraceUular concentrations of unlabelled T 3 affected the apparent Michaelis constant of uptake. In this experiment, the inhibition constant, calculated according to Dixon and Webb [13], was 24.6 nM. Discussion
T 3 is taken up into erythroeytes by a saturable, stereospecific transport system independent of Na ÷ and of metabolic energy [7,10,11]. We have previously reported [7] that the initial velocity of uptake, but not the equilibrium level was saturable. Therefore transport, but not intracellular trapping, appeared to be the ratelimiting step in zero-trans influx. Binding studies performed with erythrocyte cytosol [7] suggested that cytosol components could account for at least a part of the T 3 trapping activity. This binding appeared to dissociate in vitro extremely rapidly when equilibrium was perturbed. Therefore, under initial velocity conditions, dissociation from intracellular stores was not the rate limiting step in zero-trans efflux. Finally, efflux originated from the cell interior and not from the cell surface since it was blocked by an excess T 3 (Ref. 7 and present work). This previous characterization of the T 3 transport system of rat erythrocytes enabled us to study !
u
u
B
0.50'
.100 ~n
q, t-
.g E >
0.25"
/ 0
.,./
o io 2'o do EQUILIBRIUM UNLABELLED T3 (nM)
E
.3 '50
u
~
0.25 0.50 v/S (rain -1)
im~
0.75
>
0
Fig. 3. Trans-inhibition of T3 influx in rat erythrocytes. Washed rat erythrocytes were preincubated with urdabelled "1"3, centrifuged and the supernatant discarded. Equilibrium concentrations of T 3 were determined on parallel incubations as described under Materials and Methods. (A) loading concentrations of unlabelled T 3 were from 0 to 100 nM (equilibrium concentration range: 0-29.6 nM). Initial velocities of uptake were measured by resuspending the cells in buffer B containing 0.2 nM [125I]T3. Data (mean of triplicates) are plotted according to the Dixon linearization. (B) Eadie-Hofstee plots of uptake data (mean of triplicates) performed with various concentrations of [12SI]T3 (0.2-500 nM) at three equilibrium concentrations of unlabelled "Is: 0 nM (m); 15.8 nM (,); 32.5 nM (O), corresponding to loading concentrations of: 0, 50, 100 nM, respectively.
50 influx and efflux kinetics under initial velocity conditions. The present work was performed to distinguish between transport models having a single free carrier state which can interact with T 3 at both sides of the membrane (simple pore model) and those having two free carrier conformations, each of which can interact with T 3 at only one face of the m e m b r a n e (simple carrier model) [8,9]. Inside and outside trans-inhibition studies of labelled T 3 transport by unlabelled T 3 indicated that the outside (125 nM) and inside (21 nM) trans-inhibition constants were similar to the respective outside (119 nM) and inside (18 nM) Michaelis constants for unidirectional fluxes. There is, thus, true trans-inhibition by unlabelled T3, ruling out simple competitive cis-inhibition due to unlabelled T 3 rapidly crossing over the membrane. These results further justify the use of 10 -5 M unlabelled T 3 as a stop solution for blocking both influx and efflux of labelled T 3 and as a wash solution for preventing the loss of internalized T 3. The complete trans-inhibifion, made it impossible to perform detailed infinite-trans (it) kinetic studies (measurement of the unidirectional flux at various concentrations of labelled T 3, while trans-unlabelled T 3 is present at a limitingly high level), which would have given infinite values of K it and zero values of V it. These results exclude a simple carrier model in which the loaded carrier would have a higher mobility than the empty cartier (R °° > R ee) [14]. The fact that the inside trans-inhibition is competitive is also consistent with this conclusion [15]. The experimental procedures used in the present study (zero-trans influx, zero-trans efflux and equilibrium-exchange) are theoretically sufficient to rule out the simple pore model and also a simple cartier model for which the reorientation of the free carrier would not be the rate limiting step of transport [8,14]. Such a methodology has been used previously to reject the pore model for the transport of uridine [16], c A M P [17], leucine and tryptophan [18] and chloroquine [19] into erythrocytes. In contrast, our results do not contradict with the predictions of the pore model. When trans-inhibition occurs in both the inward and outward directions, it is clear that the maximal velocity of transport at equilibrium-exchange should be lower than in each of the zero-trans conditions. The results obtained agree with this prediction, although the measured V e* was not statistically lower than V2]t. Similarly, K ee is smaller than both KI~t and K2~. The terms R °° and 1 / K °° are not statistically different from zero (and therefore K °° is infinite) in accordance with the simple pore model (rather than the simple carrier model which would yield a non-zero value of R °° and a finite value of K°°). The simple pore transport parameters, according to Lieb and Stein [8] are then: Q ( = 1 / 1 I ) = 0.888 ± 0.109 rain ( n = 12), R ~ = ( 7 . 8 4 + 0.12). 10 -3 m i n .
n M - l ( n = 4 ) and R~tl = (5.09 +_ 0.74) .10 2 m i n . n M - 1 (n = 4). The first order rate constants ( H ) for exchange, zero-trans influx and zero-tram exit are similar, indicating the internal consistency of the data and of the methods used to determine the kinetic parameters. The T 3 carrier shows directional asymmetry, the apparent affinity in the zero-trans modes being 6-7-times higher inside than outside the cells, whereas the reverse holds for maximal velocities of transport. This may reflect differences either in the directional mobility of the carrier-substrate complex through the m e m b r a n e or in the rate constants of dissociation of the complex at the membrane surfaces. The terms 'simple pore' and 'simple carrier' refer to kinetic models and not to physical models. Various physical models may reduce to the same kinetic description [15]. Despite its kinetic behaviour, the T 3 carrier is probably not related to pores or channels responsible of ion movements because of its high stereospecificity, which suggests a tight interaction with a binding site in the transport system. Stein [14] showed that the simple carrier model reverts to the simple pore model when the rate constants for the interconversion of the two conformations of the unloaded carrier become very large compared to the other rate constants of the transport process. In this case, l / K ° ° = 0, and the kinetic equations of the simple pore and of the simple cartier become identical in form. In conclusion, the available data are consistent with T a being transported by an asymmetric cartier which the substrate can reach simultaneously from both sides of the membrane, either because there is only one form of the free carrier, or because interconversion between the outward-facing and inward-facing forms is very rapid compared to the movement or dissociation of the loaded carrier. The T 3 carrier system can be subject neither to trans-stimulation nor to countertransport, and T 3 always flows down its concentration gradient. The limiting steps of the transport are the translocation or the dissociation of the T3-carrier complex. Acknowledgement
We thank A. Leftvre for secretarial assistance. References
1 Kenning, E.P., Docter, R., Bernard, H.F., Visser, T.J. and Hennemann, G. (1978) FEBS I.~tt. 91, 113-116. 2 Holm, A.C., Wong, K.Y., Pliam, N.B., Jorgenscn, E.C. and Goldfine, I.D. (1980) Acta EndocrinoL 95, 350-358. 3 Cheng, S.Y. (1983) Endocrinology 112, 1754-1762. 4 Pontcxzorvi, A. and Robbins, J. (1986) Endocrinology 119, 2755-2761. 5 Blondeau, J.P., Osty, J. and Francon, J. (1988) J. Biol. Chem. 263, 2685-2692.
51 6 Krenning, E., Dotter, R., Bernard, B., Visser, T. and Hennemann, G. (1980) FEBS Lett. 119, 279-282. 7 0 s t y , J., Jego, L., Francon, J. and Blondeau, J.P. (1988) Endocrinology 123, 2303-2311. 8 Lieb, W.R. and Stein, W.D. (1974) Biochim. Biophys. Acta 373, 165-177. 9 Lieb, W.R. and Stein, W.D. (1974) Biochim. Biophys. Acta 373, 178-196. 10 Docter, R., Krenning, E.P., Bos, G., Fekkes, D.F. and Hennemann, G. (1982) Biochem. J. 208, 27-34. 11 Galton, V.A., St Germain, D.L. and Whittemore, S. (1986) Endocrinology 118, 1918-1923. 12 Snedecor, G.W. and Cochran, W.G. (1967) Statistical Methods, 6th Edn., Iowa State University Press, Ames, IA.
13 Dixon, M. and Webb, E.C. (1979) Enzymes, 3rd Edn., pp. 332-467, Longman, London. 14 Stein, W.D. (1986) Transport and Diffusion Across Cell Membranes, pp. 231-361, Academic Press, New York. 15 Dev6s, R. and Krupka, R.M. (1978) Biochim. Biophys. Acta 513, 156-172. 16 Cabantchik, Z.I. and Ginsburg, H. (1977) J. Gen. Physiol. 69, 75-96. 17 Hoiman, G.D. (1978) Biochim. Biophys. Acta 508, 174-183. 18 Rosenberg, R. (1981) Biochim. Biophys. Acta 649, 262-268. 19 Yayon, A. and Ginsburg, H. (1982) Biochim. Biophys. Acta 686, 197-203.
Biochimica et Biophysica Acta, 1051 (1990) 46 51
Elsevier BBAMCR 12613
The triiodothyronine carrier of rat erythrocytes: asymmetry and mechanisms of trans-inhibition Jeannine Osty, Ya Zhou, Francoise Chantoux, Jacques Francon and Jean-Paul Blondeau Unitd de Recherche sur la Glande Thyro~de et la Rdgulation Hormonale (U.96), lnstitut National de la Santd et de la Recherche M~dicale, Kremlin-Bic~tre (France)
(Received 19 June 1989)
Key words: Thyroid hormone; Triiodothyronine; Cell membrane; Transport kinetics; (Rat erythrocyte)
The kinetic properties of the carrier-mediated transport of 3,5,3'-triiodo-L-thyronine (T3) in washed rat erythrocytes were investigated (1) by studying the effects of trans unlahelled T3 on influx and efflux of labelled substrate and (2) by testing some predictions of the theory of Lieb and Stein ((1974) Biochim. Biophys. Acta 373, 165-177). The carrier was trans-inhibited by T 3 on both sides of the membrane. Under zero-trans conditions, the carrier displayed asymmetrical properties, the Michaelis constant and the maximal velocity being more than 6-times higher for influx than for efflux. Under equilibrium-exchange conditions, the Michaelis constant was lower than the zero-trans values, as expected when tram-inhibition occurs. This kinetic behaviour is consistent with a carrier which is accessible to T3 simultaneously from both sides of the erythrocyte membrane.
Introduction There is now a great deal of experimental evidence for the existence of specific, saturable systems transporting thyroid hormones across cell membranes [1-6]. These carriers are thought to work by facilitated diffusion, allowing the rapid entry of biologically active iodothyronines and restricting that of other iodothyronines [5], or by generating a concentration gradient of hormones by active, energy-dependent transport [6]. However, there have been no detailed studies of the kinetic behaviour of these carriers in terms of hypothetical transport mechanisms. We have recently shown [7] that 3,5,3'-triiodo-Lthyronine (T3) is transported across the plasma membrane of rat erythrocytes by a stereospecific, Na+-inde pendent, carrier-mediated process, and is accumulated by intracellular trapping. We suggested that, since erythrocytes are not considered to be target cells for thyroid hormones, the rat erythrocyte carrier might play a role in the blood transport of T3. This study also showed that excess unlabelled T 3 in the extracellular medium prevented the efflux of labelled T3 from preloaded cells, but not whether this effect was due to direct trans-inhibition by unlabelled T 3 or to cis-inhibi-
Abbreviation: T3, 3,5,3'-triiodo-L-thyronine. Correspondence: J.P. Blondeau, INSERM U.96, 78, rue du Gtntral Leclerc, 94275 Kremlin-BicStre, France.
tion following the rapid entry of unlabelled T 3 into the cells. The present study was designed to ascertain whether unlabelled substrate acted on the trans side of the membrane and to determine the kinetic characteristics of the T 3 carrier in terms of the formalism developed by Lieb and Stein [8,9]. The kinetic tests proposed by these authors can be used to distinguish between two transport mechanisms: one for which the access of substrate to the carrier occurs simultaneously from both sides of the membrane (simple pore model) and the other for which the access occurs alternately (simple carrier model). Only the second model can account for countertransport [9]. This difference is biologically important, since, in the case of countertransport, the driven substrate (i.e., T3) is coupled to the downhill flow of other intracellular substrates and would be pumped against its concentration gradient. Materials and Methods Chemicals. [3'-125I]T3 (SA 3 m C i / # g ) was obtained from Amersham Corp. (England) and diluted to the desired specific radioactivities with unlabeUed T 3 purchased from Sigma Chemicals (St. Louis, MO). Since T 3 binds avidly to various types of plasticware, all plastic tubes and pipette tips were siliconized (Sigmacote, Sigma). Rat erythrocytes. Male Wistar rats (250 g, Iffa Credo, Lyon, France) were decapitated. Blood was collected on
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
47 heparin (20 U / m l blood), filtered through gauze and centrifuged (2200 x g, 10 min). The plasma and buffy coat were discarded, and the erythrocytes were washed three times with 7 vol. buffer A (137 mM NaCI, 2.7 mM KC1, 8.1 mM N a 2 H P O 4, 1.5 mM K H 2 P O 4 (pH 7.5)), once with 7 vol. buffer B (125 mM NaC1, 20 mM KCI, 4 mM MgC12, 10 mM glucose, 4.05 mM N a 2 H P O 4, 0.95 mM N a H 2 P O 4 (pH 7.4)) and suspended in 1 vol. buffer B. Washed erythrocytes were kept on ice and used within 48 h of their preparation. They were equilibrated for 10-15 min at 2 5 ° C before experiments. Cell concentrations were determined in a Coulter counter. Measurement of T3 fluxes. All assays were performed in triplicate at 25 ° C and pH 7.4 in siliconized plastic tubes. Since the equilibrium level is highly temperaturedependent [7], a constant temperature was maintained throughout the experimental period until addition of the stop solution. Influx experiments were performed in a final volume of 1 ml containing approx. 105 cpm [125I]T3 and 108 erythrocytes. (a) Zero-trans influx: experiments were initiated by mixing erythrocytes with medium (preequilibrated at 25 ° C) containing labelled T 3 and various concentrations of unlabeUed T 3. (b) Inside trans-inlfibition and equilibrium-exchange influx: erythrocytes were preincubated at 2 5 ° C with various concentrations of unlabelled T 3 until equilibrium was reached (10 min). The cells were centrifuged at 250C and the supernatants were either discarded (trans-inhibition) or supplemented with 10 5 cpm [ 125i]Ts (equilibrium-exchange). Washing of the pellets was avoided because it would induce a rapid efflux of internalized T 3 even at 0 ° C [7]. T 3 adsorbed at the cell surface represented less than 4% of the equilibrium values. Uptake was initiated by resuspending the erythrocyte pellets in either 1 ml buffer B supplemented with 0.2 nM T 3 and 10 5 c p m / m l [125I]T3 (trans-inhibition) or in 1 ml of erythrocyte supernatant supplemented with labelled T 3 (equilibrium-exchange). Efflux experiments were performed as follows: 10 s erythrocytes were preincubated for 10 min at 25 ° C with a tracer amount of labelled T 3 (105 cpm) and either various concentrations of unlabelled T 3 (zero-trans procedure) or 0.2 nM unlabelled T 3 (outside trans-inhibition) (final volume 0.25 ml). The cells were centrifuged at 25 ° C, and the supernatants were saved for radioactivity measurements. Cells were used without further washing. T 3 exit was initiated by resuspending the erythrocyte pellets in 2 ml of buffer B (preequilibrated at 25°C) containing no unlabeUed T 3 (zero-trans) or containing various concentrations of unlabelled T 3
(trans-inhibition). Influx and efflux experiments were terminated by the rapid addition of 1 / 3 vol ice-cold stop solution (buffer B containing 3 . 1 0 -5 M unlabelled T3) and subsequent manipulations were done at 0 - 2 ° C . Assay times were
monitored audibly with a metronome set at 60 beats/min. Zero-time assays were performed by adding the stop solution to the cells immediately prior to adding the initiating solution. The cells were centrifuged (2600 x g, 2 min) and the pellets were washed two times with 1.5 ml ice-cold buffer A containing 10 -5 M unlabelled T 3. The radioactivity of the pellets was measured with an efficiency of 80%. Influx or efflux were measured over the first 5 s (initial velocity conditions). These values were either subtracted from the zero-time values (efflux experiments) or the zero-time values were subtracted from them (influx experiments). In the latter case, the zerotime assays contained approx. 10% of the counts present in the cell pellet after 5 s of uptake or exchange.
Determination of the equilibrium concentration of T3. We previously reported that T 3 uptake by rat erythrocytes was Na+-independent and that the accumulation of T 3 by the cells at 25 ° C was due to intracellular trapping [7]. Also, others reported evidence that T 3 uptake in human [10] and rat [11] erythrocytes was not dependent on metabolic energy. Therefore, intra- and extracellular concentrations of free T 3 were assumed to be equal at equilibrium. In the efflux experiments (zero-trans efflux and outside trans-inhibition), aliquots of the preincubation mixture and of the supernatant (after removal of the cells) were assayed for radioactivity, allowing the determination of the equilibrium T 3 concentration in the medium (and hence the free T 3 concentration in the cells). In influx experiments (inside trans-inhibition and equilibrium-exchange), this was done in parallel incubations performed under the same conditions as those of the preincubation step, except that [~25I]T3 tracer was present. The free T 3 concentration at equilibrium was 25-30% of the starting concentration. Test of the kinetic models. The outside of the erythrocytes was arbitrarily designated side 1 and the cytoplasmic side as side 2. Maximal velocities (V) and apparent Michaelis-Menten constant ( K ) were determined in the three transport modes: zero-tram influx (zt,12), zero-trans efflux (zt,21) and equilibrium-exchange (ee). R terms are the reciprocals of the maximal velocities in each of the three modes studied and thus represent 'resistance' factors. For the simple pore kinetic model [8] R ee = R ~ + R~tl and 1 / r ee = ( 1 / r ~ t g ) + ( 1 / K ~ )
whereas for the simple carrier model [9] the relations are:
R ee + R
oo
-
zt R I 2 + R~tl
and
( 1 / K ' * ) + ( 1 / K °°) = (1/K~t~) + ( 1 / K ~ I )
48 TABLE I
150~
Kinetic parameters of Ts transport in rat erythrocytes
The kinetic parameters K (Michaelis constant), V (maximal velocity) and /7 (first-order rate constant = V / K ) were obtained from EadieHofstee plots by linear regression as described in the legend to Fig. 2. Values are mean =t:S.D. of four independent experiments performed with four different batches of rat erythrocytes. The means were compared by the paired t-test. The values of the kinetic constants are statistically different (P <1%), from one experimental protocol to another, except for those marked with an asterisk (P > 5%). A is the asymmetry parameter in the zero-trans modes. Experimental protocol zt 21
'
lOO. I
~X~o-transinflux
A 50"
ee
zt 12
K (nM) 18.1+1.2 119+6 15.6+1.9 6.6+0.6 V (nM/min) 20.2+2.7 * 130+20 18.7+2.0 * 6.4+0.2 /7 (rain -1) 1.12+0.14 * 1.10+0.15 * 1.21+0.14 * -
~trans
efflux
ecluilil~um-exchange [ ~ = ' = ~ j ~ _ 0
,
0
~, ,'""Dxn'e
0.5
1.0 v / S (rnin-1)
where R °° is the resistance experienced b y the e m p t y carrier ( c o n f o r m a t i o n a l changes of the e m p t y carrier) a n d K °° is the simple carrier affinity parameter. S t a t i s t i c a l t r e a t m e n t o f data. S t a n d a r d deviations a n d paired t-test for c o m p a r i s o n s of m e a n s were calculated according to Snedecor a n d C o c h r a n [12].
Fig. 2. Zero-trans influx and efflux and equilibrium-exchangeof 1"3 in rat erythrocytes. Flux measurements were performed as described under Materials and Methods using the same population of cells. Initial velocity data (v, mean of triplicates) are plotted according to the Eadie-Hofstee linearization v vs. v/Is], where [s] is the initial (zero-trans influx) or equilibrium (zero-trans efflux and equilibrium exchange) concentration of T3. The slopes are equal to - K (Michaelis constants) and the y-intercepts to maximal velocities of transport. (o) zero-trans influx; (A) zero-trans efflux (o); equilibrium-exchange.
Results
m e d i u m . This i n h i b i t i o n was studied at various conc e n t r a t i o n s of t r a n s u n l a b e l l e d substrate, a n d the outside i n h i b i t i o n c o n s t a n t was calculated from a D i x o n plot (Fig. lb). T h e m e a n value from two i n d e p e n d e n t experiments was 125 n M (range: 1 1 8 - 1 3 0 nM). T h e Michaelis c o n s t a n t (K21) zt a n d the m a x i m u m velocity (V2~t) of efflux were d e t e r m i n e d by m e a s u r i n g the initial velocity of T 3 efflux i n t o b u f f e r from erythrocytes preloaded with increasing c o n c e n t r a t i o n s of
Zero-trans and infinite-trans efflux
Fig. l a shows the time-course of labelled T 3 efflux f r o m preloaded erythrocytes. Efflux i n t o buffer (zerot r a n s efflux) was linear for 5 s a n d reached a n e w e q u i l i b r i u m after 1 rain. T h e logarithmic t r a n s f o r m a t i o n of efflux d a t a (not shown) indicates that there was o n l y o n e c o m p o n e n t of efflux with a half-life of 11 s. Efflux of labelled T 3 was almost completely i n h i b i t e d b y the presence of 10 btM u n l a b e l l e d T 3 in the extracellular
100,AA~A~A"
'A
B
/e
c~
o/
t~J
~'
<
•
o~
•
•
O
o
i
i
TIME (rain)
~
o
~o
2oo
o
EXTRACELLUt.AR UNLABELLED T3 (nM)
Fig. 1. Trans-inhibifion of T3 efflux in rat erythrocytes. Washed erythrocytes were preincubated with [125I]T3,centrifuged and the supernatant was discarded. (A) the cells were re~uspended in buffer B (u) or in buffer B containing 10 ~M unlabelled T3 (A). Influx was stopped at the indicated times and the cell-associated radioactivity was determined (see Materials and Methods). (B) Dixon plot of trans-inhibition. The cells were resuspended in buffer B containing various concentrations of unlabelled T3. Initial velocities of efflux (mean of triplicates) were determined as described under Materials and Methods.
49 labelled T 3. Under these conditions, re-uptake of hormone was negligible. The kinetic parameters were calculated by linear regression from an Eadie-Hofstee plot of the data (Fig. 2) and the mean values of four independent experiments are given in Table I. Zero-trans and equilibrium-exchange influx In Fig. 2 are shown typical Eadie-Hofstee plots of uptake data obtained by the zero-trans and equilibrium-exchange procedures. The kinetic constants ( K ~t and V(~ for zero-trans uptake and K ~ and V ~ for equilibrium-exchange uptake) are given in Table I (mean of four separate experiments). T 3 transport displayed asymmetrical properties since the asymmetry factor (A = V12/V21 zt zt -_ "'12/"21, I," zt/r,- zt was approx. 6.5 K ~ was significantly lower than K ~t and K2~ ( P < 1%) and Vee was significantly lower than V~] ( P < 1%) but not statistically different from V2~t ( P > 5%). The limiting permeabilities ( H ) (pseudo-first-order rate constants) were not statistically different from each other. The mean /7 value was 1.14 + 0.14 rain -~ (n = 12). R ¢~ and 1 / K ~ were obtained from the data used to calculate the mean values of V and K in Table I and were tested for equality with, respectively, the sums 2:R zt = R~t~ + R ~ and ~ ( 1 / K zt) = 1/KI~ + 1/K2~. By the paired t-test (n = 4, P > 5%), R ~ (53.9 + 5.5 min/xM -1) is not significantly different from ~ R zt (58.0-48.0 m i n - # M -1) and 1 / K ~¢ (65.0+7.5 # M -~) is not significantly different from , ~ ( 1 / ( K zt) ( 6 4 . 0 + 4 . 0 # M - ~ ) . This means that the calculated values of R °°, the resistance of the empty simple cartier, and 1 / K °°, the reciprocal of the simple carrier affinity parameter, are not significantly different from zero.
Inside trans-inhibition The trans-inlfibition of labelled T 3 influx was studied with erythrocytes preloaded with increasing concentrations of unlabelled T 3 (Fig. 3a). The mean inside inhibition constant from two independent experiments was 21 nM (range: 19.2-22.2 nM). The apparent K zt and V zt were determined at several intracellular concentrations of unlabelled T 3. Fig. 3b shows that the inside trans-inhibition was competitive, since increasing intraceUular concentrations of unlabelled T 3 affected the apparent Michaelis constant of uptake. In this experiment, the inhibition constant, calculated according to Dixon and Webb [13], was 24.6 nM. Discussion
T 3 is taken up into erythroeytes by a saturable, stereospecific transport system independent of Na ÷ and of metabolic energy [7,10,11]. We have previously reported [7] that the initial velocity of uptake, but not the equilibrium level was saturable. Therefore transport, but not intracellular trapping, appeared to be the ratelimiting step in zero-trans influx. Binding studies performed with erythrocyte cytosol [7] suggested that cytosol components could account for at least a part of the T 3 trapping activity. This binding appeared to dissociate in vitro extremely rapidly when equilibrium was perturbed. Therefore, under initial velocity conditions, dissociation from intracellular stores was not the rate limiting step in zero-trans efflux. Finally, efflux originated from the cell interior and not from the cell surface since it was blocked by an excess T 3 (Ref. 7 and present work). This previous characterization of the T 3 transport system of rat erythrocytes enabled us to study !
u
u
B
0.50'
.100 ~n
q, t-
.g E >
0.25"
/ 0
.,./
o io 2'o do EQUILIBRIUM UNLABELLED T3 (nM)
E
.3 '50
u
~
0.25 0.50 v/S (rain -1)
im~
0.75
>
0
Fig. 3. Trans-inhibition of T3 influx in rat erythrocytes. Washed rat erythrocytes were preincubated with urdabelled "1"3, centrifuged and the supernatant discarded. Equilibrium concentrations of T 3 were determined on parallel incubations as described under Materials and Methods. (A) loading concentrations of unlabelled T 3 were from 0 to 100 nM (equilibrium concentration range: 0-29.6 nM). Initial velocities of uptake were measured by resuspending the cells in buffer B containing 0.2 nM [125I]T3. Data (mean of triplicates) are plotted according to the Dixon linearization. (B) Eadie-Hofstee plots of uptake data (mean of triplicates) performed with various concentrations of [12SI]T3 (0.2-500 nM) at three equilibrium concentrations of unlabelled "Is: 0 nM (m); 15.8 nM (,); 32.5 nM (O), corresponding to loading concentrations of: 0, 50, 100 nM, respectively.
50 influx and efflux kinetics under initial velocity conditions. The present work was performed to distinguish between transport models having a single free carrier state which can interact with T 3 at both sides of the membrane (simple pore model) and those having two free carrier conformations, each of which can interact with T 3 at only one face of the m e m b r a n e (simple carrier model) [8,9]. Inside and outside trans-inhibition studies of labelled T 3 transport by unlabelled T 3 indicated that the outside (125 nM) and inside (21 nM) trans-inhibition constants were similar to the respective outside (119 nM) and inside (18 nM) Michaelis constants for unidirectional fluxes. There is, thus, true trans-inhibition by unlabelled T3, ruling out simple competitive cis-inhibition due to unlabelled T 3 rapidly crossing over the membrane. These results further justify the use of 10 -5 M unlabelled T 3 as a stop solution for blocking both influx and efflux of labelled T 3 and as a wash solution for preventing the loss of internalized T 3. The complete trans-inhibifion, made it impossible to perform detailed infinite-trans (it) kinetic studies (measurement of the unidirectional flux at various concentrations of labelled T 3, while trans-unlabelled T 3 is present at a limitingly high level), which would have given infinite values of K it and zero values of V it. These results exclude a simple carrier model in which the loaded carrier would have a higher mobility than the empty cartier (R °° > R ee) [14]. The fact that the inside trans-inhibition is competitive is also consistent with this conclusion [15]. The experimental procedures used in the present study (zero-trans influx, zero-trans efflux and equilibrium-exchange) are theoretically sufficient to rule out the simple pore model and also a simple cartier model for which the reorientation of the free carrier would not be the rate limiting step of transport [8,14]. Such a methodology has been used previously to reject the pore model for the transport of uridine [16], c A M P [17], leucine and tryptophan [18] and chloroquine [19] into erythrocytes. In contrast, our results do not contradict with the predictions of the pore model. When trans-inhibition occurs in both the inward and outward directions, it is clear that the maximal velocity of transport at equilibrium-exchange should be lower than in each of the zero-trans conditions. The results obtained agree with this prediction, although the measured V e* was not statistically lower than V2]t. Similarly, K ee is smaller than both KI~t and K2~. The terms R °° and 1 / K °° are not statistically different from zero (and therefore K °° is infinite) in accordance with the simple pore model (rather than the simple carrier model which would yield a non-zero value of R °° and a finite value of K°°). The simple pore transport parameters, according to Lieb and Stein [8] are then: Q ( = 1 / 1 I ) = 0.888 ± 0.109 rain ( n = 12), R ~ = ( 7 . 8 4 + 0.12). 10 -3 m i n .
n M - l ( n = 4 ) and R~tl = (5.09 +_ 0.74) .10 2 m i n . n M - 1 (n = 4). The first order rate constants ( H ) for exchange, zero-trans influx and zero-tram exit are similar, indicating the internal consistency of the data and of the methods used to determine the kinetic parameters. The T 3 carrier shows directional asymmetry, the apparent affinity in the zero-trans modes being 6-7-times higher inside than outside the cells, whereas the reverse holds for maximal velocities of transport. This may reflect differences either in the directional mobility of the carrier-substrate complex through the m e m b r a n e or in the rate constants of dissociation of the complex at the membrane surfaces. The terms 'simple pore' and 'simple carrier' refer to kinetic models and not to physical models. Various physical models may reduce to the same kinetic description [15]. Despite its kinetic behaviour, the T 3 carrier is probably not related to pores or channels responsible of ion movements because of its high stereospecificity, which suggests a tight interaction with a binding site in the transport system. Stein [14] showed that the simple carrier model reverts to the simple pore model when the rate constants for the interconversion of the two conformations of the unloaded carrier become very large compared to the other rate constants of the transport process. In this case, l / K ° ° = 0, and the kinetic equations of the simple pore and of the simple cartier become identical in form. In conclusion, the available data are consistent with T a being transported by an asymmetric cartier which the substrate can reach simultaneously from both sides of the membrane, either because there is only one form of the free carrier, or because interconversion between the outward-facing and inward-facing forms is very rapid compared to the movement or dissociation of the loaded carrier. The T 3 carrier system can be subject neither to trans-stimulation nor to countertransport, and T 3 always flows down its concentration gradient. The limiting steps of the transport are the translocation or the dissociation of the T3-carrier complex. Acknowledgement
We thank A. Leftvre for secretarial assistance. References
1 Kenning, E.P., Docter, R., Bernard, H.F., Visser, T.J. and Hennemann, G. (1978) FEBS I.~tt. 91, 113-116. 2 Holm, A.C., Wong, K.Y., Pliam, N.B., Jorgenscn, E.C. and Goldfine, I.D. (1980) Acta EndocrinoL 95, 350-358. 3 Cheng, S.Y. (1983) Endocrinology 112, 1754-1762. 4 Pontcxzorvi, A. and Robbins, J. (1986) Endocrinology 119, 2755-2761. 5 Blondeau, J.P., Osty, J. and Francon, J. (1988) J. Biol. Chem. 263, 2685-2692.
51 6 Krenning, E., Dotter, R., Bernard, B., Visser, T. and Hennemann, G. (1980) FEBS Lett. 119, 279-282. 7 0 s t y , J., Jego, L., Francon, J. and Blondeau, J.P. (1988) Endocrinology 123, 2303-2311. 8 Lieb, W.R. and Stein, W.D. (1974) Biochim. Biophys. Acta 373, 165-177. 9 Lieb, W.R. and Stein, W.D. (1974) Biochim. Biophys. Acta 373, 178-196. 10 Docter, R., Krenning, E.P., Bos, G., Fekkes, D.F. and Hennemann, G. (1982) Biochem. J. 208, 27-34. 11 Galton, V.A., St Germain, D.L. and Whittemore, S. (1986) Endocrinology 118, 1918-1923. 12 Snedecor, G.W. and Cochran, W.G. (1967) Statistical Methods, 6th Edn., Iowa State University Press, Ames, IA.
13 Dixon, M. and Webb, E.C. (1979) Enzymes, 3rd Edn., pp. 332-467, Longman, London. 14 Stein, W.D. (1986) Transport and Diffusion Across Cell Membranes, pp. 231-361, Academic Press, New York. 15 Dev6s, R. and Krupka, R.M. (1978) Biochim. Biophys. Acta 513, 156-172. 16 Cabantchik, Z.I. and Ginsburg, H. (1977) J. Gen. Physiol. 69, 75-96. 17 Hoiman, G.D. (1978) Biochim. Biophys. Acta 508, 174-183. 18 Rosenberg, R. (1981) Biochim. Biophys. Acta 649, 262-268. 19 Yayon, A. and Ginsburg, H. (1982) Biochim. Biophys. Acta 686, 197-203.