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Nuclear triiodothyronine receptors in rat brain during maturation
Brain Research, 177 (1979) 551-554 ~ Elsevxer/North-Holland Biomedical Press
551
Short Communications
Nuclear triiodothyronine receptors in rat brain during maturation
B. DOZlN-van ROYE and Ph. DE NAYER Lahoratoo' o/Genet al Pathology, I C P , UCL 7529, A vemte Hlppocrate, 75, B-1200 Brussels ( Be[gtum)
(Accepted August 9th, 1979)
Numerous studies have shown that the central nervous system ~s markedly dependent on thyroid hormones for its overall growth and its biochemical and morphological developmenta, 6. Indeed, thyroid hormone deficiency at birth results in irreversible brain damage if not recogmzed at an early age. Significantly, the subsequent defects are amenable to hormone therapy only during an early critical age period. These observations indicate that the developing brain becomes, for a hmlted period of t~me, a specific target for thyroid hormones. We therefore stud~ed the brain nuclear receptors for trilodothyronme in rats ageing fi'om 5 to 50 days, to determine a possible correlation between the efficacy of the hormone and the properties of its receptors. The liver nuclear receptors were simultaneously analyzed to compare the evolution of the nuclear receptors for T a m both organs. A nuclear suspension was prepared from cerebrum and hver homogenates according to the method described for the liver by Spindler et al. 1° This method involves two washes of the nuclear preparation with Triton X-100 (0.5°0). DNA content in the homogenate and m the nuclear suspension was measured according to the method of Schneider 8. The D N A recovery after nuclear purification was about 60 ° o. The time course of 1"3 binding to brain nuclear receptors and the reversibility of the reaction were ascertained : the binding increased with incubation time and reached a plateau after 2 h at 22 ~C, leading to the binding of 15 ° o of the tracer hormone. The reversibility was verified by adding an excess amount of unlabeled T3 (10 -6 M) at the eqmllbrlum of the reaction. Half-dissociation was observed after 120 rain, which is in agreement with data of Spindler et al. on hver, where a half-dissociation of 108 mm was reported ~°. All experiments were conducted m conditions where the binding of 1"3 was proportional to the amount of D N A in the incubation medium. The binding of 3', 3,5-trllodothyromne to the hver and cerebrum nuclear receptors was stud~ed as follows' 0.5 ml of the nuclei suspension (200 ,ug DNA/ml) was incubated with labeled hormone ([v"~l]Ta, spec. act. 573 mCi/mg, final concentration 2 > 10-10 M) and
552 r-
015
U15
AGE ~ d •
'
k
,
5
• 11
D
o •
A'~
o,0f
50 o uJ
~ 115
005 !
005
l\ 5
10
BOUND T, 11o ~t'moles/mg DNA)
BOUNO T bL
r,c,~,,
qq DNA ~
Fig. 1. Scatchard plots of the binding of T3 to nuclei prepared from cerebral tissue from rats of different ages. Nuclei (200 fig DNA/ml) were incubated with labeled hormone ([l~51lTs), and increasing amounts of Ts, ranging from 10 10 M to 10- 6 M. Binding of [lZ~IIT3at 10- 6 M T3 concentration was considered as non-specific, and subtracted from the radioactivity observed at lower concentrations. After 2 h at 22 °C, the bound hormone was separated from the free hormone by centnfugation at 800 y g for 7 min. The pellet was washed twice with 0 5 % Triton X-100 and assayed for radioactivity. Fig. 2. Scatchard plots of the binding of T3 to nuclei prepared from the liver of rats of different ages. For details see Fag. 1
increasing a m o u n t s ofT3, ranging from 10-1° M to I0 -(> M. Binding o f [lZSl]T3 at 10 -6 M "1"3concentration was considered as non-specific and subtracted from the radloactwity observed at lower concentrations. Incubations were performed in polystyrene tubes m a final vol. of 1 ml. The incubation medium contained 20 m M Tris (pH 7.6), 0.25 M sucrose, 1 m M MgC12, 2 m M E D T A , 0.1 m M dithiothreitol, 50 m M NaCI and 5 % glycerol. After 2 h at 22 ~C, the samples were chilled m an ice bath and then centrifuged at 800 × g for 7 rain. The pellet was washed twice with 0.5 % Triton X-100 and finally assayed for radioactivity in a gamma-spectrometer. Affinity constants (K,) and m a x i m u m binding capacity (Ro) were derived from the Scatchard analysis o f the data. Figs. 1 and 2 show the results obtained respectwely with cerebrum and liver nuclear receptors. Scatchard plots o f the binding o f T3 by brain receptors are strlkmgly different with age (see Fig. 1). The Ks increases between days 5 and 12 to reach a m a x i m u m at 20 days, and then declines in older rats. The observed differences between the Kas in the 3 groups (group I, 5 and 50 days; group II, 11 and 12: and group III, 20 days) are statistically significant (P <: 0.001). A marked discrepancy is observed in the behavior o f brain and liver receptors during maturation. Indeed, liver receptors do not show an increase in their affinity for T3 with age (Fig. 2); all the slopes on the graph are almost parallel and the K~s are not statistically different. Table I summarizes the values o f Kas and the n u m b e r o f binding sites per nucleus.
553 This latter number was calculated from the values of Ro and D N A concentration of the nuclear suspension, assuming that one liver nucleus contains approxlmatively 6.5 pg DNA. This table shows that the number of binding sites also undergoes different evolution w~th age. In brain, th~s number tends to decrease whereas in the liver, the number of s~tes tends to increase. Our data on liver receptors during maturation are m agreement with those observed by D e G r o o t et al. 2. Concerning the brain receptors, on the other hand, we are at variance with the results of Valcana and Timiras 11, the difference being the peak of K, values observed during the second and third weeks of life. This development of the nuclear receptor affinity for the thyroid hormone does not seem to be related to serum T3 concentrations. Schwartz and Oppenheimer, indeed, have reported that the increase in nuclear receptor concentration in whole brain precedes the rise m plasma T3 concentration 9. The increase in c~rculating T3 could affect the binding properties of the receptors inasmuch as the nuclear-bound Tz increases, and this fraction is not exchangeable with the labeled T3 used m the test. Based on kinetic data however, it has been shown that in the test conditions 70-90%o of the endogenously-bound T3 would dissociate 1°. Moreover, if anything, the rise in circulating T3 would rather depress than enhance the Ka of the receptors. As far as the Ro is concerned, this number will not be affected by changes in the T3 concentrations, except if in vivo "1"3has a regulatory role on its own binding sites. No changes were observed in the properties of the nuclear receptors in the liver during the postnatal period. This indicates that the modulation of the status of the T3 receptors may vary from organ to organ, and that this modulation is not d~rectly related to T3 serum levels, but could depend on other factors as Dillman et al. have demonstrated for hver T3 receptors 3. Our studies of nuclear thyroid hormone receptors could provide reformation on the mechanism(s) by which thyroid hormones regulate the maturation of the developing rat brain. In this organ, the T3 effects are age-dependent and it is sigmficant that TABLE I Alfintt) constants ( K,,) and number o f binding ~ttes per nucleus (N) Age
K~ 10 7 M 1
N
Bram
5 11 12 20 50
6 28 12 04 12.52 17.15 7 15
4264 4032 4892 3247 2681
Liver
5 11 20 50
87 78 6.1 10.5
2032 3365 3171 3625
554 the affinity c o n s t a n t ~s highest when cellular proliferation a n d m i g r a t i o n are maximal a n d cytodifferentiation ~s proceeding rapidly 7. This relationship suggests a correlation between the role of T3 a n d the development of the b r a i n : it also Jmphes that the regulation of gene expression may come u n d e r h o r m o n a l control. It is interesting to m e n t i o n that cytosohc receptors for 1"3 also exhibit a transient increase m the K,, for T3 d u r i n g the early postnatal period 4. A l t h o u g h up untd now n o relationship has been established between these b i n d i n g sites and the nuclear receptors, the possibility o f mterconvers~on between these two sets of receptors has been suggested j . This work was supported by F . R . S . M . (Belgmm) G r a n t 3.4522.76,
l Baxter, J D., Eberhardt, N. L, Aprilettl, J. W., Johnson, L. K., lvarle, R D , Schachter, B. S. Morns, J'. A., Seeburg, P. H , Goodman, H M., Latham, K. R., Polansky, L R and Martial, 5 A , Thyroid hormone receptors and responses, Recent Progr. Hormone Res., in press 2 DeGroot, L. J. and Torresani, J Trilodothyronin¢ binding to isolated hver cell nuclei, Endocrinology, 96 (1975) 357-369. 3 Dillman, W. H., Schwartz, H L. and Oppenheimer, J. H , Selective alteraUons m hepatic enzyme responses after reduction of nuclear Ta receptor sites by partial hepatectomy and starvation, Btochem. btophys. Res Commun , 80 (1978) 259-266 4 Dozin-van Roye, B and de Nayer, Ph, Trnodothyronine binding to brain cytosol receptor~ during maturation, FEBS Lett., 96 (1978) 152-154 5 Eayrs, J. T , Thyroid and central nervous development. In The Scientt~c Ba~i,s o/ Medicine, Athlone Press (University of London), London, 1966, pp. 317-339 6 Hamburgh, M., The role of thyroid and growth hormones in neurogenesJs, Curr. lop. dev. Biol., 4 (1966) 109-148. 7 Oklund, S. and Tlmlras, P S, Influence of thyroid levels m brain ontogenesis m wvo and in vitro. In G D. Grave (Ed), Thyroid Hormones and Brain Development, Raven Press, N Y, t 977, pp 33-47 8 Schneider, W. C , Determination of nucleic acids m t~ssues by pentose analysis In S P Colowlck and N. O. Kaplan (Eds.), Methods in Enzymology, Vol III, Academic Press, N.Y, pp 680-684 9 Schwartz, H L. and Oppenheimer, J. H., Ontogenesis of 3,5,3'-triiodothyronine receptors in neonatal rat brain, Endocrinology, 103 (1978) 943-948. 10 Spindler, B. J , McLeod, K. M., Ring, J. and Baxter, J. D., Thyroid hormone receptors Binding characteristics and lack of hormonal dependency for nuclear localization, J. biol Chem., 250 (1975) 4113 4119 11 Valcana, T and Timlras, P. S., Nuclear triiodothyronine receptors m developing ~at brain, Mot cell. EndocrinoL, 101 (1978) 31-41
551
Short Communications
Nuclear triiodothyronine receptors in rat brain during maturation
B. DOZlN-van ROYE and Ph. DE NAYER Lahoratoo' o/Genet al Pathology, I C P , UCL 7529, A vemte Hlppocrate, 75, B-1200 Brussels ( Be[gtum)
(Accepted August 9th, 1979)
Numerous studies have shown that the central nervous system ~s markedly dependent on thyroid hormones for its overall growth and its biochemical and morphological developmenta, 6. Indeed, thyroid hormone deficiency at birth results in irreversible brain damage if not recogmzed at an early age. Significantly, the subsequent defects are amenable to hormone therapy only during an early critical age period. These observations indicate that the developing brain becomes, for a hmlted period of t~me, a specific target for thyroid hormones. We therefore stud~ed the brain nuclear receptors for trilodothyronme in rats ageing fi'om 5 to 50 days, to determine a possible correlation between the efficacy of the hormone and the properties of its receptors. The liver nuclear receptors were simultaneously analyzed to compare the evolution of the nuclear receptors for T a m both organs. A nuclear suspension was prepared from cerebrum and hver homogenates according to the method described for the liver by Spindler et al. 1° This method involves two washes of the nuclear preparation with Triton X-100 (0.5°0). DNA content in the homogenate and m the nuclear suspension was measured according to the method of Schneider 8. The D N A recovery after nuclear purification was about 60 ° o. The time course of 1"3 binding to brain nuclear receptors and the reversibility of the reaction were ascertained : the binding increased with incubation time and reached a plateau after 2 h at 22 ~C, leading to the binding of 15 ° o of the tracer hormone. The reversibility was verified by adding an excess amount of unlabeled T3 (10 -6 M) at the eqmllbrlum of the reaction. Half-dissociation was observed after 120 rain, which is in agreement with data of Spindler et al. on hver, where a half-dissociation of 108 mm was reported ~°. All experiments were conducted m conditions where the binding of 1"3 was proportional to the amount of D N A in the incubation medium. The binding of 3', 3,5-trllodothyromne to the hver and cerebrum nuclear receptors was stud~ed as follows' 0.5 ml of the nuclei suspension (200 ,ug DNA/ml) was incubated with labeled hormone ([v"~l]Ta, spec. act. 573 mCi/mg, final concentration 2 > 10-10 M) and
552 r-
015
U15
AGE ~ d •
'
k
,
5
• 11
D
o •
A'~
o,0f
50 o uJ
~ 115
005 !
005
l\ 5
10
BOUND T, 11o ~t'moles/mg DNA)
BOUNO T bL
r,c,~,,
qq DNA ~
Fig. 1. Scatchard plots of the binding of T3 to nuclei prepared from cerebral tissue from rats of different ages. Nuclei (200 fig DNA/ml) were incubated with labeled hormone ([l~51lTs), and increasing amounts of Ts, ranging from 10 10 M to 10- 6 M. Binding of [lZ~IIT3at 10- 6 M T3 concentration was considered as non-specific, and subtracted from the radioactivity observed at lower concentrations. After 2 h at 22 °C, the bound hormone was separated from the free hormone by centnfugation at 800 y g for 7 min. The pellet was washed twice with 0 5 % Triton X-100 and assayed for radioactivity. Fig. 2. Scatchard plots of the binding of T3 to nuclei prepared from the liver of rats of different ages. For details see Fag. 1
increasing a m o u n t s ofT3, ranging from 10-1° M to I0 -(> M. Binding o f [lZSl]T3 at 10 -6 M "1"3concentration was considered as non-specific and subtracted from the radloactwity observed at lower concentrations. Incubations were performed in polystyrene tubes m a final vol. of 1 ml. The incubation medium contained 20 m M Tris (pH 7.6), 0.25 M sucrose, 1 m M MgC12, 2 m M E D T A , 0.1 m M dithiothreitol, 50 m M NaCI and 5 % glycerol. After 2 h at 22 ~C, the samples were chilled m an ice bath and then centrifuged at 800 × g for 7 rain. The pellet was washed twice with 0.5 % Triton X-100 and finally assayed for radioactivity in a gamma-spectrometer. Affinity constants (K,) and m a x i m u m binding capacity (Ro) were derived from the Scatchard analysis o f the data. Figs. 1 and 2 show the results obtained respectwely with cerebrum and liver nuclear receptors. Scatchard plots o f the binding o f T3 by brain receptors are strlkmgly different with age (see Fig. 1). The Ks increases between days 5 and 12 to reach a m a x i m u m at 20 days, and then declines in older rats. The observed differences between the Kas in the 3 groups (group I, 5 and 50 days; group II, 11 and 12: and group III, 20 days) are statistically significant (P <: 0.001). A marked discrepancy is observed in the behavior o f brain and liver receptors during maturation. Indeed, liver receptors do not show an increase in their affinity for T3 with age (Fig. 2); all the slopes on the graph are almost parallel and the K~s are not statistically different. Table I summarizes the values o f Kas and the n u m b e r o f binding sites per nucleus.
553 This latter number was calculated from the values of Ro and D N A concentration of the nuclear suspension, assuming that one liver nucleus contains approxlmatively 6.5 pg DNA. This table shows that the number of binding sites also undergoes different evolution w~th age. In brain, th~s number tends to decrease whereas in the liver, the number of s~tes tends to increase. Our data on liver receptors during maturation are m agreement with those observed by D e G r o o t et al. 2. Concerning the brain receptors, on the other hand, we are at variance with the results of Valcana and Timiras 11, the difference being the peak of K, values observed during the second and third weeks of life. This development of the nuclear receptor affinity for the thyroid hormone does not seem to be related to serum T3 concentrations. Schwartz and Oppenheimer, indeed, have reported that the increase in nuclear receptor concentration in whole brain precedes the rise m plasma T3 concentration 9. The increase in c~rculating T3 could affect the binding properties of the receptors inasmuch as the nuclear-bound Tz increases, and this fraction is not exchangeable with the labeled T3 used m the test. Based on kinetic data however, it has been shown that in the test conditions 70-90%o of the endogenously-bound T3 would dissociate 1°. Moreover, if anything, the rise in circulating T3 would rather depress than enhance the Ka of the receptors. As far as the Ro is concerned, this number will not be affected by changes in the T3 concentrations, except if in vivo "1"3has a regulatory role on its own binding sites. No changes were observed in the properties of the nuclear receptors in the liver during the postnatal period. This indicates that the modulation of the status of the T3 receptors may vary from organ to organ, and that this modulation is not d~rectly related to T3 serum levels, but could depend on other factors as Dillman et al. have demonstrated for hver T3 receptors 3. Our studies of nuclear thyroid hormone receptors could provide reformation on the mechanism(s) by which thyroid hormones regulate the maturation of the developing rat brain. In this organ, the T3 effects are age-dependent and it is sigmficant that TABLE I Alfintt) constants ( K,,) and number o f binding ~ttes per nucleus (N) Age
K~ 10 7 M 1
N
Bram
5 11 12 20 50
6 28 12 04 12.52 17.15 7 15
4264 4032 4892 3247 2681
Liver
5 11 20 50
87 78 6.1 10.5
2032 3365 3171 3625
554 the affinity c o n s t a n t ~s highest when cellular proliferation a n d m i g r a t i o n are maximal a n d cytodifferentiation ~s proceeding rapidly 7. This relationship suggests a correlation between the role of T3 a n d the development of the b r a i n : it also Jmphes that the regulation of gene expression may come u n d e r h o r m o n a l control. It is interesting to m e n t i o n that cytosohc receptors for 1"3 also exhibit a transient increase m the K,, for T3 d u r i n g the early postnatal period 4. A l t h o u g h up untd now n o relationship has been established between these b i n d i n g sites and the nuclear receptors, the possibility o f mterconvers~on between these two sets of receptors has been suggested j . This work was supported by F . R . S . M . (Belgmm) G r a n t 3.4522.76,
l Baxter, J D., Eberhardt, N. L, Aprilettl, J. W., Johnson, L. K., lvarle, R D , Schachter, B. S. Morns, J'. A., Seeburg, P. H , Goodman, H M., Latham, K. R., Polansky, L R and Martial, 5 A , Thyroid hormone receptors and responses, Recent Progr. Hormone Res., in press 2 DeGroot, L. J. and Torresani, J Trilodothyronin¢ binding to isolated hver cell nuclei, Endocrinology, 96 (1975) 357-369. 3 Dillman, W. H., Schwartz, H L. and Oppenheimer, J. H , Selective alteraUons m hepatic enzyme responses after reduction of nuclear Ta receptor sites by partial hepatectomy and starvation, Btochem. btophys. Res Commun , 80 (1978) 259-266 4 Dozin-van Roye, B and de Nayer, Ph, Trnodothyronine binding to brain cytosol receptor~ during maturation, FEBS Lett., 96 (1978) 152-154 5 Eayrs, J. T , Thyroid and central nervous development. In The Scientt~c Ba~i,s o/ Medicine, Athlone Press (University of London), London, 1966, pp. 317-339 6 Hamburgh, M., The role of thyroid and growth hormones in neurogenesJs, Curr. lop. dev. Biol., 4 (1966) 109-148. 7 Oklund, S. and Tlmlras, P S, Influence of thyroid levels m brain ontogenesis m wvo and in vitro. In G D. Grave (Ed), Thyroid Hormones and Brain Development, Raven Press, N Y, t 977, pp 33-47 8 Schneider, W. C , Determination of nucleic acids m t~ssues by pentose analysis In S P Colowlck and N. O. Kaplan (Eds.), Methods in Enzymology, Vol III, Academic Press, N.Y, pp 680-684 9 Schwartz, H L. and Oppenheimer, J. H., Ontogenesis of 3,5,3'-triiodothyronine receptors in neonatal rat brain, Endocrinology, 103 (1978) 943-948. 10 Spindler, B. J , McLeod, K. M., Ring, J. and Baxter, J. D., Thyroid hormone receptors Binding characteristics and lack of hormonal dependency for nuclear localization, J. biol Chem., 250 (1975) 4113 4119 11 Valcana, T and Timlras, P. S., Nuclear triiodothyronine receptors m developing ~at brain, Mot cell. EndocrinoL, 101 (1978) 31-41