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In vivo autoregulation of rat adenohypophyseal thyrotropin-releasing hormone receptor
Life Sciences, Vol. 30, pp. 2293-2299 Printed in the U.S.A.
IN VIVO A U T O R E G U L A T I O N OF RAT ADENOHYPOPHYSEAL RELEASING HORMONE RECEPTOR
Pergamon Press
THYROTROPIN-
Arnold Banerji and Chandan Prasad* Departments of Medieine (Seetion of Endocrinology) and Biochemistry, Louisiana State University Medical Center, N e w Orleans, L A 70112 (Received in final form April 16, 1982) Summary The possible mechanism of attenuation of thyrotropin response to exogenous thyrotropin-releasing hormone in vivo after repeated administrations of the releasing hormone has been studied. To this end, the effect of prolonged hormone treatment on the binding of hormone to its receptor in the anterior pituitary gland has been evaluated. The data show that prolonged hormonal treatment resulted in a reduction in the number (B_^..) but not the binding affinity (K~) of the receptor. The effect was r e ~ i b l e and depended on the durationUof treatment. This phenomenon of down regulation or the decrease in the receptor number was found not to be due to either the metabolism of releasing hormone or its ability to activate pituitary-thyroid-axis. The tissue concentration of hormone receptors is not constant, and it can be influenced by a variety of hormonal agents and physiological stimuli (1-6). For example, chronic exposure of rats to luteinizing hormone (LH) leads to a decrease in testicular LHreceptor (1). This phenomenon of receptor down regulation is one major mechanism by which the sensitivity of target tissues to its tropic hormones is modulated in vivo. Although the in vivo down regulation of rat adenohypophyseal (anterior pR'uii~'y) thyrotropin-rel~sing hormone (TRH) receptor has not yet been studied, a number of physiological studies suggest that T R H may reduce the number of its own receptors in vivo (2-6). The results of these studies are: i) a significant attenuation of T R H stimulation of T S H secretion and ii) a fall in the basal thyroid-stimulating hormone (TSH) level after prolongod T R H administration in rats and humans (2-6). Therefore, we decided to study the potential down regulation of rat adenohypophyseal TRH-rcceptor by T R H in vivo. The results of these studies show that indeed T R H reduces the number of its ow'~ receptors, and its effect is not mediated via TSH, thyroxine (TA) or T R H metabolites, by acid TRH, or by histidyl-proline diketopiperazine (cyclo (His-Pro'~). Materials and Methods Materials - TRH, TA, bTSH, and P T U were purchased from Sigma Chemical Co. (St. Louis, ~O). GF/B~ilters were obtained from Scientific Products, while [L-Proline2,3,4,5-"H(N)]-TRH (100-115 Ci/m tool) was a product of N e w England Nuclear (Boston, MA). Acid T R H was generously provided by Dr. Arthur Felix of Hofmann-LaRoche (NuUey, N.J.), whereas cyclo (His-Pro) was synthesized as described elsewhere (7).
*To w h o m correspondence should be addressed.
0024-3205/82/262293-07503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
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Animals - Adult male Sprague-Dawley r a t s (ARS-Sprague-Dawley Company, Madison, WI), weighing 200-300 g were housed in a t e m p e r a t u r e - and l i g h t - c o n t r o l l e d room (24 _+ 2°C, 14h light - 10h dark) and provided with Purina r a t chow and w a t e r ad libitum. To induce hypothyroidism, rats were t r e a t e d for t h r e e months with 0.0596 6-n-propyl-2-thiouracil (PTU) in their drinking water before the experiment. The hypothyroid s t a t u s was confirmed by plasma thyroxine m e a s u r e m e n t s . Animals t r e a t e d with TRH, acid TRH, TA, TSH or cyelo (His-Pro) were injected once or twice daily for a specified number of day~, as indicated in individual e x p e r i m e n t s . T~ was dissolved in a small volume of 0.1 N NaOH and then diluted with 1% gelatin in 0.9%~aline. All other r e a g e n t s were dissolved in 0.9% saline. T R H - r e c e p t o r assay - [ L - P r o l i n e - 2 , 3 , 4 , 5 - 3 H ( N ) ] - T R H binding was measured as follows. Rats were d e c a p i t a t e d , the a n t e r i o r p i t u i t a r i e s were removed and then rinsed twice in ice-cold 0.9% saline. The a n t e r i o r p i t u i t a r i e s were homogenized manually (5 strokes) in 40 mM Tris-HCl, pH 7.4 + 7.5 mM KC1 + 2 mM Mg Clo + 250 mM sucrose (TMKS buffer) (10 mg tissue wet weight/200 ~1 buffer) using a 2.0 ml~glass homogenizer (Wheaton Glass Company). A f t e r this procedure, an additional 400 ~l of TMKS buffer was added and then mixed gently. For t~e r e c e p t o r assay, 30 ~l of crude p i t u i t a r y homogenate (about 0.15 mg protein), 20 ~1 of [ ~ H - P r o ] - T R H (1 p mol, 60-70,000 cpm) and 18 tll of nonradioactive TRH (0-4.0 pmol) were incubated in the presence or absence of an 800-fold excess of nonradioactive TRH a t 4°C (over ice) for 30 minutes. Before incubation, additional TMK buffer (TMKS buffer minus sucrose) was added to make up the t o t a l volume of 200 ~1. The r e a c t i o n was stopped by the addition of 2 ml ice-cold TMK buffer and the membrane bound [°H-Pro] TRH was i m m e d i a t e l y c o l l e c t e d by suction through a GF/B (2.5cm alia.) filter, followed by t h r e e 2-ml washings with TMK buffer. The filters were dried overnight in a mini-scintillation vial and then 4 ml of scintillation fluid (PCS, Amersham) was added. The vials were then vortexed until the glass-fiber filters were c o m p l e t e l y dispersed in the scintillation fluid. ~ a d i o a c t i v i t y was measured by counting each vial for 10 minutes in a Packard T r l - c a r b Liqmd Scintillation Counter set a t 60% counting e f h c l e n c y . All assays were done in t r i p l i c a t e . Calculations - S t a t i s t i c a l significance of the d a t a was evaluated by Student's t - t e s t (8). Binding d a t a were analyzed according to S c a t c h a r d (9). The d a t a a r e presented as mean + SEM of a t l e a s t t r i p l i c a t e assays. RESULTS Optimal Conditions for T R H - R e c e p t o r A ~ a y - The c r u d e 3 a n t e r i o r p i t u i t a r y e x t r a c t s p r e p a r e d in TMK buffer showed v e r y low specific binding of [ H - P r o ] - T R H ( 3.0 fmol TRH bound/rag protein) to the p i t u i t a r y e x t r a c t s . However, the addition of 250 mM of sucrose to the homogenization buffer (TKMS buffer) significantly improved the specific binding of TRH (91.2 fmol TRH bound/mg protein). The specific binding increased linearly with t i m e and protein c o n c e n t r a t i o n , to a maximum a t 30 minutes and 150-200 Bg protein/assay respectively. Time-Dependent Decrease in Pituitary TRH-receptor Number after TRH Administration - To d e t e r m i n e the p o t e n t i a l down regnlation of adenohypophyseal TRHr e c e p t o r , the following e x p e r i m e n t was performed: Rats were injected subcutaneously (s/c) with 100 pg TRH in 0.25 ml saline or saline alone, twice daily, for 1-4 days. T w e n t y four hours a f t e r the last injection, the animals were d e c a p i t a t e d , the a n t e r i o r p i t u i t a r i e s were removed, and the number of T R H - r e e e p t o r s was determined, as detailed under "Experimental Procedure." Figure 1 shows the time course of T R H - m e d i a t e d loss of the T R H - r e c e p t o r in the adenohypophysis. When c o m p a r e d with the number of T R H - r e c e p t o r s in the u n t r e a t e d a n t e r i o r p i t u i t a r i e s (44.0 + 4.6 f m o l / m g protein), the r e c e p t o r density d e c r e a s e d by 61% a f t e r one day and 6596 a f t e r t h r e e days of t r e a t m e n t , which was the maximal d e c r e a s e observed during the course of e x p e r i m e n t a t i o n . Under similar conditions, saline t r e a t m e n t did not a l t e r the r e c e p t o r number.
Vol. 30, No. 26, 1982
Autoregulation of TRH-receptor
40 ~
o E
2295
SALINE
10
I
0
I
I
I
2 3 4 DAYSOFTREATMENT l
FIG. 1 T i m e course of decrease in adenohypophyseal T R H - r e c e p t o r a f t e r TRH-treatment
Reduction in TRH-Receptor Density by TRH Is Reversible - Four groups (3 rats/group) of rats were treated twiee daily with TRH (100 pg TRH/rat, s/c) while two control groups received saline. After 5 days, the treatment was discontinued, rats were killed at designated times, and the adenohypophysealTRH-reeeptor density was measured. The data presented in Table 1 show that the decrease in the receptor number persisted for at least 2 weeks after the termination of the TRH treatment. The decrease in the receptor number after the TRH treatment was not due to the reeeptor occupancy because the half-life for the dissociation of the TRH binding to adenohypophysealextracts was 510 minutes (27-30), whereas the deerease in the reeeptor density could be demonstrated whether the receptor assay was performed 1 day or 2 weeks after the last injection.
TABLE I Reversibility of TRH-mediated decrease in adenohypophysealTRH-reeeptor. Six groups (3 rats/group) of male Sprague-Dawley rats (250-270 g) were treated for 5 days with either TRH or saline. After five days, the treatment was discontinuedand the reemergenee of anterior pituitary TRHreeeptor was evaluated as described under "Experimental Procedure." Protein concentration was determined by the method of Lowry et al. (25). Treatment Saline Saline TRH TRH TRH TRH
Weeks After Termination of Treatment
Receptor Density fmol/mg protein
0 10 0 1 2 10
34.6 35.0 13.7 6.0 18.3 30.2
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Autoregulatlon of TPd{-receptor
Vol. 30, No. 26, 1982
Characteristics of TRH Binding to Pituitary Extracts - The binding of [ 3H-Pro]TRH to pituitary extracts was saturable and of high affinity. Because TRH treatment causes an apparent decrease in the number of its binding sites, we decided to determine whether this decrease was actually due to the decrease in receptor numb~3r" or a change in the affinity of the receptor for TRH, or both. The specific binding of [ H-Pro]-TRH to pituitary extracts prepared from saline or TRH-treated rats (100 ~g x 2/day for 4 days s/c) was performed using TRH concentration of 0.1-50 nM. The resulting binding data were used to calculate the Bmo v and K n according to Scatehard (9). The data presented in Figure 2 show that chronid'TRH t r e a t m e n t resulted in significant decreases in B (ma 27.1%, control = 46.0 and T R H - t r e a t e d = 32.5 fmoles/mg protein) but no change in ~ D (control = 16.7 nM, T R H - t r e a t e d = 15.0 nM). Potential Involvement of TSHt T4 and TRH-Metabolites in TRH-Receptor Down Regulation by TRH - TRH administration stimulates TSH release, which in turn increases T~ synthesis and release (10). TA administration also decreases the number of a~enohyphyseal TRH-reeeptor in hypo1'hyroidrats (11). It became obvious, therefore, that the role of TSH and T4 in this process be determined. The data presented in Table 2 show that the treatment ofeuthyroid rats with TRH but not TSH or TAsignificantly (P < 0.01) decreased the TRH-receptor density. However, both TRH and T ~ treatments led to a significant decrease in pituitary TRH-reeeptor density in hypothyroidrats. Furthermore, the reduction in pituitary TRH-receptor number by T~treatment may not be mediated via increase in brain TRH concentration since altered thyroid status has been shown not to affect the levels of hypothalamic TRH in rats (31) or plasma TRH in humans (32). Thus, these data suggest that neither TSH nor T4 is responsible for TRH-mediated down regulation of the pituitary TRH-receptor.
25 2O u_ -. i
D I0
l
10
l
I
20
30
l
40
I
50
B
FIG. 2 Effect of TRH treatment on binding of [3H]-TRH to pituitary extracts. Male rats were treated with 0.1 mg TRH (s/e, twice daily)or 0.1 ml saline for 4 days. Twenty-fourhours after last injection, rats were killed by decapitation, pituitary extracts ~ere prepared as described under "Materials and Methods." The binding of [°H]-TRH by pituitary extraets from saline and TRH-treated rats were determined using TRH concentrations of 0.1 to 50 nM. The Seatehard analysis (9) of the binding data show a significant reduction in Bmax but not KD after TRH-treatment (see text for details).
Vol. 30, No. 26, 1982
Autoregulat±on of TRH-receptor
2297
The possibility that TRH-mediated decrease in its receptor may be due to its metabolism (12-13) to acid TRH and eyelo (His-Pro) was evaluated by treating rats for 4 days with these peptides (2 x 100 B g/day, s/c), followed by the measurement of the pituitary TRH-receptor number. Treatment with either of these two peptides did not significantly a f f e c t anterior pituitary TRH-receptor density. DISCUSSION The anterior pituitary gland consists of several different endocrine cell types associated with the synthesis and secretion of one or more of several protein hormones, viz., TSH, growth hormone (GH), prolactin (PRL) (14). The action of TRH on pituitary thyrotrophs stimulates TSH synthesis and secretion (15). This TSH response to TRH requires the presence of functional TRH-receptors in thyrotroph ceils (16). Prolonged exposure of animals and humans to TRH has been shown to result in attenuation of TSHresponse to exogenous TRH (2, 3, 5). This physiological phenomenon may be due to the down regulation of pituitary TRH-receptor number. Receptor downregulation has been shown in the ease of LH (1), GH (17), lysergic acid diethylamide (LSD) (18) and dopamine (19) but not adrenoeorticotropic hormone (ACTH) (20), TSH (21), and LH-releasing hormone (LRH) (22). We have shown for the first time the down regulation of pituitary TRH-receptors in vivo. The analysis of the kinetics of binding showed a decrease in B but not K D ~-i'gure 2). This decrease was reversible and dependent on the duration o ~ r ~ a t m e n t . data also show that TRH-mediated decrease in its receptor number was due to neither its metabolism nor its ability to activate the pituitary-thyroid-axis. Table II Effect o~ t r e a t m e n t with TRH t TSH or T. on the binding of [ H-Pro] -TRH to anterior pituita~'y extracts from euth~n'oid and hypothyroid rats. Euthyroid and hypothyroid male rats were treated for 4 days as described under "Experimental Procedure." T 4 was dissolved in 0.1N NaOH containing 0.1% BSA while the others were dissolved in saline. The biological activity of bTSH was 1 I.U./mg. The data are presented as mean + SEM from 3-8 different experiments. *P< 0.01 as compared with saline control, **P< 0.01 as compared with euthyroid control. N.D. = Not determined. Treatment Saline TRH bTSH
T4
Dosage
fmol [ 3H-Pro] -TRH hotrod/rag protein Euthyroid Hypothyroid
--
111.2 + 4.7
2 x 100~g lx100~g 2 x 50~ g
6 1 . 2 _+ 1 . 5 " 138.0_+ 1 0 . 3 104.3 _+ 9 . 6
172.2 + 8 . 2 * * 128.4 -+ 5 . 7 * N.D. 5 9 . 9 -+ 9 . 8 *
Our observation of the T.-mediated reduction in adenohypophyseal TRH-receptor number in hypothyroid but not e~lthyroid rats, as well as increased receptor density during the development of the hypothyroid status, confirms the earlier observation of De Lean et al (11). Although the mechanism of differential regulation of the TRH-receptor n u m b ~ by T ~ d u r i n g altered thyroid status is not clear, we would like to speculate on a mech/mism for this phenomenon based on the following information: i) Euthyroid pituitary consists of 50% somatotroph (St) and 10% thyrotroph (Tt) cells (23). ii) During the development of hypothyroidism St cells decrease at the rate of 1.2-1.3%/day, whereas Tt eells inerease at the rate of 5-10%/day. Fully hypothyroid pituitary contains
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Autoregulation of TRH-receptor
Vol. 30, No. 26, 1982
2096 St and 40% Tt cells (23). iii) Although the relative density of the TRH-reeeptors on different pituitary cell types in vivo is not available, the receptor density in rodent thyrotropic tumor cells is much higher than that of adrenoeortieotrophie or mammotropie tumor cells (24). Also thyrotropic TRH-reeeptor is much more sensitive to thyroxine suppression than adrenoeorticotropie or mammotropic. However, these three tumor cells (24) do not differ in their ability to respond to TRH suppression of TRH-receptor number. Therefore, on the basis of the above information, we suggest that in euthyroid pituitary T~ does not decrease TRH-reeeptor number because it is mostly composed of lessre~Sponsive somatotropie cells, whereas hypothyroid pituitary, composed mostly of thyrotropic cells, shows greater sensitivity toward inhibition by T 4. A signifieant decrease of receptor number by TRH irrespective of the thyroid status of the animal is consistent with the observation that TRH acts equally well on all cell types. ACKNOWLEDGMENT We thank Charles F. Chapman and his staff of the Editorial Office, LSU School of Medicine, for excellent editorial assistance. This research was supported in part by Office of Naval Research (contract # N00014-80-C-0416). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
TSURAHARA, T., DUFAU, M.L., and GIGORAAGA, S., J. Biol. Chem. 252, 9002 (1977). S N Y D E R , P.J., and UTIGER, R.D., J. Clin. Invest. 52, 2305-2312 (1973). N A K A G A W A , K., Hormone Metabol. Res. 7, 217-222-'~1975). FREY, H.M.M., and H A U G , E. Aeta Endoer. 85, 744-752 (1977). STAUB, J.J., GII~ARD, J., MULLER-BRAND, J., NOELPP, B., WERNER-ZODROW, I., BAUR, U., HEITZ, P., and GEMSENHAEGER, F., J. Clin. Endoer. Metab. 46, 260 (1978). NEMEROFF, C.B., BISSETTE, G., MARTIN, J.B., BRAZEAU, P., VALE, W., KIZER, J.S., and PRANGE, JR., A.J., Neuroendoer. 30, 193-199 (1980). PRASAD, C., MATSUI, T., and PETERKOFSKY, A., Nature 268, 142-144 (1977). SNEDECOR, G.W., and COCHRAN, W.G., Statistical Methods, Sixth Edition, pp. 59-65, The Iowa University Press, Ames, Iowa (1978). SCATCHARD, G., Ann. N.Y. Acad. Sci. 51, 660-672 (1949). CATT, K.J., An ABC of Endocrinology, Little, Brown, Boston, MA (1971). De LEAN, A., FERLAND, L., DROUIN, J., KELLY, P.A., and LABRIE, F., Endoer. 100, 1496-1504 (1977). MATSUI, T., PRASAD, C., and PETERKOFSKY, A., J. Biol. Chem. 254, 2439 (1979). PRASAD, C., and PETERKOFSKY, A., J. Biol. Chem. 251, 3229-3234-'~1976). ONTJES, D.A., WALTON, J., and NEY, R.L., In Metabolic Control and Disease (Bondy, P.K., and Rosenberg, L.E., eds.), Eighth Ed'~tion, pp. 1168-1169 (1980). W.B. Saunders Co., Philadelphia. GUILLEMIN, R., In Metabolic Control and Disease (Bondy, P.K., and Rosenberg, L.E., eds.), EighthE'dition, pp. 1160-1161 0980). W.B. Saunders Co., Philadelphia. HINKEL, P.M, WOROCH, E.L., and TASHJIAN, A.H., J. Biol. Chem. 249, 3085 (1974). LESNIAK, M.A., and ROTH, J., J. Biol. Chem. 251, 3720-3729 (1976). TRULSON, M.E., and JACOBS, B.L., Life Sci. 24, 2053-2062 (1979). FRIEND, W.C., BROWN, G.M., JAWAHIR, G., LEE, T., and SEEMAN, P., Am. J. Psychiatry 135, 839-841 (1978). MORERA, A.M., and SAE, J.M., Cr. Aead. Sci. (D) 285, 1471-1474 (1977). FIELD, J.B., BLOOM, G., and CHOW, C.Y., J. Clin.Invest. 59, 659-665 (1977). CLAYTON, R.N., SOLANO, A.R., GARCIA-VELA, A., DUFAU, M.L., and CATT, K.J., Endocr. 107, 699-706 (1980). DeFESI, C.R.,--A"~TIER, H.S., and SURKS, M.L., Endoer. 10._~4,1172-1180 (1979).
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Autoregulation of TRH-receptor
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24.
PECK, V., and G E R S H E N G O R N ,
25. 26.
TELL, G.P., HAOUR, F., and SAEZ, J.M., Metabolism 27, 1566-1592 (1978). LOWRY,O.H., ROSEBROUGH, N.J., FARR, A.L., and RANDALL, R.J., J. Biol. Chem. 193, 265-275 (1951). STEINER,H., Experientia 29, 759 (1973). GRANT,G., VALE, W., and GUILLEMIN, R., Biochem. Biophys. Res. Commun. 46, 28-34 (1972). BARDEN,N., and LABRIE, F., J. Biol. Chem. 248, 7601-7606 (1973). VALE, W., BLACKWELL, R., GRA~T, G., and GUILLEMIN, R., Endocrinology 93, 26-33 (1973). PRASAD, C., MORI, M., WILBER, J.F., PIERSON, W., PEGUES, J., and JAYARAMAN, A., Peptides, Submitted for Publication (1982). MALLIK,T., RICHARDS, V., SEIBEL, M.J., PRASAD, C., and WILBER, J.F., Abs. The Endocrine Soe. Mtg., Washington, D.C. (1980).
27. 28. 29. 30. 31. 32.
M.C., J. Clin. Endoer. Metab. 50, 1144-1148 (1960).
IN VIVO A U T O R E G U L A T I O N OF RAT ADENOHYPOPHYSEAL RELEASING HORMONE RECEPTOR
Pergamon Press
THYROTROPIN-
Arnold Banerji and Chandan Prasad* Departments of Medieine (Seetion of Endocrinology) and Biochemistry, Louisiana State University Medical Center, N e w Orleans, L A 70112 (Received in final form April 16, 1982) Summary The possible mechanism of attenuation of thyrotropin response to exogenous thyrotropin-releasing hormone in vivo after repeated administrations of the releasing hormone has been studied. To this end, the effect of prolonged hormone treatment on the binding of hormone to its receptor in the anterior pituitary gland has been evaluated. The data show that prolonged hormonal treatment resulted in a reduction in the number (B_^..) but not the binding affinity (K~) of the receptor. The effect was r e ~ i b l e and depended on the durationUof treatment. This phenomenon of down regulation or the decrease in the receptor number was found not to be due to either the metabolism of releasing hormone or its ability to activate pituitary-thyroid-axis. The tissue concentration of hormone receptors is not constant, and it can be influenced by a variety of hormonal agents and physiological stimuli (1-6). For example, chronic exposure of rats to luteinizing hormone (LH) leads to a decrease in testicular LHreceptor (1). This phenomenon of receptor down regulation is one major mechanism by which the sensitivity of target tissues to its tropic hormones is modulated in vivo. Although the in vivo down regulation of rat adenohypophyseal (anterior pR'uii~'y) thyrotropin-rel~sing hormone (TRH) receptor has not yet been studied, a number of physiological studies suggest that T R H may reduce the number of its own receptors in vivo (2-6). The results of these studies are: i) a significant attenuation of T R H stimulation of T S H secretion and ii) a fall in the basal thyroid-stimulating hormone (TSH) level after prolongod T R H administration in rats and humans (2-6). Therefore, we decided to study the potential down regulation of rat adenohypophyseal TRH-rcceptor by T R H in vivo. The results of these studies show that indeed T R H reduces the number of its ow'~ receptors, and its effect is not mediated via TSH, thyroxine (TA) or T R H metabolites, by acid TRH, or by histidyl-proline diketopiperazine (cyclo (His-Pro'~). Materials and Methods Materials - TRH, TA, bTSH, and P T U were purchased from Sigma Chemical Co. (St. Louis, ~O). GF/B~ilters were obtained from Scientific Products, while [L-Proline2,3,4,5-"H(N)]-TRH (100-115 Ci/m tool) was a product of N e w England Nuclear (Boston, MA). Acid T R H was generously provided by Dr. Arthur Felix of Hofmann-LaRoche (NuUey, N.J.), whereas cyclo (His-Pro) was synthesized as described elsewhere (7).
*To w h o m correspondence should be addressed.
0024-3205/82/262293-07503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2294
Autoregulation of TRH-receptor
Vol. 30, No. 26, 1982
Animals - Adult male Sprague-Dawley r a t s (ARS-Sprague-Dawley Company, Madison, WI), weighing 200-300 g were housed in a t e m p e r a t u r e - and l i g h t - c o n t r o l l e d room (24 _+ 2°C, 14h light - 10h dark) and provided with Purina r a t chow and w a t e r ad libitum. To induce hypothyroidism, rats were t r e a t e d for t h r e e months with 0.0596 6-n-propyl-2-thiouracil (PTU) in their drinking water before the experiment. The hypothyroid s t a t u s was confirmed by plasma thyroxine m e a s u r e m e n t s . Animals t r e a t e d with TRH, acid TRH, TA, TSH or cyelo (His-Pro) were injected once or twice daily for a specified number of day~, as indicated in individual e x p e r i m e n t s . T~ was dissolved in a small volume of 0.1 N NaOH and then diluted with 1% gelatin in 0.9%~aline. All other r e a g e n t s were dissolved in 0.9% saline. T R H - r e c e p t o r assay - [ L - P r o l i n e - 2 , 3 , 4 , 5 - 3 H ( N ) ] - T R H binding was measured as follows. Rats were d e c a p i t a t e d , the a n t e r i o r p i t u i t a r i e s were removed and then rinsed twice in ice-cold 0.9% saline. The a n t e r i o r p i t u i t a r i e s were homogenized manually (5 strokes) in 40 mM Tris-HCl, pH 7.4 + 7.5 mM KC1 + 2 mM Mg Clo + 250 mM sucrose (TMKS buffer) (10 mg tissue wet weight/200 ~1 buffer) using a 2.0 ml~glass homogenizer (Wheaton Glass Company). A f t e r this procedure, an additional 400 ~l of TMKS buffer was added and then mixed gently. For t~e r e c e p t o r assay, 30 ~l of crude p i t u i t a r y homogenate (about 0.15 mg protein), 20 ~1 of [ ~ H - P r o ] - T R H (1 p mol, 60-70,000 cpm) and 18 tll of nonradioactive TRH (0-4.0 pmol) were incubated in the presence or absence of an 800-fold excess of nonradioactive TRH a t 4°C (over ice) for 30 minutes. Before incubation, additional TMK buffer (TMKS buffer minus sucrose) was added to make up the t o t a l volume of 200 ~1. The r e a c t i o n was stopped by the addition of 2 ml ice-cold TMK buffer and the membrane bound [°H-Pro] TRH was i m m e d i a t e l y c o l l e c t e d by suction through a GF/B (2.5cm alia.) filter, followed by t h r e e 2-ml washings with TMK buffer. The filters were dried overnight in a mini-scintillation vial and then 4 ml of scintillation fluid (PCS, Amersham) was added. The vials were then vortexed until the glass-fiber filters were c o m p l e t e l y dispersed in the scintillation fluid. ~ a d i o a c t i v i t y was measured by counting each vial for 10 minutes in a Packard T r l - c a r b Liqmd Scintillation Counter set a t 60% counting e f h c l e n c y . All assays were done in t r i p l i c a t e . Calculations - S t a t i s t i c a l significance of the d a t a was evaluated by Student's t - t e s t (8). Binding d a t a were analyzed according to S c a t c h a r d (9). The d a t a a r e presented as mean + SEM of a t l e a s t t r i p l i c a t e assays. RESULTS Optimal Conditions for T R H - R e c e p t o r A ~ a y - The c r u d e 3 a n t e r i o r p i t u i t a r y e x t r a c t s p r e p a r e d in TMK buffer showed v e r y low specific binding of [ H - P r o ] - T R H ( 3.0 fmol TRH bound/rag protein) to the p i t u i t a r y e x t r a c t s . However, the addition of 250 mM of sucrose to the homogenization buffer (TKMS buffer) significantly improved the specific binding of TRH (91.2 fmol TRH bound/mg protein). The specific binding increased linearly with t i m e and protein c o n c e n t r a t i o n , to a maximum a t 30 minutes and 150-200 Bg protein/assay respectively. Time-Dependent Decrease in Pituitary TRH-receptor Number after TRH Administration - To d e t e r m i n e the p o t e n t i a l down regnlation of adenohypophyseal TRHr e c e p t o r , the following e x p e r i m e n t was performed: Rats were injected subcutaneously (s/c) with 100 pg TRH in 0.25 ml saline or saline alone, twice daily, for 1-4 days. T w e n t y four hours a f t e r the last injection, the animals were d e c a p i t a t e d , the a n t e r i o r p i t u i t a r i e s were removed, and the number of T R H - r e e e p t o r s was determined, as detailed under "Experimental Procedure." Figure 1 shows the time course of T R H - m e d i a t e d loss of the T R H - r e c e p t o r in the adenohypophysis. When c o m p a r e d with the number of T R H - r e c e p t o r s in the u n t r e a t e d a n t e r i o r p i t u i t a r i e s (44.0 + 4.6 f m o l / m g protein), the r e c e p t o r density d e c r e a s e d by 61% a f t e r one day and 6596 a f t e r t h r e e days of t r e a t m e n t , which was the maximal d e c r e a s e observed during the course of e x p e r i m e n t a t i o n . Under similar conditions, saline t r e a t m e n t did not a l t e r the r e c e p t o r number.
Vol. 30, No. 26, 1982
Autoregulation of TRH-receptor
40 ~
o E
2295
SALINE
10
I
0
I
I
I
2 3 4 DAYSOFTREATMENT l
FIG. 1 T i m e course of decrease in adenohypophyseal T R H - r e c e p t o r a f t e r TRH-treatment
Reduction in TRH-Receptor Density by TRH Is Reversible - Four groups (3 rats/group) of rats were treated twiee daily with TRH (100 pg TRH/rat, s/c) while two control groups received saline. After 5 days, the treatment was discontinued, rats were killed at designated times, and the adenohypophysealTRH-reeeptor density was measured. The data presented in Table 1 show that the decrease in the receptor number persisted for at least 2 weeks after the termination of the TRH treatment. The decrease in the receptor number after the TRH treatment was not due to the reeeptor occupancy because the half-life for the dissociation of the TRH binding to adenohypophysealextracts was 510 minutes (27-30), whereas the deerease in the reeeptor density could be demonstrated whether the receptor assay was performed 1 day or 2 weeks after the last injection.
TABLE I Reversibility of TRH-mediated decrease in adenohypophysealTRH-reeeptor. Six groups (3 rats/group) of male Sprague-Dawley rats (250-270 g) were treated for 5 days with either TRH or saline. After five days, the treatment was discontinuedand the reemergenee of anterior pituitary TRHreeeptor was evaluated as described under "Experimental Procedure." Protein concentration was determined by the method of Lowry et al. (25). Treatment Saline Saline TRH TRH TRH TRH
Weeks After Termination of Treatment
Receptor Density fmol/mg protein
0 10 0 1 2 10
34.6 35.0 13.7 6.0 18.3 30.2
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Autoregulatlon of TPd{-receptor
Vol. 30, No. 26, 1982
Characteristics of TRH Binding to Pituitary Extracts - The binding of [ 3H-Pro]TRH to pituitary extracts was saturable and of high affinity. Because TRH treatment causes an apparent decrease in the number of its binding sites, we decided to determine whether this decrease was actually due to the decrease in receptor numb~3r" or a change in the affinity of the receptor for TRH, or both. The specific binding of [ H-Pro]-TRH to pituitary extracts prepared from saline or TRH-treated rats (100 ~g x 2/day for 4 days s/c) was performed using TRH concentration of 0.1-50 nM. The resulting binding data were used to calculate the Bmo v and K n according to Scatehard (9). The data presented in Figure 2 show that chronid'TRH t r e a t m e n t resulted in significant decreases in B (ma 27.1%, control = 46.0 and T R H - t r e a t e d = 32.5 fmoles/mg protein) but no change in ~ D (control = 16.7 nM, T R H - t r e a t e d = 15.0 nM). Potential Involvement of TSHt T4 and TRH-Metabolites in TRH-Receptor Down Regulation by TRH - TRH administration stimulates TSH release, which in turn increases T~ synthesis and release (10). TA administration also decreases the number of a~enohyphyseal TRH-reeeptor in hypo1'hyroidrats (11). It became obvious, therefore, that the role of TSH and T4 in this process be determined. The data presented in Table 2 show that the treatment ofeuthyroid rats with TRH but not TSH or TAsignificantly (P < 0.01) decreased the TRH-receptor density. However, both TRH and T ~ treatments led to a significant decrease in pituitary TRH-reeeptor density in hypothyroidrats. Furthermore, the reduction in pituitary TRH-receptor number by T~treatment may not be mediated via increase in brain TRH concentration since altered thyroid status has been shown not to affect the levels of hypothalamic TRH in rats (31) or plasma TRH in humans (32). Thus, these data suggest that neither TSH nor T4 is responsible for TRH-mediated down regulation of the pituitary TRH-receptor.
25 2O u_ -. i
D I0
l
10
l
I
20
30
l
40
I
50
B
FIG. 2 Effect of TRH treatment on binding of [3H]-TRH to pituitary extracts. Male rats were treated with 0.1 mg TRH (s/e, twice daily)or 0.1 ml saline for 4 days. Twenty-fourhours after last injection, rats were killed by decapitation, pituitary extracts ~ere prepared as described under "Materials and Methods." The binding of [°H]-TRH by pituitary extraets from saline and TRH-treated rats were determined using TRH concentrations of 0.1 to 50 nM. The Seatehard analysis (9) of the binding data show a significant reduction in Bmax but not KD after TRH-treatment (see text for details).
Vol. 30, No. 26, 1982
Autoregulat±on of TRH-receptor
2297
The possibility that TRH-mediated decrease in its receptor may be due to its metabolism (12-13) to acid TRH and eyelo (His-Pro) was evaluated by treating rats for 4 days with these peptides (2 x 100 B g/day, s/c), followed by the measurement of the pituitary TRH-receptor number. Treatment with either of these two peptides did not significantly a f f e c t anterior pituitary TRH-receptor density. DISCUSSION The anterior pituitary gland consists of several different endocrine cell types associated with the synthesis and secretion of one or more of several protein hormones, viz., TSH, growth hormone (GH), prolactin (PRL) (14). The action of TRH on pituitary thyrotrophs stimulates TSH synthesis and secretion (15). This TSH response to TRH requires the presence of functional TRH-receptors in thyrotroph ceils (16). Prolonged exposure of animals and humans to TRH has been shown to result in attenuation of TSHresponse to exogenous TRH (2, 3, 5). This physiological phenomenon may be due to the down regulation of pituitary TRH-receptor number. Receptor downregulation has been shown in the ease of LH (1), GH (17), lysergic acid diethylamide (LSD) (18) and dopamine (19) but not adrenoeorticotropic hormone (ACTH) (20), TSH (21), and LH-releasing hormone (LRH) (22). We have shown for the first time the down regulation of pituitary TRH-receptors in vivo. The analysis of the kinetics of binding showed a decrease in B but not K D ~-i'gure 2). This decrease was reversible and dependent on the duration o ~ r ~ a t m e n t . data also show that TRH-mediated decrease in its receptor number was due to neither its metabolism nor its ability to activate the pituitary-thyroid-axis. Table II Effect o~ t r e a t m e n t with TRH t TSH or T. on the binding of [ H-Pro] -TRH to anterior pituita~'y extracts from euth~n'oid and hypothyroid rats. Euthyroid and hypothyroid male rats were treated for 4 days as described under "Experimental Procedure." T 4 was dissolved in 0.1N NaOH containing 0.1% BSA while the others were dissolved in saline. The biological activity of bTSH was 1 I.U./mg. The data are presented as mean + SEM from 3-8 different experiments. *P< 0.01 as compared with saline control, **P< 0.01 as compared with euthyroid control. N.D. = Not determined. Treatment Saline TRH bTSH
T4
Dosage
fmol [ 3H-Pro] -TRH hotrod/rag protein Euthyroid Hypothyroid
--
111.2 + 4.7
2 x 100~g lx100~g 2 x 50~ g
6 1 . 2 _+ 1 . 5 " 138.0_+ 1 0 . 3 104.3 _+ 9 . 6
172.2 + 8 . 2 * * 128.4 -+ 5 . 7 * N.D. 5 9 . 9 -+ 9 . 8 *
Our observation of the T.-mediated reduction in adenohypophyseal TRH-receptor number in hypothyroid but not e~lthyroid rats, as well as increased receptor density during the development of the hypothyroid status, confirms the earlier observation of De Lean et al (11). Although the mechanism of differential regulation of the TRH-receptor n u m b ~ by T ~ d u r i n g altered thyroid status is not clear, we would like to speculate on a mech/mism for this phenomenon based on the following information: i) Euthyroid pituitary consists of 50% somatotroph (St) and 10% thyrotroph (Tt) cells (23). ii) During the development of hypothyroidism St cells decrease at the rate of 1.2-1.3%/day, whereas Tt eells inerease at the rate of 5-10%/day. Fully hypothyroid pituitary contains
2298
Autoregulation of TRH-receptor
Vol. 30, No. 26, 1982
2096 St and 40% Tt cells (23). iii) Although the relative density of the TRH-reeeptors on different pituitary cell types in vivo is not available, the receptor density in rodent thyrotropic tumor cells is much higher than that of adrenoeortieotrophie or mammotropie tumor cells (24). Also thyrotropic TRH-reeeptor is much more sensitive to thyroxine suppression than adrenoeorticotropie or mammotropic. However, these three tumor cells (24) do not differ in their ability to respond to TRH suppression of TRH-receptor number. Therefore, on the basis of the above information, we suggest that in euthyroid pituitary T~ does not decrease TRH-reeeptor number because it is mostly composed of lessre~Sponsive somatotropie cells, whereas hypothyroid pituitary, composed mostly of thyrotropic cells, shows greater sensitivity toward inhibition by T 4. A signifieant decrease of receptor number by TRH irrespective of the thyroid status of the animal is consistent with the observation that TRH acts equally well on all cell types. ACKNOWLEDGMENT We thank Charles F. Chapman and his staff of the Editorial Office, LSU School of Medicine, for excellent editorial assistance. This research was supported in part by Office of Naval Research (contract # N00014-80-C-0416). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
TSURAHARA, T., DUFAU, M.L., and GIGORAAGA, S., J. Biol. Chem. 252, 9002 (1977). S N Y D E R , P.J., and UTIGER, R.D., J. Clin. Invest. 52, 2305-2312 (1973). N A K A G A W A , K., Hormone Metabol. Res. 7, 217-222-'~1975). FREY, H.M.M., and H A U G , E. Aeta Endoer. 85, 744-752 (1977). STAUB, J.J., GII~ARD, J., MULLER-BRAND, J., NOELPP, B., WERNER-ZODROW, I., BAUR, U., HEITZ, P., and GEMSENHAEGER, F., J. Clin. Endoer. Metab. 46, 260 (1978). NEMEROFF, C.B., BISSETTE, G., MARTIN, J.B., BRAZEAU, P., VALE, W., KIZER, J.S., and PRANGE, JR., A.J., Neuroendoer. 30, 193-199 (1980). PRASAD, C., MATSUI, T., and PETERKOFSKY, A., Nature 268, 142-144 (1977). SNEDECOR, G.W., and COCHRAN, W.G., Statistical Methods, Sixth Edition, pp. 59-65, The Iowa University Press, Ames, Iowa (1978). SCATCHARD, G., Ann. N.Y. Acad. Sci. 51, 660-672 (1949). CATT, K.J., An ABC of Endocrinology, Little, Brown, Boston, MA (1971). De LEAN, A., FERLAND, L., DROUIN, J., KELLY, P.A., and LABRIE, F., Endoer. 100, 1496-1504 (1977). MATSUI, T., PRASAD, C., and PETERKOFSKY, A., J. Biol. Chem. 254, 2439 (1979). PRASAD, C., and PETERKOFSKY, A., J. Biol. Chem. 251, 3229-3234-'~1976). ONTJES, D.A., WALTON, J., and NEY, R.L., In Metabolic Control and Disease (Bondy, P.K., and Rosenberg, L.E., eds.), Eighth Ed'~tion, pp. 1168-1169 (1980). W.B. Saunders Co., Philadelphia. GUILLEMIN, R., In Metabolic Control and Disease (Bondy, P.K., and Rosenberg, L.E., eds.), EighthE'dition, pp. 1160-1161 0980). W.B. Saunders Co., Philadelphia. HINKEL, P.M, WOROCH, E.L., and TASHJIAN, A.H., J. Biol. Chem. 249, 3085 (1974). LESNIAK, M.A., and ROTH, J., J. Biol. Chem. 251, 3720-3729 (1976). TRULSON, M.E., and JACOBS, B.L., Life Sci. 24, 2053-2062 (1979). FRIEND, W.C., BROWN, G.M., JAWAHIR, G., LEE, T., and SEEMAN, P., Am. J. Psychiatry 135, 839-841 (1978). MORERA, A.M., and SAE, J.M., Cr. Aead. Sci. (D) 285, 1471-1474 (1977). FIELD, J.B., BLOOM, G., and CHOW, C.Y., J. Clin.Invest. 59, 659-665 (1977). CLAYTON, R.N., SOLANO, A.R., GARCIA-VELA, A., DUFAU, M.L., and CATT, K.J., Endocr. 107, 699-706 (1980). DeFESI, C.R.,--A"~TIER, H.S., and SURKS, M.L., Endoer. 10._~4,1172-1180 (1979).
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Autoregulation of TRH-receptor
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24.
PECK, V., and G E R S H E N G O R N ,
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TELL, G.P., HAOUR, F., and SAEZ, J.M., Metabolism 27, 1566-1592 (1978). LOWRY,O.H., ROSEBROUGH, N.J., FARR, A.L., and RANDALL, R.J., J. Biol. Chem. 193, 265-275 (1951). STEINER,H., Experientia 29, 759 (1973). GRANT,G., VALE, W., and GUILLEMIN, R., Biochem. Biophys. Res. Commun. 46, 28-34 (1972). BARDEN,N., and LABRIE, F., J. Biol. Chem. 248, 7601-7606 (1973). VALE, W., BLACKWELL, R., GRA~T, G., and GUILLEMIN, R., Endocrinology 93, 26-33 (1973). PRASAD, C., MORI, M., WILBER, J.F., PIERSON, W., PEGUES, J., and JAYARAMAN, A., Peptides, Submitted for Publication (1982). MALLIK,T., RICHARDS, V., SEIBEL, M.J., PRASAD, C., and WILBER, J.F., Abs. The Endocrine Soe. Mtg., Washington, D.C. (1980).
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M.C., J. Clin. Endoer. Metab. 50, 1144-1148 (1960).