202 Horm. Metab. Res. 8 (1976) 202-206
© Georg Thieme Verlag Stuttgart
Cyclic AMP Level of Human Thyroid Cells in Monolayer Culture TSH Induced Refractoriness to TSH Action Y. Kaneko
Summary Thyroid cells from euthyroid patients with Graves' disease were cultured in a chemically defined medium. The cells preserved the ability to respond to TSH with 8-fold increase in cyclic AMP concentration. This cyclic AMP response to TSH was diminished by prior exposure of cells to TSH. The decrease in cyclic AMP response to TSH induced by TSH was reversible, was not associated with a similar decrease in cyclic AMP response to PGE., and could not be attributed to increased phosphodiesterase activity or to decreased ade ny I cyclase activity. The partial resistence to TSH stimulation of thyroid cells previously exposed to TSH may be due to changes in the TSH receptor, possibly caused by TSH itself. Key-Words: Thyroid Cells - Cell Cullure - cAMP - TSH - PGE.
Introduction The action of many peptide hormone is mediated by ehanges in eellular levels of adenosine 3',5'-monophosphate. The magnitude and duration of the cycHe AMP response depend not only on the dose of stimulating hormone but also on the degree of activation of adenylate cyclase and on the amount of phosphodiesterase activity in the target cells (Rohison, Butcher, Oye, Morgan and Sutherland 1968, Kakiuchi, Rall and Mac/llwain 1969, Kuo and DeRenzo 1969). Cells da not always respond to the same amount of stimulation with the same degree of eyelie AMP elevation. One of many possible factors which might influenee the magnitude of eyclic AMP response is previously exposure to the stimulating hormone. Ho and Sutherland found that stimulation of isolated rat adipocytes with epinephrine was followed by refractory period du ring which exposure to additional epinephrine did not result in elevation of eyclic AMP levels (Ho and Sutherland 1971). A similar phenomenon was then reported in other kind of cells (Franklin and Foster 1973). This desensitization was eonsidered due either to the appearanee of antagonistic substanees in the target cells (Ho and Sutherland 1971), or to agonist-induced conformational changes in the receptors (Franklin and Foster 1973). Neither proposal, however, is sufficient to account for many aspects of the refraetoriness. Received: 15 Sept. 1975
Accepted: 5 Jan. 1976
Thyroid cells, tao, may exhibit different sensitivity to TSH according to the presence or absence of prior exposure to TSH, as well as to differences in other extra- and intracellular factors (Shishiba, Takaishi, Miyachi and Ozawa 1975). Knowledge of the underlying mechanisms involved in the refractory period seems important in the understanding of the mechanism of TSH action. The present study was conducted to ex amine these cellular regulatory mechanisms using cultured thyroid cells from patients with hyperthyroidism. Materials and Methods
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The First Department of Medicine. University of Tokyo, Hongo, TOkyo, Japan
Cell Cullure. Human thyroid tissue was obtained at surgery from patients with Graves' disease who were euthyroid after treatment for three to four months with thiourea. The tissue was cut into small fragments which were digested with 0.25% of trypsin (Difco, 1:250) according to the method of Jons· son et al. (Jonsson and Fagraeus 1969). lsolated cells were suspended in Ham's FI2 medium (Nissui Seiyaku, Tokyo) supplemented with 10% calf serum (Chiba Kessei, Chiba), 100 p.U/ml of penicillin and 100 #li/mi of streptomycin, and were seeded at the density of 7 x 1()4 cells/cm 2 in plastic culture dishes (60 mm, Falcon Plastics). They were cultured at 37°C in humidified air containing 5% CO 2 • Twenty-four hours after see ding cells were washed three times with Ham's F12 medium and then maintained in the medium with or without bovine thyrotropin (TSH, Armour). Medium was renewed every two days. Incubalion and Washing Procedure. Thc cells maintained in
TSH-free medium were preincubated far various times at 31 0 C in Ham's F12 medium with or without various concentration of TSH (first incubation). Cells were next washed three times with 5 ml of TSH-free medium. They were then incubated for two 30 min periods in TSH-free medium (second incubation) to ensure removal of TSH (Gallin, Ro/h, NelliJle, Mey/s and BuelI974). Some monolayers at this stage were utilized for determining the effect of NaF on adenylate cyclase activity. Others were further incubated for 30 min with 100 mU/ml of TSH, 100 mU/ml of TSH + 1 mM theophylline or 10 pg/ml of PGEI (Upjohn Co.) to know the cyclic AMP response of cells to the stimulators (third incubation). Cyclic AMP Assay. Cyclic AMP was prepared from the cells by the method of Ollen et al. (Ollen, Johnson and Paslan
1972) and assayed by a competitive pro tein binding method (GilTTIIZn 1972) using rat Iiver protein kinase (Kumon, YaTTIIZ· mura and Nishizuka 1970). Adenyl Cycl4se AClillity. Monolayers wcre harvestcd with a rubber policeman. Cells were homogenized in 50 mM potassium phosphate buffer pH 7.2 and centrifuged at 10,000 x g. The effect of NaF on the adenylate cyclase activity of thc particulate fraction was determined by the method of Salo· mon et al. (Salomon, Londos and Rodbell 1914).
203
Cyclic AMP Level in Human Thyroid Cells
Results Maintained in serum-free chemically defined medium, cAMP 80 thyroid cells enlarged their cytoplasm without detectable cell death and cell division. Monolayers formed within three days of seeding. Basal levels of cyclic pmoles 60 AMP were 6.5 ± 0.7,10.0 ± 1.2 and 9.8 ± 0.8 pmoles/.o....::---106 cells at 2, 4 and 6 days of culture, respectively 106cells 40 (Fig. la). Cyclic AMP levels rose more than 8-fold
20
b
o
100
o
cAMP 80
0.5 1
5 10 TSH
PSoles 60 10 cetls
40
r r r~ ~ r r 02460246
20
50100 mUlmt
Fig. 2. Effect of varying concentration of TSH on cyclic AMP level. Thyroid cells cultured 4 days in medium without TSH were challenged with different concentration of TSH (abscissa) for 30 minutes at 37 0 C. Each point is the mean ± SEM of triplicates.
o r+
CULTURE
TI M E (days)
Fig. 1. Refractoriness to TSH of thyroid cells maintained with TSH. Cells were cultured in Ham's F12 medium with (b) or without (a) 100 mU/mI of TSH. Medium was changed at 24, 72 and 120 hours after the seeding. 24 hours after each medium change, cells were washed and reincubated with (hatched bars) or without (open bars) TSH (100 mU/ mI). 30 minutes later cyclic AMP was measured as described in the Methods. Each point is the mean ± SEM of triplicates.
pmoles 1(}6cells 4
20 O~'~~--~~L-~~~~~
o
(hours) on addition of TSH to the medium at 2, 4 and 6 days of culture (Fig. la). The cells preserved the ability to respond to TSH for at least two weeks. The minimum dose of TSH required to induce a cycHc AMP response was 1 mV/mi and a maximal increase in cyclic AMP was obtained with from 50 mV/mi to 100 mV/mi of TSH. Between these doses the magnitude of cyclic AMP response was proportional to the concentration of TSH and demonstrated the usual sigmoid dose response curve (Fig. 2). Increase in cycHc AMP was detectable within 5 min of the addition of TSH and the response was maximal 30 min after TSH was added to the culture medium (Fig. 3). CycHc AMP levels then decreased, but were still higher at 6 and 12 hr in the presence of TSH than in the absence of TSH (Fig. 3). Even 48 hours after the addition of TSH cyclic AMP levels were still higher in the presence of TSH (20.0 ± 2.5 pmoles/106 cells) than in the absence of TSH (10.0 ± 1.1 pmoles/106 cells), indicating that cells maintained with TSH synthesize more cyclic AMP than those. kept in TSHfree medium throughout the culture.
Fig. 3. Time course of cyclic AMP level of thyroid cells stimulated with TSH. Thyroid cells cultured for 4 days in the medium without TSH were washed once in Ham's F12 medium, and incubated with (dosed cirde) or without (open cirde) 100 mU/ml of TSH. At each indicated time (abscissa) cyclic AMP was measured. Each point is the mean ± SEM of triplicates.
100 cAMP 80 pmoles 60
106cells 40 20 O'~~~~~~~~~~~
o
lÖ6
,6 5 164 1& 102 lÖ1 TSH
U/ml
Fig. 4. Cyclic AMP response of thyroid cells preincubated Prior incubation of cells with TSH decreased the ca- with different concentration of TSH. Cells of four-day-old culture were incubated in fresh medium with various amount pacity for the subsequent addition of TSH to elevate of TSH (abscissa) for 24 hours. They werc then washed fulcellular levels of cyclic AMP (Fig. 1b). A decrease in Iy as described in the Methods and challcnged for 30 minthe cyclic AMP response to TSH was detectable when utes with 100 mU/ml of TSH at 37 0 C. Each point is the mean ± SEM of triplicates. cells were preincubated in medium containing TSH
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a
204
Y. Kaneko
100
cAMP 80
cAMP
60
pmoles l06cells 40 20
rt
80
~
pmoles 60
O~~~~~~~~~LL~
o
3
6
15
106cells 40 ~f 20 ~
24
o
PREINCUBATION TIME hours Fig. S. Relationship between duration of preincubation with TSH and the degree of refractoriness to TSH. Cells of fourday-old culture were incubated with 100 mU/ml of TSH for different time (abscissa). At the end of each preincubation, ceUs were washed as described in the Methods. These cells were then incubated in Ham's FI2 medium with (hutched bars) or without (open bars) 100 mU/ml of TSH, and 30 minutes later cyclic AMP level was measured. Each point is the mean ± SEM of triplicates.
at a concentration of 1 mU/ml or greater (Fig. 4). The duration of exposure to TSH required to produce a decrease in cyclic AMP response to subse: quent TSH stimulation is depicted in Figure 5. Basal value at different preincubation time with TSH shown by the open bars were similar indicating that the washing procedure described in Methods decreased the initial rise in cyclic AMP levels caused by addition of TSH. On subsequent TSH stimulation, these cells showed decreasing cyclic AMP response to TSH dependent on the duration of prior exposure to TSH (Fig. 5, hutched bars). These data indicates that prolonged exposure of cells to TSH is necessary to produce a decrease in cyclic .AMP res~onse. The time co~rse o~ the onset of thlS ~efractive state to TSH action ~ld not parallel the time co~rse of the onset of cychc AMP response to TSH (Flg. 3). While refractoriness to TSH was most marked in cells preincubated with TSH for a long time, cells maintained with TSH for as long as six days still retained the ability to respond with at least a 2-fold increase in cyclic AMP. In addition, the partial refractoriness was transient and cells regained their uso ual sensitivity to TSH when they were washed and transferred to TSH-free medium. Maximum return of sensitivity required at least 6 hr incubation (Fig. 6). These phenomena could be explained by changes in adenylate cyclase activity or phosphodiesterase activity induced by prior exposure to TSH. To investigate this, adenylate cyclase activity and the cyclic AMP response to TSH in the presence or absence of theophylline, a phosphodiesterase inhibitor, were measured using cells preincubated in medium with or without TSH. Results shown in Table 1 indieate that prior incubation with TSH did not decrease the
+
.
+ 3
6 12 TIME
2t. 48 hours
Fig. 6. Recovery from refractoriness of cyclic AMP response to TSH of thyroid cells. Cells of four-day-old culture were preincubated for 24 ho urs with 100 mU/ml of TSH. Then, cells were washed as described in the Methods, and incuhated in TSH-free medium for various times (abscissa). At the indicated time they were challenged for 30 minutes with 100 mU/ml of TSH and cyclic AMP levels were measured. Each point is the mean ± SEM of triplicates.
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100
basal activity of cellular adenylate cyclase or the capacity for NaF to stimulate adenylate cyclase activity. Prior incubation with TSH decreased subsequent TSH stimulation of cellular cyclic AMP levels in the presence or absence of theophylline (Table 1). The presence of theophylline doubled levels of cyclic AMP indieating inhibition of phosphodiesterase but such inhibition of phosphodiesterase did not effect the diminished responsiveness to TSH. TSH inhibition of subsequent TSH action, therefore, cannot be explained by decrease in total adenylate cyclase activity or increase in phosphodiesterase activity. Other possible" explanation of TSH induced refractoriness to TSH action might include TSH induced changes in the cell membrane and the receptors it was of interest to investigate whether the thyroid cell cyclic AMP response to PGE) is also altered by prior exposure to TSH. There was, however, no difference in the cyclic AMP response to 10 1oI8/ml of PGE) of cells preincubated with or without 100 mU/ ml of TSH (58.8 ± 4.2 and 52.4 ± 3.4 pmoles/106 cells, respectively). This indieates that if prior exposure to TSH inhibits further cyclic AMP response to TSH stimulation by alterations in the cell membrane these alterations are specific for the TSH receptor or TSH-dependent moiety of adenyl cyclase. Discussion
After exposure to TSH for several hours to days, monolayer of thyroid cells showed a decreased cyclic AMP response to subsequent TSH stimulation. This phenomenon is reversible and has no direct relationship with intracellular cyclic AMP levels. This latter fact is consistent with a previous report showing that thyroid cells cultured in the medium supplemented
Cyclic AMP Level in Human Thyroid Cells
205
Table 1. Adenylate cyclase activity and effects of theophylline on cyclic AMP response to TSH of monolayer cells in the medium with or without TSH preincubation
adenylate cyclase activity (pmoies cyclic AMP formed /5min/l0 6 cells)
cyclic AMP response to TSH (pmoles/ 10 6 cells)
basal
NaF 00 lJg/ml)
theophylline (-)
theophylline (+)
A. medium alone
12.4 ± 1.3
65.4 ± 2.0
85.5 ± 4.5
185 ± 19
B. medium + TSH
10.4 ± 1.1
63.2 ± 2.5
36.4 ± 4.9
54.0 ± 7.6
with dibutyryl cyclic AMP have similar TSH binding properties to those cultured without dibutyryl cyclic AMP (Lissizky, Fayet and Varrier 1973). Furthermore, the decrease in cyclic AMP response cannot be attributed to a decrease in adenylate cyclase activity and probably not to an increase in phosphodiesterase activity, indicating that the phenomenon may be related to changes in the cell surface.
an alteration in the effect of TSH at the binding site. Theoretically this could be caused by changes in the hormone, in the receptor or in the TSH dependent moiety of adenyl cyclase. Previous reports (Ho and Sutherland 1971, Pohl, Krans, Birnbaumer and Rodbell 1972) have suggested the presence of hormone degrading enzymes at the receptor site. Such enzymes when activated by excessive hormone concentrations might reduce the magnitude of hormone bindRecent reports have shown that thyroid cells have ing. Gavin et a1. on the other hand have reported dedifferent receptors, specific to TSH and to PGE, on crease in the number of cell receptors when incutheir plasma membrane, and that clear correlation bated with increasing concentration of insulin and exists between the amount of hormone bound to rehave postulated that insulin controls the number of ceptors and magnitude of increase in adenyl cyclase activity (Amir, Carraway alld Kahn 1973, Moore and its own receptors (Gavin, Roth, Neville, Meyts and Buell 1974). These workers have not proposed a speWolf! 1973). If TSH and PGE, receptors share an adenylate cyclase system as has been proposed (Birn- cific mechanism for this reduction in receptor numbers but arecent report (Hung and Cuatrecasas 1975) baumer, Pohl, Krans and Rodbell 1970), possible suggests that insulin (and perhaps other polypeptide changes causing refractoriness must be restricted to hormones) possesses intrinsic proteolytic activity and TSH receptors or membrane components ne ar them, can thereby destroy their receptors. Although it is as cells did not show any decrease in sensitivity to possible that relatively sm all changes in the TSH dePGE, after incubation with TSH. pendent moiety of adenyl cyclase have not been reA possible explanation for the refractoriness is that cognized under the experimental condition used the TSH remains on the cell surface in a biologically acresults of NaF stimulation indicate that major changes tive form. Evidence for persistent binding of active in adenylate cyclase activity are not caused by prior TSH was reported recently (DeRurertis, Chayoth, incubation with TSH. Zar and Field 1975). If this is true, very few recepRegardless of the mechanism it is likely that expotors will be available for newly added TSH. Other sure of human thyroid cells to TSH is associated with reports have shown, however, that [3H)-TSH bound a reduction in the number of TSH receptors, just as to receptors could be readily displaced by subseprior incubation of lymphocytes or fat cells with inquently added cold TSH within few minutes (Amir, sulin is associated with reduced number of insulin reCarraway and Kohn 1973). From this, a continuous ceptors. process of association-dissociation can be visualized and newly added TSH should be able to bind to the Acknowledgement receptors by displacing previously bound hormone. The absence of refractoriness in cells subject to short I am grateful to Dr. K. Ito, Ito Hospital, Tokyo for his gepreincubation time during which almost all available nerous supply of thyroid tissues from his surgical materials. I also wish to express my gratitude to Drs. H. Suzuki, T. Jureceptors would be occupied by excessive TSH added to the medium is an additional argument against ji (Dep. of Medicine and Blood Transfusion Service, University of Tokyo), N. Fleischer and R. Mortimcr (Dep. of Mepersistent receptor bin ding of TSH as a cause of redicine, Albert Einstein College of Medicinc) for their help fractoriness. and constructive remarks. An alternative explanation for the refractoriness is
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Thyroid cells, preincubated for 24 hours in medium with (B) or without (A) 100 mU/ml of TSH, were utilized. Adenylate cyclasc activity was assayed according to the procedure of Solmon ct al. (Solmon, Londos, Rodbel 1974). To dctcrmine the effect of theophylline on the cyclic AMP response to TSH, cells were stimulated with 100 mU/ml of TSH for 30 minutes at 37 0 C in the presence or absence of 1 mM theophylline.
206
N. Takasu, S. Sato, T. Yamada, M. Makiuchi, R. Furihata and M. Miyakawa
References
Kumon, A .. H. Yamamura, Y. Nishizuka: Mode of action of adenosine 3',5'-cyclic monophosphate on protein kinase from rat liver. Biochem.Biophys.Res.Commun. 41: 12901297 (1970) Kuo, J.F., E.C. DeRenzo: A comparison of the effects of lipolytic and antilipolytic agents on adenosine 3',5'-monophosphate levels in adipose cells as determined by prior labeling with adenosinc-8-14 C. J.Bio!.Chem. 244: 146-148 (1973) Lissitzky, S., G. Fayet, B. Varrier: Thyroid-stimulating hormone binding to cultured thyroid cells. FEBS Letters 29: 20-24 (1973) Moore, W. V., J. Wolf: Binding of prostagiandin EI to beef thyroid membranes. J.Bio!.Chem. 248: 5705-5711 (1973) Otten, J., G.S. Johnson, I. Pastan: Regulation of cell growth by cyclic adenosine 3',5'-monophosphate. Effect of cell density and agents which alter cell growth on cyclic adenosine 3',5'-monophosphate levels in fibroblasts. J.Bio!. Chem. 247: 7082-7087 (1974) Pohl, S.L., M.J. Krans, L. Birnbaumer, M. Rodbell: Inactivation of glucagon by plasma membrane of rat Iiver. J.Bio!. Chem. 247: 2295-2301 (1972) Robison, G.A., R. W. Butcher, I. Oye, H. W. Morgan, E. W. Sutherland: The effect of epinephrine on adenosine 3',5'phosphate levels in the isolated perfused rat heart. Molec.Pharmac. 1: 168-177 (1965) Salomon, Y., C. Londos, M. Rodbell: A high sensitive ade ny I cyclase assay. Analytical Biochemistry 58: 541-548 (1974) Shishiba, Y., M. Takaishi, Y. Miyachi, Y. Ozawa: Alteration of thyroid responsiveness to TSH under the influence of circulating thyroid hormone: Short feed-back regulatory effect. Endocrinologia Japonica 22: 367-370 (1975)
Requests for reprints should be addressed to: YOshiyasu Kaneko, Dep. of Medicine, AECOM, 1945-20D, Eastchester Rd, Bronx, NY 10461 (USA)
Horm. Metab. Res. 8 (1976) 206-211
© Georg Thieme Verlag Stuttgart
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Amir, S.M., T.F. Carraway Jr., L.D. Kohn: The binding of thyrotropin to isolated bovine thyroid plasma membranes. J.Bio!.Chem. 248: 4092-4100 (1973) Birnbaumer, F.R., S.L. Pohl, M.L. Krans, M. Rodbell: Action of hormones on the adenyl cyclase system. Advances in Biochemical Psychopharmacology 3: 185-208 (1970) DeRubertis, F.R., R. Chayoth, U. Zor, J.B. Field: Evidence for persistent binding of biologically active thyrotropin to thyroid in vitro. Endocrinology 96: 1579-1586 (1975) FrankIin, T.J., S.J. Foster: Hormone-induced desensitization of hormonal control of cyclic AMP level in human diploid fibroblast. Nature New Bio!. 246: 146-148 (1973) Gavin, J.R. 111, J. Roth, D.M. Neville Jr., P.D. Mayts, D.N. Buell: Insulin-dependellt regulation of insulin receptor concentrations: A direct demonstration in cell culture. Proc.Nat.Acad.Sci. USA 71: 84-88 (1974) Gilman, A.G.: A pro tein binding assay for adenosine 3',5'cyclic monophosphate. Proc.Nat!.Acad.Sci. USA 67: 305312 (1972) Ho, R..J., E. W. Sutherland: Formation and release of hormone antagonist by rat adipocytes. J.Biol.Chem. 246: 6822-6827 (1971) Huang, D., P. Cuatrecasas: Insulin-induced reduction of membrane receptor concentrations in isolated fat cells and Iymphocytes. J.BioI.Chem. 250: 8251-8259 (1975) Jonsson, J., A. Fagraeus: On the mechanism of the ring zone effect obtained with the mixed haemoadsorption technique. Studies with human anti-thyroid sera reacting with thyroid monolayer cultures. Immunology 17: 385-411 (1969) Kakiuchi, s., T. W. Rall, H. Mcllwain: The effect of electrical stimulation upon the accumulation of adenosine 3',5'monophosphate in isolated cerebral tissue. J.Neurochem. 16: 485-491 (1969)
The Different Modes of Action of Thyrotropin and Prostaglandin E I on Cyclic Adenosine 3',5'-Monophosphate Synthesis in Human Thyroid, as Studied by Sequential Stimulations* N. Takasu, S. Sato, T. Yamada, M. Makiuchi, R. Furihata and M. Miyakawa Department of Medicine, Institute of Adaptation Medicine. and Second Department of Surgery. School of Medicine, Shinshu University. Matsumoto. Japan
Summary PGE I was equally effective in increasing 3 H-cyclic AMP in normal and in toxie thyroids, whereas TSH was less effective but over a longer time in the toxie thyroids. Stimulation by a large second dose of TSH could not be elicited after prior stimulation by large doses of TSH. Similar results *Supported by a Grant from thc Ministry of Education of Japan Received: 15 Sept. 1975
Accepted: 5 Jan. 1976
were obtained with regard to the effect of PGE l' However, stimulation by a large dose of PGE I was still effcctive after the slices be ca me refractory to TSH. Similarly, stimulation by a large dose of TSH was still effective after the slices became refractory to PGE I' It is suggested that the site and/or mode of action of TSH is quite different from that of PGE l '
Key-Words: Thyrotropin - Prostagiandin EI - Cyc1ic AMP - Human Thyroid
© Georg Thieme Verlag Stuttgart
Cyclic AMP Level of Human Thyroid Cells in Monolayer Culture TSH Induced Refractoriness to TSH Action Y. Kaneko
Summary Thyroid cells from euthyroid patients with Graves' disease were cultured in a chemically defined medium. The cells preserved the ability to respond to TSH with 8-fold increase in cyclic AMP concentration. This cyclic AMP response to TSH was diminished by prior exposure of cells to TSH. The decrease in cyclic AMP response to TSH induced by TSH was reversible, was not associated with a similar decrease in cyclic AMP response to PGE., and could not be attributed to increased phosphodiesterase activity or to decreased ade ny I cyclase activity. The partial resistence to TSH stimulation of thyroid cells previously exposed to TSH may be due to changes in the TSH receptor, possibly caused by TSH itself. Key-Words: Thyroid Cells - Cell Cullure - cAMP - TSH - PGE.
Introduction The action of many peptide hormone is mediated by ehanges in eellular levels of adenosine 3',5'-monophosphate. The magnitude and duration of the cycHe AMP response depend not only on the dose of stimulating hormone but also on the degree of activation of adenylate cyclase and on the amount of phosphodiesterase activity in the target cells (Rohison, Butcher, Oye, Morgan and Sutherland 1968, Kakiuchi, Rall and Mac/llwain 1969, Kuo and DeRenzo 1969). Cells da not always respond to the same amount of stimulation with the same degree of eyelie AMP elevation. One of many possible factors which might influenee the magnitude of eyclic AMP response is previously exposure to the stimulating hormone. Ho and Sutherland found that stimulation of isolated rat adipocytes with epinephrine was followed by refractory period du ring which exposure to additional epinephrine did not result in elevation of eyclic AMP levels (Ho and Sutherland 1971). A similar phenomenon was then reported in other kind of cells (Franklin and Foster 1973). This desensitization was eonsidered due either to the appearanee of antagonistic substanees in the target cells (Ho and Sutherland 1971), or to agonist-induced conformational changes in the receptors (Franklin and Foster 1973). Neither proposal, however, is sufficient to account for many aspects of the refraetoriness. Received: 15 Sept. 1975
Accepted: 5 Jan. 1976
Thyroid cells, tao, may exhibit different sensitivity to TSH according to the presence or absence of prior exposure to TSH, as well as to differences in other extra- and intracellular factors (Shishiba, Takaishi, Miyachi and Ozawa 1975). Knowledge of the underlying mechanisms involved in the refractory period seems important in the understanding of the mechanism of TSH action. The present study was conducted to ex amine these cellular regulatory mechanisms using cultured thyroid cells from patients with hyperthyroidism. Materials and Methods
Downloaded by: York University libraries. Copyrighted material.
The First Department of Medicine. University of Tokyo, Hongo, TOkyo, Japan
Cell Cullure. Human thyroid tissue was obtained at surgery from patients with Graves' disease who were euthyroid after treatment for three to four months with thiourea. The tissue was cut into small fragments which were digested with 0.25% of trypsin (Difco, 1:250) according to the method of Jons· son et al. (Jonsson and Fagraeus 1969). lsolated cells were suspended in Ham's FI2 medium (Nissui Seiyaku, Tokyo) supplemented with 10% calf serum (Chiba Kessei, Chiba), 100 p.U/ml of penicillin and 100 #li/mi of streptomycin, and were seeded at the density of 7 x 1()4 cells/cm 2 in plastic culture dishes (60 mm, Falcon Plastics). They were cultured at 37°C in humidified air containing 5% CO 2 • Twenty-four hours after see ding cells were washed three times with Ham's F12 medium and then maintained in the medium with or without bovine thyrotropin (TSH, Armour). Medium was renewed every two days. Incubalion and Washing Procedure. Thc cells maintained in
TSH-free medium were preincubated far various times at 31 0 C in Ham's F12 medium with or without various concentration of TSH (first incubation). Cells were next washed three times with 5 ml of TSH-free medium. They were then incubated for two 30 min periods in TSH-free medium (second incubation) to ensure removal of TSH (Gallin, Ro/h, NelliJle, Mey/s and BuelI974). Some monolayers at this stage were utilized for determining the effect of NaF on adenylate cyclase activity. Others were further incubated for 30 min with 100 mU/ml of TSH, 100 mU/ml of TSH + 1 mM theophylline or 10 pg/ml of PGEI (Upjohn Co.) to know the cyclic AMP response of cells to the stimulators (third incubation). Cyclic AMP Assay. Cyclic AMP was prepared from the cells by the method of Ollen et al. (Ollen, Johnson and Paslan
1972) and assayed by a competitive pro tein binding method (GilTTIIZn 1972) using rat Iiver protein kinase (Kumon, YaTTIIZ· mura and Nishizuka 1970). Adenyl Cycl4se AClillity. Monolayers wcre harvestcd with a rubber policeman. Cells were homogenized in 50 mM potassium phosphate buffer pH 7.2 and centrifuged at 10,000 x g. The effect of NaF on the adenylate cyclase activity of thc particulate fraction was determined by the method of Salo· mon et al. (Salomon, Londos and Rodbell 1914).
203
Cyclic AMP Level in Human Thyroid Cells
Results Maintained in serum-free chemically defined medium, cAMP 80 thyroid cells enlarged their cytoplasm without detectable cell death and cell division. Monolayers formed within three days of seeding. Basal levels of cyclic pmoles 60 AMP were 6.5 ± 0.7,10.0 ± 1.2 and 9.8 ± 0.8 pmoles/.o....::---106 cells at 2, 4 and 6 days of culture, respectively 106cells 40 (Fig. la). Cyclic AMP levels rose more than 8-fold
20
b
o
100
o
cAMP 80
0.5 1
5 10 TSH
PSoles 60 10 cetls
40
r r r~ ~ r r 02460246
20
50100 mUlmt
Fig. 2. Effect of varying concentration of TSH on cyclic AMP level. Thyroid cells cultured 4 days in medium without TSH were challenged with different concentration of TSH (abscissa) for 30 minutes at 37 0 C. Each point is the mean ± SEM of triplicates.
o r+
CULTURE
TI M E (days)
Fig. 1. Refractoriness to TSH of thyroid cells maintained with TSH. Cells were cultured in Ham's F12 medium with (b) or without (a) 100 mU/mI of TSH. Medium was changed at 24, 72 and 120 hours after the seeding. 24 hours after each medium change, cells were washed and reincubated with (hatched bars) or without (open bars) TSH (100 mU/ mI). 30 minutes later cyclic AMP was measured as described in the Methods. Each point is the mean ± SEM of triplicates.
pmoles 1(}6cells 4
20 O~'~~--~~L-~~~~~
o
(hours) on addition of TSH to the medium at 2, 4 and 6 days of culture (Fig. la). The cells preserved the ability to respond to TSH for at least two weeks. The minimum dose of TSH required to induce a cycHc AMP response was 1 mV/mi and a maximal increase in cyclic AMP was obtained with from 50 mV/mi to 100 mV/mi of TSH. Between these doses the magnitude of cyclic AMP response was proportional to the concentration of TSH and demonstrated the usual sigmoid dose response curve (Fig. 2). Increase in cycHc AMP was detectable within 5 min of the addition of TSH and the response was maximal 30 min after TSH was added to the culture medium (Fig. 3). CycHc AMP levels then decreased, but were still higher at 6 and 12 hr in the presence of TSH than in the absence of TSH (Fig. 3). Even 48 hours after the addition of TSH cyclic AMP levels were still higher in the presence of TSH (20.0 ± 2.5 pmoles/106 cells) than in the absence of TSH (10.0 ± 1.1 pmoles/106 cells), indicating that cells maintained with TSH synthesize more cyclic AMP than those. kept in TSHfree medium throughout the culture.
Fig. 3. Time course of cyclic AMP level of thyroid cells stimulated with TSH. Thyroid cells cultured for 4 days in the medium without TSH were washed once in Ham's F12 medium, and incubated with (dosed cirde) or without (open cirde) 100 mU/ml of TSH. At each indicated time (abscissa) cyclic AMP was measured. Each point is the mean ± SEM of triplicates.
100 cAMP 80 pmoles 60
106cells 40 20 O'~~~~~~~~~~~
o
lÖ6
,6 5 164 1& 102 lÖ1 TSH
U/ml
Fig. 4. Cyclic AMP response of thyroid cells preincubated Prior incubation of cells with TSH decreased the ca- with different concentration of TSH. Cells of four-day-old culture were incubated in fresh medium with various amount pacity for the subsequent addition of TSH to elevate of TSH (abscissa) for 24 hours. They werc then washed fulcellular levels of cyclic AMP (Fig. 1b). A decrease in Iy as described in the Methods and challcnged for 30 minthe cyclic AMP response to TSH was detectable when utes with 100 mU/ml of TSH at 37 0 C. Each point is the mean ± SEM of triplicates. cells were preincubated in medium containing TSH
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a
204
Y. Kaneko
100
cAMP 80
cAMP
60
pmoles l06cells 40 20
rt
80
~
pmoles 60
O~~~~~~~~~LL~
o
3
6
15
106cells 40 ~f 20 ~
24
o
PREINCUBATION TIME hours Fig. S. Relationship between duration of preincubation with TSH and the degree of refractoriness to TSH. Cells of fourday-old culture were incubated with 100 mU/ml of TSH for different time (abscissa). At the end of each preincubation, ceUs were washed as described in the Methods. These cells were then incubated in Ham's FI2 medium with (hutched bars) or without (open bars) 100 mU/ml of TSH, and 30 minutes later cyclic AMP level was measured. Each point is the mean ± SEM of triplicates.
at a concentration of 1 mU/ml or greater (Fig. 4). The duration of exposure to TSH required to produce a decrease in cyclic AMP response to subse: quent TSH stimulation is depicted in Figure 5. Basal value at different preincubation time with TSH shown by the open bars were similar indicating that the washing procedure described in Methods decreased the initial rise in cyclic AMP levels caused by addition of TSH. On subsequent TSH stimulation, these cells showed decreasing cyclic AMP response to TSH dependent on the duration of prior exposure to TSH (Fig. 5, hutched bars). These data indicates that prolonged exposure of cells to TSH is necessary to produce a decrease in cyclic .AMP res~onse. The time co~rse o~ the onset of thlS ~efractive state to TSH action ~ld not parallel the time co~rse of the onset of cychc AMP response to TSH (Flg. 3). While refractoriness to TSH was most marked in cells preincubated with TSH for a long time, cells maintained with TSH for as long as six days still retained the ability to respond with at least a 2-fold increase in cyclic AMP. In addition, the partial refractoriness was transient and cells regained their uso ual sensitivity to TSH when they were washed and transferred to TSH-free medium. Maximum return of sensitivity required at least 6 hr incubation (Fig. 6). These phenomena could be explained by changes in adenylate cyclase activity or phosphodiesterase activity induced by prior exposure to TSH. To investigate this, adenylate cyclase activity and the cyclic AMP response to TSH in the presence or absence of theophylline, a phosphodiesterase inhibitor, were measured using cells preincubated in medium with or without TSH. Results shown in Table 1 indieate that prior incubation with TSH did not decrease the
+
.
+ 3
6 12 TIME
2t. 48 hours
Fig. 6. Recovery from refractoriness of cyclic AMP response to TSH of thyroid cells. Cells of four-day-old culture were preincubated for 24 ho urs with 100 mU/ml of TSH. Then, cells were washed as described in the Methods, and incuhated in TSH-free medium for various times (abscissa). At the indicated time they were challenged for 30 minutes with 100 mU/ml of TSH and cyclic AMP levels were measured. Each point is the mean ± SEM of triplicates.
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100
basal activity of cellular adenylate cyclase or the capacity for NaF to stimulate adenylate cyclase activity. Prior incubation with TSH decreased subsequent TSH stimulation of cellular cyclic AMP levels in the presence or absence of theophylline (Table 1). The presence of theophylline doubled levels of cyclic AMP indieating inhibition of phosphodiesterase but such inhibition of phosphodiesterase did not effect the diminished responsiveness to TSH. TSH inhibition of subsequent TSH action, therefore, cannot be explained by decrease in total adenylate cyclase activity or increase in phosphodiesterase activity. Other possible" explanation of TSH induced refractoriness to TSH action might include TSH induced changes in the cell membrane and the receptors it was of interest to investigate whether the thyroid cell cyclic AMP response to PGE) is also altered by prior exposure to TSH. There was, however, no difference in the cyclic AMP response to 10 1oI8/ml of PGE) of cells preincubated with or without 100 mU/ ml of TSH (58.8 ± 4.2 and 52.4 ± 3.4 pmoles/106 cells, respectively). This indieates that if prior exposure to TSH inhibits further cyclic AMP response to TSH stimulation by alterations in the cell membrane these alterations are specific for the TSH receptor or TSH-dependent moiety of adenyl cyclase. Discussion
After exposure to TSH for several hours to days, monolayer of thyroid cells showed a decreased cyclic AMP response to subsequent TSH stimulation. This phenomenon is reversible and has no direct relationship with intracellular cyclic AMP levels. This latter fact is consistent with a previous report showing that thyroid cells cultured in the medium supplemented
Cyclic AMP Level in Human Thyroid Cells
205
Table 1. Adenylate cyclase activity and effects of theophylline on cyclic AMP response to TSH of monolayer cells in the medium with or without TSH preincubation
adenylate cyclase activity (pmoies cyclic AMP formed /5min/l0 6 cells)
cyclic AMP response to TSH (pmoles/ 10 6 cells)
basal
NaF 00 lJg/ml)
theophylline (-)
theophylline (+)
A. medium alone
12.4 ± 1.3
65.4 ± 2.0
85.5 ± 4.5
185 ± 19
B. medium + TSH
10.4 ± 1.1
63.2 ± 2.5
36.4 ± 4.9
54.0 ± 7.6
with dibutyryl cyclic AMP have similar TSH binding properties to those cultured without dibutyryl cyclic AMP (Lissizky, Fayet and Varrier 1973). Furthermore, the decrease in cyclic AMP response cannot be attributed to a decrease in adenylate cyclase activity and probably not to an increase in phosphodiesterase activity, indicating that the phenomenon may be related to changes in the cell surface.
an alteration in the effect of TSH at the binding site. Theoretically this could be caused by changes in the hormone, in the receptor or in the TSH dependent moiety of adenyl cyclase. Previous reports (Ho and Sutherland 1971, Pohl, Krans, Birnbaumer and Rodbell 1972) have suggested the presence of hormone degrading enzymes at the receptor site. Such enzymes when activated by excessive hormone concentrations might reduce the magnitude of hormone bindRecent reports have shown that thyroid cells have ing. Gavin et a1. on the other hand have reported dedifferent receptors, specific to TSH and to PGE, on crease in the number of cell receptors when incutheir plasma membrane, and that clear correlation bated with increasing concentration of insulin and exists between the amount of hormone bound to rehave postulated that insulin controls the number of ceptors and magnitude of increase in adenyl cyclase activity (Amir, Carraway alld Kahn 1973, Moore and its own receptors (Gavin, Roth, Neville, Meyts and Buell 1974). These workers have not proposed a speWolf! 1973). If TSH and PGE, receptors share an adenylate cyclase system as has been proposed (Birn- cific mechanism for this reduction in receptor numbers but arecent report (Hung and Cuatrecasas 1975) baumer, Pohl, Krans and Rodbell 1970), possible suggests that insulin (and perhaps other polypeptide changes causing refractoriness must be restricted to hormones) possesses intrinsic proteolytic activity and TSH receptors or membrane components ne ar them, can thereby destroy their receptors. Although it is as cells did not show any decrease in sensitivity to possible that relatively sm all changes in the TSH dePGE, after incubation with TSH. pendent moiety of adenyl cyclase have not been reA possible explanation for the refractoriness is that cognized under the experimental condition used the TSH remains on the cell surface in a biologically acresults of NaF stimulation indicate that major changes tive form. Evidence for persistent binding of active in adenylate cyclase activity are not caused by prior TSH was reported recently (DeRurertis, Chayoth, incubation with TSH. Zar and Field 1975). If this is true, very few recepRegardless of the mechanism it is likely that expotors will be available for newly added TSH. Other sure of human thyroid cells to TSH is associated with reports have shown, however, that [3H)-TSH bound a reduction in the number of TSH receptors, just as to receptors could be readily displaced by subseprior incubation of lymphocytes or fat cells with inquently added cold TSH within few minutes (Amir, sulin is associated with reduced number of insulin reCarraway and Kohn 1973). From this, a continuous ceptors. process of association-dissociation can be visualized and newly added TSH should be able to bind to the Acknowledgement receptors by displacing previously bound hormone. The absence of refractoriness in cells subject to short I am grateful to Dr. K. Ito, Ito Hospital, Tokyo for his gepreincubation time during which almost all available nerous supply of thyroid tissues from his surgical materials. I also wish to express my gratitude to Drs. H. Suzuki, T. Jureceptors would be occupied by excessive TSH added to the medium is an additional argument against ji (Dep. of Medicine and Blood Transfusion Service, University of Tokyo), N. Fleischer and R. Mortimcr (Dep. of Mepersistent receptor bin ding of TSH as a cause of redicine, Albert Einstein College of Medicinc) for their help fractoriness. and constructive remarks. An alternative explanation for the refractoriness is
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Thyroid cells, preincubated for 24 hours in medium with (B) or without (A) 100 mU/ml of TSH, were utilized. Adenylate cyclasc activity was assayed according to the procedure of Solmon ct al. (Solmon, Londos, Rodbel 1974). To dctcrmine the effect of theophylline on the cyclic AMP response to TSH, cells were stimulated with 100 mU/ml of TSH for 30 minutes at 37 0 C in the presence or absence of 1 mM theophylline.
206
N. Takasu, S. Sato, T. Yamada, M. Makiuchi, R. Furihata and M. Miyakawa
References
Kumon, A .. H. Yamamura, Y. Nishizuka: Mode of action of adenosine 3',5'-cyclic monophosphate on protein kinase from rat liver. Biochem.Biophys.Res.Commun. 41: 12901297 (1970) Kuo, J.F., E.C. DeRenzo: A comparison of the effects of lipolytic and antilipolytic agents on adenosine 3',5'-monophosphate levels in adipose cells as determined by prior labeling with adenosinc-8-14 C. J.Bio!.Chem. 244: 146-148 (1973) Lissitzky, S., G. Fayet, B. Varrier: Thyroid-stimulating hormone binding to cultured thyroid cells. FEBS Letters 29: 20-24 (1973) Moore, W. V., J. Wolf: Binding of prostagiandin EI to beef thyroid membranes. J.Bio!.Chem. 248: 5705-5711 (1973) Otten, J., G.S. Johnson, I. Pastan: Regulation of cell growth by cyclic adenosine 3',5'-monophosphate. Effect of cell density and agents which alter cell growth on cyclic adenosine 3',5'-monophosphate levels in fibroblasts. J.Bio!. Chem. 247: 7082-7087 (1974) Pohl, S.L., M.J. Krans, L. Birnbaumer, M. Rodbell: Inactivation of glucagon by plasma membrane of rat Iiver. J.Bio!. Chem. 247: 2295-2301 (1972) Robison, G.A., R. W. Butcher, I. Oye, H. W. Morgan, E. W. Sutherland: The effect of epinephrine on adenosine 3',5'phosphate levels in the isolated perfused rat heart. Molec.Pharmac. 1: 168-177 (1965) Salomon, Y., C. Londos, M. Rodbell: A high sensitive ade ny I cyclase assay. Analytical Biochemistry 58: 541-548 (1974) Shishiba, Y., M. Takaishi, Y. Miyachi, Y. Ozawa: Alteration of thyroid responsiveness to TSH under the influence of circulating thyroid hormone: Short feed-back regulatory effect. Endocrinologia Japonica 22: 367-370 (1975)
Requests for reprints should be addressed to: YOshiyasu Kaneko, Dep. of Medicine, AECOM, 1945-20D, Eastchester Rd, Bronx, NY 10461 (USA)
Horm. Metab. Res. 8 (1976) 206-211
© Georg Thieme Verlag Stuttgart
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Amir, S.M., T.F. Carraway Jr., L.D. Kohn: The binding of thyrotropin to isolated bovine thyroid plasma membranes. J.Bio!.Chem. 248: 4092-4100 (1973) Birnbaumer, F.R., S.L. Pohl, M.L. Krans, M. Rodbell: Action of hormones on the adenyl cyclase system. Advances in Biochemical Psychopharmacology 3: 185-208 (1970) DeRubertis, F.R., R. Chayoth, U. Zor, J.B. Field: Evidence for persistent binding of biologically active thyrotropin to thyroid in vitro. Endocrinology 96: 1579-1586 (1975) FrankIin, T.J., S.J. Foster: Hormone-induced desensitization of hormonal control of cyclic AMP level in human diploid fibroblast. Nature New Bio!. 246: 146-148 (1973) Gavin, J.R. 111, J. Roth, D.M. Neville Jr., P.D. Mayts, D.N. Buell: Insulin-dependellt regulation of insulin receptor concentrations: A direct demonstration in cell culture. Proc.Nat.Acad.Sci. USA 71: 84-88 (1974) Gilman, A.G.: A pro tein binding assay for adenosine 3',5'cyclic monophosphate. Proc.Nat!.Acad.Sci. USA 67: 305312 (1972) Ho, R..J., E. W. Sutherland: Formation and release of hormone antagonist by rat adipocytes. J.Biol.Chem. 246: 6822-6827 (1971) Huang, D., P. Cuatrecasas: Insulin-induced reduction of membrane receptor concentrations in isolated fat cells and Iymphocytes. J.BioI.Chem. 250: 8251-8259 (1975) Jonsson, J., A. Fagraeus: On the mechanism of the ring zone effect obtained with the mixed haemoadsorption technique. Studies with human anti-thyroid sera reacting with thyroid monolayer cultures. Immunology 17: 385-411 (1969) Kakiuchi, s., T. W. Rall, H. Mcllwain: The effect of electrical stimulation upon the accumulation of adenosine 3',5'monophosphate in isolated cerebral tissue. J.Neurochem. 16: 485-491 (1969)
The Different Modes of Action of Thyrotropin and Prostaglandin E I on Cyclic Adenosine 3',5'-Monophosphate Synthesis in Human Thyroid, as Studied by Sequential Stimulations* N. Takasu, S. Sato, T. Yamada, M. Makiuchi, R. Furihata and M. Miyakawa Department of Medicine, Institute of Adaptation Medicine. and Second Department of Surgery. School of Medicine, Shinshu University. Matsumoto. Japan
Summary PGE I was equally effective in increasing 3 H-cyclic AMP in normal and in toxie thyroids, whereas TSH was less effective but over a longer time in the toxie thyroids. Stimulation by a large second dose of TSH could not be elicited after prior stimulation by large doses of TSH. Similar results *Supported by a Grant from thc Ministry of Education of Japan Received: 15 Sept. 1975
Accepted: 5 Jan. 1976
were obtained with regard to the effect of PGE l' However, stimulation by a large dose of PGE I was still effcctive after the slices be ca me refractory to TSH. Similarly, stimulation by a large dose of TSH was still effective after the slices became refractory to PGE I' It is suggested that the site and/or mode of action of TSH is quite different from that of PGE l '
Key-Words: Thyrotropin - Prostagiandin EI - Cyc1ic AMP - Human Thyroid
