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Studies on the mechanism of desensitization of the cyclic amp response to tsh stimulation in a cloned rat thyroid cell line
Molecular and CeNular Endocrinology, 42 (1985) 21-27 Elsevier Scientific Publishers Ireland, Ltd.
21
MCE 01346
Studies on the mechanism of desensitization of the cyclic AMP response to TSH stimulation in a cloned rat thyroid cell line Hideshi Hirayu, Ronald P. Magnusson and Basil Rapoport The Departments of Medicine, V.A. Medical Center, Metabolism (IIIF), 4150 Clement Street, San Francisco, CA 94121, and the University of California, San Francisco, CA (U.S.A.) (Received
Keywork
TSH; thyroid
8 March 1985; accepted
22 April 1985)
cells; cyclic AMP; desensitization.
We examined aspects of the mechanism of desensitization of adenylate cyclase activation by TSH in a cloned line of rat thyroid cells (FRTL). Increasing FRTL intracellular CAMP concentrations by preincubation for 6 h in either 1 mM dBcAMP or 100 PM forskolin did not induce TSH desensitization. Forskolin stimulation was unimpaired in TSH-desensitized cells, indicating ‘uncoupling’ of the adenylate cyclase catalytic unit from the TSH receptor. Stimulation by the Ni inhibitory pathway of adenylate cyclase by epinephrine (10e6 M-10e4 M in the presence of low4 M propranolol) was unaltered in cells previously desensitized to TSH. That is, Ni-mediated inhibition of adenylate cyclase was additive to TSH desensitization. Pre-exposure of FRTL cells for 18 h to 50 ng/ml pertussis toxin did not prevent the induction of TSH desensitization. TSH desensitization was prevented by cycloheximide or actinomycin D added during the last 3-4 h of a 6 h period of TSH stimulation. The rates of turnover of the putative desensitization protein and its mRNA therefore appear to be similar.
Prior exposure of thyroid tissue to TSH in vitro (Kaneko, 1976; Rapoport, 1976; Rapoport and Adams, 1976; Shuman et al., 1976; Takasu et al., 1976), and in vivo (Field et al., 1979; Holmes et al., 1980; Zakarija et al., 1980; Gerber et al., 1981; Laurberg, 1981; Ahren et al., 1982) leads to partial desensitization to further TSH stimulation. Data from most (Kaneko, 1976; Rapoport, 1976; Rapoport and Adams, 1976; Shuman et al., 1976; Takasu et al., 1976), but not all (Levasseur et al., 1982), studies have indicated that TSH desensitization involves, at least in part, reduced activation of thyroid adenylate cyclase. Many questions exist regarding the mechanism by which desensitization of adenylate cyclase activation by TSH is induced. For example, previous studies from our laboratory 0303-7207/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
suggested that TSH desensitization in cultured thyroid cells involves decreased coupling of the TSH receptor with the stimulatory regulatory protein N, (Rapoport et al., 1982). The functional activity of N, and the adenylate cyclase catalytic unit, however, remained intact (Rapoport et al., 1982). Totsuka et al. (1983), on the other hand, provided evidence that N, is indeed involved in TSH desensitization. In recent years an inhibitory adenylate cyclase regulatory protein (Ni) has been discovered (Katada and Ui, 1982; Hildebrandt et al., 1983; Murayama and Ui, 1983; Katada et al., 1984), and appears to be functionally active in the thyroid (Yamashita et al. 1977; Cochaux et al., 1982; Filetti and Rapoport, 1983). There has therefore been speculation that Ni may be inLtd.
22
volved in the process of hormone desensitization in a variety of tissues, including the thyroid. Among other questions regarding the mechanism of TSH desensitization is whether or not the induction of this process is CAMP-dependent. Avivi et al., using FRTL, rat thyroid cells have recently reported that CAMP does induce TSH desensitization (Avivi et al., 1981). These data contrast with those obtained with cultured dog (Rapoport and Adams, 1976), and porcine (Takasu et al., 1978) thyroid cells which suggest that the mechanism of TSH desensitization is CAMP-independent. Inhibition of protein synthesis by cycloheximide or histidinol in dog (Rapoport and Adams, 1976) human (Filetti and Rapoport, 1982) and porcine (Takasu et al., 1978) thyroid cells in monolayer culture, prevents the induction of TSH desensitization, suggesting the possible existence of an inhibitory protein that modulates adenylate cyclase Inhibition of RNA synthesis by activation. actinomycin D similarly prevents the induction of TSH desensitization (Takasu et al., 1978; Rapoport et al., 1983), possibly by reducing the amount of mRNA available for the translation of this inhibitory protein. No data are available on the relative turnover rates of this putative protein and its mRNA. In the present studies we addressed some of these issues using a clonal line of rat thyroid cells (FRTL). These cells provide the important advantage of not being ‘contaminated’ with nonthyroidal cells. All previous studies using primary cultures of dog, human or pig thyroid cells, thyroid slices of plasma membranes have also contained a variety of other cell types including fibroblasts and endothelial cells. These nonthyroidal cells also contain adenylate cyclase that can be activated by agents such as fluoride, GTP or forskolin, and therefore complicate interpretation of the data obtained. The present data suggest that (1) in contrast to prior data in cloned rat thyroid cells, TSH desensitization is not induced by CAMP, (2) Ni does not play a role in the mechanism of desensitization of adenylate cyclase activation by TSH, and (3) in cultured thyroid cells the rates of turnover of the functional activities of the putative inhibitory protein and its mRNA are similar and relatively rapid.
Methods and materials Thyroid cell cultures FRTL rat thyroid cells were kindly provided by Dr. Hayden Coon. These cells were propagated as described by Ambesi-Impiombato et al., (1980) with minor modifications. Coon’s modified F-12 medium was supplemented with 2% fetal calf serum, 5 mu/ml bovine TSH, 10 mu/ml insulin and 5 pg/ml transferrin. Somatostatin, hydrocortisone and glycyl-L-histidyl-r_-lysine acetate were omitted. Cells were subcultured using Minimum Essential Medium, without Ca2’ or Mg*+, supplemented with 0.05% trypsin and 0.2% EDTA. For individual experiments, FRTL cells were passed from 100 mm diameter into 35 mm diameter culture dishes. Cells were allowed to adhere to the dishes and to recover from trypsinization for at least 3 days prior to use. Unless indicated otherwise, cells were rinsed 3 times with phosphatebuffered saline, pH 7.4, in order to remove the TSH, and they were then incubated overnight in TSH-free Coon’s modified F-12 medium. On the following day, first incubations of up to 6 h were performed in Ml99 medium containing 10% fetal calf serum and 20 mM Hepes, pH 7.4, in a waterjacketed incubator at 37°C. Where indicated, second (30 min) incubations were conducted in Leibovitz-15 medium containing 0.5 mM 3-isobutyl-l-methylxanthine and 20 mM Hepes, pH 7.4. in room air in a water bath at 37°C. CAMP radioimmunoassay Intracellular CAMP was measured by a modification of the method previously reported by our laboratory (Rapoport, 1976) and Steiner et al., 1972. Immediately after aspiration of the medium, 1 ml of 95% ethanol, cooled with dry ice, was added to the cell monolayers. Cold ethanol was added in order to prevent a possible ethanolinduced burst of CAMP generation before killing of the cells. After 15-30 min at 4°C or room temperature the ethanol in the dishes was well mixed and then transferred to 13 x 100 mm glass tubes. The ethanol was evaporated in a stream of air in a water bath at 60°C. The CAMP was then resuspended in 50 mM sodium acetate, pH 6.2, for radioimmunoassay. In preliminary experiments we determined that ethanol extraction of CAMP was
as efficient as extraction with trichloroacetic acid, the method previously used in our laboratory, and has the advantage of not requiring ether extraction of the sample. Radioimmunoassay of CAMP was performed as previously described (Steiner et al., 1972; Rapoport, 1976), with the exception that bound and free ligand was separated using 2.5 ml of 95% ethanol, chilled with dry ice. After approximately 15 min at 4°C the tubes were centrifuged for 15 min at 3000 rpm in a IEC PR-6000 centrifuge. Materials Materials were obtained from the following sources: bovine TSH from Armour Pharmaceutical Company, Kankakee, IL; cycloheximide, actinomycin D, 3-isobutyl-l-methylxanthine, dibutyryl CAMP, epinephrine and propranolol from Sigma Chemical Co., St. Louis, MO. Forskolin from Calbiochem, La Jolla, CA; antibody for the radioimmunoassay from Becton-Dickinson Co., Orangeburg, NY. Tissue culture media and additives were obtained from the Cell Culture Facility, University of California San Francisco. Pertussis toxin was kindly provided by Dr. E. Hewlett, University of Virginia, Charlottesville, VA. Results Because the FRTL cells are propagated in medium containing TSH, it was first necessary to determine the optimal period of withdrawal from TSH prior to the reinduction of TSH desensitization. Recovery from TSH desensitization was found to have occurred within 3-4 h of TSH withdrawal (Table 1). In subsequent studies cells were therefore prepared by incubation overnight in TSH-free medium. The rate of induction of TSH desensitization in resensitized FRTL cells was also rapid. In a representative experiment (Fig. l), desensitization of 48% was present within 60 min of TSH stimulation, and desensitization of 77% after 4 h. Role of CAMP in the induction of TSH desensitization Two approaches were attempted in order to determine whether or not CAMP is involved in the
TABLE
1
TIME PERIOD OF RECOVERY ZATION IN FRTL CELLS Time period of recovery (h) 6 (basal) 0 1 2 3 4 5 6
FROM
a Cellular CAMP (pmol/dish) 2.4+ 1.1 ’ 27.0+0.8 26.6 f 0.9 46.9 f 3.0 58.7+2.5 74.6+4.2 66.2f4.9 68.0 f 8.9
TSH DESENSITI-
% of maximum
b
_ 39.7 39.1 69.0 86.3 109.7 97.4 100.0
a FRTL cells cultured in Coon’s modified F-12 medium were stimulated for 18 h with 50 mu/ml TSH. After 3 washes in phosphate-buffered saline, pH 7.4, the cells were incubated for up to 6 h in the same medium without TSH. All cells (except basal) were then incubated for 30 min at 37°C in L-15 medium, 20 mM Hepes, pH 7.4, containing 50 mu/ml TSH and 0.5 mM 3-isobutyl-l-methylxanthine. Cellular CAMP was measured by RIA, as described in Methods and materials. b Based on cellular CAMP content after 6 h of recovery, at which time maximum recovery from desensitization has clearly occurred. ’ Mean rfrSEM of values obtained in triplicate dishes of cells, each containing approximately 100 pg of cellular protein.
induction of TSH desensitization. First, resensitized cells were incubated in 1 mM dBcAMP for 6 h in an attempt to mimic TSH-induced desensitization. Second, resensitized cells were treated for a similar time period with 10 PM or 100 PM forskolin, a potent stimulator of CAMP generation in intact cells. Neither dBcAMP nor forskolin induced TSH desensitization in FRPL cells (Fig. 2). Indeed, forskolin actually enhanced the subsequent CAMP response to TSH stimulation. As previously reported with cultured human thyroid cells (Rapoport et al., 1982), preincubation in 50 mu/ml TSH for 6 h did not reduce (but instead increased) the subsequent CAMP response to forskolin stimulation in FRTL cells. Role of N, in TSH desensitization There has been speculation that the newly discovered regulatory protein of adenylate cyclase complex (Ni) may be involved in the mechanism of induction of TSH desensitization. In order to test this hypothesis, the activity of the Ni inhibitory pathway during TSH desensitization was
24
0
1
2
3
4
Time (h)
Fig. 1. Time course of desensitization of the CAMP response to TSH stimulation. FRTL thyroid cells were incubated overnight without TSH. The cells were then exposed for the indicated time periods in L-15 medium containing 0.5 mM 3-isobutyl-lmethylxanthine and 50 mu/ml bTSH. Cellular CAMP was extracted and measured by RIA as described in Methods and materials. Each point represents mean f SEM of data obtained in triplicate dishes of cells.
probed by cY,-adrenergic stimulation. Inhibition of the CAMP response to TSH by 10-6M-10-4 M epinephrine in the presence of lop4 M proprano101 (a,-adrenergic stimulation) was unaltered in FRTL thyroid cells already desensitized to TSH (Fig. 3). That is Ni activation and TSH desensitization were additive. We also examined the role of Ni in TSH desensitization by attempting to inactive Ni using pertussis toxin. Thus, if Ni plays a role in the induction of TSH desensitization, its inactivation would be expected to prevent TSH desensitization. Preincubation of FRTL cells for 18 h in 50 ng/ml pertussis toxin was without effect on subsequent TSH stimulation of cellular CAMP generation, or on desensitization of the CAMP response to TSH stimulation (data not shown). However, pre-exposure of FRTL cells to pertussigen did not block the inhibitory effect of ol,-adrenergic stimulation (lop4 M epinephrine + lop4 M propranolol) on the CAMP response to TSH stimulation (data not shown). In contrast, the same pertussigen preparation was a potent inhibitor of GTP-induced inhibition in opossum kidney cells (A. Teitelbaum, personal communication).
Fig. 2. Role of CAMP in the induction of TSH desensitization. FRTL thyroid cells were preincubated for 6 h in either control (CON) medium or in medium supplemented with 50 mU/ml TSH, 1 mM dRcAMP, or in the indicated concentrations of forskolin (FORSK). The cells were then washed 3 times with phosphate-buffered saline, pH 7.4, after which they were incubated for 30 mm in L-15 medium supplemented with 0.5 mM 3-isobutyl-1-methylxanthine and the indicated additives. Cellular CAMP was extracted and measured by RIA as described in Methods and materials. Each bar represents the mean*SEM of values obtained in triplicate dishes of cells.
Roles of protein and RNA synthesis in TSH desensitization Support for the concept that synthesis of an inhibitory protein is necessary for the induction of TSH desensitization has been provided by the ability of cycloheximide or actinomycin D to prevent the induction of TSH desensitization in cultured thyroid cells (Takasu et al., 1978; Rapoport et al., 1983). TSH could enhance the translation of this protein, or the transcription of its mRNA. In order to help distinguish between these two alternatives we examined the relative turnover rates of the activities of the inhibitory protein and its mRNA by the addition of lop4 M cycloheximide or 1 pg/ml actinomycin D to the culture medium at different time intervals during a continual 6 h period of TSH stimulation. TSH desensitization was prevented by the presence of either agent for at least the last 4 h of the 6 h TSH stimulation period (Fig. 4). As a corollary, TSH desensitization was still apparent when cycloheximide or
25
150
2 4
100
1 g ,P n. 3 50
0
D
Fig. 4. Relationship between the duration of inhibition of protein and RNA synthesis and the prevention of TSH desensitization. FRTL thyroid cells were preincubated for 6 h in either control medium (CON), or in medium containing 50 mu/ml TSH. During the time intervals represented by the hatched bars 10m4 M cycloheximide or 1 pg/ml actinomycin D were also present. At the end of this 6 h preincubation period, the cells underwent a second (30 min) incubation in L-15 medium containing 0.5 mM 3-isobutyl-1-methylxanthine with or without (CON) 50 mu/ml TSH. Cellular CAMP levels were extracted and measured as described in Methods and materials. Each bar represents the meanf SEM of values obtained in triplicate dishes of cells.
100
E z ._
75
$ B $
50
25
0 0
10-6
10-4
1o-s Epinephrine
(AA)
Fig. 3. The inhibitory (Ni) pathway of adenylate cyclase activation in TSH desensitization. Thyroid cells were preincubated for 6 h in either control (CONTROL) medium or in medium containing 50 mu/ml TSH. All cells then underwent a second incubation (30 min) in L-15 medium containing 50 mu/ml TSH, 0.5 mM 3-isobutyl-l-methylxanthine, lo-’ M proprano101, and the indicated concentrations of epinephrine. Cellular CAMP was extracted and measured as described in Methods and materials. In the upper panel each point represents the mean f SEM of CAMP values determined in triplicate dishes of cells. In the lower panel the same data are expressed as a percentage of the maximum CAMP values observed in the absence of a,-adrenergic inhibition by epinephrine + propranolol.
actinomycin D was present during the last 3 h of the 6 h period. In 3 experiments this latter time period varied between 2 and 3 h. These data
indicate that in FRTL cells the functional activities of the putative inhibitory protein and its mRNA each persist for approximately 3 h after their synthesis has been inhibited. That is their rates of disappearance are similar. Discussion The present data provide further insight into our understanding of the mechanism of desensitization of adenylate cyclase activation by TSH in thyroid cells. First, an important and puzzling discrepancy in the literature is the role of CAMP in the mechanism of TSH desensitization (Rapoport and Adams, 1976; Takasu et al., 1978; Avivi et al., 1981). It is surprising that conflicting data should be obtained with FRTL (present study) and FRTL, (Avivi et al., 1981), cells, which are closely related
26
variants of the same cell line. Our data support the thesis that TSH desensitization is homologous (Shuman et al., 1976), and that CAMP is not directly involved in this process. The reason for these discrepant results is unknown, but is possibly methodological. Thus Avivi et al. (1981) first prelabeled the intracellular ATP pool with [3H]adenosine, then incubated the cells in the presence or absence of dBcAMP, and finally, in a third incubation, stimulated the cells with TSH, prior to extraction and measurement of radioactive CAMP. It is therefore possible that the specific activity of the labeled ATP precursor was altered by preincubation in dBcAMP. In contrast, in the present study, CAMP was measured directly by radioimmunoassay, which is not subject to such an artifact. Even stronger evidence that CAMP is not involved in the induction of TSH desensitization was obtained using forskolin. Although the action of this compound is complex, and is modulated by the adenylate cyclase regulatory proteins, it is nevertheless a potent direct activator of the catalytic subunit of adenylate cyclase (Seamon and Daly, 1981; Pfeuffer and Metzger, 1982). Forskolin is extremely useful in that, unlike fluoride and GTP analogues such as Gpp(NH)p, it acts on intact cells. Our data demonstrate that despite elevation of intracellular CAMP to levels greater than those achieved by TSH stimulation, desensitization to TSH was not induced. These data suggest that CAMP does not play a direct role in the induction of TSH desensitization. In support of data obtained with human thyroid cells (Rapoport et al., 1982), and bovine thyroid slices (Totsuka et al., 1983), our results also demonstrate in another cell line (FRTL) that forskolin activation is unaltered in TSH-desensitized thyroid cells. This finding is strong evidence that the alteration in the adenylate cyclase complex in TSH desensitization is proximal to, and does not involve, the catalytic unit itself. Finally our data (Fig. 2) indicate that in FRTL cells, unlike in guinea pig thyroid tissue (Fradkin et al., 1982), preincubation in forskolin enhances the subsequent CAMP response to TSH stimulation. This finding is in agreement with the original observation of Daly et al. (1982) that forskolin enhances receptor-mediated responses. Adenylate cyclase activation by hormones is
regulated by a stimulatory regulatory protein (N,) and a more recently recognized inhibitory regulatory protein (Ni) (Yamashita et al., 1977; Katada and Ui, 1982; Hildebrandt et al., 1983; Murayama and Ui, 1983; Katada et al., 1984). Previous data from our laboratory suggested that the N, pathway is unaltered in TSH desensitization (Rapoport et al., 1982). With the discovery of the Ni pathway an attractive hypothesis was that TSH desensitization involved activation of the Ni pathway. Our data demonstrate that this is not likely to be the case. Thus control and TSH desensitized thyroid cells are equally sensitive to cy,-adrenergic inhibition of adenylate cyclase activation. That is CQadrenergic stimulation is additive with that of TSH desensitization, suggesting that these two actions involve separate pathways. More direct evidence against involvement of the N, pathway in TSH desensitization would be that TSH desensitization still occurs even when this pathway is inactivated by pertussis toxin. Pre-exposure of thyroid cells to pertussis toxin did not prevent the subsequent induction of TSH desensitization, however this evidence was inconclusive. This was because the pertussis toxin used, even though potent in another cell system, did not prevent a,-adrenergic inhibition of TSH stimulation in FRTL cells. The reason for this difference between tissues in their susceptibility to pertussis toxin is unclear. Nevertheless such differences have been reported (Katada et al., 1983), and may involve the ability of different tissues to activate the pertussis toxin by separation into A and B promoters. It is therefore possible that cultured thyroid cells belong to that group of cells that lack the ability to activate pertussigen. It is also interesting that pretreatment with pertussis toxin did not alter the CAMP response to TSH stimulation in FRTL cells, unlike the enhancement reported in dog thyroid slices (Katori and Yamashita, 1982). The ability of inhibitors of protein and RNA synthesis to prevent desensitization of activation by TSH of adenylate cyclase in thyroid cell cultures suggests the existence of a protein that is capable of modulating the activity of this enzyme (Rapoport and Adams, 1976). TSH could regulate the synthesis of this putative protein at the ribosomal level or by influencing transcription of its mRNA. Once synthesized, the activity of this pro-
27
tein could also be modulated by post-translational modifications, such as ADP-ribosylation (Filetti and Rapoport, 1981; Filetti et al. 1981) or phosphorylation. The present data suggest that, in FRTL thyroid cells, the rates of turnover of the functional activities of this protein and its mRNA are similar, and relatively rapid. Thus their functional activities persist for approximately 3 h after their synthesis has been inhibited. This time period is consistent with the time period of recovery from desensitization observed following withdrawal of TSH (Table 1). From the foregoing information it is not possible to deduce whether the primary action of TSH in the induction of desensitization is at the transcriptional or translational level. A turnover rate for the mRNA slower than that of the protein would have been evidence supporting an action of TSH at the translational or posttranslational level. Experiments of a different approach will be necessary to resolve this issue. Acknowledgements These studies were supported by NIH Grant AM 19289 and the Medical Research Service of the Veterans’ Administration. The expert secretarial assistance of Peggy Mathews is gratefully acknowledged. References Ahren, B., Gustafson, A. and Hedner, P. (1982) Life Sci. 31, 2583. Ambesi-Impiombato, F.S., Parks, L.A.M. and Coon, H.G. (1980) Proc. Nat. Acad. Sci. (U.S.A.) 77, 3455-3459. Avivi, A., Tramontano, D., Ambesi-Impiombato, F.S. and Schlessinger, .I. (1981) Science 214, 1237-1239. Cochaux, P., Van Sande, J. and Dumont, J.E. (1982) B&him. Biophys. Acta 721, 39-46. Daly, J.W., Padgett, W. and Seamon, K.B. (1982) J. Neurothem. 38, 532-544. Field, J.B., Dekker, A., Titus, G., Kerins, M.E., Worden, W. and Frumess, R. (1979) J. Clin. Invest. 64, 265-271. Filetti, S. and Rapoport, B. (1981) J. Clin. Invest. 68, 461-467.
Filetti, S. and Rapoport, B. (1982) J. Biol. Chem. 257; 1342-1346. Filetti, S. and Rapoport, B. (1983) Endocrinology 113, 1608-1615. Filetti, S., Takai, N.A. and Rapoport, B. (1981) J. Biol. Chem. 256, 1072-1075. Fradkin, J.E., Cook, G.H., Kilhoffer, M.-C. and Wolff, J. (1982) Endocrinology 111, 849-856. Gerber, H., Studer, H., Conti, A., Engler, H., Kohler, H. and Haeberli, A. (1981) J. Clin. Invest. 68, 1138-1347. Hildebrandt, J.D., Sekura, R.D., Codina, J., lyengar, R., Manclark, C.R. and Birnbaumer, L. (1983) Nature (Lond.) 302, 706-709. Holmes, S.D., Gitlin, J., Titus, G. and Field, J.B. (1980) Endocrinology 106, 1892-1899. Kaneko, Y. (1976) Horm. Metab. Res. 8, 202-206. Katada, T. and Ui, M. (1982) J. Biol. Chem. 257. 7210-7216. Katada, T., Tamura, M. and Ui, M. (1983) Arch. B&hem. Biophys. 224, 290-298. Katada, T., Northup, J.K., Bokoch, G.M., Ui, M. and Gilman, A.G. (1984) J. Biol. Chem. 259, 3578-3585. Katori, A. and Yamashita, K. (1982) Endocrinol. Jpn. 29, 261-263. Laurberg, P. (1981) Endocrinology 109, 1560-1565. Levasseur, S., Kostelec, M. and Burke, G. (1982) Clin. Res. 30, 721A. Murayama, T. and Ui, M. (1983) J. Biol. Chem. 258,3319-3326. Pfeuffer, T. and Metzger, H. (1982) FEBS Lett. 146, 369-375. Rapoport, B. (1976) Endocrinology 98, 1189-1197. Rapoport, B. and Adams, R.J. (1976) J. Biol. Chem. 251, 6653-6661. Rapoport, B., Filetti, S., Takai, N. and Seto, P. (1982) FEBS Lett. 146, 23-27. Rapoport, B., Filetti, S. and Takai, N.A. (1983) Endocrinology 112, 1874-1876. Seamon, K. and Daly, J.W. (1981) J. Biol. Chem. 256, 9799-9801. Shuman, S.J., Zor, U., Chayoth, R. and Field, J.B. (1976) J. Clin. Invest. 57, 1132-1141. Steiner, A.L., Paghara, A.S., Chase, L.R. and Kipnis, D.M. (1972) J. Biol. Chem. 247, 1114-1120. Takasu, N., Sato, S., Yamada, T., Makiuchi, M., Furihata, R. and Miyakawa, M. (1976) Horm. Metab. Res. 8, 206-211. Takasu, N., Chartier, B., Mauchamp, J. and Lissitzky, S. (1978) Eur. J. B&hem. 90, 131-138. Totsuka, Y., Nielsen, T.B. and Field, J.B. (1983) Endocrinology 113, 1088-1095. Yamashita, K., Yamashita, S. and Ogata, E. (1977) Life Sci. 21, 607-611. Zakarija, M., Witte, A. and McKenzie, J.M. (1980) Endocrinology 197, 2045-2050.
21
MCE 01346
Studies on the mechanism of desensitization of the cyclic AMP response to TSH stimulation in a cloned rat thyroid cell line Hideshi Hirayu, Ronald P. Magnusson and Basil Rapoport The Departments of Medicine, V.A. Medical Center, Metabolism (IIIF), 4150 Clement Street, San Francisco, CA 94121, and the University of California, San Francisco, CA (U.S.A.) (Received
Keywork
TSH; thyroid
8 March 1985; accepted
22 April 1985)
cells; cyclic AMP; desensitization.
We examined aspects of the mechanism of desensitization of adenylate cyclase activation by TSH in a cloned line of rat thyroid cells (FRTL). Increasing FRTL intracellular CAMP concentrations by preincubation for 6 h in either 1 mM dBcAMP or 100 PM forskolin did not induce TSH desensitization. Forskolin stimulation was unimpaired in TSH-desensitized cells, indicating ‘uncoupling’ of the adenylate cyclase catalytic unit from the TSH receptor. Stimulation by the Ni inhibitory pathway of adenylate cyclase by epinephrine (10e6 M-10e4 M in the presence of low4 M propranolol) was unaltered in cells previously desensitized to TSH. That is, Ni-mediated inhibition of adenylate cyclase was additive to TSH desensitization. Pre-exposure of FRTL cells for 18 h to 50 ng/ml pertussis toxin did not prevent the induction of TSH desensitization. TSH desensitization was prevented by cycloheximide or actinomycin D added during the last 3-4 h of a 6 h period of TSH stimulation. The rates of turnover of the putative desensitization protein and its mRNA therefore appear to be similar.
Prior exposure of thyroid tissue to TSH in vitro (Kaneko, 1976; Rapoport, 1976; Rapoport and Adams, 1976; Shuman et al., 1976; Takasu et al., 1976), and in vivo (Field et al., 1979; Holmes et al., 1980; Zakarija et al., 1980; Gerber et al., 1981; Laurberg, 1981; Ahren et al., 1982) leads to partial desensitization to further TSH stimulation. Data from most (Kaneko, 1976; Rapoport, 1976; Rapoport and Adams, 1976; Shuman et al., 1976; Takasu et al., 1976), but not all (Levasseur et al., 1982), studies have indicated that TSH desensitization involves, at least in part, reduced activation of thyroid adenylate cyclase. Many questions exist regarding the mechanism by which desensitization of adenylate cyclase activation by TSH is induced. For example, previous studies from our laboratory 0303-7207/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
suggested that TSH desensitization in cultured thyroid cells involves decreased coupling of the TSH receptor with the stimulatory regulatory protein N, (Rapoport et al., 1982). The functional activity of N, and the adenylate cyclase catalytic unit, however, remained intact (Rapoport et al., 1982). Totsuka et al. (1983), on the other hand, provided evidence that N, is indeed involved in TSH desensitization. In recent years an inhibitory adenylate cyclase regulatory protein (Ni) has been discovered (Katada and Ui, 1982; Hildebrandt et al., 1983; Murayama and Ui, 1983; Katada et al., 1984), and appears to be functionally active in the thyroid (Yamashita et al. 1977; Cochaux et al., 1982; Filetti and Rapoport, 1983). There has therefore been speculation that Ni may be inLtd.
22
volved in the process of hormone desensitization in a variety of tissues, including the thyroid. Among other questions regarding the mechanism of TSH desensitization is whether or not the induction of this process is CAMP-dependent. Avivi et al., using FRTL, rat thyroid cells have recently reported that CAMP does induce TSH desensitization (Avivi et al., 1981). These data contrast with those obtained with cultured dog (Rapoport and Adams, 1976), and porcine (Takasu et al., 1978) thyroid cells which suggest that the mechanism of TSH desensitization is CAMP-independent. Inhibition of protein synthesis by cycloheximide or histidinol in dog (Rapoport and Adams, 1976) human (Filetti and Rapoport, 1982) and porcine (Takasu et al., 1978) thyroid cells in monolayer culture, prevents the induction of TSH desensitization, suggesting the possible existence of an inhibitory protein that modulates adenylate cyclase Inhibition of RNA synthesis by activation. actinomycin D similarly prevents the induction of TSH desensitization (Takasu et al., 1978; Rapoport et al., 1983), possibly by reducing the amount of mRNA available for the translation of this inhibitory protein. No data are available on the relative turnover rates of this putative protein and its mRNA. In the present studies we addressed some of these issues using a clonal line of rat thyroid cells (FRTL). These cells provide the important advantage of not being ‘contaminated’ with nonthyroidal cells. All previous studies using primary cultures of dog, human or pig thyroid cells, thyroid slices of plasma membranes have also contained a variety of other cell types including fibroblasts and endothelial cells. These nonthyroidal cells also contain adenylate cyclase that can be activated by agents such as fluoride, GTP or forskolin, and therefore complicate interpretation of the data obtained. The present data suggest that (1) in contrast to prior data in cloned rat thyroid cells, TSH desensitization is not induced by CAMP, (2) Ni does not play a role in the mechanism of desensitization of adenylate cyclase activation by TSH, and (3) in cultured thyroid cells the rates of turnover of the functional activities of the putative inhibitory protein and its mRNA are similar and relatively rapid.
Methods and materials Thyroid cell cultures FRTL rat thyroid cells were kindly provided by Dr. Hayden Coon. These cells were propagated as described by Ambesi-Impiombato et al., (1980) with minor modifications. Coon’s modified F-12 medium was supplemented with 2% fetal calf serum, 5 mu/ml bovine TSH, 10 mu/ml insulin and 5 pg/ml transferrin. Somatostatin, hydrocortisone and glycyl-L-histidyl-r_-lysine acetate were omitted. Cells were subcultured using Minimum Essential Medium, without Ca2’ or Mg*+, supplemented with 0.05% trypsin and 0.2% EDTA. For individual experiments, FRTL cells were passed from 100 mm diameter into 35 mm diameter culture dishes. Cells were allowed to adhere to the dishes and to recover from trypsinization for at least 3 days prior to use. Unless indicated otherwise, cells were rinsed 3 times with phosphatebuffered saline, pH 7.4, in order to remove the TSH, and they were then incubated overnight in TSH-free Coon’s modified F-12 medium. On the following day, first incubations of up to 6 h were performed in Ml99 medium containing 10% fetal calf serum and 20 mM Hepes, pH 7.4, in a waterjacketed incubator at 37°C. Where indicated, second (30 min) incubations were conducted in Leibovitz-15 medium containing 0.5 mM 3-isobutyl-l-methylxanthine and 20 mM Hepes, pH 7.4. in room air in a water bath at 37°C. CAMP radioimmunoassay Intracellular CAMP was measured by a modification of the method previously reported by our laboratory (Rapoport, 1976) and Steiner et al., 1972. Immediately after aspiration of the medium, 1 ml of 95% ethanol, cooled with dry ice, was added to the cell monolayers. Cold ethanol was added in order to prevent a possible ethanolinduced burst of CAMP generation before killing of the cells. After 15-30 min at 4°C or room temperature the ethanol in the dishes was well mixed and then transferred to 13 x 100 mm glass tubes. The ethanol was evaporated in a stream of air in a water bath at 60°C. The CAMP was then resuspended in 50 mM sodium acetate, pH 6.2, for radioimmunoassay. In preliminary experiments we determined that ethanol extraction of CAMP was
as efficient as extraction with trichloroacetic acid, the method previously used in our laboratory, and has the advantage of not requiring ether extraction of the sample. Radioimmunoassay of CAMP was performed as previously described (Steiner et al., 1972; Rapoport, 1976), with the exception that bound and free ligand was separated using 2.5 ml of 95% ethanol, chilled with dry ice. After approximately 15 min at 4°C the tubes were centrifuged for 15 min at 3000 rpm in a IEC PR-6000 centrifuge. Materials Materials were obtained from the following sources: bovine TSH from Armour Pharmaceutical Company, Kankakee, IL; cycloheximide, actinomycin D, 3-isobutyl-l-methylxanthine, dibutyryl CAMP, epinephrine and propranolol from Sigma Chemical Co., St. Louis, MO. Forskolin from Calbiochem, La Jolla, CA; antibody for the radioimmunoassay from Becton-Dickinson Co., Orangeburg, NY. Tissue culture media and additives were obtained from the Cell Culture Facility, University of California San Francisco. Pertussis toxin was kindly provided by Dr. E. Hewlett, University of Virginia, Charlottesville, VA. Results Because the FRTL cells are propagated in medium containing TSH, it was first necessary to determine the optimal period of withdrawal from TSH prior to the reinduction of TSH desensitization. Recovery from TSH desensitization was found to have occurred within 3-4 h of TSH withdrawal (Table 1). In subsequent studies cells were therefore prepared by incubation overnight in TSH-free medium. The rate of induction of TSH desensitization in resensitized FRTL cells was also rapid. In a representative experiment (Fig. l), desensitization of 48% was present within 60 min of TSH stimulation, and desensitization of 77% after 4 h. Role of CAMP in the induction of TSH desensitization Two approaches were attempted in order to determine whether or not CAMP is involved in the
TABLE
1
TIME PERIOD OF RECOVERY ZATION IN FRTL CELLS Time period of recovery (h) 6 (basal) 0 1 2 3 4 5 6
FROM
a Cellular CAMP (pmol/dish) 2.4+ 1.1 ’ 27.0+0.8 26.6 f 0.9 46.9 f 3.0 58.7+2.5 74.6+4.2 66.2f4.9 68.0 f 8.9
TSH DESENSITI-
% of maximum
b
_ 39.7 39.1 69.0 86.3 109.7 97.4 100.0
a FRTL cells cultured in Coon’s modified F-12 medium were stimulated for 18 h with 50 mu/ml TSH. After 3 washes in phosphate-buffered saline, pH 7.4, the cells were incubated for up to 6 h in the same medium without TSH. All cells (except basal) were then incubated for 30 min at 37°C in L-15 medium, 20 mM Hepes, pH 7.4, containing 50 mu/ml TSH and 0.5 mM 3-isobutyl-l-methylxanthine. Cellular CAMP was measured by RIA, as described in Methods and materials. b Based on cellular CAMP content after 6 h of recovery, at which time maximum recovery from desensitization has clearly occurred. ’ Mean rfrSEM of values obtained in triplicate dishes of cells, each containing approximately 100 pg of cellular protein.
induction of TSH desensitization. First, resensitized cells were incubated in 1 mM dBcAMP for 6 h in an attempt to mimic TSH-induced desensitization. Second, resensitized cells were treated for a similar time period with 10 PM or 100 PM forskolin, a potent stimulator of CAMP generation in intact cells. Neither dBcAMP nor forskolin induced TSH desensitization in FRPL cells (Fig. 2). Indeed, forskolin actually enhanced the subsequent CAMP response to TSH stimulation. As previously reported with cultured human thyroid cells (Rapoport et al., 1982), preincubation in 50 mu/ml TSH for 6 h did not reduce (but instead increased) the subsequent CAMP response to forskolin stimulation in FRTL cells. Role of N, in TSH desensitization There has been speculation that the newly discovered regulatory protein of adenylate cyclase complex (Ni) may be involved in the mechanism of induction of TSH desensitization. In order to test this hypothesis, the activity of the Ni inhibitory pathway during TSH desensitization was
24
0
1
2
3
4
Time (h)
Fig. 1. Time course of desensitization of the CAMP response to TSH stimulation. FRTL thyroid cells were incubated overnight without TSH. The cells were then exposed for the indicated time periods in L-15 medium containing 0.5 mM 3-isobutyl-lmethylxanthine and 50 mu/ml bTSH. Cellular CAMP was extracted and measured by RIA as described in Methods and materials. Each point represents mean f SEM of data obtained in triplicate dishes of cells.
probed by cY,-adrenergic stimulation. Inhibition of the CAMP response to TSH by 10-6M-10-4 M epinephrine in the presence of lop4 M proprano101 (a,-adrenergic stimulation) was unaltered in FRTL thyroid cells already desensitized to TSH (Fig. 3). That is Ni activation and TSH desensitization were additive. We also examined the role of Ni in TSH desensitization by attempting to inactive Ni using pertussis toxin. Thus, if Ni plays a role in the induction of TSH desensitization, its inactivation would be expected to prevent TSH desensitization. Preincubation of FRTL cells for 18 h in 50 ng/ml pertussis toxin was without effect on subsequent TSH stimulation of cellular CAMP generation, or on desensitization of the CAMP response to TSH stimulation (data not shown). However, pre-exposure of FRTL cells to pertussigen did not block the inhibitory effect of ol,-adrenergic stimulation (lop4 M epinephrine + lop4 M propranolol) on the CAMP response to TSH stimulation (data not shown). In contrast, the same pertussigen preparation was a potent inhibitor of GTP-induced inhibition in opossum kidney cells (A. Teitelbaum, personal communication).
Fig. 2. Role of CAMP in the induction of TSH desensitization. FRTL thyroid cells were preincubated for 6 h in either control (CON) medium or in medium supplemented with 50 mU/ml TSH, 1 mM dRcAMP, or in the indicated concentrations of forskolin (FORSK). The cells were then washed 3 times with phosphate-buffered saline, pH 7.4, after which they were incubated for 30 mm in L-15 medium supplemented with 0.5 mM 3-isobutyl-1-methylxanthine and the indicated additives. Cellular CAMP was extracted and measured by RIA as described in Methods and materials. Each bar represents the mean*SEM of values obtained in triplicate dishes of cells.
Roles of protein and RNA synthesis in TSH desensitization Support for the concept that synthesis of an inhibitory protein is necessary for the induction of TSH desensitization has been provided by the ability of cycloheximide or actinomycin D to prevent the induction of TSH desensitization in cultured thyroid cells (Takasu et al., 1978; Rapoport et al., 1983). TSH could enhance the translation of this protein, or the transcription of its mRNA. In order to help distinguish between these two alternatives we examined the relative turnover rates of the activities of the inhibitory protein and its mRNA by the addition of lop4 M cycloheximide or 1 pg/ml actinomycin D to the culture medium at different time intervals during a continual 6 h period of TSH stimulation. TSH desensitization was prevented by the presence of either agent for at least the last 4 h of the 6 h TSH stimulation period (Fig. 4). As a corollary, TSH desensitization was still apparent when cycloheximide or
25
150
2 4
100
1 g ,P n. 3 50
0
D
Fig. 4. Relationship between the duration of inhibition of protein and RNA synthesis and the prevention of TSH desensitization. FRTL thyroid cells were preincubated for 6 h in either control medium (CON), or in medium containing 50 mu/ml TSH. During the time intervals represented by the hatched bars 10m4 M cycloheximide or 1 pg/ml actinomycin D were also present. At the end of this 6 h preincubation period, the cells underwent a second (30 min) incubation in L-15 medium containing 0.5 mM 3-isobutyl-1-methylxanthine with or without (CON) 50 mu/ml TSH. Cellular CAMP levels were extracted and measured as described in Methods and materials. Each bar represents the meanf SEM of values obtained in triplicate dishes of cells.
100
E z ._
75
$ B $
50
25
0 0
10-6
10-4
1o-s Epinephrine
(AA)
Fig. 3. The inhibitory (Ni) pathway of adenylate cyclase activation in TSH desensitization. Thyroid cells were preincubated for 6 h in either control (CONTROL) medium or in medium containing 50 mu/ml TSH. All cells then underwent a second incubation (30 min) in L-15 medium containing 50 mu/ml TSH, 0.5 mM 3-isobutyl-l-methylxanthine, lo-’ M proprano101, and the indicated concentrations of epinephrine. Cellular CAMP was extracted and measured as described in Methods and materials. In the upper panel each point represents the mean f SEM of CAMP values determined in triplicate dishes of cells. In the lower panel the same data are expressed as a percentage of the maximum CAMP values observed in the absence of a,-adrenergic inhibition by epinephrine + propranolol.
actinomycin D was present during the last 3 h of the 6 h period. In 3 experiments this latter time period varied between 2 and 3 h. These data
indicate that in FRTL cells the functional activities of the putative inhibitory protein and its mRNA each persist for approximately 3 h after their synthesis has been inhibited. That is their rates of disappearance are similar. Discussion The present data provide further insight into our understanding of the mechanism of desensitization of adenylate cyclase activation by TSH in thyroid cells. First, an important and puzzling discrepancy in the literature is the role of CAMP in the mechanism of TSH desensitization (Rapoport and Adams, 1976; Takasu et al., 1978; Avivi et al., 1981). It is surprising that conflicting data should be obtained with FRTL (present study) and FRTL, (Avivi et al., 1981), cells, which are closely related
26
variants of the same cell line. Our data support the thesis that TSH desensitization is homologous (Shuman et al., 1976), and that CAMP is not directly involved in this process. The reason for these discrepant results is unknown, but is possibly methodological. Thus Avivi et al. (1981) first prelabeled the intracellular ATP pool with [3H]adenosine, then incubated the cells in the presence or absence of dBcAMP, and finally, in a third incubation, stimulated the cells with TSH, prior to extraction and measurement of radioactive CAMP. It is therefore possible that the specific activity of the labeled ATP precursor was altered by preincubation in dBcAMP. In contrast, in the present study, CAMP was measured directly by radioimmunoassay, which is not subject to such an artifact. Even stronger evidence that CAMP is not involved in the induction of TSH desensitization was obtained using forskolin. Although the action of this compound is complex, and is modulated by the adenylate cyclase regulatory proteins, it is nevertheless a potent direct activator of the catalytic subunit of adenylate cyclase (Seamon and Daly, 1981; Pfeuffer and Metzger, 1982). Forskolin is extremely useful in that, unlike fluoride and GTP analogues such as Gpp(NH)p, it acts on intact cells. Our data demonstrate that despite elevation of intracellular CAMP to levels greater than those achieved by TSH stimulation, desensitization to TSH was not induced. These data suggest that CAMP does not play a direct role in the induction of TSH desensitization. In support of data obtained with human thyroid cells (Rapoport et al., 1982), and bovine thyroid slices (Totsuka et al., 1983), our results also demonstrate in another cell line (FRTL) that forskolin activation is unaltered in TSH-desensitized thyroid cells. This finding is strong evidence that the alteration in the adenylate cyclase complex in TSH desensitization is proximal to, and does not involve, the catalytic unit itself. Finally our data (Fig. 2) indicate that in FRTL cells, unlike in guinea pig thyroid tissue (Fradkin et al., 1982), preincubation in forskolin enhances the subsequent CAMP response to TSH stimulation. This finding is in agreement with the original observation of Daly et al. (1982) that forskolin enhances receptor-mediated responses. Adenylate cyclase activation by hormones is
regulated by a stimulatory regulatory protein (N,) and a more recently recognized inhibitory regulatory protein (Ni) (Yamashita et al., 1977; Katada and Ui, 1982; Hildebrandt et al., 1983; Murayama and Ui, 1983; Katada et al., 1984). Previous data from our laboratory suggested that the N, pathway is unaltered in TSH desensitization (Rapoport et al., 1982). With the discovery of the Ni pathway an attractive hypothesis was that TSH desensitization involved activation of the Ni pathway. Our data demonstrate that this is not likely to be the case. Thus control and TSH desensitized thyroid cells are equally sensitive to cy,-adrenergic inhibition of adenylate cyclase activation. That is CQadrenergic stimulation is additive with that of TSH desensitization, suggesting that these two actions involve separate pathways. More direct evidence against involvement of the N, pathway in TSH desensitization would be that TSH desensitization still occurs even when this pathway is inactivated by pertussis toxin. Pre-exposure of thyroid cells to pertussis toxin did not prevent the subsequent induction of TSH desensitization, however this evidence was inconclusive. This was because the pertussis toxin used, even though potent in another cell system, did not prevent a,-adrenergic inhibition of TSH stimulation in FRTL cells. The reason for this difference between tissues in their susceptibility to pertussis toxin is unclear. Nevertheless such differences have been reported (Katada et al., 1983), and may involve the ability of different tissues to activate the pertussis toxin by separation into A and B promoters. It is therefore possible that cultured thyroid cells belong to that group of cells that lack the ability to activate pertussigen. It is also interesting that pretreatment with pertussis toxin did not alter the CAMP response to TSH stimulation in FRTL cells, unlike the enhancement reported in dog thyroid slices (Katori and Yamashita, 1982). The ability of inhibitors of protein and RNA synthesis to prevent desensitization of activation by TSH of adenylate cyclase in thyroid cell cultures suggests the existence of a protein that is capable of modulating the activity of this enzyme (Rapoport and Adams, 1976). TSH could regulate the synthesis of this putative protein at the ribosomal level or by influencing transcription of its mRNA. Once synthesized, the activity of this pro-
27
tein could also be modulated by post-translational modifications, such as ADP-ribosylation (Filetti and Rapoport, 1981; Filetti et al. 1981) or phosphorylation. The present data suggest that, in FRTL thyroid cells, the rates of turnover of the functional activities of this protein and its mRNA are similar, and relatively rapid. Thus their functional activities persist for approximately 3 h after their synthesis has been inhibited. This time period is consistent with the time period of recovery from desensitization observed following withdrawal of TSH (Table 1). From the foregoing information it is not possible to deduce whether the primary action of TSH in the induction of desensitization is at the transcriptional or translational level. A turnover rate for the mRNA slower than that of the protein would have been evidence supporting an action of TSH at the translational or posttranslational level. Experiments of a different approach will be necessary to resolve this issue. Acknowledgements These studies were supported by NIH Grant AM 19289 and the Medical Research Service of the Veterans’ Administration. The expert secretarial assistance of Peggy Mathews is gratefully acknowledged. References Ahren, B., Gustafson, A. and Hedner, P. (1982) Life Sci. 31, 2583. Ambesi-Impiombato, F.S., Parks, L.A.M. and Coon, H.G. (1980) Proc. Nat. Acad. Sci. (U.S.A.) 77, 3455-3459. Avivi, A., Tramontano, D., Ambesi-Impiombato, F.S. and Schlessinger, .I. (1981) Science 214, 1237-1239. Cochaux, P., Van Sande, J. and Dumont, J.E. (1982) B&him. Biophys. Acta 721, 39-46. Daly, J.W., Padgett, W. and Seamon, K.B. (1982) J. Neurothem. 38, 532-544. Field, J.B., Dekker, A., Titus, G., Kerins, M.E., Worden, W. and Frumess, R. (1979) J. Clin. Invest. 64, 265-271. Filetti, S. and Rapoport, B. (1981) J. Clin. Invest. 68, 461-467.
Filetti, S. and Rapoport, B. (1982) J. Biol. Chem. 257; 1342-1346. Filetti, S. and Rapoport, B. (1983) Endocrinology 113, 1608-1615. Filetti, S., Takai, N.A. and Rapoport, B. (1981) J. Biol. Chem. 256, 1072-1075. Fradkin, J.E., Cook, G.H., Kilhoffer, M.-C. and Wolff, J. (1982) Endocrinology 111, 849-856. Gerber, H., Studer, H., Conti, A., Engler, H., Kohler, H. and Haeberli, A. (1981) J. Clin. Invest. 68, 1138-1347. Hildebrandt, J.D., Sekura, R.D., Codina, J., lyengar, R., Manclark, C.R. and Birnbaumer, L. (1983) Nature (Lond.) 302, 706-709. Holmes, S.D., Gitlin, J., Titus, G. and Field, J.B. (1980) Endocrinology 106, 1892-1899. Kaneko, Y. (1976) Horm. Metab. Res. 8, 202-206. Katada, T. and Ui, M. (1982) J. Biol. Chem. 257. 7210-7216. Katada, T., Tamura, M. and Ui, M. (1983) Arch. B&hem. Biophys. 224, 290-298. Katada, T., Northup, J.K., Bokoch, G.M., Ui, M. and Gilman, A.G. (1984) J. Biol. Chem. 259, 3578-3585. Katori, A. and Yamashita, K. (1982) Endocrinol. Jpn. 29, 261-263. Laurberg, P. (1981) Endocrinology 109, 1560-1565. Levasseur, S., Kostelec, M. and Burke, G. (1982) Clin. Res. 30, 721A. Murayama, T. and Ui, M. (1983) J. Biol. Chem. 258,3319-3326. Pfeuffer, T. and Metzger, H. (1982) FEBS Lett. 146, 369-375. Rapoport, B. (1976) Endocrinology 98, 1189-1197. Rapoport, B. and Adams, R.J. (1976) J. Biol. Chem. 251, 6653-6661. Rapoport, B., Filetti, S., Takai, N. and Seto, P. (1982) FEBS Lett. 146, 23-27. Rapoport, B., Filetti, S. and Takai, N.A. (1983) Endocrinology 112, 1874-1876. Seamon, K. and Daly, J.W. (1981) J. Biol. Chem. 256, 9799-9801. Shuman, S.J., Zor, U., Chayoth, R. and Field, J.B. (1976) J. Clin. Invest. 57, 1132-1141. Steiner, A.L., Paghara, A.S., Chase, L.R. and Kipnis, D.M. (1972) J. Biol. Chem. 247, 1114-1120. Takasu, N., Sato, S., Yamada, T., Makiuchi, M., Furihata, R. and Miyakawa, M. (1976) Horm. Metab. Res. 8, 206-211. Takasu, N., Chartier, B., Mauchamp, J. and Lissitzky, S. (1978) Eur. J. B&hem. 90, 131-138. Totsuka, Y., Nielsen, T.B. and Field, J.B. (1983) Endocrinology 113, 1088-1095. Yamashita, K., Yamashita, S. and Ogata, E. (1977) Life Sci. 21, 607-611. Zakarija, M., Witte, A. and McKenzie, J.M. (1980) Endocrinology 197, 2045-2050.