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Acute effect of triiodothyronine on the dynamics of thyrotropin release from superfused anterior pituitary cells
79
Molecular and Cellular Endocrinology, 41 (1985) 79-84
Elsevier Scientific Publishers Ireland, Ltd. MCE 01317
Acute effect of triiodothyronine on the dynamics of thyrotropin release from superfused anterior pituitary cells Kunio Shiota, Keiji Yoshida, Masahiro Kawase, Tsuneo Masaki and Katsuichi Sudo Central Research Division, Takeda Chemical Industries, Ltd., Juso - Honmachi, Yodogawa - ku, Osaka 532 (Japan)
(Received 3 January 1985; accepted 4 March 1985)
Keywords: thyrotrop~n; thyrotropin-releasing
hormone; anterior pituitary; trii~othyronine;
protein synthesis; negative feedback.
Summary
The effects of triiodothyronine (T3) on the dynamics of thyrotropin (TSH) release induced by TSH-releasing hormone (TRH) were examined in the presence or absence of a protein synthesis inhibitor, cycloheximide (CX), in a superfusion system using primarily cultured cells of the rat anterior pituitary gland on microcarrier beads. When the cells were continuously stimulated with TRH (10 nM, 180 mm), TSH release occurred in a biphasic manner and the profile of TSH release was characterized by an initial sharp peak (phase I), followed by a lower plateau form phase (phase II). Both phase-I and phase-II releases were significantly suppressed in the presence of T3 (1 ng/ml), which was added to the superfusion medium 1 h before initiation of TRH stimulation. The biphasic nature of the release profile was maintained in the presence of T,, su~esting that the site of the T3 action may be common between phase-I and phase-II release. We have already suggested that phase-f release is protein synthesis-independent and phase-II release protein synthesis-dependent using CX in TRH-stimulated cells. In the presence of CX, phase-I release was not suppressed by T3, while phase-II release was still suppressed by T3. The inability of CX to reverse the T,-induced suppression of phase-II release may be masked by the direct CX effect on phase-II release of TSH. The present study indicates that each component (phase I and phase II) of the biphasic release of TSH induced by TRH stimulation was acutely suppressed by T3 and suggests that the T3 action is mediated through protein synthesis.
It is generally accepted that TSH secretion is under the negative control of thyroid hormones, as deduced from in vivo (Fukuda et al., 1975; Garcia et al., 1976; Spira et al., 1979; Larsen and Silva, 1983) and in vitro studies (Vale et al., 1973; Lewis et al., 1977; Chopra et al., 1978; Chin et al., 1981; Marshall et al., 1981; Padmanabhan et al., 1981; Hinkle and Goh, 1982). Protein synthesis inhibitors have been found to block the suppression of TRH-induced TSH release by thyroxine or T3 in in vitro studies using pituitary fragments or in vivo studies (Bakke and Lawrence, 1968; Bowers et al.,
1968; Vale et al., 1968; Gard et al., 1981), suggesting that the suppressive action of thyroid hormones mediated the protein synthesis. In these experiments, protein synthesis inhibitors did not alter the basal or TRH-induced TSH release. We reported that the mechanism of TRH-induced TSH release involved the process of protein synthesis other than de novo synthesis of TSH (Shiota et al., 1984b). In superfusion studies using dispersed pituitary cells, Schrey et al. (1977), Connors et al. (1981) and Shiota et al. (1984a, b) noted a biphasic response of TSH release during con-
0303-7207/85/~03.30 0 1985 Elsevier Scientific Publishers Ireland, Ltd,
x0
stant stimulation of the cells with TRH. The pattern of TSH release was characterized by an initial high peak (phase I), followed by a lower plateau form phase (phase II); phase-11 release but not phase-1 release was decreased in the presence of cycloheximide (CX). suggesting that phase-I release was protein synthesis-independent while phase-11 release was protein synthesis-dependent (Shiota et al., 1984h). Therefore, it was of interest to investigate: (1) the effect of T, on the dynamics of TSH release, and (2) the relationship between TRH-induced TSH release and its suppression by thyroid hormone in the light of the protein synthesis.
Materials and methods Male Sprague-Dawley rats (aged 8-9 weeks, Charles River Japan Inc.) were maintained in a controlled environment at 24 i 1°C in a 14 h light-10 h dark cycle (Iight on at 07 : 30 h); they had free access to water and standard laboratory chow. TRH was synthesized in the Chemistry Laboratories of the Takeda Central Research Division (Fukuda et al., 1980). TX was obtained from Sigma Chemical Co. (St. Louis, MO); cycloheximide was obtained from Wako Pure Chemical Industries, Ltd. (Japan). Other materials required for dispersion of the rat anterior pituitary cells and primary beads were culture on Cytodex@- 1 microcarrier obtained from the sources described in Shiota et al. (1984a, b). The primary cultures of the rat anterior pituitary cells on microcarrier beads and superfusion of the cells were performed as described (Shiota et al., 1984a, b). Briefly, rat anterior pituitary glands were digested with 0.4% collagenase, 0.4% BSA, 10 pg/ml DNAse, and then with 10% pancreatin. Dulbecco’s modified Eagle medium (DMEM, pH 7.3) supplemented with 10% fetal bovine serum, 20 mM Hepes, 50 U/ml penicillin and 50 pg/ml stre tomycin, was the culture medium used. Cytodex & -1 was added to the cell suspension and the preparations were incubated for 4-6 days under a saturated atmosphere of 5% CO,/95% air in a CO, incubator. The amounts of thyroxine and T3 in the culture medium before the incubation were 6.5
ng/ml and 21.6 pg/ml. respectively. measured using a RIA kit (Mallinckrodt Inc., St. Louis, MO). Six columns were run in parallel in an experiment. Cells (9 x lO~/coIun?n) attached to the Cytodex@-1 were introduced into the column. DNA content in the column was practically identical, about 80-90 pg/column throughout the experiments, and the intra-experimental coefficient of variation was less than 5%, as reported (Shiota et al., 1984b). The superfusion medium consisted of DMEM (pH 7.3) containing 1.85 g NaHCO,/ liter supplemented with 20 mM Hepes, without serum or antibiotics. Flow rate of the medium was 0.25 ml/min and the effluent was collected every 10 min. A lag period of 13.5 + 0.6 min (n = 4) was needed for the TRH to reach the fraction cohector. The cells were superfused for 2 h before sample collection. In some experiments, cells were harvested at the end of the superfusion for measurement of cellular TSH. All these samples were stored at - 20°C until assay. TRH and CX were freshly dissolved in the superfusion medium to give final concentrations of nM and 7 PM, respectively. The dose of CX inhibited [methyl-3H]methionine incorporation of primary culture of pituitary cells into trichloroacetic acid precipitable material by about 80% in static incubation for 2 and 4 h (Shiota et al., 1984b). Ti was dissolved in 0.01 N NaOH and then diluted with the medium to the final concentration of 1 ng/ml. TSH in the medium and cells was measured by radioimmunoassay using a kit provided by the MIAMDD Rat Pituitary Hormone Distribution Program. The results were expressed in terms of NIAMDD rat TSH-RP-1 standard. Samples from each experiment were measured within one assay. The intra-assay coefficient of variation was 5.7% and the interassay coefficient of variation was less than 10% according to the method previously described (Shiota et al.. 1984a. b). TSH release (ng/min) was plotted against time and ail results were expressed as means + SE. The significance of the difference among the 3 groups was calculated by Duncan’s multiple range test (Dunnet, 1970) and the difference between 2 groups was calculated by Student’s r-test. P values of less than 0.05 were considered significant unless otherwise described.
81
Results T3 effects on TSH secretion during intermittent stimulation with TRH To examine the effect of Tj on pulsatile release of TSH and to determine the time required for the T3 suppressive effect on TSH release, the cells were intermittently exposed to TRH (10 nM for 20 min) every 60 min, in the presence or absence of 1 ng/ml Tj. The results of 2 independent experiments are shown in Fig. 1A and B, in which the
TIME,
II 0
1
2 0
3
1 TIME,hr
hr
4 2
3
Fig. 1. Inhibitory effect of Ts on TSH release caused by intermittent stimulation with TRH in the superfusion study using the primary cultured cells attached to Cytodex @l. Results of the 2 independent experiments are shown (A and B). In both experiments, cells were repeatedly exposed to TRH (10 nM) for 20 min at 60-min intervals. Ts (1 &ml) was present from the start of the second exposure to TRH. In each experiment, 6 columns were run in parallel and 3 columns were used for each treatment: TRH alone or TRH plus Ts. The flow rate was 0.25 ml/mm and the effluent was collected every 10 min. Time scales indicate the period after the start of either sample collection or Ts treatment. Each time point is the mean + SE.
cells were stimulated with TRH 7 times (A) or 4 times (B). In the absence of T3, there were pulsatile releases of TSH corresponding to each stimulation with TRH, and the magnitudes of the responses were similar during the experiment, except for the release by the first stimulation with TRH shown in Fig. 1B. Before the addition of T3, there was no significant difference in TRH-induced TSH release between the 2 groups of treatment in each experiment. In both experiments, Tj was present in the medium from the start of the second TRH stimulation. Basal TSH release was not affected by Tj throughout. On the second stimulation with TRH, TSH release was not suppressed by T3, while TSH release caused by the third stimulation - 1 h after the addition of Tj - was decreased significantly by T3. Therefore, pretreatment with Tj for about 1 h seems to be required for the inhibitory action seen in our superfusion system. The degree of the T3 suppressive effect on TSH release increased time-dependently. In the present experimental condition, the amount of T3 (1 ng/ml) seems to be the maximal suppressive dose, because further suppression was not achieved by increasing the concentration of T3 up to 20 ng/ml (data not shown). At the end of the experiment shown in Fig. lA, the immunoreactive TSH remaining was 120.7 5 5.2 pg/column in the TRH-treated cells and 145.1 k 6.4 pg/column in TRH plus TXtreated cells, respectively, with a significant difference. Effects of cycloheximide on TSH secretion induced by continuous stimulation with TRH The cells were continuously exposed to TRH (10 nM) for 3 h in the presence or absence of a protein synthesis inhibitor, CX (7 PM). Since a similar experiment had been done using a maximally effective concentration of TRH (100 nM) (Shiota et al., 1984b), the results from a single experiment are presented in Fig. 2. During continuous exposure to TRH, TSH release occurred in a biphasic manner. The profile of TSH release was characterized by an initial sharp peak, followed by a lower plateau form phase. In this report, we termed TSH release during the initial 50 min as phase I and the TSH subsequently released as phase II. On withdrawal of TRH, TSH release decreased to a basal level.
H2
I
I
CX TRH
I
2
TIME,
3
1
2
, 4 t 3
hr
Fig. 2. TSH release in response to continuous exposure to TRH in the presence or absence of ~y~lohex~mide (CX) in the superfusion study. CX (7 PM) was added 1 h before the start of continuous exposure to TRH (10 nM) for 3 h. Time scales indicate the period after the start of either sample collection, CX or TRH treatment. Six columns were run in parallel and 3 columns were used for each treatment: TRH alone or TRH plus CX. Each time point is the mean&SE.
When CX was added 1 h before the start of TRH exposure, basal (or unstimulated) release and phase-I release were not suppressed, while phase-11 release was significantly reduced. Thus, we confirmed our previous findings of a biphasic release and the possible dependency of phase-II release on protein synthesis (Shiota et al., 1984a, h). TSH release in the late part of phase I and initial part of phase II, during a time of 40-60 min, was significantly higher in CX-treated cells than that in the control. At the end of the experiment, 193.1 i: 5.2 pg/column and 165.1 $9.9 pg/column of immunoreactive TSH remained in the control and CX-treated cells, respectively. Thus, a large amount of TSH remained, even in the phase-11 blocked cells, suggesting that only a small percentage of immunoreactive TSH in the cells is releasable. T” effects on biphasic release of TSH and dependemy on protein synthesis T, effects on biphasic release induced by continuous stimulation with TRH were examined. The data from 3 independent experiments were com-
1
TRH
I
1
qTR i $ytrol) ATR
lTR H+T,+CX
n:3
\
1 I 0
or T3+CX
T3
I 0 TRH(control) .TRH+CX
!
t 0
I
ri:6
11
0
I 0
1
2 1 I 0
, 2 1
3
4 3 I 2
t 4 I 3
5
TIME,hr Fig. 3. Inhibitory effects of T, on biphasic release of TSH and possible dependency on protein synthesis. T, (I ng/ml) or T, plus cycloheximide (7 pM. CX) was present I h before the start of continuous exposure to TRH (10 nM) for 3 h. The data from 3 independent experiments were combined. In each experiment. 6 columns were run in parallel and 2 columns were used for each treatment: TRH alone. TRH plus Ts, or TRH plus Ta plus CX. Each time point is the mean i SE.
bined and the mean values are shown in Fig. 3. The total amount of TSH released during the phase-l and phase-11 periods is shown in Tabfe 1. The cells were continuously exposed to TRH (10 nM) for 3 h in the presence or absence of 1 ng/ml T,. Exposure to T3 was started 1 h before the start of continuous exposure to TRH. Continuous exposure to TRH caused a biphasic release of TSH, and the phase-I and phase-II releases were significantly lower in the T,-treated cells, compared with those in cells treated with TRH alone. To evaluate the inhibitory effect of T,, a possible dependency of T3 action on protein synthesis was examined (Fig. 3, Table 1). T3 (1 ng/ml) and CX (7 ,nM) were added 1 h before continuous stimulation with TRH (10 nM). During the constant exposure to TRH, TSH release during the phase-I period was not decreased by T, in the presence of CX, while phase-II release of TSH was significantly less in CX- plus T,-treated cells. Thus, the inhibitory effect of T3 on phase-1 release was completely blocked.
83 TABLE
1
EFFECTS OF Ts (1 ng/ml) WITH OR WITHOUT CX (7 FM) ON TSH RELEASE DURING CONSTANT STIMULATION WITH TRH (10 nM) TSH released pg/phase II (130 min)
gg/pbase I (150 min) TRH (control) TRH + T, TRH+T,+CX
11.19*0.53 6.08kO.17 11.94kO.47
B b c
18.43 f 1.07 d 11.12~0.39 e 8.57 k 0.48 f
Total amount of TSH released was calculated from the experiment shown in Fig. 3. Values are meansf SE (n = 6) and expressed by NIAMDD rat TSH-RP-1. Statistical significance (by Duncan’s multiple range test, P c 0.01): a vs. b, b vs. c, d vs. e, and d vs. f.
The 2 protein synthesis-dependent events, TRH-induced TSH release (phase II) and Ts-induced suppression of TSH release (phases I and II), were chronologically separated using a superfusion system. We confirmed that TSH release occurs in a biphasic manner in response to continuous stimulation with TRH (Schrey et al., 1977; Connors et al., 1981; Shiota et al., 1984a, b) and that phase-I release is protein synthesis-independent while phase-II release is protein synthesis-dependent (Shiota et al., 1984b). Both phases of the biphasic release of TSH were suppressed by T3. A common process in the TSH release mechanism between phase I and phase II may be affected by T3 treatment, since the biphasic profile of the response was well preserved. During phase-I release, not only the amount of TSH released but also the release profile were in~stinguishable between TRH-treated and TRH- plus Ts- plus CXtreated cells, suggesting that the acute T3 suppressive action totally depends on mechanisms involving protein synthesis. On the other hand, phase-II TSH release, which was also suppressed by Ts, was not reversed in the presence of CX. The failure to block T3 action by CX may be masked by the effect of CX on phase-II reiease since phase-II release itself is suppressed by CX. These results confirmed and extended previous studies (Bakke and Lawrence, 1968; Bowers et al., 1968; Vale et
al., 1968; Gard et al., 1981), suggesting that Ts suppresses TRH-induced TSH release through a protein synthesis mechanism. In these experiments, pituitary fragments were exposed to TRH for shorter periods than 30 min (Vale et al., 1968; Gard et al., 1981). Judging from the length of the incubation periods, the previous results may be referring to phase-I release. We suggested that there was a protein synthesis-independent TSH release immediately after TRH stimulation in which the previously prepared pool of releasable TSH (&iota et al., 1984b) was released (phase-I release). If this is the case, the acute inhibition by Tj of TSH release might be defined as the stimulation of the protein synthesis responsible for the blockade of the pooled TSH to be released. Though there have been reports indicating that thyroid hormones inhibit TSH synthesis (Spira et al., 1979; Chin et al., 1981; Marshall et al., 1981), T3 does not seem to suppress de novo TSH synthesis as far as the acute inhibitory action of T, is concerned. This agrees with the observation that suppression of TSH release by T3 resulted in an increase in intracellular TSH contents. We found that a low dose of Ts (1 ng/ml) acutely - within 60 min of the pretreatment inhibited TRH-induced TSH release in vitro. The period required for the ?; inhibitory action was close to that observed in in vivo time-course studies of plasma TSH suppression (Fukuda et al., 1975; Garcia et al., 1976; Spira et al., 1979; Larsen and Silva, 1983) and pituitary nuclear T3 accumulation after an intravenous injection of T3 in rats (Larsen and Silva, 1983). Moreover, the circulating concentration of T3 in vivo ranged between 0.5 and 0.7 ng/ml in the rat, and the dose of Ts used in the present study is considered to be within a physiolo~cal range. In the superfusion studies performed by others, relatively high doses of T, were required to demonstrate the effects (Schrey et al., 1977; Connors et al., 1981; Gard et al., 1981). In the studies of Connors et al. (1981), 20 ng/ml Ts did not inhibit TSH release from acutely dispersed cells though the same treatment did inhibit TSH release from pituitary fragments. The differences between inhibitory TX doses used in the present study and others may reside in the responsiveness of dispersed cells. The cells used in our study were cultured for 4 or 5 days before use to allow them
84
to recover from the damage of the enzymatic dispersion. When the cells were intermittently stimulated with TRH, pulsatile or phase-I-like releases were obtained. The present results showed that the degree of the T3 suppressive effect on the phase-I-like releases was time-dependently increased. Since the anterior pituitary hormones including TSH are secreted intermittently rather than continuously (Willoughby et al., 19773, the effects of T3 on the phase-I-like release may be physiologically important. We noted that CX treatment slightly augmented TSH release in the late part of phase I. Protein synthesis might be involved in the mechanisms of shift of TSH release from phase I to phase II. Recently, a similar phenomenon was observed in the case of growth hormone release (GH) (Cronin et al., 1984). Treatment with CX potentiated human pancreatic GH-releasing factor-induced CAMP accumulation and GH release in primary cultures of anterior pituitary ceils (Cronin et al., 1984). Whether or not there is a similar mechanism involved in the release of TSH remains to be determined. In conclusion, each component of the biphasic release of TSH (phase I and phase II) induced by TRH stimulation was acutely inhibited by T3 in vitro, and the action of T3 seems to be mediated by a mechanism involving protein synthesis.
We thank Dr. M. Takahashi (Tokyo University) for his valuable suggestions, Dr. A.F. Parlow and the NIAMDD Rat Pituitary Hormone Distribution Program for the gift of the rat TSH RIA kit. We are also grateful to Ms. Y. Akinaga for technical assistance, and to M. Ohara for reading the manuscript. References Bakke. J.L. and 308-314.
Lawrence,
N. (1968)
Eur. J. Pharmacol.
2.
Bowers. C.Y.. Lee, K.L. and Schally. A.V. (1968) Endocrinology 82. 75-82. Chin. W.W.. Maloof. F., Martorano, M.A.. Pierce. J.G. and Ridgway, E.C. (1981) Endo~rinoiogy 108. 387394. Chopra, I.J., Carlson, H.E. and Solomon, D.H. (197X) Eadocrinology 103, 3933402. Connors, J.M., Wright, K.C., Judd, A.M.. Liu, C.M. and Hedge. G..4. (1981) Horm. Res. 14, 1-17. Cronin, M.J.. Hewlett. EL.. Evans. W.S., Thorner. M.O. and Rogol. A.D. (1984) Endocrinology 114. 904913. Dunnet. C.W. (1970) In: Statistics in Endocrinology. Eds.: J.W. McArthur and T. Cohen (MIT Press. Cambridge, MA) pp. 799103. Fukuda. H., Yasuda, N. and Greer, M.A. (1975) Endocrinology 97. 924-931. Fukuda, N.. Nishimura. 0.. Shikata. M., Hatanaka, C., Miyamoto. M., Saji, Y., Nakayama, R., Fujino, M. and Nagawa, Y. (1980) Chem. Pharm. Bull. 28, 1667-1672. Garcia, M.D., Escobar del Rey. F. and Morreale de Escobar, G. (1976) Endocrinology 98. 203-213. Gard, T.G.. Bernstein, B. and Larsen, P.R. (1981) Endocrinology 108, 2046-2053. Hinkle, P.M. and Goh. K.B.C. (1982) Endocrinology 110, 1725-1731. Larsen, P.R. and Silva, J.E. (1983) In: Molecular Basis of Thyroid Hormone Action, Eds.: J.H. Oppenheimer and H.H. Samuels (Academic Press, New York) pp. 351-385. Lewis. M., Yeo, P.P.B., Green. E. and Evered, D.C. (1977) J. Endocrinol. 74, 405-414. Marshall, Jr., M.C.. Williams, D. and Weintraub, B.D. (1981) End~~~rinology 108. 908-915. Padmanabhan, V., Kesner. J.S. and Convey, EM. (1981) Endocrinology 108. 226.-231. Schrey, M.P.,
Brown, B.L.. and Ekins. R.P. (1977) Mol. Cell. Endocrinol. 8, 271-282. Shiota. K., Yoshida, K., Noguchi. C. and Nakayama, R. (1984a) Acta Endocrinol. (Kbh.) 106, 71-78. Shiota, K., Yoshida, K., Masaki, T.. Kawase, M., Makayama, R. and Sudo. K. (1984b) Endocrinol. Jpn. 31. 165-175. Spira, O., Birkenfeld. A., Avni, A.. Gross, J. and Gordon, A. (1979) Acta Endocrinol. (Kbh.) 92, 502-511. Vale, W., Burgus. R. and Guillemin. R. (1968) Neuroendocrinology 3. 34-46. Vale, W.. Blackwell. R., Grant, G. and Guillemin, R. (1973) endocrinology 93, 26-33. Will~~ughby. J.O., Terry, L.C.. Brazeau, P. and Martin, J.B. (1977) Brain Res. 127. 137-152.
Molecular and Cellular Endocrinology, 41 (1985) 79-84
Elsevier Scientific Publishers Ireland, Ltd. MCE 01317
Acute effect of triiodothyronine on the dynamics of thyrotropin release from superfused anterior pituitary cells Kunio Shiota, Keiji Yoshida, Masahiro Kawase, Tsuneo Masaki and Katsuichi Sudo Central Research Division, Takeda Chemical Industries, Ltd., Juso - Honmachi, Yodogawa - ku, Osaka 532 (Japan)
(Received 3 January 1985; accepted 4 March 1985)
Keywords: thyrotrop~n; thyrotropin-releasing
hormone; anterior pituitary; trii~othyronine;
protein synthesis; negative feedback.
Summary
The effects of triiodothyronine (T3) on the dynamics of thyrotropin (TSH) release induced by TSH-releasing hormone (TRH) were examined in the presence or absence of a protein synthesis inhibitor, cycloheximide (CX), in a superfusion system using primarily cultured cells of the rat anterior pituitary gland on microcarrier beads. When the cells were continuously stimulated with TRH (10 nM, 180 mm), TSH release occurred in a biphasic manner and the profile of TSH release was characterized by an initial sharp peak (phase I), followed by a lower plateau form phase (phase II). Both phase-I and phase-II releases were significantly suppressed in the presence of T3 (1 ng/ml), which was added to the superfusion medium 1 h before initiation of TRH stimulation. The biphasic nature of the release profile was maintained in the presence of T,, su~esting that the site of the T3 action may be common between phase-I and phase-II release. We have already suggested that phase-f release is protein synthesis-independent and phase-II release protein synthesis-dependent using CX in TRH-stimulated cells. In the presence of CX, phase-I release was not suppressed by T3, while phase-II release was still suppressed by T3. The inability of CX to reverse the T,-induced suppression of phase-II release may be masked by the direct CX effect on phase-II release of TSH. The present study indicates that each component (phase I and phase II) of the biphasic release of TSH induced by TRH stimulation was acutely suppressed by T3 and suggests that the T3 action is mediated through protein synthesis.
It is generally accepted that TSH secretion is under the negative control of thyroid hormones, as deduced from in vivo (Fukuda et al., 1975; Garcia et al., 1976; Spira et al., 1979; Larsen and Silva, 1983) and in vitro studies (Vale et al., 1973; Lewis et al., 1977; Chopra et al., 1978; Chin et al., 1981; Marshall et al., 1981; Padmanabhan et al., 1981; Hinkle and Goh, 1982). Protein synthesis inhibitors have been found to block the suppression of TRH-induced TSH release by thyroxine or T3 in in vitro studies using pituitary fragments or in vivo studies (Bakke and Lawrence, 1968; Bowers et al.,
1968; Vale et al., 1968; Gard et al., 1981), suggesting that the suppressive action of thyroid hormones mediated the protein synthesis. In these experiments, protein synthesis inhibitors did not alter the basal or TRH-induced TSH release. We reported that the mechanism of TRH-induced TSH release involved the process of protein synthesis other than de novo synthesis of TSH (Shiota et al., 1984b). In superfusion studies using dispersed pituitary cells, Schrey et al. (1977), Connors et al. (1981) and Shiota et al. (1984a, b) noted a biphasic response of TSH release during con-
0303-7207/85/~03.30 0 1985 Elsevier Scientific Publishers Ireland, Ltd,
x0
stant stimulation of the cells with TRH. The pattern of TSH release was characterized by an initial high peak (phase I), followed by a lower plateau form phase (phase II); phase-11 release but not phase-1 release was decreased in the presence of cycloheximide (CX). suggesting that phase-I release was protein synthesis-independent while phase-11 release was protein synthesis-dependent (Shiota et al., 1984h). Therefore, it was of interest to investigate: (1) the effect of T, on the dynamics of TSH release, and (2) the relationship between TRH-induced TSH release and its suppression by thyroid hormone in the light of the protein synthesis.
Materials and methods Male Sprague-Dawley rats (aged 8-9 weeks, Charles River Japan Inc.) were maintained in a controlled environment at 24 i 1°C in a 14 h light-10 h dark cycle (Iight on at 07 : 30 h); they had free access to water and standard laboratory chow. TRH was synthesized in the Chemistry Laboratories of the Takeda Central Research Division (Fukuda et al., 1980). TX was obtained from Sigma Chemical Co. (St. Louis, MO); cycloheximide was obtained from Wako Pure Chemical Industries, Ltd. (Japan). Other materials required for dispersion of the rat anterior pituitary cells and primary beads were culture on Cytodex@- 1 microcarrier obtained from the sources described in Shiota et al. (1984a, b). The primary cultures of the rat anterior pituitary cells on microcarrier beads and superfusion of the cells were performed as described (Shiota et al., 1984a, b). Briefly, rat anterior pituitary glands were digested with 0.4% collagenase, 0.4% BSA, 10 pg/ml DNAse, and then with 10% pancreatin. Dulbecco’s modified Eagle medium (DMEM, pH 7.3) supplemented with 10% fetal bovine serum, 20 mM Hepes, 50 U/ml penicillin and 50 pg/ml stre tomycin, was the culture medium used. Cytodex & -1 was added to the cell suspension and the preparations were incubated for 4-6 days under a saturated atmosphere of 5% CO,/95% air in a CO, incubator. The amounts of thyroxine and T3 in the culture medium before the incubation were 6.5
ng/ml and 21.6 pg/ml. respectively. measured using a RIA kit (Mallinckrodt Inc., St. Louis, MO). Six columns were run in parallel in an experiment. Cells (9 x lO~/coIun?n) attached to the Cytodex@-1 were introduced into the column. DNA content in the column was practically identical, about 80-90 pg/column throughout the experiments, and the intra-experimental coefficient of variation was less than 5%, as reported (Shiota et al., 1984b). The superfusion medium consisted of DMEM (pH 7.3) containing 1.85 g NaHCO,/ liter supplemented with 20 mM Hepes, without serum or antibiotics. Flow rate of the medium was 0.25 ml/min and the effluent was collected every 10 min. A lag period of 13.5 + 0.6 min (n = 4) was needed for the TRH to reach the fraction cohector. The cells were superfused for 2 h before sample collection. In some experiments, cells were harvested at the end of the superfusion for measurement of cellular TSH. All these samples were stored at - 20°C until assay. TRH and CX were freshly dissolved in the superfusion medium to give final concentrations of nM and 7 PM, respectively. The dose of CX inhibited [methyl-3H]methionine incorporation of primary culture of pituitary cells into trichloroacetic acid precipitable material by about 80% in static incubation for 2 and 4 h (Shiota et al., 1984b). Ti was dissolved in 0.01 N NaOH and then diluted with the medium to the final concentration of 1 ng/ml. TSH in the medium and cells was measured by radioimmunoassay using a kit provided by the MIAMDD Rat Pituitary Hormone Distribution Program. The results were expressed in terms of NIAMDD rat TSH-RP-1 standard. Samples from each experiment were measured within one assay. The intra-assay coefficient of variation was 5.7% and the interassay coefficient of variation was less than 10% according to the method previously described (Shiota et al.. 1984a. b). TSH release (ng/min) was plotted against time and ail results were expressed as means + SE. The significance of the difference among the 3 groups was calculated by Duncan’s multiple range test (Dunnet, 1970) and the difference between 2 groups was calculated by Student’s r-test. P values of less than 0.05 were considered significant unless otherwise described.
81
Results T3 effects on TSH secretion during intermittent stimulation with TRH To examine the effect of Tj on pulsatile release of TSH and to determine the time required for the T3 suppressive effect on TSH release, the cells were intermittently exposed to TRH (10 nM for 20 min) every 60 min, in the presence or absence of 1 ng/ml Tj. The results of 2 independent experiments are shown in Fig. 1A and B, in which the
TIME,
II 0
1
2 0
3
1 TIME,hr
hr
4 2
3
Fig. 1. Inhibitory effect of Ts on TSH release caused by intermittent stimulation with TRH in the superfusion study using the primary cultured cells attached to Cytodex @l. Results of the 2 independent experiments are shown (A and B). In both experiments, cells were repeatedly exposed to TRH (10 nM) for 20 min at 60-min intervals. Ts (1 &ml) was present from the start of the second exposure to TRH. In each experiment, 6 columns were run in parallel and 3 columns were used for each treatment: TRH alone or TRH plus Ts. The flow rate was 0.25 ml/mm and the effluent was collected every 10 min. Time scales indicate the period after the start of either sample collection or Ts treatment. Each time point is the mean + SE.
cells were stimulated with TRH 7 times (A) or 4 times (B). In the absence of T3, there were pulsatile releases of TSH corresponding to each stimulation with TRH, and the magnitudes of the responses were similar during the experiment, except for the release by the first stimulation with TRH shown in Fig. 1B. Before the addition of T3, there was no significant difference in TRH-induced TSH release between the 2 groups of treatment in each experiment. In both experiments, Tj was present in the medium from the start of the second TRH stimulation. Basal TSH release was not affected by Tj throughout. On the second stimulation with TRH, TSH release was not suppressed by T3, while TSH release caused by the third stimulation - 1 h after the addition of Tj - was decreased significantly by T3. Therefore, pretreatment with Tj for about 1 h seems to be required for the inhibitory action seen in our superfusion system. The degree of the T3 suppressive effect on TSH release increased time-dependently. In the present experimental condition, the amount of T3 (1 ng/ml) seems to be the maximal suppressive dose, because further suppression was not achieved by increasing the concentration of T3 up to 20 ng/ml (data not shown). At the end of the experiment shown in Fig. lA, the immunoreactive TSH remaining was 120.7 5 5.2 pg/column in the TRH-treated cells and 145.1 k 6.4 pg/column in TRH plus TXtreated cells, respectively, with a significant difference. Effects of cycloheximide on TSH secretion induced by continuous stimulation with TRH The cells were continuously exposed to TRH (10 nM) for 3 h in the presence or absence of a protein synthesis inhibitor, CX (7 PM). Since a similar experiment had been done using a maximally effective concentration of TRH (100 nM) (Shiota et al., 1984b), the results from a single experiment are presented in Fig. 2. During continuous exposure to TRH, TSH release occurred in a biphasic manner. The profile of TSH release was characterized by an initial sharp peak, followed by a lower plateau form phase. In this report, we termed TSH release during the initial 50 min as phase I and the TSH subsequently released as phase II. On withdrawal of TRH, TSH release decreased to a basal level.
H2
I
I
CX TRH
I
2
TIME,
3
1
2
, 4 t 3
hr
Fig. 2. TSH release in response to continuous exposure to TRH in the presence or absence of ~y~lohex~mide (CX) in the superfusion study. CX (7 PM) was added 1 h before the start of continuous exposure to TRH (10 nM) for 3 h. Time scales indicate the period after the start of either sample collection, CX or TRH treatment. Six columns were run in parallel and 3 columns were used for each treatment: TRH alone or TRH plus CX. Each time point is the mean&SE.
When CX was added 1 h before the start of TRH exposure, basal (or unstimulated) release and phase-I release were not suppressed, while phase-11 release was significantly reduced. Thus, we confirmed our previous findings of a biphasic release and the possible dependency of phase-II release on protein synthesis (Shiota et al., 1984a, h). TSH release in the late part of phase I and initial part of phase II, during a time of 40-60 min, was significantly higher in CX-treated cells than that in the control. At the end of the experiment, 193.1 i: 5.2 pg/column and 165.1 $9.9 pg/column of immunoreactive TSH remained in the control and CX-treated cells, respectively. Thus, a large amount of TSH remained, even in the phase-11 blocked cells, suggesting that only a small percentage of immunoreactive TSH in the cells is releasable. T” effects on biphasic release of TSH and dependemy on protein synthesis T, effects on biphasic release induced by continuous stimulation with TRH were examined. The data from 3 independent experiments were com-
1
TRH
I
1
qTR i $ytrol) ATR
lTR H+T,+CX
n:3
\
1 I 0
or T3+CX
T3
I 0 TRH(control) .TRH+CX
!
t 0
I
ri:6
11
0
I 0
1
2 1 I 0
, 2 1
3
4 3 I 2
t 4 I 3
5
TIME,hr Fig. 3. Inhibitory effects of T, on biphasic release of TSH and possible dependency on protein synthesis. T, (I ng/ml) or T, plus cycloheximide (7 pM. CX) was present I h before the start of continuous exposure to TRH (10 nM) for 3 h. The data from 3 independent experiments were combined. In each experiment. 6 columns were run in parallel and 2 columns were used for each treatment: TRH alone. TRH plus Ts, or TRH plus Ta plus CX. Each time point is the mean i SE.
bined and the mean values are shown in Fig. 3. The total amount of TSH released during the phase-l and phase-11 periods is shown in Tabfe 1. The cells were continuously exposed to TRH (10 nM) for 3 h in the presence or absence of 1 ng/ml T,. Exposure to T3 was started 1 h before the start of continuous exposure to TRH. Continuous exposure to TRH caused a biphasic release of TSH, and the phase-I and phase-II releases were significantly lower in the T,-treated cells, compared with those in cells treated with TRH alone. To evaluate the inhibitory effect of T,, a possible dependency of T3 action on protein synthesis was examined (Fig. 3, Table 1). T3 (1 ng/ml) and CX (7 ,nM) were added 1 h before continuous stimulation with TRH (10 nM). During the constant exposure to TRH, TSH release during the phase-I period was not decreased by T, in the presence of CX, while phase-II release of TSH was significantly less in CX- plus T,-treated cells. Thus, the inhibitory effect of T3 on phase-1 release was completely blocked.
83 TABLE
1
EFFECTS OF Ts (1 ng/ml) WITH OR WITHOUT CX (7 FM) ON TSH RELEASE DURING CONSTANT STIMULATION WITH TRH (10 nM) TSH released pg/phase II (130 min)
gg/pbase I (150 min) TRH (control) TRH + T, TRH+T,+CX
11.19*0.53 6.08kO.17 11.94kO.47
B b c
18.43 f 1.07 d 11.12~0.39 e 8.57 k 0.48 f
Total amount of TSH released was calculated from the experiment shown in Fig. 3. Values are meansf SE (n = 6) and expressed by NIAMDD rat TSH-RP-1. Statistical significance (by Duncan’s multiple range test, P c 0.01): a vs. b, b vs. c, d vs. e, and d vs. f.
The 2 protein synthesis-dependent events, TRH-induced TSH release (phase II) and Ts-induced suppression of TSH release (phases I and II), were chronologically separated using a superfusion system. We confirmed that TSH release occurs in a biphasic manner in response to continuous stimulation with TRH (Schrey et al., 1977; Connors et al., 1981; Shiota et al., 1984a, b) and that phase-I release is protein synthesis-independent while phase-II release is protein synthesis-dependent (Shiota et al., 1984b). Both phases of the biphasic release of TSH were suppressed by T3. A common process in the TSH release mechanism between phase I and phase II may be affected by T3 treatment, since the biphasic profile of the response was well preserved. During phase-I release, not only the amount of TSH released but also the release profile were in~stinguishable between TRH-treated and TRH- plus Ts- plus CXtreated cells, suggesting that the acute T3 suppressive action totally depends on mechanisms involving protein synthesis. On the other hand, phase-II TSH release, which was also suppressed by Ts, was not reversed in the presence of CX. The failure to block T3 action by CX may be masked by the effect of CX on phase-II reiease since phase-II release itself is suppressed by CX. These results confirmed and extended previous studies (Bakke and Lawrence, 1968; Bowers et al., 1968; Vale et
al., 1968; Gard et al., 1981), suggesting that Ts suppresses TRH-induced TSH release through a protein synthesis mechanism. In these experiments, pituitary fragments were exposed to TRH for shorter periods than 30 min (Vale et al., 1968; Gard et al., 1981). Judging from the length of the incubation periods, the previous results may be referring to phase-I release. We suggested that there was a protein synthesis-independent TSH release immediately after TRH stimulation in which the previously prepared pool of releasable TSH (&iota et al., 1984b) was released (phase-I release). If this is the case, the acute inhibition by Tj of TSH release might be defined as the stimulation of the protein synthesis responsible for the blockade of the pooled TSH to be released. Though there have been reports indicating that thyroid hormones inhibit TSH synthesis (Spira et al., 1979; Chin et al., 1981; Marshall et al., 1981), T3 does not seem to suppress de novo TSH synthesis as far as the acute inhibitory action of T, is concerned. This agrees with the observation that suppression of TSH release by T3 resulted in an increase in intracellular TSH contents. We found that a low dose of Ts (1 ng/ml) acutely - within 60 min of the pretreatment inhibited TRH-induced TSH release in vitro. The period required for the ?; inhibitory action was close to that observed in in vivo time-course studies of plasma TSH suppression (Fukuda et al., 1975; Garcia et al., 1976; Spira et al., 1979; Larsen and Silva, 1983) and pituitary nuclear T3 accumulation after an intravenous injection of T3 in rats (Larsen and Silva, 1983). Moreover, the circulating concentration of T3 in vivo ranged between 0.5 and 0.7 ng/ml in the rat, and the dose of Ts used in the present study is considered to be within a physiolo~cal range. In the superfusion studies performed by others, relatively high doses of T, were required to demonstrate the effects (Schrey et al., 1977; Connors et al., 1981; Gard et al., 1981). In the studies of Connors et al. (1981), 20 ng/ml Ts did not inhibit TSH release from acutely dispersed cells though the same treatment did inhibit TSH release from pituitary fragments. The differences between inhibitory TX doses used in the present study and others may reside in the responsiveness of dispersed cells. The cells used in our study were cultured for 4 or 5 days before use to allow them
84
to recover from the damage of the enzymatic dispersion. When the cells were intermittently stimulated with TRH, pulsatile or phase-I-like releases were obtained. The present results showed that the degree of the T3 suppressive effect on the phase-I-like releases was time-dependently increased. Since the anterior pituitary hormones including TSH are secreted intermittently rather than continuously (Willoughby et al., 19773, the effects of T3 on the phase-I-like release may be physiologically important. We noted that CX treatment slightly augmented TSH release in the late part of phase I. Protein synthesis might be involved in the mechanisms of shift of TSH release from phase I to phase II. Recently, a similar phenomenon was observed in the case of growth hormone release (GH) (Cronin et al., 1984). Treatment with CX potentiated human pancreatic GH-releasing factor-induced CAMP accumulation and GH release in primary cultures of anterior pituitary ceils (Cronin et al., 1984). Whether or not there is a similar mechanism involved in the release of TSH remains to be determined. In conclusion, each component of the biphasic release of TSH (phase I and phase II) induced by TRH stimulation was acutely inhibited by T3 in vitro, and the action of T3 seems to be mediated by a mechanism involving protein synthesis.
We thank Dr. M. Takahashi (Tokyo University) for his valuable suggestions, Dr. A.F. Parlow and the NIAMDD Rat Pituitary Hormone Distribution Program for the gift of the rat TSH RIA kit. We are also grateful to Ms. Y. Akinaga for technical assistance, and to M. Ohara for reading the manuscript. References Bakke. J.L. and 308-314.
Lawrence,
N. (1968)
Eur. J. Pharmacol.
2.
Bowers. C.Y.. Lee, K.L. and Schally. A.V. (1968) Endocrinology 82. 75-82. Chin. W.W.. Maloof. F., Martorano, M.A.. Pierce. J.G. and Ridgway, E.C. (1981) Endo~rinoiogy 108. 387394. Chopra, I.J., Carlson, H.E. and Solomon, D.H. (197X) Eadocrinology 103, 3933402. Connors, J.M., Wright, K.C., Judd, A.M.. Liu, C.M. and Hedge. G..4. (1981) Horm. Res. 14, 1-17. Cronin, M.J.. Hewlett. EL.. Evans. W.S., Thorner. M.O. and Rogol. A.D. (1984) Endocrinology 114. 904913. Dunnet. C.W. (1970) In: Statistics in Endocrinology. Eds.: J.W. McArthur and T. Cohen (MIT Press. Cambridge, MA) pp. 799103. Fukuda. H., Yasuda, N. and Greer, M.A. (1975) Endocrinology 97. 924-931. Fukuda, N.. Nishimura. 0.. Shikata. M., Hatanaka, C., Miyamoto. M., Saji, Y., Nakayama, R., Fujino, M. and Nagawa, Y. (1980) Chem. Pharm. Bull. 28, 1667-1672. Garcia, M.D., Escobar del Rey. F. and Morreale de Escobar, G. (1976) Endocrinology 98. 203-213. Gard, T.G.. Bernstein, B. and Larsen, P.R. (1981) Endocrinology 108, 2046-2053. Hinkle, P.M. and Goh. K.B.C. (1982) Endocrinology 110, 1725-1731. Larsen, P.R. and Silva, J.E. (1983) In: Molecular Basis of Thyroid Hormone Action, Eds.: J.H. Oppenheimer and H.H. Samuels (Academic Press, New York) pp. 351-385. Lewis. M., Yeo, P.P.B., Green. E. and Evered, D.C. (1977) J. Endocrinol. 74, 405-414. Marshall, Jr., M.C.. Williams, D. and Weintraub, B.D. (1981) End~~~rinology 108. 908-915. Padmanabhan, V., Kesner. J.S. and Convey, EM. (1981) Endocrinology 108. 226.-231. Schrey, M.P.,
Brown, B.L.. and Ekins. R.P. (1977) Mol. Cell. Endocrinol. 8, 271-282. Shiota. K., Yoshida, K., Noguchi. C. and Nakayama, R. (1984a) Acta Endocrinol. (Kbh.) 106, 71-78. Shiota, K., Yoshida, K., Masaki, T.. Kawase, M., Makayama, R. and Sudo. K. (1984b) Endocrinol. Jpn. 31. 165-175. Spira, O., Birkenfeld. A., Avni, A.. Gross, J. and Gordon, A. (1979) Acta Endocrinol. (Kbh.) 92, 502-511. Vale, W., Burgus. R. and Guillemin. R. (1968) Neuroendocrinology 3. 34-46. Vale, W.. Blackwell. R., Grant, G. and Guillemin, R. (1973) endocrinology 93, 26-33. Will~~ughby. J.O., Terry, L.C.. Brazeau, P. and Martin, J.B. (1977) Brain Res. 127. 137-152.