Regulation of pituitary receptors for thyrotropin-releasing hormone by thyroid hormones.

Thyroid hormones regulate the concentration of receptors for thyrotropin-releasing hormone (TRH) on GH3 pituitary tumor cells. The addition of 10 nM Ltriiodothyronine (T3) to hypothyroid culture medium caused a decrease in TRH receptors from 0.8 to 0.4 pmol/mg of cell protein, and half of this decrease was obtained with 0.2 11~ L-triiodothyronine. TRH receptor concentrations were significantly lower 12 h after addition of L-triiodothyronine and reached a minimum after 72 h. These effects were reversible within 72 h if L-triiodothyronine was removed. Both L-triiodothyronine and L-thyroxine caused the same loss of TRH receptors, and these effects did not depend on cell growth. Half-maximal saturation of receptors was obtained with 8 to 10 no [3H]TRH using cultures grown in hypothyroid or L-triiodothyronine-supplemented media, but maximal 13H]TRH binding was reduced 50% in cells grown with L-triiodothyronine. Membranes prepared from cells incubated with L-triiodothyronine exhibited the same & (5 to 6 nM at O’C) as those prepared from hypothyroid cells, but the maximal number of TRH binding sites was decreased. The effects of thyroid hormones on TRH receptors were additive to the loss of TRH receptors caused by TRH alone. TRH stimulates the synthesis and secretion of prolactin by the GH3 cell line. In hypothyroid cultures, TRH caused increases of 550 and 110% in prolactin synthesis and release, respectively. These responses were only 100 and 30% in L-triiodothyronine-treated cultures. In cells incubated with thyroid hormones the maximal effects of TRH on prolactin synthesis and release were reduced, but the dose-response curves for TRH effects were not shifted. The data indicate that thyroid hormones regulate the number of TRH receptors on pituitary mammotrophs without altering the affinity of receptors for TRH. These changes in the number of receptors per cell may be partly responsible for the decreased responses to TRH observed after L-triiodothyronine treatment.

The levels of thyroid hormones in tissues and plasma are tightly controlled.
Release of L-thyroxine and L-triiodothyronine from the thyroid gland is stimulated by the pituitary hormone thyrotropin.
Thyrotropin secretion is in turn stimulated by the hypothalamic peptide thyrotropin-releasing hormone, pGlu-His-ProNHz (1,2 (3,5,7) and in vitro (4,8). The prolactinstimulating activity of TRH is inhibited by thyroid hormones in animals and in cell culture experiments (3,4), although the inhibition is less marked than inhibition of thyrotropin release. Clonal lines of rat pituitary tumor cells have been used extensively to study the actions of TRH and thyroid hormones. We have used one of these lines, GH.1, which secretes prolactin and growth hormone, to study the mechanism of feedback inhibition by L-triiodothyronine and L-thyroxine; the system offers the advantage of a homogeneous cell population in which the TRH and L-triiodothyronine responses have been characterized.
In GHa cells TRH stimulates prolactin release (9) and synthesis (8,10). The peptide binds to a limited number of high affinity receptor sites which are found only on responsive cell lines (11)(12)(13)(14). The affinity of TRH and structural analogs for these receptors correlates well with their ability to stimulate prolactin, suggesting that receptor binding is an early event in TRH-mediated stimulation of hormone production (12,14). The effects of TRH on prolactin are reduced by thyroid hormones in a similar system (15). Physiological concentrations of L-triiodothyronine increase the rate of cell growth and markedly stimulate growth hormone synthesis (16-18). Thyroid hormones bind to nuclear receptor sites in pituitary tumor cells (19,20).
In this communication we report that L-triiodothyronine and L-thyroxine control the concentration of TRH receptors in GHB cells and that the concentration of receptors correlates with the ability of the cells to respond to the peptide. A preliminary account of this work has appeared (21). Rates of Changes in TRH Receptor Concentration-The rate at which L-triiodothyronine causes a decrease in receptors is shown in Fig. 2. GH:s cells were grown in hypothyroid medium for 3 days. At the start of the experiment fresh medium with or without 50 nM L-triiodothyronine was added, and TRH receptors were determined at intervals. TRH receptor concentrations were significantly lower after 12 h with Ltriiodothyronine and decreased gradually to 55% of hypothyroid values. This loss of receptors is reversible. When cultures were treated with L-triiodothyronine for 3 days and then switched to hypothyroid medium, receptor levels slowly increased to the level observed in hypothyroid cultures (Fig. 3). If cultures were alternately incubated with L-triiodothyronine- FIG supplemented and hypothyroid media, the number of TRH receptors per dish increased after each 48-h period in hypothyroid medium and decreased after L-triiodothyronine treatment, while values for protein per dish followed an opposite pattern (Fig. 4) Kd values at 0°C were 6 and 5 nM for hypothyroid and L-triiodothyronine-treated cells (Fig. 6). There was negligible binding of ["H]TRH to 4,000 x g supernatant fractions in both cases, confirming earlier studies showing that TRH receptors are localized on membranes (11,23). Therefore L-triiodothyronine does not simply cause a migration of active TRH receptors to the cytoplasm. We have also found (data not shown) that 50 nM L-thyroxine decreases the concentration of available TRH receptors on GHS cells without altering the affinity of these sites for the peptide. Additive Effects of TRH and Thyroid Hormones-Hinkle and Tashjian (23) reported that exposure of GH3 cells to TRH caused a slow, reversible loss of TRH receptors. To determine whether the effects of Mriiodothyronine and TRH were additive, hypothyroid or L-triiodothyronine-treated cultures were incubated with various concentrations of TRH for 72 h. Available TRH receptors were then measured as described previously (23) using conditions in which ["H]TRH exchanges with any bound unlabeled peptide so that all available binding sites are detected. Receptors were decreased 47% by L-triiodothyronine and 71% by the combination of L-triiodothyronine and TRH; L-thyroxine alone caused a 61% receptor loss, while L-thyroxine and TRH lowered the values by 80% (Fig.  7). In the experiments shown in Fig. 7 two different lots of hypothyroid calf serum were used and these sera may have contained different concentrations of thyroid binding globulins or traces of thyroid hormones. When tested in a single experiment, L-triiodothyronine and L-thyroxine caused the same degree of TRH receptor loss with or without TRH (Table I). It is possible that metabolism of L-thyroxine to Ltriiodothyronine may account for some of the activity we have attributed to L-thyroxine. For comparison, Table I also shows data obtained with the culture medium used by most investigators which contains sera from euthyroid animals; in this   were determined in a 72-h incubation.
Dannies and Tashjian (10) have shown that prolactin is not degraded intracehularly or extracehularly in this system, so that prolactin accumulation in the medium is a measure of the rate of de uouo prolactin synthesis. Again the TRH response was blunted by L-triiodothyronine (Fig. 8). Increases in prolactin synthesis were 550 and 100% in hypothyroid and high L-triiodothyronine conditions. Table II gives the results of a different experiment in which the effects of 100 and 1000 no TRH were tested. The effect of L-triiodothyronine is to reduce the maximal response to TRH. Similar data were obtained using L-thyroxine (data not shown). We found that the effects of L-triiodothyronine alone on prolactin are variable; however, L-triiodothyronine inhibits the TRH response whether basal prolactin synthesis is lower or higher than in hypothyroid cells.

DISCUSSION
The experiments described in this communication demonstrate that thyroid hormones regulate the number of TRH receptors on GH3 pituitary cells. This conclusion is based on the fact that a single cell type is present and that L-triiodothyronine and L-thyroxine do not alter the apparent affinity of TRH for receptors. The mechanism of the L-triiodothyronine-induced TRH receptor loss is unknown. Samuela and his co-workers have characterized nuclear L-triiodothyronine receptors in the related GH1 pituitary line (19,20).
The Kd value for the L-triiodothyronine.receptor complex, 0.5 nM, is close to the concentration of L-triiodothyronine which caused a half-maximal loss of TRH receptors, 0.2 11~. These data support the idea that binding of L-triiodothyronine to a nuclear site may be an initial step in decreasing the number of TRH receptor molecules on the cell membrane.
The L-triiodothyronine-mediated receptor loss seen in the GH, cell system is in agreement with results obtained from in uiuo studies in which rats were thyroidectomized and some animals were then injected with L-thyroxine.
Homogenates of the anterior pituitary glands of hypothyroid rata bound more ['H]TRH than those obtained from animals given L-thyroxine (25,26). The affinity of the tissue for ["H]TRH was the same in both cases (26). Interpretation of these experiments is complicated because the number of thyrotrophic and mammotrophic cells changes when thyroid status is altered (27). Receptors may also have been occupied by endogenous TRH, and it is unclear whether exchange between endogenous and radioactive TRH would have occurred during the assays. The present in vitro results, taken together with the in uiuo data, indicate that thyroid hormone levels can affect the number of TRH receptors per responsive cell.
The concentration of TRH receptors on GH3 cells is also regulated by TRH. Incubation of cultures for 2 days with TRH causes a loss of 60 to 75% of receptors (23). Since the effects of TRH and thyroid hormones on TRH receptors were additive (Fig. 7), regulation by these hormones may occur through different mechanisms. Hydrocortisone increases the number of TRH receptors on GH,, cells (28) and total ["H]TRH binding is increased in animals treated with estradiol (26). We have found that the effects of both hydrocortisone and estradiol on TRH receptors in GHa cells require the presence of thyroid hormones." Other peptides such as insulin (29) and growth hormone (30) induce losses of binding sites for the homologous hormone. In all of these examples of modulation of receptor concentration the physiological significance of the changes is unclear. We have tried to examine the relationship between the concentration of TRH receptors, as regulated by L-triiodothyronine, and the responsiveness of the cells. TRH increased the rates of prolactin release and synthesis by 110 and 550%, respectively, in hypothyroid cells; these responses were reduced to 30 and 100% in L-triiodothyronine-treated cultures. Thus the loss of TRH receptor sites was accompanied by a decreased ability to respond to the peptide. In early studies (31) with partially purified TRH preparations, the actions of thyroid hormones and TRH on thyrotropin release appeared to be competitive in nature, i.e. high concentrations of TRH could overcome the L-triiodothyronine blockade of thyrotropin release and vice versa. These results differ from those we obtained with prolactin-secreting tumor cells. This may reflect differences between control of thyrotropin secretion in uivo and control of prolactin secretion by cultured GHB cells. Alternatively, differences in the purity, timing, and doses of drugs may be responsible.
The studies reported here show that thyroid hormones reduce the number of TRH receptors on GHB mammotrophs.
The decreased number of receptors may be partially responsible for decreased responsiveness to TRH as measured by prolactin synthesis and release.