TSH crosslinks to the TSH receptor through the beta subunit

TSH binds specifically to the TSH receptor present on the surface of thyroid follicular cells and consequently activates adenylate cyclase. It was not known, however, which of the two subunits of the TSH bound to the receptor. By covalently crosslinking TSH to its receptor and characterizing the product with antisera specific for either the alpha or beta subunit of TSH, we have shown that the TSH beta subunit but not the TSH alpha subunit crosslinks to the TSH receptor.     

The pituitary TSH response to TRH is inversely related to the plasma TSH concentration and directly related to the pituitary TSH content during hypothyroidism in the rat

We studied the effects of degree and duration of hypothyroidism on the pituitary TSH concentration and the pituitary TSH secretory response to TRH. Varying degrees of hypothyroidism were achieved in thyroparathyroidectomized rats (THYREX) by continuous sc infusion of T3 (0.2, 0.3, 0.4, or 0.5 microgram/100 g X day) or T4 (0.6, 1.2, or 1.8 microgram/100 g X day). While T3 was more potent than T4, both resulted in a dose-dependent suppression of the post-thyroidectomy rise in TSH. After 7 or 14 days of severe hypothyroidism (nonreplaced THYREX rats) the pituitary TSH secretory response to TRH (250 ng/100 g body weight, iv) was found to be decreased when compared to that of euthyroid rats. Decreasing the degree of hypothyroidism increased the pituitary secretory response to TRH and the pituitary TSH content. The results indicate that in the hypothyroid rat: severe hypothyroidism results in a blunted pituitary TSH response to TRH through 14 days after thyroidectomy, at 7 and 14 days after thyroidectomy the pituitary TSH response to exogenous TRH is inversely related to the basal plasma TSH concentration, the pituitary TSH concentration increases with the duration of hypothyroidism, the pituitary TSH content is increased by low rates of thyroid hormone replacement, and the pituitary TSH response to exogenous TRH is directly related to the pituitary TSH content.     

Serum reduces the TSH content in rat pituitary cells in culture

Euthyroid rat pituitary cells were plated on a natural extracellular matrix (ECM) and cultured in either a medium supplemented with hormones and growth factors (SM) or in an unsupplemented medium (USM). Hormone supplementation resulted in a marked increase in the number of cells and in a less pronounced increase in the TSH content. Addition of 10% dehormonized serum (DHS) to the medium caused a further increase in the cell number as well as a significant elevation in the GH content but, surprisingly, it caused also a dramatic decrease in the TSH content. Time course experiments revealed that the serum doubled the rate of suppression of the TSH. DHS prepared from rat serum was as effective as that prepared from foetal calf serum and heat-inactivation of the serum did not change its suppressive effect on the production of TSH. This effect was confined to the serum fraction which was precipitable with 45% ammonium sulphate but did not bind to Con-A. It is suggested that a high molecular weight protein fraction of serum reduces the number of thyrotrophs in the culture and/or decreases the rate of TSH production by these cells.     

Minimal quantities of FSH beta subunit are released by rat anterior pituitary cells in primary culture

FSH beta in the medium and extracts of cultured rat anterior pituitary cells was quantitated with a heterologous radioimmunoassay utilizing anti-rFSH beta and hFSH beta as the standard and tracer. After 4 days in culture, extracts were prepared from washed cells, cells incubated for 6 h or 24 h in fresh medium or cells incubated for 6 h in the presence of 10(-7) M GnRH. FSH beta/rFSH molar ratios were approximately 0.05, 0.04, 0.21 and 0.35, respectively. The elevation in FSH beta/rFSH molar ratios in 24 h basal and GnRH-treated cultures was due in part to an increase in intracellular FSH beta. Both unstimulated and GnRH-treated cultures contained non-detectable quantities of FSH beta in the medium (less than 0.063 ng/100 000 cells; FSH beta/rFSH molar ratio less than 0.024). Thus, it appears that cultured rat anterior pituitary cells contain a small amount of uncombined FSH beta but that minimal quantities are released. Furthermore, GnRH may increase intracellular quantities of FSH beta but does not induce its release.     

Discordant changes in pituitary concentrations of LH and TSH subunits following hormonal manipulation

We have recently shown that mouse pituitary concentrations of free TSH-beta and alpha-subunits respond divergently to thyroid hormone manipulations in vivo. This study contrasts the changes in free LH-beta and alpha-subunits after castration and polyestradiol phosphate (E2) administration with those in free TSH-beta and alpha-subunits after thyroidectomy and thyroxine (T4) administration. Target hormone depletion (castration or thyroidectomy) resulted in an increase in plasma concentrations of the respective tropic hormones and their subunits; target hormone administration (E2 or T4) resulted in a decrease in plasma concentrations. In contrast, pituitary subunit concentrations of LH and TSH responded discordantly to target hormone depletion or administration, and the nature of the discordance for alpha-subunit and LH-beta was opposite to that for alpha-subunit and TSH-beta. Hypogonadism caused a decrease in pituitary LH and LH-beta concentrations to 65% (P less than 0.01) and 48% (P less than 0.001) of values in control (eugonadal) mice, respectively, while alpha-subunit was unchanged. Hypothyroidism caused a two-fold (P less than 0.001) and 20-fold (P less than 0.001) increase in pituitary TSH and TSH-beta concentrations, respectively, while alpha-subunit fell to 68% (P less than 0.01) of values in control (euthyroid) mice. Administration of E2 to hypogonadal mice resulted in a two-fold increase in pituitary LH and LH-beta after 11 days, while alpha-subunit fell to 55% (P less than 0.01) of values in control (untreated hypogonadal) mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyrotropin (TSH)-releasing hormone regulation of TSH subunit biosynthesis and glycosylation in normal and hypothyroid rat pituitaries

The regulation of TSH apoprotein and carbohydrate biosynthesis by TRH was studied by incubating pituitaries from normal and hypothyroid (3 weeks postthyroidectomy) rats in medium containing varying doses of TRH, [14C] alanine or [35S]methionine, and [3H]glucosamine. Samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to remove LH and free LH beta, and anti-LH alpha to precipitate free alpha-subunits. Total proteins were acid precipitated. All precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In hypothyroid samples, acute TRH (6 h) stimulated [3H] glucosamine incorporation into secreted combined alpha-subunit to 204% and secreted combined beta-subunit to 227% of control values (P less than 0.01), and stimulated [14C]alanine incorporation into secreted combined alpha-subunit to 201% and secreted combined beta-subunit to 258% of control values (P less than 0.01); pituitary content was not altered by TRH. In hypothyroid incubates, the half-maximal response was 8 X 10(-10) M TRH for both labeled precursors. In contrast, in normal samples, acute TRH (6 H) did not stimulate TSH subunit carbohydrate and apoprotein synthesis, but after 24 h, TRH stimulated [3H]glucosamine incorporation into both subunits of TSH to 270% of control values (P less than 0.02), with no change in [14C]alanine incorporation. Free alpha-subunit synthesis was not altered by TRH in normal or hypothyroid incubates. The glucosamine to alanine ratio of total newly synthesized TSH, reflecting its relative glycosylation, was increased by TRH in both combined subunits in hypothyroid samples as early as 6 h (P less than 0.05) and in normal samples only at 24 h (P less than 0.01). In summary, 1) TRH in hypothyroid incubates stimulated apoprotein and carbohydrate synthesis in combined alpha- and beta-subunits, but not free alpha-subunits, at 6 and 24 h. 2) In normal pituitary incubates, TRH stimulated TSH subunit carbohydrate, but not apoprotein, synthesis only at 24 h. 3) TRH increased the relative glycosylation of TSH in hypothyroid and normal rat pituitary incubates. Such alterations in TSH glycosylation may be due to structural changes in the carbohydrate moiety and may be important for hormone release, biological activity, or clearance.     

Studies of human pituitary LH containing internally cleaved beta subunit

We have investigated the origin of the internal peptide bond cleavage found in the beta subunit of a proportion of pituitary human LH (hLH) molecules, as well as the effects of this cleavage (or nick) on the interaction between alpha and beta subunits and on binding of hLH to its receptor. The content of cleaved beta subunit, assessed by the intensity of staining of an approximately 10 kDa component on sodium dodecyl sulphate-polyacrylamide gel electrophoresis of reduced hLH, was variable in batches of hLH prepared from pooled acetone-preserved human pituitary glands. There was evidence of a similar cleavage in purified hTSH, but not in the purified hFSH or human chorionic gonadotrophin examined. Although intact hLH was relatively resistant to cleavage in solution, urea dissociation of hormone followed by dialysis resulted in an increased content of nicked beta subunit, which was largely prevented by incorporation of the proteolytic enzyme inhibitor phenylmethylsulphonyl fluoride (PMSF) and the metal-chelating agent EDTA. Hormone that was virtually free of nicked beta subunit was obtained by urea dissociation of hLH subunits in the presence of PMSF and EDTA followed by dialysis to remove urea, reassociation of subunits at 37 degrees C (pH 7) and purification of reassociated hLH dimer by gel filtration on Sephadex G-100 in the presence of 1-anilinonaphthalene-8-sulphonic acid (ANS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental and patterns of pituitary and plasma TSH in the normal and hypothyroid female rat.

The developmental patterns of thyroid-stimulating hormone (TSH) was examined in female Long-Evans rats by radioimmunoassay of pituitary and plasma from 5 through 80 days of age. The effects of hypothyroidism on normal TSH levels were examined in animals radiothyroidectomized at birth. Control animals showed a peak in circulating TSH levels at day 12 followed by lower levels through day 30, and even lower levels at day 80 when values were those of adult animals. Hypothyroid females had higher plasma TSH levels than controls from days 10 through 30 with no distinct peak at day 12. Pituitary content reached a peak at day 18 and was always higher in control than hypothyroid animals.     

Role of naloxone and opioid peptides on thyrotrophin, alpha subunit and beta thyrotrophin by dispersed rat pituitary cells

This study was undertaken to determine the effect of opioid peptides and naloxone on the secretion of thyrotrophin (TSH), alpha subunit (alpha subunit) and beta thyrotrophin (TSH-beta) from rat pituitary dispersed cells in primary culture. Naloxone (NAL) 10(-5) M was found to increase basal TSH, alpha subunit and TSH-beta secretion. This effect of NAL was not blocked by human beta-endorphin (beta h-End) 10(-7) M. Concurrent treatment with triiodothyronine (T3) 10(-7) M significantly decreased NAL stimulated secretion of TSH and its subunits. Thyrotrophin releasing hormone (TRH) stimulation of secretion of TSH and its subunits was not further augmented by NAL. In contrast, 10(-7) M of beta h-End, methionine-enkephalin (Met-Enk) and D-ala2-met-enkephalinamide (DALA) had no effect on secretion of TSH and subunits. A time course study confirmed no change in TSH secretion following pre-treatment with beta h-End at 4, 10, 24 and 48 h. These findings go against a direct action of beta h-End, Met-Enk and DALA on TSH secretion. The response of TSH and its subunits to NAL and the lack of interaction with beta h-End might be explained by the existence of different types of opiate receptors. Counteraction of this effect by T3 suggests other possible mechanisms.     

Sex steroids modulate the pituitary parameters involved in the regulation of TSH secretion in the rat

The sex-related differences observed in the regulation of TSH secretion was further investigated by determination of the densities of T3 nuclear and TRH membrane receptors as well as the activity of 5'-deiodinase (5'D) in the anterior pituitary gland of adult male and female rats. The respective modulatory roles of androgens and estrogens on these parameters were evaluated by similar determinations carried out in castrated and in estrogen-treated male rats. The density of pituitary T3 and TRH receptors and the activity of 5'D type II were significantly greater in the female than in the male rats. The E2-treated male rats disclosed a female profile, viz. also greater densities of T3 and TRH receptors when compared with control male rats (2.3 +/- 0.2 vs 1.8 +/- 0.2 fmol T3/mg gland and 9.4 +/- 0.8 vs 6.0 +/- 0.8 fmol TRH/mg gland, mean +/- SEM), whereas no changes were found in the castrated rats. The E2-treated rats and the castrated rats exhibited an increased pituitary activity of 5'D, type II (0.87 +/- 0.10 and 0.66 +/- 0.05, respectively, vs control 0.34 +/- 0.07 pmol rT3.h-1.(mg protein)-1), suggestive of a stimulatory effect of E2 and of an inhibitory effect of androgens on this parameter. In contrast, no differences in hepatic 5'D were found between all groups, illustrating the well-known tissue-specific regulation of 5'D. These results demonstrate that the sex difference in the density of pituitary T3 and TRH receptors and the activity of 5'D in the adult rat is mainly due to a modulatory effect of estrogens, which may be responsible for the sex-dependent regulation of TSH secretion.     

Discordant effects of thyrotropin (TSH)-releasing hormone on pre- and posttranslational regulation of TSH biosynthesis in rat pituitary

To investigate whether TRH regulates TSH production through a pre- or posttranslational mechanism, we determined the pituitary levels of mRNAs for alpha-subunit and TSH beta in male Sprague-Dawley rats given TRH in the presence or absence of thyroid hormones, with or without hypothalamic influence. In normal rats, serum TSH increased 6-fold after a single sc injection of TRH (7 micrograms/kg BW), but the levels of mRNA for both TSH subunits did not differ from the control values. Infusion of TRH, achieved by osmotic minipumps that were implanted sc, increased serum TSH for 3 days. Conversely, the pituitary content of TSH dropped to and remained 35% of that in the controls. In these normal rats, throughout the TRH infusion, the pituitary levels of mRNA for the TSH subunits did not differ from those in the controls. Thyroidectomy increased, by 27 and 75 times, the normal levels of mRNAs for alpha and TSH beta, respectively. TRH, given either as a single injection or a 3-day infusion, did not further elevate these levels. We then studied thyroidectomized animals whose pituitaries were transplanted under their renal capsules. These pituitaries responded to TRH infusion by releasing TSH. T4 injection inhibited this response significantly, but not completely. In spite of this evidence of normal responsiveness to TRH, infusion of TRH for a week did not increase the level of mRNAs for either TSH subunit in transplanted pituitaries. We conclude that in the presence or absence of thyroid hormones, with or without concurrent hypothalamic influence, TRH did not affect rat pituitary level of mRNA for either TSH subunit despite persistent high levels of serum TSH. Therefore, TRH does not regulate TSH production through a pretranslational mechanism. Although a translational regulation cannot be completely excluded, the present data, in conjunction with previous findings, support the hypothesis that TRH regulates TSH production primarily by stimulating both posttranslational carbohydrate processing and secretion of this hormone.     

实验性甲状腺功能低下大鼠下丘脑神经递质的变化及其对TSH分泌的调节

本文目的在于探讨甲状腺低功大鼠下丘脑神经递质的变化及其对血清 TSH 水平的影响。结果表明:低甲大鼠下丘脑神经递质的代谢比较活跃,NE 及5-HIAA 较对照组增高;下丘脑 NE、5-HIAA、5-HIAA/5-HT 与血清 TSH 呈线性正相关,NE 与 T_4,5-HIAA 与 T_3、T_4均呈线性负相关。由此推测,NE 及5-HT 系统能够促进 TSH 的分泌和释放。甲状腺激素对腺垂体 TSH的负反馈调节作用可能由两方面的参与,其一是甲状腺激素对腺垂体 TSH 的直接负反馈调节作用,其二是甲状腺激素可能通过神经递质的改变发挥其对 TSH 的负反馈调节作用。