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Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham b15 2th
received 1 November 1987
Introduction
Thyrotrophin (TSH) is one of a family of glycoprotein hormones which includes the pituitary hormones follicle-stimulating hormone and luteinizing hormone, and placental chorionic gonadotrophin. Each hormone is composed of two dissimilar, non-covalently linked glycosylated subunits, α and β. The mammalian genome contains a single gene encoding the α-subunit which is common to each of the glycoprotein hormones (Fiddes & Goodman, 1981). In contrast, the β-subunits of each hormone are encoded by different genes and confer biological and immunological specificity upon the intact dimer.
The gene encoding the β-subunit of TSH has been assigned to chromosome 1 in man (Fukushige, Murotsu & Matsubara, 1986) and chromosome 3 in the mouse (Kourides, Barker, Gurr et al. 1984). The α-gene, unlike the β-gene, has been assigned in the mouse to chromosome 4 (Kourides et al.
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ABSTRACT
Amplification of desensitization of TSH response to thyrotrophin-releasing hormone (TRH) may be important mechanisms in the regulation of its secretion. We have investigated this possibility in vitro, using monolayer culture of rat anterior pituitary cells. Cells (1–1·5 × 105/250 μl per well) were cultured for 72 h, exposed to TRH or dibutyryl cyclic AMP (dbcAMP) for 6 or 8 h, washed, and then treated for 4 h with various doses of TRH, or with K+ (55 mmol/l) as a non-specific secretagogue. Pretreatment with TRH (20 nmol/l) for 8 h reduced subsequent TSH release: basal release fell to 64% of the control value (1·01±0·10 μg/l pretreated, 1·58 ± 0·16 control) and release in response to TRH (100 nmol/l) to 69% of the control (2·7 ± 0·19 μg/l vs 3·98 ± 0·22); K+ response was reduced to 86% of the control (3·77 ± 0·21 μg/l vs 4·39 ± 0·20), significantly less than the other reductions. The extent of the parallel downward shift of the TRH dose–response curve was proportional to dose and duration of prior TRH exposure. There was no significant change in the dose of TRH required to cause half-maximal TSH release (ED50: pretreated 4·8, control 2·8 nmol TRH/l) suggesting depletion of an intracellular pool of TSH rather than 'desensitization'. After 6-h pretreatment with dbcAMP, subsequent TSH responses were augmented: basal release was 130% of the control, response to TRH (100 nmol/l) was 137% and to K+ it was 132% of the control, with a parallel upward shift of the TRH dose–response curve but no change in cellular TSH content. We suggest that an intracellular pool of TSH exists which can be depleted by prior TRH exposure without a desensitization effect. The size of this pool may be increased by dbcAMP, indicating that cyclic nucleotides may modulate the availability of TSH to an acutely releasable intracellular pool.
J. Endocr. (1986) 108, 211–217
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ABSTRACT
We have studied the secretion of TSH and prolactin from perifused rat anterior pituitary glands in vitro in response to single pulses of thyrotrophin releasing hormone (TRH) and KCl after prior exposure to TRH. Anterior pituitary fragments were incubated in normal medium or in medium containing 28 nmol TRH/1 for 20 h before perifusion. Thyrotrophin releasing hormone (28 nmol/l), administered as a 3-min pulse, stimulated TSH and prolactin release from control tissue to a peak value four or five times that of basal. After exposure of the pituitary tissue to TRH for 20 h, the subsequent response of TSH to a 3-min pulse of TRH was, however, markedly reduced; in contrast, the prolactin response was not significantly reduced. In a similar series of experiments KCl (60 nmol/l) was administered to both control and TRH-'treated' pituitary tissue as a 3-min pulse; no significant differences in TSH responses or prolactin responses were observed. These data indicate that TRH desensitizes the pituitary thyrotroph to a subsequent TRH stimulus but has very little effect on prolactin secretion.
J. Endocr. (1984) 101, 101–105
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ABSTRACT
We have studied the effects of cyclic AMP (cAMP) on TSH secretion by cultured rat pituitary cells, using forskolin and dibutyryl cAMP (dbcAMP) to raise the cellular cAMP content by different mechanisms. Forskolin (10 μmol/l), a stimulator of adenylate cyclase, raised the cAMP content within 10 min, but had a more delayed effect on TSH release, with no significant stimulation for at least 6 h, but a clear dose-dependent effect at 24 h. Incubation with dbcAMP likewise increased TSH release after 6–24 h. By contrast, high cellular cAMP levels induced by either forskolin or dbcAMP augmented the TSH response to TRH at an early stage, before any detectable change in unstimulated TSH release. Pretreatment of cells with forskolin led to a parallel upward shift in the subsequent TRH dose-response curve, without a significant change in median effective dose or any change in cellular TSH content.
These findings suggest that cAMP acts to increase the availability of TSH for acute release by TRH by modulation of an intracellular releasable hormone pool, and indicate synergistic interactions between the adenylate cyclase system and the phospholipid-calcium stimulus-release coupling mechanism of TRH.
J. Endocr. (1986) 109, 365–369
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Neurotensin is a hypothalamic peptide which inhibits secretion of TSH in the rat in vivo. We have demonstrated a calcium-dependent release of neurotensin from incubated rat hypothalamus in response to depolarizing stimuli, as well as a dose-dependent stimulatory effect of tri-iodothyronine (T3) on neurotensin secretion. We suggest that part of the neuroendocrine control of TSH secretion involves the interaction of T3, neurotensin and TSH; the presence of neurotensin in extracts of anterior pituitary gland is further evidence for its hypophysiotrophic role.
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Abstract
Epidermal growth factor (EGF) is a potent mitogen for sheep pituitary cells but the factors controlling the binding and expression of EGF and its receptor (EGFR) in the pituitary are poorly understood. Regulation of EGF binding and EGFR gene expression may determine cellular responsiveness to EGF and could play a role in neoplastic development. Scatchard analysis of 125I-EGF binding in cultured sheep pituitary cells revealed two receptor binding sites (high affinity class of 2·5± 0·5 × 103 receptors/cell with a dissociation affinity constant (Kd) of 3·2± 0·7 × 1010 m and low affinity class of 3·3 ±1·0 × 104 receptors/cell with a Kd of 71 ±1·3 × 10−9 m). Exposure of the cultured cells to some target gland hormones of the pituitary (oestrogen, tri-iodothyronine and hydrocortisone), pituitary growth factors (EGF, basic fibroblast growth factor, transforming growth factor-β and a tumour-promoting phorbol ester (TPA) resulted in an increase in the binding affinity of the high affinity receptors while reducing the receptor number and also a reduction of EGFR mRNA levels, shown by Northern blot analysis. In contrast, forskolin, an activator of adenylate cyclase, showed no significant effect on EGF binding and receptor gene expression. We conclude that the EGFR in normal pituitary cells can be modulated by several hormones and other growth factors at both receptor binding and mRNA levels. Transmodulation of EGFR by hormones and growth factors in the pituitary may be one of the regulatory mechanisms controlling the balance of normal pituitary growth and function. Defects in this regulatory system could have a role in the multistep process of pituitary tumourigenesis.
Journal of Endocrinology (1994) 143, 489–496
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ABSTRACT
Serum thyroglobulin (Tg), measured by radioimmunoassay, was high in 6-propylthiouracil (PTU)-treated rats but low in thyroxine (T4)-treated animals compared with euthyroid controls. Thyroid-stimulating hormone (TSH) stimulated Tg release in vitro from enzymatically dispersed normal rat thyroid cells in a dose-dependent manner. Thyroid cells prepared from T4-treated animals behaved similarly to cells from control rats, whereas in vitro basal release of Tg from thyroid cells prepared from PTU-treated animals was high and the response to TSH was lost. Our data confirm the TSH dependency of Tg release in vivo and in vitro and our system provides a means of studying the control of Tg secretion in vitro.
J. Endocr. (1984) 101, 107–111
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ABSTRACT
Thyroid hormones may regulate prolactin gene transcription. We have previously found that phenytoin inhibits tri-iodothyronine (T3) nuclear binding, and have suggested that phenytoin may act as a partial T3 agonist. We have therefore investigated the effects of phenytoin and T3 on prolactin release and gene transcription, using the technique of cytoplasmic dot hybridization with complementary DNA probes to estimate prolactin messenger (m) RNA concentrations in cytoplasm from cultured rat pituitary cells.
Tri-iodothyronine treatment led to a small but significant fall in prolactin release by 72 h, but caused marked dose- and time-dependent reductions in prolactin mRNA levels at 48–72 h. Phenytoin, however, caused more rapid falls in both prolactin release and mRNA concentrations. Neither T3 nor phenytoin significantly altered GH mRNA levels. These studies suggest effects of phenytoin similar, but not identical, to those of T3 in the lactotroph.
J. Endocr. (1986) 109, 359–364
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ABSTRACT
Effects of thyroid status on expression of a variety of myocardial genes, such as those encoding contractile proteins, have been reported, as well as interactions between thyroid hormones and developmental and haemodynamic regulation of contractile protein synthesis. In addition, it is clear that developmental and haemodynamic factors regulate expression of specific proto-oncogenes, including c-myc, c-fos and H-ras, in the myocardium but the effect of thyroid status on such proto-oncogene products, which are proposed to play a critical signal-transducing role in the heart, has been previously unexplored.
In order to determine whether changes in thyroid status are associated with changes in expression of these putative intracellular signals, we examined the effect of hypothyroidism and tri-iodothyronine (T3) treatment on myocardial levels of c-myc, c-fos and H-ras mRNAs in the rat. The induction of hypothyroidism was associated with a marked increase in myocardial c-myc, c-fos and H-ras mRNAs, changes reversed by 72 h of T3 replacement. Administration of T3 to euthyroid rats had no significant effect on myocardial c-myc or c-fos mRNAs, but inhibition of H-ras mRNA by T3 was evident. These observations demonstrating influences of thyroid status on expression of specific proto-oncogenes suggest that thyroid hormones, as well as exerting direct effects on expression of functionally important myocardial genes, also interact with the cellular transduction pathways mediated by the products of the c-myc, c-fos and H-ras genes.
Journal of Endocrinology (1991) 130, 239–244
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ABSTRACT
We have used a recombinant human basic fibroblast growth factor (basic FGF) to study its effects on cell proliferation, gene expression and accumulation of cyclic AMP (cAMP) and inositol phosphates in two well-characterized endocrine cell lines, FRTL-5 rat thyroid and GH3 rat pituitary cells. Basic FGF induced a dose-dependent increase in mitogenesis (assessed by measuring incorporation of [3H]thymidine) in FRTL-5 cells (40 ng basic FGF/ml increased mitogenesis above the control value by 2148±108% (mean ± s.e.m.), but inhibited mitogenesis in GH3 cells at all doses (85±4% of control with 40 ng basic FGF/ml)). Thyroglobulin mRNA concentration was increased in FRTL-5 cells (126±6% of control with 40 ng basic FGF/ml) as was prolactin mRNA in GH3 cells (246±11% of control with 40 ng basic FGF/ml), but GH mRNA in GH3 cells was not significantly affected by any dose of basic FGF. Intracellular cAMP was reduced by basic FGF in both FRTL-5 and GH3 cells (40 ng bFGF/ml giving 80±5% of the control value in FRTL-5, and 67±15% of the control value in GH3 cells) despite increased levels when FRTL-5 cells were stimulated with 150 μU TSH/ml (5645±484% of control) or GH3 cells were stimulated by 10 μmol forskolin/1 (3347±396% of control). In both FRTL-5 and GH3 cells, accumulation of [3H]inositol phosphates was increased by 40 ng basic FGF/ml (201±6 and 330±51% of control values respectively).
We have shown that basic FGF has different effects on mitogenesis in the two cell lines; gene expression and accumulation of inositol phosphates were increased in both, whereas the intracellular concentration of cAMP was decreased. The actions of basic FGF may be mediated through both inhibition of adenylate cyclase and hydrolysis of phosphatidyl inositol bisphosphate as has been proposed for 3T3 fibroblasts. Our data suggest that there may be a physiological role for basic FGF in both thyroid and pituitary tissue.
Journal of Endocrinology (1990) 127, 39–46
