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  • Author: Egil Haug x
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Egil Haug and Peter Torjesen

ABSTRACT

Four normal male subjects received iv injections of synthetic luteinizing hormone- and follicle stimulating hormone-releasing hormone (LH/FSH-RH) in doses of 12.5, 25, 100, 200 and 400 μg, respectively. A dose of 12.5 μg of LH/FSH-RH caused a significant increase in serum FSH, and 25 μg significantly increased the serum LH.

The peak responses occurred 15 to 30 min after the LH/FSH-RH injections in most of the experiments. The increase in the mean maximum serum LH and FSH levels was 2 to 4 fold. There was great variation in response between the subjects, but when tested repeatedly with the same dose of LH/FSH-RH a given individual responded in a consistent manner. The log dose-response curve between LH/FSH-RH and serum LH, and between LH/FSH-RH and serum FSH was approximately linear.

A small but significant (P < 0.05) rise in serum thyrotrophin (TSH) was found after LH/FSH-RH in doses ranging from 25 to 400 μg. There was no significant rise in serum growth hormone (HGH).

On the basis of the present study a standard 100 μg iv LH/FSH-RH test is suggested.

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Egil Haug, Harald Frey and Terje Sand

ABSTRACT

Seventeen subjects without any clinical or laboratory evidence of thyroidal or pituitary disease were given 1.0 mg thyrotrophin-releasing hormone (TRH) as a rapid iv injection 48 hours after an oral dose of 50 μCi 131I-. In all subjects there was a clear rise in serum PB131I. The elevation in the mean serum PB131I was significant (P<0.01) one hour after TRH, and the mean peak response was noted at 4 hours. It is suggested that this elevation in serum PB131I following TRH administration reflects the effect of the TSH released. In order to find the most suitable method of administration, 1.0 mg TRH was given iv, im, or as a 1 hour infusion. The maximal responses seemed to be independent of the mode of administration. Six subjects were given 3.0 mg TRH iv and 4 others 6.0 mg TRH iv. It was not possible to demonstrate a clear dose-response relationship. In five subjects the serum PB127I and the serum PB131I were measured at the same times following administration of TRH. This showed that the serum PB131I was a more sensitive index of TSH release than the serum PB127I. Twenty-four hours after the TRH injection the same subjects were given 5 IU TSH as a rapid iv or im injection. All subjects responded with a significant rise in serum PB131I. In the subjects who did not respond to TRH the response to TSH allows the differentiation between pituitary and thyroid disease.

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Oddvar Naess, Egil Haug and Kaare Gautvik

Abstract.

The effect of corticosterone and dexamethasone on the production of growth hormone and prolactin was studied in rat pituitary tumour cells (GH3-cells) in culture. Corticosterone and dexamethasone caused a dose-dependent stimulation of growth hormone synthesis, and the highest concentration (10−6 mol/l) increased growth hormone levels to 250% of controls. This concentration, however, decreased prolactin synthesis to 25% of the control values.

The cytosol fractions from monolayer cultures as well as from tumours of GH3-cells were found to possess receptor molecules for glucocorticoid hormones, having a sedimentation constant close to 8 S in a salt-free buffer and 4 S in the presence of 0.5 mol/l KCL. Isoelectric point of the receptor was 5.8. Scatchard analysis showed one single class of binding sites with high affinity (Kd 2.1 ± 0.4 (sd × 10−9 mol/l). Studies on the steroid specificity revealed that dexamethasone had the highest affinity for the receptor. Corticosterone, cortisol and progesterone had also high affinity, whereas testosterone and oestradiol-17β had no significant affinity for the receptors. After in vivo administration of [3H]dexamethasone to GH3 tumour-bearing rats, radioactivity could be extracted from purified nuclei bound to 4 S macromolecules.

The presence of receptors for glucocorticosteroid hormones in the GH3-cells, suggests that these hormones may alter growth hormone and prolactin production at the anterior pituitary level.

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Harald M. M. Frey and Egil Haug

ABSTRACT

Forty mg TRH/day was given orally for 3 weeks to 10 euthyroid women and 10 women with primary hypothyroidism on low replacement doses of thyroxine. Once weekly oral TRH was replaced by an iv TRH-test (0.4 mg) with measurement of serum concentration of TSH, prolactin (PRL), thyroxine (T4), triiodothyronine (T3) and cholesterol.

In the normal group, mean serum T4 concentration increased after one week and remained elevated. Serum TSH concentration showed a slight tendency to decline. Maximal rise in TSH concentration after iv TRH (ΔTSH) fell from a mean of 4.0 ng/ml to 1.4 ng/ml within one week and stayed low. T3, cholesterol, PRL and ΔPRL were normal and unchanged throughout.

In the hypothyroid group T4, T3, cholesterol, PRL and ΔPRL were not influenced by the TRH administration. In 2 patients (with the highest serum T4 concentrations) serum TSH concentration was normal and resistant to iv TRH. Of the 8 patients with elevated TSH, basal level and ΔTSH did not change in 2 (with subnormal T4 levels and the highest TSH levels). In the other 6 (with intermediate T4 levels) basal TSH fell from a mean of 10.1 ng/ml to 4.2 ng/ml, and ΔTSH from 10.0 ng/ml to 3.3 ng/ml after three weeks.

It is concluded that in addition to feed-back effect of thyroid hormones, the pituitary response to long-term administration of TRH is determined by other factors. Among these may be reduced pituitary TRH receptor capacity and the activity of the TSH producing cells.

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Harald M. M. Frey and Egil Haug

Abstract. Forty mg TRH/day given orally for 3 weeks to 8 patients with mild primary hypothyroidism decreased serum TSH from a mean of 4.0 ng/ml ± 1.2 (se) to 2.0 ng/ml ± 0.4 (49%), and their mean incremental TSH response to iv TRH was equally reduced from 8.6 ng/ml ± 2.5 to 4.0 ng/ml ± 1.9 (46%). In the same patients serum Prl was 8.2 ng/ml ± 2.2 before oral TRH treatment and 6.6 ng/ml ± 1.5 (81%) after treatment, and the mean incremetal Prl response to iv TRH was reduced from 43.5 ng/ml ± 5.0 to 35.9 ng/ml ± 7.5(83%).

The oral administration of 10 mg of the dopamine antagonist metoclopramide increased mean serum TSH from 0.6 ng/ml ± 0.1 (se) to 0.7 ng/ml ± 0.1 (120%) in euthyroid subjects and from 4.0 ng/ml ±1.2 to 5.7 ng/ml ± 1.6 (145%) in patients with primary hypothyroidism, and mean serum Prl from 8.6 ng/ml ± 0.8 to 109.5 ng/ml ± 24.3 (1251%) and from 8.2 ng/ml ± 2.2 to 119.6 ng/ml ± 45.5(1460%), respectively.

The incremental TSH responses to iv TRH increased 2.3-fold in euthyroid subjects pre-treated with metoclopramide, while no change was observed in the TSH responsiveness in patients with primary hypothyroidism following metoclopramide pre-treatment. In the euthyroid subjects metoclopramide treatment had no effect on the Prl response to iv TRH. In the primary hypothyroid group metoclopramide pre-treatment caused a reduced Prl response to iv TRH in more than 50% of the patients. It is concluded that long-term TRH treatment decreased the serum levels of TSH and Prl as well as the incremental increases in TSH and Prl to iv TRH stimulation in patients with primary hypothyroidism. Long-term TRH treatment did not change the TSH and Prl responses to the dopamine antagonist metoclopramide.

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Arne Attramadal, Oddvar Naess, Egil Haug, Vidar Hansson and Ken Purvis

ABSTRACT

The androgen receptor system in prolactin secreting oestrogen induced pituitary tumours has been studied. The tumour cytosol was found to contain specific androgen receptors binding [3H]5α-dihydrotestosterone (DHT) and [3H] testosterone (T) with high affinity and low capacity. Scatchard analysis of the saturation data for T revealed one class of high affinity binding sites. The equilibrium constant of dissociation (Kd) was ∼ 4 × 10−10 m and the number of binding sites was calculated to be 12.8 femtomoles/mg protein. The sedimentation coefficient of the androgen receptor complex in low salt sucrose gradients was ∼ 7 S, the electrophoretic mobility (R F) in 3.25 % polyacrylamide gels ∼ 0.5 and the isoelectric point 5.8. The protein nature of the receptor was indicated by the finding that protease, but not DNase and RNase, eliminated androgen binding. Furthermore, the receptor was thermolabile and functionally dependent on free SH-groups since androgen binding was eliminated by heating 45°C for 30 min) and treatment with p-chloromercuriphenyl sulphonate (1 mm). Steroid specificity was tested in vitro by examining the competing efficiency of different unlabelled steroids for the binding of [3H]T. The affinity of DHT for the receptor was approximately twice that of testosterone while the binding affinity of oestradiol-17β and progesterone was very low. Cortisol had no affinity for the androgen receptor. The dissociation of the androgen receptor complex was very slow at 0°C (t ½ > 48 h). Thus, the characteristics of the cytoplasmic androgen receptors of the prolactin producing pituitary tumours are very similar to those of the androgen receptors earlier demonstrated in the anterior pituitary, hypothalamus, ventral prostate, epididymis and testis. The presence of specific androgen receptors in prolactin producing pituitary tumours indicates that androgen is involved in the regulation of synthesis and release of prolactin.

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Kjersti Sletholt, Jan Gordeladze, Egil Haug and Kaare M. Gautvik

Abstract. In GH4C1 cells, the calmodulin antagonist trifluoperazine (TFP) showed a dose-dependent, biphasic effect on the basal release of PRL. An inhibition of PRL release was observed with 15–50 μmol/l TFP, whereas a concentration of 100 μmol/l and above had a stimulatory effect. The increase in basal hormone release evoked by TRH (1 μmol/l) and high extracellular concentration of K+ (50 mmol/l) was eliminated by 30 μmol/l TFP. The stimulatory effect of 100 μmol/l TFP on basal hormone release was not affected by addition of TRH (1 μmol/l) or K+ (50 mmol/l). The Ca2+ antagonists Co2+ (5 mmol/l) and verapamil (100 μmol/l), and the Ca2+ chelator EgTA (4 mmol/l) abolished the stimulatory effect of TRH (1 μmol/l) and of K+ (50 mmol/l on PRL release, whereas only Co2+ inhibited the stimulation caused by 100 μmol/l TFP. TFP (75 μmol/l) caused a transient increase in the concentration of cellular cAMP. Incubation of intact GH4C1 cells with TFP (75 μmol/l), had an inhibitory effect on both the low and the high affinity form of cAMP phosphodiesterase. Basal as well as TRH-stimulated adenyl cyclase activity were inhibited by TFP, and this effect was counteracted by addition of calmodulin.

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Oddvar Naess, Egil Haug, Arne Attramadal and Kaare M. Gautvik

Abstract.

Progesterone and corticosterone have a similar effect on the production of growth hormone (GH) and prolactin (Prl) by pituitary tumour cells (GH3 cells) in culture. Previously we have shown that progesterone has a high affinity for the glucocorticoid receptors in these cells. Progesterone may therefore exert its effects through binding to the glucocorticoid receptor. The aim of the present study was to investigate if the GH3 tumour cells and an oestrogen induced pituitary tumour, which also produce GH and Prl, possess specific receptors for progesterone. Both the GH3 tumours and the oestrogen induced pituitary tumour were in fact found to possess cytoplasmatic receptor molecules for progesterone by using the potent progestin R5020 as a marker. Isoelectric focusing revealed one binding component (pH 5.9), which was of protein nature. The binding was of high affinity (Kd 2 × 10−9 mol/l). In the oestrogen induced tumour, the maximal binding was 70 fmol/mg cytosol protein. In female rats with GH3 tumours the binding was 55 fmol/mg cytosol protein. Priming of the animals with 1 mg oestradiol-valerate increased the binding to 116 fmol/mg cytosol protein, whereas very little binding was found in GH3 tumours from rats castrated 7 days before sacrifice. The receptors in the oestrogen induced pituitary tumour and the GH3 tumours exhibited high affinity for R5020 and progesterone, whereas corticosterone had no significant affinity for the receptors. Using exchange assay, it was demonstrated that the cytoplasmic progestin receptors could be translocated to the nucleus after administration of progesterone to the animals. Thus, the presence of specific progesterone receptors, different from the glucocorticoid receptors, strongly indicates that the effects of progesterone on GH and Prl production are mediated through the progesterone receptors.

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Kjersti Sletholt, Claes Magnusson, Egil Haug and Kaare M. Gautvik

Abstract. The metabolic inhibitors antimycin A (2 μmol/l), dinitrophenol (0.5 mmol/l), and iodoacetate (6 mmol/l) were tested for their effects on hormone release, cAMP levels, and oxygen consumption in clonal strains of rat pituitary cells (GH3 cells). Basal release of growth hormone (GH) and prolactin (PRL) was reduced by all three inhibitors, and thyrotropin-releasing hormone (TRH) (l μmol/l) and K+ (50 mmol/l) stimulated hormone release were blocked. Trifluoperazine, a calmodulin antagonist, inhibited basal GH and PRL release at concentrations up to 30 μmol/l and stimulated above 50 μmol/l. The stimulatory effect of 80 μmol/l trifluoperazine on basal hormone release was eliminated by antimycin A, dinitrophenol, and iodoacetate, whereas the inhibitory effect of antimycin A, dinitrophenol and iodoacetate on basal hormone was not affected by 30 μmol/l trifluoperazine. None of the inhibitors had any effect on the level of cellular cAMP (i.e. intracellular plus extracellular). Oxygen consumption of GH3 cells was blocked by antimycin A, reduced by 25% by iodoacetate and increased by about 100% by dinitrophenol. In contrast, hormone secretion stimulated by TRH and K+ was not accompanied by any measurable alteration in oxygen consumption. Trifluoperazine (⩾ 80 μmol/l) reduced the basal oxygen consumption and blocked the stimulatory effect of dinitrophenol on oxygen consumption. In conclusion, inhibition of the energy generation of GH and PRL-producing cells severely affects the action of secretagogues, although stimulated hormone secretion may not be accompanied by any measurable increase in oygen consumption. The cellular energy supporting hormone secretion is mostly generated via oxidative phosphorylation.

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Jens P. Berg, Peter A. Torjesen and Egil Haug

Abstract.

The FRTL-5 cell line is widely used as a model for normal thyroid follicular cells. These cells have retained their ability to alter cAMP production, cell proliferation, iodine uptake, and thyroglobulin synthesis in response to thyrotropin. We have previously shown that calcitriol attenuated both basal and TSH stimulated cAMP production dose-dependently in FRTL-5 cells. Cytosol fractions (105000 g, 60 min, 4°C) prepared from FRTL-5 cell homogenates possessed calcitriol-binding components with a sedimentation coefficient of approximately 3.7 S in high salt (0.3 mol/l KCl) sucrose gradients (5–20%). At 4°C, specific binding increased rapidly during the first 4 h and reached a plateau after 8 h. The specific binding (18 h, 4°C) was maximal at a [3H]calcitriol concentration of approximately 0.5 nmol/l. Scatchard analysis of the binding data indicated one single class of high affinity binding sites with Kd = 105±2 pmol/l and Bmax=38.5±4.7 pmol/g cytosol protein (mean ± sd, N=6). In conclusion, our results suggest that the FRTL-5 cells possess functional receptors for calcitriol with the same physicochemical properties as the receptors found in normal rat tissues.