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SUMMARY
Studies of the rate of aldosterone production in vitro of adrenals of rats hypophysectomized before dietary sodium restriction showed that hypophysectomy not only prevented the increases in aldosterone production observed in intact, Na-deprived rats, but also depressed the level of aldosterone production to below that of intact rats maintained on a normal diet. Rats hypophysectomized for a similar period of time but maintained on the normal diet showed a similar decrease.
Experiments on adeno- and neuro-hypophysectomized rats indicated that the pituitary factor required for the normal mineralocorticoid response to dietary sodium restriction resides in the anterior pituitary.
Treatment of hypophysectomized rats during dietary sodium restriction with doses of a long-acting corticotrophin (ACTH) prevented adrenal atrophy and maintained a normal glucocorticoid response to intravenous injections of ACTH, but failed to increase aldosterone production rates in vitro to levels above that of intact rats on a normal diet; it also failed to restore the enhanced adrenocortical sensitivity to the stimulating effect of aldosterone production of intravenously injected ACTH which is characteristic of acutely hypophysectomized, Na-deficient rats. Treatment with anterior pituitary powder (8–12 mg./day) for similar periods, however, restored the aldosterone production of adrenals in vitro of hypophysectomized, Na-deprived rats to levels nearly indistinguishable from those of acutely hypophysectomized, Na-deprived controls. The same doses of anterior pituitary powder were shown not to have any demonstrable effect on the aldosterone production of adrenals in vitro of intact rats on a normal diet.
These results are interpreted as indicating the existence of a pituitary factor other than ACTH which stimulates aldosterone secretion. This factor does not appear to act directly on the adrenal cortex or to stimulate the secretion of specific glomerulotropic substances, but probably exerts its effect by maintaining the normal functional capacity of some as yet undefined tissues which secrete glomerulotropic substances in response to dietary sodium restriction.
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ABSTRACT
The β-core fragment of human chorionic gonadotrophin (hCG) is a major part of the immunoreactive hCG-like material found in the urine of normal pregnant women. Patients with non-trophoblastic gynaecological malignancies have been found to have raised levels of urinary β-core. We describe the purification of β-core, the preparation of a polyclonal sheep antiserum and the development of radioimmunoassay. The minimum detection limit of this assay was 0·025 μg β-core/l. There was no significant cross-reaction with the free α-subunit, hLH, hFSH and hTSH (<0·7%), and only partial cross-reaction with intact hCG and free β-subunit of hCG (6·9 and 18%). Within-assay variability ranged from 2·03 to 12·5% and between-assay variability from 2·25 to 13·4%. The assay was applied to urine samples from 92 normal non-pregnant premenopausal women, 54 normal postmenopausal women and 65 women with active gynaecological disease (47 postmenopausal and 18 premenopausal). In normal premenopausal women the values ranged from < 0·025 to 0·62 μg β-core/l (median 0·043 μg β-core/l). The values for normal postmenopausal women ranged from <0·025 to 0·64 μg β-core/l (median 0·26 μg β-core/l). Postmenopausal women with gynaecological malignancy had values which ranged from <0·025 to 4·0 μg/β-core/l (median 0·31 μg β-core/l); premenopausal women in this group had values which ranged from < 0·025 to 1·15 μg β-core/l (median 0·12 μg β-core/l). On molecular sieve chromatography, the material found in the urine of normal postmenopausal women showed the physicochemical characteristics of authentic β-core. However, the only detectable serum gonadotrophin was LH. Considering the homology and evolution of hCG from LH, postmenopausal β-core-like material could be derived from the degradation of β-LH resulting in a fragment with essentially identical physicochemical and immunological properties. The use of β-core assay as a tumour marker must take account of the fact that this molecule occurs in the urine of normal subjects, and that the levels are substantially raised in normal postmenopausal women.
Journal of Endocrinology (1991) 130, 481–489
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ABSTRACT
Material with the immunochemical properties of the β-core of human chorionic gonadotrophin (hCG) can be found in the urine of normal postmenopausal women. However, we have been unable to detect intact hCG (using an assay which is specific for the α–β heterodimer of intact hCG) in serum of such subjects. The levels of serum LH and urinary β-core were compared in matched samples from 28 women (serum LH: median 27 U/l, range 4-70 U/l, urinary β-core: median 0·27 μg/l, range < 0·05–0·645 μg/l). Urine (4 litres) from three postmenopausal women was concentrated, dialysed and subjected to gel exclusion chromatography on Sephadex G-100. Fractions were analysed by specific assays for LH, intact hCG, total β-hCG (free β-subunit and intact hCG), free α-subunit and β-core. Material eluting at the expected position of the β-core fragment of hCG was detected in all three samples by the β-core, β-hCG and LH assays, despite the fact that the LH antibody does not recognize the authentic β-core of pregnancy. Electrophoresis and Western blotting of the concentrated urines revealed that material of the same molecular size as β-core was recognized by the antibody to LH but not by a monoclonal antibody raised to free β-hCG which also recognizes the β-core molecule of hCG. We conclude that the predominant core-like material identified in postmenopausal urine is probably derived from the β-subunit of LH.
Journal of Endocrinology (1992) 133, 459–466
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ABSTRACT
We have previously suggested that in porcine granulosa cells, a putative intermediate, 5(10)-oestrene-3,17-dione is involved in 4-oestrene-3,17-dione (19-norandrostenedione; 19-norA) and 4-oestren-17β-ol-3-one (19-nortestosterone: 19-norT) formation from C19 aromatizable androgens. In this study, luteal cells prepared from porcine, bovine and rat corpora lutea by centrifugal elutriation were used as a source of 3β-hydroxysteroid dehydrogenase/isomerase in order to investigate the role of this enzyme in the biosynthesis of 19-norsteroids. Small porcine luteal cells made mainly 19-norT and large porcine luteal cells 19-norA from 5(10)-oestrene-3β,17β-diol, the reduced product of the putative intermediate 5(10)-oestrene-3,17-dione. However, neither small nor large cells metabolized androstenedione to 19-norsteroids. Serum and serum plus LH significantly stimulated formation of both 19-norA and 19-norT from 5(10)-oestrene-3β,17β-diol, compared with controls.
Inhibitors of the 3β-hydroxysteroid dehydrogenase/isomerase (trilostane and cyanoketone) significantly reduced formation of 19-norT in small porcine luteal cells and 19-norA in large porcine luteal cells, although they were effective at different concentrations in each cell type. In parallel incubations, formation of [4-14C]androstenedione from added [4-14C]dehydroepiandrosterone was also inhibited by cyanoketone in both small and large porcine luteal cells in a dose-dependent manner; however, trilostane (up to 100 μmol/l) did not inhibit androstenedione formation in large porcine luteal cells. In addition, the decrease in progesterone synthesis induced by trilostane and cyanoketone (100 μmol/l each) was accompanied by a parallel accumulation of pregnenolone in both cell types. These results suggest that 3β-hydroxysteroid dehydrogenase/isomerase, or a closely related enzyme, present in small and large porcine luteal cells can convert added 5(10)-3β-hydroxysteroids into 19-nor-4(5)-3-kestosteroids in vitro. In the porcine ovarian follicle, therefore, formation of 19-norA from androstenedione can be envisaged as a two-step enzymatic process: 19-demethylation of androstenedione to produce the putative intermediate 5(10)-oestrene-3,17-dione, and subsequent isomerization to 19-norA. In contrast to granulosa cells, porcine luteal cells synthesized 19-norA or 19-norT only when provided with the appropriate substrate. Unfractionated rat luteal cells also metabolized 5(10)-oestrene-3β,17β-diol to a mixture of 19-norA and 19-norT; conversion was inhibited by trilostane. In addition, small bovine luteal cells synthesized mainly 19-norT and formation was also inhibited by trilostane and cyanoketone.
In addition to 19-norA, an unknown metabolite, formed in low amounts by large porcine luteal cells, appears to be related to another steroid which accumulated at high inhibitor concentrations; it may represent 5(10)-oestrene-3,17-dione postulated as a putative intermediate formed during 19-norsteroid biosynthesis.
Journal of Endocrinology (1991) 129, 233–243
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Abstract
Sex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability. The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract. We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79·0, 68·1 and 63·2% amino acid identity with the corresponding human, rat and mouse proteins respectively. Northern blot and hot-nested PCR analyses indicated that rabbit SHBG is produced from a 1·6 kilobase mRNA in the liver of both sexes and in the testis. The rabbit SHBG cDNA was inserted into pGEX-1λT for expression of a glutathione S-transferase/SHBG fusion protein in Escherichia coli. The bacterial product bound 5α-dihydrotestosterone (DHT) in the same manner as the corresponding protein in serum. The dissociation constants (Kd) for rabbit and human SHBGs produced in E. coli were 11·1 ± 1·1 nm and 2·1 ± 0·6 nm respectively, and rabbit SHBG formed a less stable protein-steroid complex (t½=5 min) than human SHBG (t½>60 min). Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity. To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5′-terminal half of SHBG from one species and 3′-terminal half of SHBG from the other species were constructed and expressed. It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species. Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex. This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site.
Journal of Endocrinology (1997) 153, 373–384
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ABSTRACT
5-Methoxytryptamine is a potent agonist of presynaptic 5-hydroxytryptamine autoreceptors modulating serotonin release in the central nervous system. This methoxyindole can be synthesized in the pineal gland, but its presence in vivo is still controversial, probably because of rapid catabolism by monoamine oxidase. An improved high-pressure liquid chromatography method, with coulometric detection, has been developed for the simultaneous measurement of melatonin, 5-methoxytryptamine, 5-methoxytryptophol and 5-methoxyindolacetic acid. We have demonstrated a day–night rhythmicity in the amount of 5-methoxytryptamine in the pineal gland of golden hamsters (Mesocricetus auratus) maintained under a long photoperiod (14 h light: 10 h darkness) and pretreated with the monoamine oxidase inhibitor pargyline. Levels of 5-methoxytryptamine were highest at 16.30 h and lowest at 00.30 h. The rhythm for 5-methoxytryptamine appears to be the same as for serotonin (opposite in phase to that of melatonin). The identification of 5-methoxytryptamine has been confirmed by analysis with gas chromatography–mass spectrometry.
J. Endocr. (1988) 118, 389–397