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ABSTRACT
The effect of etomidate (an anaesthetic), epostane (WIN 32729; an inhibitor of ovarian and adrenal steroidogenesis) and cyproterone acetate (an antiandrogen) on testosterone secretion from mouse Leydig cells stimulated with LH (5 i.u./l) was tested. The concentration of drug which inhibited testosterone secretion by 50% was 11·5±1·1 (s.e.m.) μmol/l for cyproterone acetate, 1·2 ± 0·2 μmol/l for etomidate and 0·23 ± 0·03 μmol/l for epostane.
The effect of all three drugs on testicular steroidogenesis was completely reversible. Thus testicular cells which had been washed after exposure to a >95% inhibitory dose of drug responded in a similar manner to hormone stimulation as cells similarly washed and which had not been exposed to the drug.
The sites of the antisteroidogenic effect of epostane, etomidate and cyproterone acetate were established using a method based on the sequential stimulation by the exogenous precursor steroids of the various steps leading to the biosynthesis of testosterone. It was concluded that etomidate acts at the sequence between LH binding and pregnenolone production, epostane acts at 3β-hydroxysteroid dehydrogenase and cyproterone acetate inhibits 3β-hydroxysteroid dehydrogenase and C17,20-lyase.
J. Endocr. (1987) 113, 457–461
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Department of Medicine and Department of Cell and Structural Biology, University of Manchester, Stopford Building, Oxford Road, Manchester m139pt
*Department of Clinical Biochemistry, Clinical Sciences Building, Hope Hospital, Eccles Old Road, Salford m6 8hd
received 14 April 1988
Introduction
An understanding of the mechanisms underlying the differentiation of mammalian cells represents one of the most challenging aspects of modern cell biology, and the success of investigations into such processes has been greatly influenced by the availability of experimental in-vitro systems in which the cellular environment closely resembles that encountered by the cells in vivo. For any particular cell type, however, the successful implementation of such experimental models is also dependent upon the isolation of long-term, stable cell lines having a normal phenotype and the differentiation characteristics of cells within the parent tissue.
In the absence of a stable, characterized line of thyroid epithelial cells, early studies of thyroid
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ABSTRACT
The degree of stability in vitro of bioactive and immunoreactive LH in human blood, plasma and serum was examined. Bioactivity and immunoreactivity of LH were assayed by the dispersed mouse Leydig cell assay and by standard radioimmunoassay respectively, using the same reference preparation (first international reference preparation for human pituitary LH 68/40 for immunoassay).
Bioactive and immunoreactive LH were stable in blood and plasma at 22 °C for up to 4 and 24 h respectively, and in blood at 4 °C for up to 24 h. There was no loss of biological or immunological LH activity in plasma which had been either snap-frozen and stored at −70 °C, allowed to freeze at −20 °C and stored at that temperature or kept at 4 °C for 24 h and then stored at − 70 °C. Likewise, the levels of LH in plasma and serum which had been stored at either − 20 or − 70 °C and then thawed and refrozen up to four times remained unchanged. In addition, the biological and immunological activity of LH was not affected after vortexing samples of plasma or serum for up to 60 s. Bioactive LH was also stable in plasma samples after prolonged storage (up to 9 months) at either −70 or −20 °C.
We conclude that LH bioactivity and immunoreactivity are stable in blood and plasma following a variety of treatments commonly experienced during normal handling of a blood sample after venepuncture.
J. Endocr. (1988) 117, 139–145
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ABSTRACT
We have studied cortisol and androstenedione secretion by dispersed cells of the outer zona fasciculata (ZF) plus zona glomerulosa, and the inner zona reticularis (ZR) plus medulla of the guinea-pig adrenal. The ZF and ZR were microdissected apart, the cells dispersed and incubated (200 000 cells/ml) for 90 min in the presence of adrenocorticotrophin (ACTH; 500 ng/l), dibutyryl cyclic AMP (dbcAMP; 1 mmol/l), pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol. The steroid concentrations were 5–25 μmol/l. Cortisol secretion was assayed by radioimmunoassay. There was no detectable cortisol secretion ( < 50 nmol/l) from the ZR in the controls (no additive) or after dbcAMP stimulation. Adrenocorticotrophin-stimulated cortisol secretion was also low (range < 50–340 nmol/l). In contrast the ZF secreted 177–379 (control), 828–2052 (dbcAMP) and 2863–9735 (ACTH) nmol cortisol/l. There was no detectable (i.e. < 2 nmol/l) cAMP production by ZR or ZF either basally (no ACTH) or after ACTH stimulation (500 ng/l). Challenge of the ZR cells with each cortisol precursor steroid (5 μmol/l) increased (P < 0·05) cortisol secretion over that seen with the corresponding basal and ACTH-stimulated controls. Thus pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol (converted directly to cortisol by 21-hydroxylase) gave rise to (mean ± s.d., n = 4) 406 ± 86, 680 ± 180, 1307 ± 111, 1141 ± 234 and 3160 ± 419 nmol cortisol/l respectively. The corresponding figures for the ZF prepared from the same adrenal cortex were 3919 ± 309, 4122 ± 97, 5766 ± 615, 5035 ± 260 and 6954 ± 755 nmol cortisol/l. With pregnenolone (25 μmol/l), cortisol secretion increased to 7847 ± 1424 (ZR) and 12880 ± 982 nmol/l (ZF), a ZR:ZF ratio of 0·6 compared with 0·1 for pregnenolone at 5 μmol/l. Androstenedione was secreted in the basal state by both ZF and ZR in similar quantities, i.e. 3·7 ± 0·3 and 3·7 ± 0·4 nmol/l (n = 9 and 14 respectively). Both cell types were ACTH sensitive, with androstenedione secretion increasing to 28 ± 4·8 (ZF) and 12·5 ± 0·9 (ZR) nmol/l.
There was a direct correlation (r = 0·924, P < 0·05) between total adrenal weight and body weight, between the percentage ZR in the cortex and adrenal weight (r = 0·96, P < 0·05) and between the percentage ZR and body weight (r = 0·981, P< 0·05). In a mature animal (800–1000 g) the ZR may occupy > 66% of the adrenal cortex. The mean diameters of cells from the ZF and ZR were 21 and 25 μm respectively.
J. Endocr. (1986) 109, 399–404
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The effect of administering the antioestrogenic drug, tamoxifen, on the growth of the pituitary tumour 7315a in the rat was studied. This tumour is induced by the administration of oestrogen. When administered early after the implantation of the tumour, tamoxifen prevented its growth completely but when treatment was delayed until a later stage of its development, 20 μg tamoxifen/100 g body wt each day for 7–12 days stopped further tumour growth, while 200 μ/100 g body wt each day reduced the size of the tumours. These effects of tamoxifen on tumour growth were accompanied by a decrease in the level of prolactin in the circulation, if the treatment was started at an early stage of tumour development and if the high dose of tamoxifen was administered. Bromocriptine either when given alone or together with tamoxifen was unable to inhibit growth and secretion of prolactin by these rat pituitary tumours. The high plasma concentrations of prolactin in the tumour-bearing rats are known to produce atrophy of the pituitary gland of the host and to decrease the synthesis and release of prolactin. Despite the inhibitory effect of tamoxifen on both tumour size and plasma levels of prolactin, the ability of the pituitary glands of these animals to synthesize prolactin remained suppressed. It was concluded that tamoxifen has a dual effect on this model of a transplantable pituitary tumour that secretes prolactin in the rat; it prevents and/or inhibits tumour growth and it has an inhibitory effect on the synthesis of prolactin by the pituitary gland.
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We have studied the physiological and clinical relevance of measurements of serum total and free IGF-I and IGF-binding protein-3 (IGFBP-3) in 57 previously untreated patients with active acromegaly (32 males, 25 females; mean age 47 years) as compared with sex- and age-matched normal healthy controls. Serum total and free IGF-I, but not IGFBP-3, are suitable biochemical parameters for screening for acromegaly. In acromegalics, the mean 24 h serum GH, total IGF-I and IGFBP-3 levels tend to decrease with age. However, in our series of patients, mean 24 h serum GH levels, IGFBP-3, total and free IGF-I do not correlate with disease activity in acromegaly.
Journal of Endocrinology (1997) 155, S9–S13
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Abstract
To investigate the role of cortisol in the etiology of insulin resistance in men and women, we examined 218 healthy non-hospitalized elderly, selected from the Rotterdam Study. Free cortisol was assessed by the ratio of fasting serum cortisol over corticosteroid-binding globulin (CBG), and insulin resistance was estimated by the fasting insulin level. CBG was higher in women and decreased with age. In both men and women, the early morning free cortisol level showed no association with age or waist/hip ratio. In men, an inverse association between cortisol and body mass index was observed. In women, higher cortisol levels were associated with increased insulin levels; an increase of 9·7 mU/l insulin per unit cortisol/CBG (s.e. 3·9, P=0·01). The association did not change after adjustment for age, body mass index or waist/hip ratio. The results of this study in elderly subjects suggest that in women cortisol may be implicated in the age-associated insulin resistance.
Journal of Endocrinology (1996) 149, 313–318
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ABSTRACT
Quantitative aspects of bioconversions in the testes of the African catfish (Clarias gariepinus) were studied in vitro by incubation of tissue with [3H]pregnenolone or [3H]androstenedione. During the breeding period, spawning and non-spawning animals were collected from their natural habitat, the Hula nature reserve, in northern Israel. In the same period, non-spawning animals were collected from a fish pond in the same region. It was shown that spawning was accompanied by significant changes in steroid bioconversions, i.e. a reduction in androgen synthesis, especially of 11β-hydroxyandrostenedione and 11β-hydroxytestosterone and an increase in the production of C21-steroids, especially progesterone, 17α-hydroxyprogesterone and a pregnenolone ester. These changes resulted from a decreased contribution of the cytochrome P-450 enzymes 17α-hydroxylase, C17–20-lyase and 11β-hydroxylase. A rise in plasma gonadotrophin concentration was observed only in spawning catfish. In the absence of such an increase in plasma gonadotrophin, steroid synthesis in the testes of non-spawning feral and pond catfish was primarily directed towards the production of 11-oxygenated androgens and 5β-pregnane-3α,17α,20α-triol. It is suggested that spawning is induced by gonadotrophin and the ensuing change in steroidogenesis. It is possible that husbandry conditions inhibit the necessary increase in gonadotrophin release.
J. Endocr. (1987) 112, 323–332
Endocrinology Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy
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Pituitary Center Rotterdam, Erasmus MC, Rotterdam, The Netherlands
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Department of Internal Medicine and & Medical Specialties (DIMI) and Center of Excellence for Biomedical Research (CEBR), University of Genoa, Genoa, Italy
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Pituitary Center Rotterdam, Erasmus MC, Rotterdam, The Netherlands
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Pituitary-directed medical treatment for Cushing’s disease (CD) is currently represented by membrane receptor targeting drugs (somatostatin analogs and dopamine agonists). Somatostatin and dopamine receptors are regulated by β-arrestins, which have been shown to be differentially regulated by glucocorticoids in non-neuroendocrine cells. In this study we investigated the effects of glucocorticoids on β-arrestin expression in corticotroph tumor cells. First, AtT20 cells, a mouse model of CD, were exposed to dexamethasone (Dex) at different time points and β-arrestin expression was evaluated at mRNA and protein levels. Futhermore, β-arrestin mRNA expression was evaluated in 17 human corticotroph adenoma samples and correlated to patients’ pre-operative cortisol levels. We observed that Dex treatment induced a time-dependent increase in β-arrestin 1 mRNA expression and a decrease in β-arrestin 2. The same modulation pattern was observed at protein level. Dex-mediated modulation of β-arrestins was abolished by co-treatment with mifepristone, and Dex withdrawal restored β-arrestin expression to basal levels after 72 h. The evaluation of β-arrestin mRNA in corticotroph adenomas from CD patients with variable disease activity showed a significant positive correlation between β-arrestin 1 mRNA and urinary cortisol levels. The effect of glucocorticoids on β-arrestin levels was confirmed by the analysis of two samples from a single patient, which underwent adenomectomy twice, with different pre-operative cortisol levels. In conclusion, glucocorticoids induce an inverse modulation of the two β-arrestin isofoms in corticotroph tumor cells. Since β-arrestins regulate membrane receptor functions, this finding may help to better understand the variable response to pituitary-targeting drugs in patients with Cushing’s disease.
