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I. Thomson, R. Fraser and C. J. Kenyon

ABSTRACT

We have previously reported that benzodiazepines inhibit microsomal steroid hydroxylases. We have now studied their effects at much lower drug concentrations and have also addressed the suggestion that benzodiazepines alter cellular calcium metabolism.

We investigated the in-vitro effects of midazolam on microsomal steroid hydroxylation by measuring basal and ACTH-stimulated cortisol and 17α-hydroxyprogesterone (17-OHP) synthesis. Threshold inhibition of basal cortisol production was achieved by 3·4 μmol midazolam/1 while ACTH-stimulated production required 13·6 μmol/l. This was accompanied by a biphasic response of 17-OHP production, rising to a maximum at 13·6 μmol midazolam/l for basal and 6·8 μmol midazolam/l for ACTH-stimulated synthesis suggesting a preferential inhibitory effect on 21-hydroxylase activity at < 6·8 μmol/l and additonal effects on 17α-hydroxylation at higher drug concentrations. This explains the inhibition of ACTH-stimulated cortisol synthesis by midazolam (50% inhibitor dose (IC50) 22 μmol/l). Using 21-deoxycortisol as substrate, we have demonstrated that midazolam is a competitive inhibitor of 21-hydroxylase (inhibitory constant (KI) 35 μmol/l).

Both midazolam and diazepam inhibited K+-stimulated aldosterone synthesis, with IC50 values of 1·2 μmol/l and 0·8 μmol/l respectively, which are far lower than those observed for ACTH-stimulated cortisol synthesis. With 11β-hydroxyprogesterone as substrate, the K I for the inhibition of aldosterone synthesis by midazolam was 54 μmol/l. Potassium stimulates aldosterone biosynthesis at least partly by changing intracellular free calcium levels. To investigate possible antagonistic effects of benzodiazepines on calcium metabolism, we measured 45Ca uptake in the presence of midazolam. Both basal (P < 0·01) and K+-stimulated 45Ca uptake (P < 0·05) were inhibited by the drug although the effects of K+ were not completely abolished. Comparison of the dose-dependent effects of midazolam on basal 45Ca uptake in cell suspensions prepared from different areas of the adrenal cortex indicated that zona glomerulosa cells are more sensitive to midazolam.

We confirm that benzodiazepines at low concentrations have a direct effect on microsomal steroid hydroxylase enzymes in vitro and postulate that the greater sensitivity to benzodiazepines of K+-stimulated aldosterone synthesis, when compared with either ACTH-stimulated cortisol synthesis or conversion of 21-deoxycortisol to cortisol, may be explained by additional effects of these drugs on plasma membrane calcium transport.

Journal of Endocrinology (1992) 135, 361–369

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R. FRASER and V. H. T. JAMES

SUMMARY

A double isotope derivative assay technique for the simultaneous estimation of aldosterone, corticosterone and cortisol in human peripheral plasma is described. With this method, concentrations of the corticosteroids in plasma from adrenalectomized subjects were not significantly different from zero. Further evidence of specificity for aldosterone was obtained by measuring the disappearance rate of a single intravenous injection of unlabelled aldosterone from peripheral plasma, by demonstrating a rapid rise in aldosterone concentration in normal subjects acutely depleted of sodium, and by a comparison of aldosterone levels in patients with primary aldosteronism before and after surgical treatment. A study has also been made of the variation of the ratio of cortisol to corticosterone in normal subjects and of the effect upon this ratio of corticotrophin administration and haemorrhage.

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SE Dickson, R Bicknell and HM Fraser

Vascular endothelial growth factor (VEGF) is essential for the angiogenesis required for the formation of the corpus luteum; however, its role in ongoing luteal angiogenesis and in the maintenance of the established vascular network is unknown. The aim of this study was to determine whether VEGF inhibition could intervene in ongoing luteal angiogenesis using immunoneutralisation of VEGF starting in the mid-luteal phase. In addition, the effects on endothelial cell survival and the recruitment of periendothelial support cells were examined. Treatment with a monoclonal antibody to VEGF, or mouse gamma globulin for control animals, commenced on day 7 after ovulation and continued for 3 days. Bromodeoxyuridine (BrdU), used to label proliferating cells to obtain a proliferation index, was administered one hour before collecting ovaries from control and treated animals. Ovarian sections were stained using antibodies to BrdU, the endothelial cell marker, CD31, the pericyte marker, alpha-smooth muscle actin, and 3' end DNA fragments as a marker for apoptosis. VEGF immunoneutralisation significantly suppressed endothelial cell proliferation and the area occupied by endothelial cells while increasing pericyte coverage and the incidence of endothelial cell apoptosis. Luteal function was markedly compromised by anti-VEGF treatment as judged by a 50% reduction in plasma progesterone concentration. It is concluded that ongoing angiogenesis in the mid-luteal phase is primarily driven by VEGF, and that a proportion of endothelial cells of the mid-luteal phase vasculature are dependent on VEGF support.

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D R Mann and H M Fraser

Pituitary testicular function in the neonate

The early postnatal period of male primates (including humans) is associated with activation of the hypothalamicpituitary-testicular axis. Circulating levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) begin to rise in male infants during the second week of postnatal life, reach a peak between 2 and 4 months, and decline thereafter, reaching juvenile levels by 1 year of age (Forest 1990) (Fig. 1). Total testosterone levels rise commensurate with the increase in neonatal LH, reaching peak values that approach the low normal adult male range between 1 and 3 months of age, and then fall in concert with declining LH values to juvenile levels by 6 to 8 months of age (Forest 1990). Comparable patterns of neonatal endocrine changes have been reported in the chimpanzee, rhesus monkey, cynomolgus monkey, mangabey and marmoset (Steiner & Bremner 1981, Fuller et al. 1982, Mann et al. 1983,

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S F Lunn, R Recio, K Morris and H M Fraser

Abstract

In primates, plasma testosterone concentrations are elevated for some 3 months from birth. The function of this rise is uncertain, but studies in rats suggest that its prevention by castration or administration of gonadotrophin hormone-releasing hormone (GnRH) analogues has effects on development and expression of social and sexual behaviours, and adverse long-term effects on fertility. The consequences of suppression of this rise in testosterone by treatment with the GnRH antagonist antide have been investigated in the marmoset monkey. Eight sets of male:male twins were used, one of each set receiving s.c. injections of antide (10 mg/kg), on days 0, 3 and 7, then weekly from birth to 98 days of age, with the twin receiving vehicle only. Plasma samples were taken at weekly intervals for the determination of testosterone concentrations from birth until 2 years of age. Treatment with antide completely abolished the neonatal rise in testosterone seen in control animals. The timing of the onset of the pubertal testosterone rise was not significantly affected by treatment; however, the subsequent pattern of circulating testosterone showed a tendency to decreased plasma concentrations in the neonatally treated group from weeks 25 to 42, relative to controls, and this difference was significant between 43 and 70 weeks. This was associated with a similar depression in bioactive LH concentrations around this time. Thereafter, the testosterone concentrations were similar between treated and control groups. There was no effect of treatment on growth, based on sequential body weight data. At 20 months the animals underwent behaviour tests with ovariectomized females. During these encounters, males showed the full panoply of normal sexual behaviours, with response to female proceptivity, tongue flicking, sustainable penile erections, mounts with pelvic thrusting, intromissions and intravaginal ejaculations. No significant differences were observed for any of these parameters between the control and treated animals. No changes in aggressive behaviours between groups were evident. These results show that blockade of the postnatal rise in testosterone in the male marmoset monkey may result in attenuation of the pubertal rise in testosterone but is without major effect on adult basal testosterone concentrations or reproductive behaviour.

Journal of Endocrinology (1994) 141, 439–447

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R. FRASER, G. F. JOPLIN, L. H. OPIE and D. RABINOWITZ

SUMMARY

1. An augmented insulin tolerance test and its normal response is described; after a standard preparation, 11·1 u. of soluble insulin/m.2 of body surface is injected intravenously, and the level of blood sugar followed for 2 hr.

2. The degree of insulin resistance, in such states as acromegaly, can be indexed by the sum of the blood sugar values, as mg./100 ml., at 60, 90 and 120 min. after insulin. In normal subjects the mean 'insulin resistance index' was 92 (range 62–142). The test is valid only if the fasting blood sugar is normal.

3. Results are reported in: (a) twelve 'untreated' acromegalics in all of whom the test showed an abnormal insulin resistance index (mean 173 and range 144–228); (b) fourteen treated acromegalics (after gold-198 or yttrium-90 implants), in eight of whom the insulin resistance index was normal, although only five were judged to be in full clinical remission; (c) twelve subjects with gross but 'simple' obesity in whom the test results were nearly as abnormal as those of the 'untreated' acromegalics (mean insulin resistance index 159 and range 119–221).

4. The test can be used to assess the 'activity' of acromegaly and its response to treatment, provided the subject is not also grossly obese.

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N. A. SAMAAN, W. J. DEMPSTER, R. FRASER and D. STILLMAN

SUMMARY

Insulin was infused into the portal vein of four greyhounds previously pancreatectomized; assays on their hepatic venous serum showed thereafter not only a striking rise in total insulin-like activity (ILA), but also a steady rise in 'atypical' ILA (to 200–300% of the pre-infusion level).

For controls in two other pancreatectomized greyhounds, with their livers temporarily excluded from the circulation, insulin was similarly infused into a femoral vein and the brachial vein sampled for assays; no rise in 'atypical' ILA was then seen.

The levels of 'typical' or 'atypical' ILA in the three venous samples were not found to be altered by oxygenation before their plasmas were separated for bioassay.

These experiments suggest that 'atypical' insulin is found in serum because some of the 'typical' insulin secreted by the pancreas is transformed to the 'atypical' form during its passage through the liver.

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R. M. SHARPE, H. M. FRASER and J. SANDOW

Male rats aged 30 days were injected once daily for between 1 and 7 days with 50 ng (d-serine t-butyl6, des-glycine-NH2 10) luteinizing hormone releasing hormone ethylamide (LH-RH agonist), and pituitary and testicular function were assessed. Treatment for 7 days significantly (P < 0·02) inhibited maturational increases in the pituitary content and serum concentration of gonadotrophins, testicular luteinizing hormone (LH)-receptor concentration and the testicular capacity to secrete testosterone; the pituitary content and serum concentration of prolactin, the hypothalamic content of LH-RH and testicular weight were unaffected. In rats treated with LH-RH agonist, the initial (2 to 3 days) reduction in testicular LH-receptors and the capacity to secrete testosterone probably resulted from acutely raised levels of LH in the blood, whilst later effects may have resulted from the apparently chronic reduction in serum gonadotrophin levels. The latter may reflect a decrease in pituitary responsiveness to repeated stimulation with LH-RH agonist. Despite the extensive loss of testicular LH-receptors and diminished responsiveness, the concentration of HCG which significantly (P < 0·05) increased testosterone secretion by the testis in vitro was the same (2 pmol/l) as that for testes from control rats.

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K. L. Hull, R. A. Fraser and S. Harvey

ABSTRACT

Although GH has no direct effect on GH release from chicken pituitary glands, GH receptor mRNA similar to that in the rabbit liver was identified by Northern blot analysis in extracts of adult chicken pituitaries. Complementary (c) DNA, reverse transcribed from chicken pituitary RNA, was amplified by the polymerase chain reaction (PCR) in the presence of 3′- and 5′-oligonucleotide primers coding for the extracellular domain of the chicken liver GH receptor and was found to contain an electrophoretically separable fragment of 500 bp, identical in size to that in chicken liver. Digestion of this pituitary cDNA with NcoI produced expected moities of 350 and 150 bp. Amplification of chicken pituitary cDNA in the presence of oligonucleotide primers for the intracellular sequence of the chicken liver GH receptor produced an electrophoretically separable fragment of approximately 800 bp, similar to that in chicken liver. This fragment was cut into expected moieties of 530 and 275 bp after digestion with EcoRI. These PCR fragments were identified in extracts of the pituitary caudal lobe, in which somatotrophs are confined and account for the majority of endocrine cell types, and in the cephalic lobe, in which somatotrophs are lacking. Translation of the GH receptor mRNA in the pituitary gland was indicated by the qualitative demonstration of radio-labelled GH-binding sites in plasma membrane preparations, in pituitary cytosol and in nuclear membranes. These results provide evidence for the expression and translation of the GH receptor gene in pituitary tissue, in which GH receptors appear to be widely distributed within cells and in different cell types. GH may therefore have paracrine, autocrine or intracrine effects on pituitary function.

Journal of Endocrinology (1992) 135, 459–468

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A. S. McNEILLY, R. M. SHARPE and H. M. FRASER

SUMMARY

To investigate the role of adrenal and gonadal steroids in the long-term suppression of gonadotrophin secretion induced by prolactin the effects of adrenalectomy or castration on the serum and pituitary levels of LH, FSH and prolactin and the hypothalamic content of LH releasing hormone (LH-RH) have been studied in adult male rats with hyper prolactinaemia produced by the transplantation of pituitary glands under the kidney capsule.

Levels of LH and FSH in serum were significantly suppressed in all intact pituitary-grafted rats. Adrenalectomy on the day of pituitary implantation or 20 days later did not affect this suppression. However, castration on days 0,28 or 49 after pituitary grafting resulted in a rise in levels of FSH in serum indistinguishable from that in control rats. While the rise in levels of LH after castration on day 0 was the same as the controls, this increase was significantly reduced 2 days after castration on days 28 and 49 after pituitary grafting.

Castration resulted in an increase in the pituitary content of LH and a reduction in the hypothalamic content of LH-RH but no change in the pituitary content of FSH. Hyperprolactinaemia did not appear to affect these responses.

The present results showed clearly that the gonad but not the adrenal must be present for prolactin to exert an inhibitory effect on gonadotrophin secretion.