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R.A. Fraser, K. Siminoski and S. Harvey

ABSTRACT

Specific hybridization of polyadenylated RNA, extracted from rat, rabbit and human pituitary glands with a 638 bp rabbit GH receptor (rGHR) cRNA was demonstrated by Northern analysis. In-situ hybridization of tissue sections with the probe demonstrated the localization of rGHR mRNA throughout the rat pituitary gland and its presence in the anterior lobe of the rabbit pituitary. Growth hormone binding sites on pituitary membranes were not, however, demonstrated by radioligand binding studies. Thus, although the GH receptor gene is expressed in pituitary tissue, functional GH receptors may not be inserted into pituitary plasma membranes.

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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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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.

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S G Matthews, M Fraser and J R G Challis

Abstract

Development of the fetal ovine pituitary is essential for normal maturation and initiation of the parturition process, as well as for orchestrating endocrine responses to stress in utero. Increases in the biosynthesis of ACTH and prolactin (PRL) occur in the late-gestation fetal sheep pituitary. In the anterior lobe (AL) of the pituitary, pro-opiomelanocortin (POMC) biosynthesis and processing are primarily regulated by corticotrophin-releasing hormone and vasopressin. However, POMC in the intermediate lobe (IL) and PRL in the AL are known to be primarily regulated by dopamine, via the D2 receptor, in adult sheep. Because of the importance of ACTH and PRL during gestation we have investigated a potential role of dopamine in the control of both IL melanotrophs and AL lactotrophs and corticotrophs, in late gestation.

Catheters were implanted into a maternal femoral artery and vein, fetal carotid artery and jugular vein as well as into the amniotic cavity. At day 130 of gestation, fetuses were infused intravenously with either the specific D2 receptor agonist bromocriptine (n = 5) or vehicle (n=5), for 5 days. Blood samples were taken throughout the experiment and pituitaries were removed at the end of the treatment period. Bromocriptine caused a significant decrease (>50%) in POMC mRNA levels in the IL. In contrast, bromocriptine had no significant effect on POMC mRNA levels or distribution in the AL. Fetal arterial ACTH and cortisol concentrations were unaffected by the bromocriptine infusion, compared with vehicle-infused controls. There was a dramatic decrease (>80%) in plasma PRL concentrations, compared with the control fetuses. However, PRL mRNA levels in the AL were not significantly affected by bromocriptine. In conclusion, we have found that bromocriptine inhibits aspects of both melanotroph and lactotroph function in late-gestation fetal sheep. The data indicate that the fetal pituitary possesses functional D2 receptors in late gestation.

Journal of Endocrinology (1996) 150, 187–194

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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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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. 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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JOAN D. FULLER, P. A. MASON and R. FRASER

* Zoology Department, University of St Andrews, Scotland, KY16 9TS, and † Medical Research Council Blood Pressure Research Unit, Western Infirmary, Glasgow, G11 6NT

(Received 2 April 1976)

Although several corticosteroids have been reported to be present in teleost plasma (review by Idler, 1972) relatively few studies have taken adequate steps to ensure that these compounds have been reliably characterized. Methods of establishing the identity of steroids have been discussed by Brooks, Brooks, Fotherby, Grant, Klopper & Klyne (1970) and those related to teleost studies have been reviewed and assessed by Idler (1972). This report describes the analysis of a small number of plasma samples from Salmonidae caught in Loch Lomond, Scotland. Analysis was by a highly specific method based on gas–liquid chromatography (g.l.c.) which satisfies many of the criteria suggested by the above authors.

Specimens of four salmonid species were collected by means of seine or gill-nets. Coregonus lavaretus,