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  • Author: R. Fraser x
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* 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,

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A continuous isotope infusion technique was used to measure the metabolic clearance rate (MCRB) and production rate (PRB) of oestradiol-17β in a group of six non-pregnant conscious Chinchilla rabbits. The mean MCRB of oestradiol was 162 ml/min (0·079 1/ min/weight0·75), and at equilibrium a significant proportion of the radioactivity in the circulation was present as oestrone (conversion ratio oestradiol to oestrone, 27·6%). The mean PRB of oestradiol was 4·3 ng/min, which is equivalent to about 5 μg/day. Some day-to-day variation was seen in the endogenous concentrations of oestradiol, oestrone and progesterone although in general the levels of the two oestrogens were less than 30–40 pg/ml, and progesterone levels were below 300 pg/ml apart from short peaks up to 600 pg/ml. These values were compared with previous measurements of the ovarian secretion rate of oestradiol measured directly in anaesthetized animals.

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S G Matthews, M Fraser and J R G Challis


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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H. D. Simpson, R. Shepherd, J. Shepherd, R. Fraser, A. F. Lever and C. J. Kenyon


Freshly isolated bovine adrenocortical cells were pretreated with various concentrations of cholesterol and of high- (HDL) and low-density lipoprotein (LDL) fractions of known cholesterol content and then incubated in medium alone with and without angiotensin II. Preincubation with cholesterol (323 μmol/l) caused basal aldosterone synthesis to increase from 0·89 ± 0·08 to 2·77 ± 0·22 pmol/106 cells per hour (±s.e.m.) but did not significantly affect angiotensin-stimulated synthesis. Human HDL containing cholesterol at a final concentration of 129–647 μmol/l increased both basal and angiotensin-stimulated aldosterone synthesis. In HDL-treated cells, both the threshold response and responses to increasing concentrations of angiotensin were raised. Human LDL had no effect on basal or stimulated aldosterone synthesis nor did LDL alter the effects of HDL when cells were incubated with HDL and LDL in combination. Qualitatively similar results were obtained with bovine lipoproteins.

These studies show that, in short-term incubations of fresh tissue, the supply of cholesterol may be a limiting factor in aldosterone synthesis and that HDL rather than LDL is the preferred source. These observations are discussed in relation first to the mechanisms by which cholesterol/HDL might augment steroid responses and secondly to other studies with cultured cells which have demonstrated a role for LDL.

Journal of Endocrinology (1989) 121,125–131

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R. M. Shepherd, R. Fraser, D. J. Nichols and C. J. Kenyon


Angiotensin II (AII) stimulation of steroidogenesis is known to be associated with depolarization of the adrenocortical cell membrane. In these cells, membrane permeability to potassium ions governs electrical potential. The effects of All on the rate of efflux of K+ in relation to the control of aldosterone synthesis has been investigated in bovine adrenocortical cells preloaded with 43K.

In static incubations, the pattern of 43K efflux fitted a model with two exponential components with t ½ values of 47·7±1·7 and 14·2±0·6 (s.e.m.) min. AII increased the efflux rate of the slow-exchange component (t ½ 37·1±0·6 min) and retarded efflux from the fast-exchange component. With ouabain present to prevent reuptake of the isotope, the rate of efflux for both components was increased in unstimulated cells (t ½ 28·4±1·1 and 12·0±0·7 min). AII again increased the rate of efflux from the slow component (t ½ = 24·2±1·7 min, P < 0·01) and retarded efflux from the fast component. These biphasic effects were apparent in cells treated with a range of AII concentrations (0·1 nmol/l–1 μmol/l) but the point in time at which increased efflux from the slower component predominated over retardation of the slow component was earlier for cells treated with 1 μmol AII/l than for cells treated with lower concentrations.

We suggest that decreases and increases in K+ efflux caused by AII are associated with depolarization and repolarization respectively. Changes in intracellular concentrations of Ca2+ may link these events.

Journal of Endocrinology (1991) 128, 297–304

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


GH receptor (GHR) mRNA has been identified in peripheral (liver and muscle) and central (brain and hypothalamus) tissues of sex-linked dwarf (SLD) Leghorn chickens. Total RNA was extracted from the tissues of immature (1 week, 4 week), pubertal (16 week) and adult (> 24 weeks) SLD and K (the normally growing strain) Leghorn chickens. In both groups and all tissues, an mRNA moiety of 4·4 kb hybridized with cRNA probes derived from the rabbit hepatic GHR sequence. An additional low-abundance transcript of 2·8 kb was also identified in some tissues. An age-related increase in expression was observed in K and SLD hepatic GHR mRNA, suggesting normal regulation of SLD GHR gene transcription. Amplification of cDNA from K and SLD tissues in the presence of oligonucleotide primers coding for the intracellular or extracellular domains of the chicken GHR generated electrophoretically separable fragments of expected size. Restriction enzyme digestion of the products with EcoRI, BstNI, HaeIII, NcoI or BamHI produced smaller moieties of expected sizes in both strains. These results demonstrate that, in contrast to broiler SLDs, a GHR gene deletion is not responsible for the GHR dysfunction in Leghorn SLDs. Although the actual defect in GHR gene expression in SLD Leghorns remains to be identified, this study demonstrates that sex-linked dwarfism, like Laron dwarfism, is due to a heterogeneity of lesions.

Journal of Endocrinology (1993) 137, 91–98

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F McManus, R Fraser, E Davies, J M C Connell and E M Freel

The importance of corticosteroids in cardiovascular and other chronic disease is recognised. In addition, plasma steroid precursor-to-product ratios are useful and convenient indirect indicators of efficiency of key steroidogenic enzymes (aldosterone synthase, 11β-hydroxylase and 17α-hydroxylase). The use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) has enabled measurement of numerous corticosteroid compounds simultaneously. However, normal responses to trophins and variation in salt intake are not well described. This study examined these parameters in a large group of healthy volunteers. Sixty normotensive volunteers were recruited and underwent infusion of angiotensin II (AngII) and ACTH, following low- and high-salt diet. Measurement of plasma steroids at baseline and 30 min after infusion of trophin was carried out by LC–MS. As expected, plasma mineralocorticoid levels increased in response to salt restriction and were suppressed with salt loading; ACTH infusion increased all corticosteroids, while AngII increased mineralocorticoids and suppressed glucocorticoid production. ACTH increased S:F but decreased DOC:B, thus the S:F ratio is a more appropriate index of 11β-hydroxylase efficiency. The B:F ratio increased following ACTH treatment and salt restriction. A larger proportion of plasma B than generally accepted may be derived from the zona glomerulosa and this ratio may be most informative of 17α-hydroxylase activity in salt-replete subjects. Although DOC:aldosterone, B:aldosterone and 18-hydroxyB:aldosterone should provide indices of aldosterone synthase efficiency, responses of individual compounds to trophins suggest that none of them accurately reflect this. Based on these data, aldosterone synthase activity is most accurately reflected by aldosterone concentration alone.

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B. W. Morris, S. MacNeil, K. Stanley, T. A. Gray and R. Fraser


Evidence in the literature suggests that the trace element chromium may have a role in glucose homeostasis through the regulation of insulin action. We have previously reported a significant reduction in plasma chromium levels in healthy individuals, following a 75 g oral glucose load, and after meals and glucose-dependent uptake of chromium in insulindependent tissues in vitro. However, in vivo it is unclear whether the changes in plasma chromium relate to changes in plasma glucose or insulin.

The present study describes a series of euglycaemic hyperinsulinaemic clamps designed to attempt to define the initiator of changes in plasma chromium levels in ten healthy individuals.

The data showed a significant (P<0·01) reduction in fasting plasma chromium levels following glucose infusion and an initial bolus of insulin. Significant (P<0·02) increases in post-clamp urinary chromium excretion were insufficient to explain the decrease in plasma levels. During the recovery phase of an extended two-phase clamp protocol we found plasma insulin levels decreased by 70% within 10 min, associated with an increase in plasma chromium levels of 30% and no significant change in plasma glucose level.

These data indicate that alterations in plasma glucose are unlikely to be directly related to changes in plasma chromium, whilst supporting the hypothesis that plasma insulin may influence plasma levels of this trace element. In contrast, plasma zinc was unaffected throughout these clamp studies.

Journal of Endocrinology (1993) 139, 339–345

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The urinary excretion of thyroid-stimulating hormone (TSH) has been measured by double antibody radioimmunoassay after concentration by dialysis followed by lyophilization. Among 30 normal subjects, the excretion was 5·6 ± 0·31 (s.e.m.) μu./h. No diurnal variation nor differences between sexes were discerned. In 14 primary hypothyroid subjects the urinary excretion was raised (P < 0·001) to 25·1 ± 3·3 μu./h. In 14 hyperthyroid and 7 hypopituitary subjects subnormal levels of 2·6 ± 0·2 and 2·5 ± 0·22 μu./h (P < 0·001) respectively, were found. Serum and urinary TSH concentrations were measured before, during and after an infusion of human pituitary TSH (MRC 70/9) in two subjects and showed a correlation.

Urinary TSH measurement is thus a good discriminant between normal and hyperthyroid or hypopituitary patients.

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C. J. Kenyon, L. Anyaorah, L. Woodburn, J. M. C. Connell and R. Fraser


The role of protein kinase C activation in the control of cortisol synthesis was studied using the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). Bovine zona fasciculata cells were incubated with various concentrations of TPA in the presence and absence of EGTA, verapamil or nitrendipine to see whether cortisol stimulation was dependent on extracellular calcium ions. When free extracellular concentration of Ca2+ was reduced to approximately 10 μmol/l the cortisol response at all concentrations of TPA was reduced by approximately 25% indicating that protein kinase C activation is only partially dependent on extracellular calcium ions. This is confirmed by the effects of the voltage-dependant calcium channel blocker verapamil, which partially inhibited the cortisol response to a maximally effective concentration of TPA (1 μmol/l). However, a second channel blocker, nitrendipine, proved to be ten times more potent than verapamil and totally inhibited the TPA response. The partial effects of EGTA and verapamil and the contrast between verapamil and nitrendipine do not exclude the possibility that intracellular calcium ions are important in protein kinase C activation and may indicate that nitrendipine has better access to an additional site of inhibitory action than verapamil. It is significant that the ionophore A23187, which facilitates Ca2+ entry independently of voltage-sensitive channels, failed to overcome the inhibitory effects of nitrendipine in TPA-stimulated cells.

In some other tissues, the effects of protein kinase C activation are mediated by the opening of Na+/H+ exchange ports. The involvement of this port in the cortisol response has been tested by incubating TAP-and ACTH-treated cells with amiloride, an inhibitor of Na+/H+ exchange. It would seem unlikely that the steroidogenic effects of TPA are mediated by altered Na+/H+ exchange because, at the high concentration of amiloride used to block cortisol synthesis (1 mmol/l), non-specific side effects may occur and also because amiloride at this concentration inhibited the cortisol response to ACTH whose mechanism of action is not solely dependent on protein kinase C activation.

Cells were also incubated with increasing concentration of TPA in the presence and absence of ACTH and angiotensin. TPA did not further stimulate ACTH-treated cells and at 0·32 μmol/l inhibited cortisol synthesis. The effects of angiotensin and TPA were additive which suggests that the steroidogenic effects of angiotensin II are largely independent of protein kinase C activation.

J. Endocr. (1988) 117, 423–429