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M. P. Schrey, H. J. Clark and S. Franks

ABSTRACT

A role for the regulation of cellular Ca2+ homeostasis in the dopaminergic control of prolactin secretion was investigated in rat anterior pituitary glands. Withdrawal of dopamine stimulated the uptake of 45Ca2+ into hemipituitary tissue by 48% after 3 min. Radioisotope desaturation from tissue prelabelled with 45Ca2+ was significantly retarded in the presence of dopamine. Withdrawal of dopamine rapidly stimulated 45Ca2+ efflux from prelabelled tissue by 79% and was accompanied by a three- to fourfold rise in prolactin secretion. The 45Ca2+ efflux response to dopamine withdrawal was reduced in tissue prelabelled in the presence of dopamine. Agonist displacement with metoclopramide mimicked the effect of dopamine withdrawal on 45Ca2+ efflux and prolactin secretion.

These observations demonstrate that the stimulation of prolactin release by dopamine withdrawal is accompanied by a redistribution of cellular Ca2+ and support the hypothesis that dopamine inhibits secretion by decreasing Ca2+ influx in the mammotroph cell.

J. Endocr. (1986) 108, 423–429

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P. VAN DER SCHOOT, D. W. LINCOLN and J. S. CLARK

Changes in brain activity after electrochemical stimulation of the preoptic area of prooestrous rats were studied by the measurement of the electro-encephalogram (EEG) of the frontal cortex and the recording of single neurones in the anterior hypothalamus. All rats were anaesthetized with urethane between 10.00 and 12.00 h to allow prolonged electrophysiological recording and to block the spontaneous surge of LH during the afternoon. Electrochemical stimulation was applied, between 12.00 and 14.00 h, as an anodal current through an implanted steel electrode; this caused the electrolytic deposition of iron and evoked the release of LH and ovulation.

Electrochemical stimulation of the preoptic area changed the cortical EEG, either immediately or after a delay of a few minutes, from a labile pattern with alternate periods of arousal and slow-wave sleep, to a stage of continuous arousal which persisted for the remainder of the recording period (2–3 h). Conversely, the EEG pattern of the cortex was not disturbed by electrolytic lesions placed in the preoptic area through a platinum electrode. Electrochemical stimulation of the arcuate region of the hypothalamus, the lateral septal area, the medial amygdaloid complex and the anterior parts of the thalamus caused no obvious change in the EEG patterns.

Ipsilateral anterior hypothalamic neurones, about 1 mm caudal to the focus of electrochemical stimulation, displayed an immediate decrease in electrical activity after application of the current. After 10–20 min however, the rates of discharge of action potentials in 9 out of the 16 neurones under consideration increased progressively from 0·5 to 15–25 action potentials/s and these rates were maintained until the recordings were lost after 90–230 min. No such acceleration in electrical activity was observed in neurones on the contralateral side.

Iron deposited during electrochemical stimulation was precipitated as sulphide and stained by Timm's method. There was a central damaged area of radius 0·6 mm surrounded by an 'undamaged' area with considerable infiltration of iron, up to a distance of 1·7 mm from the electrode tip. Cells within the area of infiltration did not stain for iron 10 min after electrochemical stimulation, but after 30 min, neural elements in this peripheral zone were stained in a manner similar to the Golgi method.

The concentrations of LH in the plasma remained unchanged in all rats for 10–15 min after electrochemical stimulation. Thereafter, the concentrations increased progressively and approximately in parallel to the changes in action potential activity until, after 2 h, the individual concentrations of 300–600 ng LH/ml were more than six times the values obtained before stimulation. Bilateral electrochemical stimulation resulted in appreciably higher concentrations of LH and produced values close to those observed during the pro-oestrous surge of the hormone.

Electrochemical stimulation during the afternoon of the day before pro-oestrus consistently advanced ovulation; this response occurred irrespective of whether the resultant lesions were large or small. The production of a large lesion during the afternoon of the day before pro-oestrus without concomitant deposition of iron by the use of a platinum electrode appeared to block the surge of LH on the subsequent day and the preovulatory follicles became atretic.

These results suggest that the ovulatory response to electrochemical stimulation is related primarily to an increase in the electrical activity of the hypothalamus and not to the destruction of brain tissue.

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K Walder, A Filippis, S Clark, P Zimmet and GR Collier

Leptin is secreted from adipose tissue, and is thought to act as a 'lipostat', signalling the body fat levels to the hypothalamus resulting in adjustments to food intake and energy expenditure to maintain body weight homeostasis. In addition, plasma leptin concentrations have been shown to be related to insulin sensitivity independent of body fat content, suggesting that the hyperleptinemia found in obesity could contribute to the insulin resistance. We investigated the effects of leptin on insulin binding by isolated adipocytes. Adipocytes isolated from Sprague-Dawley rats exhibited a dose-dependent reduction in the uptake of 125I-labelled insulin when incubated with various concentrations of exogenous leptin. For example, addition of 50 nM leptin reduced total insulin binding in isolated adipocytes by 19% (P < 0.05). Analysis of displacement curve binding data suggested that leptin reduced maximal insulin binding in a dose-dependent manner, but had no significant effect on the affinity of insulin for its binding site. We conclude that leptin directly inhibited insulin binding by adipocytes, and the role of leptin in the development of insulin resistance in obese individuals requires further investigation.

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M. Caleffi, I. S. Fentiman, G. M. Clark, D. Y. Wang, J. Needham, K. Clark, A. La Ville and B. Lewis

ABSTRACT

As part of a controlled trial of the use of tamoxifen for the treatment of mastalgia, some of the metabolic and haematological effects of this agent were measured. A panel of haemostatic variables including prothrombin time, kaolin cephalin clotting time, fibrinogen, euglobulin lysis time, factor VII, factor VIII, protein C and anti-thrombin III were determined. In addition, levels of sex hormone-binding globulin and both total and free oestradiol were estimated. No alteration in clotting function was found during the administration of tamoxifen, although hepatic function did alter during this period with an increase in concentration of sex hormone-binding globulin. There was a significant increase in total oestradiol and free oestradiol although the percentage of biologically available free oestradiol fell slightly during the course of tamoxifen treatment. There was a slight reduction in low-density lipoprotein cholesterol with an increase in HDL2, a subclass of high-density lipoprotein (HDL) cholesterol, consistent with an oestrogen-agonist effect. These data suggest that tamoxifen administration does not adversely influence haemostatic mechanisms or lipoprotein metabolism in the short term.

J. Endocr. (1988) 119, 335–339

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D E Clark, S K Smith, A M Sharkey, H M Sowter and D S Charnock-Jones

Abstract

Hepatocyte growth factor (HGF), also known as scatter factor, acts via the c-met receptor resulting in pronounced effects on certain epithelial cells. We hypothesised that HGF would be important in placental development where the trophoblast represents a specialised barrier of epithelial origin. In this paper we examine the expression and production of HGF and its receptor in the human placenta throughout pregnancy. In addition, RT-PCR was undertaken on human embryos to ascertain whether preimplantation embryonic or trophoblast cells were under the influence of this growth factor. In samples from the first trimester of pregnancy in situ hybridisation with a c-met antisense probe detected message expression in villous cytotrophoblast and in decidual glands but not in extravillous trophoblast. Some c-met expression was detected in cytotrophoblast from the second trimester placentae; this declined to negligible levels by term. Staining with an anti c-met antibody largely confirmed these findings but found relatively strong staining of cytotrophoblast at term. HGF was confined to the villous core throughout pregnancy when examined by both in situ hybridisation and immunohistochemistry. Trophoblast was consistently negative for HGF. Pre-implantation embryos examined by RT-PCR were negative for both c-met and HGF mRNA. These results indicate that the HGF may exert an important influence on cytotrophoblast throughout the process of placental formation and growth.

Journal of Endocrinology (1996) 151, 459–467

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MARY ELLEN STEWART, P. E. POCHI, J. S. STRAUSS, H. H. WOTIZ and S. J. CLARK

SUMMARY

The in-vitro metabolism of [3H]testosterone by human scalp and back skin was examined for possible differences in enzyme activity in skin from these two areas, both of which contain large sebaceous glands but only one of which, the back, is prone to develop acne. Punch biopsy specimens of skin, obtained from the scalp and back of adult men, were minced and incubated with [3H]testosterone. The metabolic products were diluted with carrier steroids, then separated and measured by thin-layer chromatography and by gas chromatography on an instrument equipped with a splitter. The results showed that of the 5α-reduced metabolites identified, a major one in both the scalp and back skin incubations was 5α-androstane-3β,17β-diol. Formation of the androstanediol was especially pronounced in scalp skin where it accounted for up to 50% of the 5α-reduced metabolites produced. This finding that 5α-androstane-3β,17β-diol is a major product of testosterone metabolism in vitro by human skin containing sebaceous glands, supports the possibility, previously suggested by studies in the rat, that this steroid can stimulate sebum secretion.

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R. G. Clark, L. M. S. Carlsson and I. C. A. F. Robinson

ABSTRACT

An automatic method for repetitive microsampling of blood from conscious animals was used to obtain detailed GH secretory profiles from normal female rats, which were compared with those in males and ovariectomized females. Female rats showed a highly variable GH secretory pattern, with sustained periods of low, almost continuous secretion, followed by very rapid bursts of high amplitude and short duration, occurring mostly at night. There was no clear relationship between the pattern of GH secretion and the phase of the oestrous cycle in rats continuously sampled over a 5-day period. In ovariectomized rats, the day:night difference was maintained, though the nocturnal GH surges were larger and of longer duration than in intact females. Male rats produced multicomponent GH bursts which continued unchanged throughout the day and night. This study shows for the first time that female rats switch to a rapid, highly pulsatile pattern of GH release at night, which can only be resolved by rapid blood sampling over extended periods in conscious undisturbed animals.

J. Endocr. (1987) 114, 399–407

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R. G. Clark, L. M. S. Carlsson, B. Rafferty and I. C. A. F. Robinson

ABSTRACT

We have studied the rebound secretion of GH following short-term somatostatin (SS) infusions in conscious rats, using an automatic sampling system for withdrawing frequent microsamples of blood. Intravenous infusions of SS (5–50 μg/h per rat) inhibited spontaneous GH secretion, but when SS was withdrawn there was a large burst of rebound GH secretion. A sub-anaesthetic dose of urethane reduced such rebound bursts of GH, suggesting a hypothalamic involvement in rebound GH secretion. Passive immunization with an antibody against rat GH-releasing factor (GRF) attenuated the rebound GH secretory response to the withdrawal of an SS infusion (GH concentration during rebound secretion was 26±21 μg/l vs 475 ± 127 μg/l (mean ± s.e.m.), after 0·5 ml anti-GRF serum or non-immune serum respectively). The inhibition of GH rebound secretion was related to the dose of anti-GRF serum administered. Intravenous infusions of human GH (20– 100 μg/h per rat) also reduced the size of the rebound GH secretion following SS withdrawal, in both male and female rats. We suggest that the rebound GH secretion that follows SS withdrawal in vivo is caused mainly by a hypothalamic release of GRF. Exogenous GH inhibits SS-induced rebound GH secretion in the conscious rat, possibly by inhibiting hypothalamic GRF release.

J. Endocr. (1988) 119, 397–404

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L. M. S. Carlsson, R. G. Clark and I. C. A. F. Robinson

ABSTRACT

Growth hormone inhibits its own secretion in animals and man but the mechanism for this inhibition is unclear: both stimulation of somatostatin release and inhibition of GH-releasing factor (GRF) release have been implicated. We have now studied the GRF responsiveness of conscious male and female rats under conditions of GH feedback induced by constant infusion of exogenous human GH (hGH). Intravenous infusions of hGH (60 μg/h) were maintained for 3 to 6 h whilst serial injections of GRF(1–29)NH2 (0·2–1 μg) were given at 45-min intervals. The GH responses were studied by assaying blood samples withdrawn at frequent intervals using an automatic blood sampling system. We have confirmed that male and female rats differ in their ability to respond to a series of GRF injections; female rats produced consistent GH responses for up to 13 consecutive GRF injections, whereas male rats showed a 3-hourly pattern of intermittent responsiveness. In female rats, multiple injections of GRF continued to elicit uniform GH responses during hGH infusions, whereas hGH infusions in male rats disturbed their intermittent pattern of responsiveness to GRF, and their regular 3-hourly cycle of refractoriness was prolonged. We suggest that this sex difference in GH feedback may be due to GH altering the pattern of endogenous somatostatin release differentially in male and female rats. Such a mechanism of GH autofeedback could be involved in the physiological control of the sexually differentiated pattern of GH secretion in the rat.

Journal of Endocrinology (1990) 126, 27–35

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R. G. Clark, L. M. S. Carlsson and I. C. A. F. Robinson

ABSTRACT

The negative-feedback effects of GH on its own secretion were studied in conscious male and female rats bearing indwelling double-bore venous cannulae. Intravenous infusions of human GH (hGH; 20–60 μg/h) or somatostatin (SS; 10 μg/h) were given while frequent serial microsamples of blood were withdrawn using an automatic blood-sampling system. In both sexes, i.v. infusions of hGH for 6 h inhibited endogenous GH secretory pulses, with a slow onset of the inhibition. There was no rebound GH secretion immediately following the removal of the hGH infusion, but spontaneous GH secretion gradually returned to normal. Infusions of hGH did not inhibit the pituitary GH response to repeated GH-releasing factor (GRF) injections (1 μg) given i.v. every 40 min to female rats. By contrast, infusions of SS, which also blocked spontaneous GH release, dramatically reduced the GH responses to serial GRF injections. When SS Infusions were stopped, the subsequent GRF-induced GH secretory responses were enhanced. These results show that GH can inhibit its own release when given by i.v. infusion to conscious male and female rats. Since GH responses to GRF are maintained during a GH infusion, the feedback effect of GH is unlikely to be exerted directly on the pituitary or by increasing SS release. Our results are consistent with the idea that GH feedback in the conscious rat involves an inhibition of GRF release.

J. Endocr. (1988) 119, 201–209