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Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London w12 0hs
received 30 December 1985
INTRODUCTION
Banting & Best (1922) used what is now a classical endocrinological technique in their discovery of insulin. They pulverized pancreas with buffer, filtered the crude tissue extract and found that it produced hypoglycaemia when injected into an experimental dog. One would have thought that within a few years most bodily tissues would have been treated similarly in a search for further circulating hormones. This has not been the case. As recently as 1981 de Bold and his colleagues prepared an extract of rat cardiac atria in a fashion not dissimilar to that of Banting & Best (1922) and found that injection into a donor rat produced a dramatic diuresis and natriuresis. Indeed the urine flow increased tenfold whilst sodium and chloride excretion increased more than 30-fold (de Bold, Borenstein, Veress &
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A specific radioimmunoassay for neurotensin in human plasma has been developed capable of detecting changes of 5 pmol/l with 95% confidence. Neurotensin-like immunoreactivity has been detected in human plasma in two molecular forms and rises by 27 ± 8 (s.e.m.) pmol/l (n = 9) after ingestion of food.
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ABSTRACT
The possibility that the sensitivity of the adrenal cortex to endogenous ACTH may be affected by splanchnic nerve activity has been investigated in conscious, weaned, 5- to 8-month-old lambs. The animals were atropinized (0·5 mg/kg) and tested with an i.v. infusion of noradrenaline (333 ng/kg per min for 10 min), which produced a significant rise in the mean concentration of both ACTH and cortisol in the arterial plasma. In lambs tested at least 7 days after section of both splanchnic nerves, just below the diaphragm, the rise in plasma ACTH concentration was significantly greater, and that in plasma cortisol significantly less, than in control lambs. The mean plasma ACTH and cortisol concentrations were linearly related to one another in both groups (r = 0·93 and 0·92) but the sensitivity of the adrenal cortex to the steroidogenic action of ACTH appeared to have been roughly halved 1 week after bilateral splanchnic nerve section.
J. Endocr. (1986) 110, 81–85
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ABSTRACT
The response of plasma atrial natriuretic peptide (ANP) concentration to acute intravascular volume expansion was measured in ten male Wistar rats. An infusion of 3 ml polygelene colloidal solution at 37 °C over 45 s produced peak venous pressure rises of 1·5cm water. A highly significant (P<0·001) rise of immunoreactive plasma ANP from 24·4 ± 2·2 (mean ± s.e.m.) pmol/l to a peak of 70·0±10·5 pmol/l occurred within 2·5 min. Plasma ANP concentrations had virtually returned to basal levels (32·7 ± 2·7 pmol/l) 30 min after this acute volume load. A further infusion of 10 ml polygelene colloidal solution in 2 min produced peak venous pressure rises of 10 cm water and caused a dramatic and significant (P< 0·001) increase of plasma ANP concentration to a peak of 534·8 ± 38·5 pmol/l, occurring 7·5 min after infusion. The plasma ANP concentration had fallen but remained above basal levels 30 min later (137·2 ± 26·4 pmol/l).
Similar results were obtained using an identical protocol but with whole rat blood instead of polygelene solution as the volume-expanding agent. Gel column chromatography suggested that the majority of the immunoreactive ANP in rat plasma was of similar molecular size to rat α-ANP(1–28).
These results support the hypothesis that blood volume expansion is a potent stimulus for the release of ANP into plasma.
J. Endocr. (1986) 109, 9–13
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A sensitive radioimmunoassay for the measurement of human gastric inhibitory polypeptide (GIP), using pure porcine GIP, has been developed. Cross-reactivity of the antiserum with all available mammalian gut peptide preparations was negligible with the exception of glucagon when it was approximately 1%. Two major molecular forms of GIP were detectable in plasma and tissue extracts, one of large molecular size and the other corresponding to the elution coefficient of pure porcine standard.
Concentrations of GIP in plasma from 50 normal subjects after overnight fasting were 9 ± 1·0 (s.e.m.) pmol/l rising to a peak of 34 ± 2·8 pmol/l following the ingestion of a small mixed test meal. Ingestion of glucose or fat resulted in a similar rise of plasma GIP, whereas no change was observed after the ingestion of protein.
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ABSTRACT
Laser-light scatter signals generated from living cells provide useful information with regard to both cell size (forward-angle light scatter) and granularity (ninety-degree or perpendicular light scatter). By measuring angles of light scatter and fluorescence, a fluorescence-activated cell sorter is capable of analysing and sorting cells on the basis of their size, granularity and cell-surface fluorescence. Using an electronically programmable individual cell sorter we were able to analyse single, viable, dispersed anterior pituitary cells of the female rat on the basis of their laser light scatter characteristics. Two distinct populations of differing granularity were defined: 26±2·2% (mean ± s.e.m.) were more granular and 74±3·5% less granular. Acutely dispersed anterior pituitary cells were labelled with antibodies against four of the anterior pituitary hormones, and cell size and granularity were compared amongst the different hormonal cell types. Somatotrophs were the most granular cell type, gonadotrophs were the largest and corticotrophs the smallest, whilst lactotrophs were of intermediate size. Labelling was demonstrated to be dependent upon the secretory state of the cell. Hypothalamic stimulating factors increased cell-surface labelling, whilst dopamine and somatostatin decreased labelling. These changes compare favourably with published data obtained by immunocytochemistry. Using dual-colour fluorescence cell surface labelling we were unable to define a population of cells secreting both prolactin and growth hormone (mammosomatotrophs).
Journal of Endocrinology (1990) 126, 261–268
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ABSTRACT
As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96·7 ±1·7 (s.e.m.)% and 98±1·0% respectively by immunocytochemistry, and to 95·8 ±1·1% and 97±0·8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 ±3·4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin.
Journal of Endocrinology (1990) 126, 269–274
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ABSTRACT
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalami and so called because of its ability to stimulate pituitary adenylate cyclase activity. Alternative amidation and proteolytic processing of prepro-PACAP gives rise to two bioactive-amidated forms, PACAP-NH2(1–38) (PACAP-38) and PACAP-NH2(1–27) (PACAP-27). 7B2 is a polypeptide of 185 amino acids which is predominantly found in secretory granules and is widely distributed in rat and human tissues. We investigated the ability of the two forms of PACAP to stimulate GH, prolactin and 7B2 release by the rat pituitary clonal cell line GH3, and ACTH and 7B2 by the mouse pituitary clonal cell line AtT-20. PACAP-38 and PACAP-27 stimulated 7B2 and GH/prolactin or ACTH secretion with a similar efficacy over the 2-h incubation period from GH3 and AtT-20 cells respectively. 7B2 secretion was also stimulated by corticotrophin-releasing factor (CRF-41) and vasoactive intestinal polypeptide (VIP) in AtT-20 cells, and thyrotrophin-releasing hormone (TRH) and VIP in GH3 cells. Addition of PACAP to CRF-41 resulted in an additive effect on ACTH secretion and a synergistic effect on 7B2 secretion in AtT-20 cells. No synergism was observed when PACAP was added together with TRH, either on GH and prolactin secretion or on 7B2 release from GH3 cells. PACAP-mediated 7B2 secretion from both cell lines and PACAP-stimulated ACTH release from AtT-20 cells were reduced by 5 mg octapeptide synthetic somatostatin analogue/l (5 mg SMS 201-995/1).
Journal of Endocrinology (1992) 132, 107–113
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ABSTRACT
Plasma glucagon concentrations were measured in chronically catheterized fetal pigs during the last third of gestation and compared with the values observed in anaesthetized fetuses of similar gestational age. The mean plasma concentration of glucagon in the chronically catheterized fetuses was 10·0 ± 1·4 (s.e.m.) pmol/l (n = 11; term = 114 ± 2 days). Concentrations were increased after catheterization and fell to baseline values within 48 h of surgery. Arginine infusion evoked a rapid release of glucagon in chronically catheterized fetuses between 105 and 108 days of gestation; the mean maximum increment in plasma glucagon was 15·4 ± 4·5 pmol/l (n = 5). Plasma glucagon concentrations increased with increasing gestational age in both anaesthetized and chronically catheterized fetuses. Between 95 and 110 days of gestation, glucagon levels were significantly higher in anaesthetized fetuses than in chronically catheterized animals with similar normal pH values. Catheterization and prematurity had no apparent effect on plasma glucagon levels at birth. The plasma concentrations at birth were similar to those observed in the chronically catheterized fetuses in utero provided the piglets did not become acidotic during delivery. Significantly higher plasma levels of glucagon were found in newborn piglets with acidaemia (pH < 7·3) than in piglets with normal pH values at birth (pH > 7·3). When all the data from the newborn piglets were combined, there was a significant negative correlation (r= − 0·79, n = 39, P < 0·01) between blood pH and the plasma concentration of glucagon at birth. These observations demonstrate that the fetal α cells are functional and responsive in utero and at birth.
J. Endocr. (1986) 108, 137–142
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SUMMARY
The role of calcitonin (CT) in postprandial calcium homeostasis and the possibility of a gastroentero-thyroid C-cell system was studied in young pigs. When pigs were fasted for more than 36 h and then fed, the plasma calcium concentration decreased by 6·1% over a period of 60–120 min after a meal. Since in thyroidectomized pigs the plasma calcium concentration increased by 7·2% when they were fed after a fast of 36 h it is suggested that increased CT secretion assists in the control of postprandial hypercalcaemia. Direct measurement of CT in peripheral plasma supported this suggestion. Because the plasma calcium concentration in an intact pig on a normal feeding regime does not change after a meal, the possibility of the involvement of one or more humoral factors in the stimulation of thyroid C-cells was investigated. Exogenous gastrin and endogenous gastrin stimulated by meat extract were both previously shown by us to increase CT secretion rate. This observation has now been extended to include other stimuli to endogenous gastrin, e.g. glycine and gastric distension. Furthermore, partially purified enteroglucagon increased CT secretion rate from perfused thyroid glands, isolated in situ. Stimulation of endogenous entero-glucagon by the intraduodenal administration of glucose, and probable stimulation of endogenous pancreozymin by intraduodenal fat, were both associated with an increased CT secretion rate from the thyroid gland.
These results support the concept of a gastroentero-thyroid C-cell system which serves to stimulate CT secretion and thus to protect the skeleton from excessive bone resorption during periods of dietary sufficiency.
