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C. J. Ashworth
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I. Wilmut
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A. J. Springbett
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R. Webb
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ABSTRACT

The effect of an inhibitor of 3β-hydroxysteroid dehydrogenase on peripheral progesterone concentration during the luteal phase of the oestrous cycle and on embryo survival was determined in sheep. Following administration of 10, 50, 100 or 250 mg epostane (4,5-epoxy-17-hydroxy-4,17,dimethyl-3-oxo-androstane-2-carbonitrile) progesterone concentrations were significantly lower than control levels 4 h after injection, from 2·5 to 22 h, 1·5 to 24 h and 1 to 24 h after injection respectively. There appeared to be no effect on peripheral oestradiol concentrations. Adrenal progesterone production was small and not influenced by epostane treatment.

Epostane was administered on day 9 of the oestrous cycle to cause a reduction in progesterone concentrations for approximately 12-18 h on day 9 only (group 1, 250 mg epostane on day 9), or a series of such reductions on 3 consecutive days (group 2, 50 mg epostane on days 9, 10 and 11) or a continuous reduction for 3 days (group 3, 250 mg epostane on days 9, 10 and 11). The proportion of ewes that were pregnant was significantly (P<0·05) lower in ewes treated to give a continuously low progesterone concentration for 3 days than in either controls or ewes in which progesterone concentration was reduced for less than 24 h (in controls and groups 1, 2 and 3 the proportion was 85, 92, 54 and 18% of ewes treated respectively). Embryo survival was not affected by administration of 250 mg epostane on days 9, 10 and 11 if luteal phase levels of progesterone were maintained by insertion of a silicone elastomer implant of the steroid. The proportion of embryos surviving was 72% in controls compared with 78% in the treated animals.

J. Endocr. (1987) 112, 205–213

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R J Ashworth
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J Ham
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S M Cockle
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Abstract

Pyroglutamylglutamylprolineamide, which was first discovered in mammalian prostate, differs from thyrotrophin-releasing hormone (TRH) by substitution of glutamic acid for histidine at position two of the tripeptide. Recently, the newly discovered peptide has been identified in substantial concentrations in the rat anterior pituitary gland and, in this study, we have investigated the effects of the peptide on rat anterior pituitary cells in culture. GH3 cells were chosen to examine the possible effects of the new peptide, particularly in relation to its effects on the TRH receptor. This cell-type was deficient, in comparison with normal rat pituitary cells, in the new TRH-related peptide and appeared to be an ideal model cell in which to study the effects of pGlu-Glu-ProNH2. TRH (0·01–100 nm) was found to stimulate the secretion of both GH and prolactin from GH3 cells whereas pGlu-Glu-ProNH2 had no effect within the same concentration ranges. In contrast, at micromolar concentrations pGlu-Glu-ProNH2 exhibited intrinsic TRH-like activity causing stimulation of both GH and prolactin release from GH3 cells. Both TRH and pGlu-Glu-ProNH2 appeared to act through the same intracellular signalling mechanism, causing significant increases in intracellular inositol phosphate within the expected concentration ranges. However, pGlu-Glu-ProNH2 (up to 1 mm) displaced neither [3H]TRH nor [3H]MeTRH from membrane-binding sites on GH3 cells, suggesting that the effects of the new peptide were mediated through a second receptor. The physiological relevance of these effects of pGlu-Glu-ProNH2 requires further investigation.

Journal of Endocrinology (1994) 142, 111–118

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R. D. G. MILNER
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A. J. BARSON
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M. A. ASHWORTH
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SUMMARY

Pieces of human foetal pancreas were incubated under control conditions and in media containing different stimuli of insulin release. Insulin secretion was stimulated from the pancreases of foetuses (83–625 g body weight) which were of 16–24 weeks gestational age. Potassium (60 mmol/l), barium (2·54 mmol/l) and ouabain (10−5 mol/l) were effective stimuli in all experiments. Glucagon (5 μg/ml), theophylline (1 mmol/l) and dibutyryl 3′,5′-cyclic adenosine monophosphate (1 mmol/l) stimulated insulin secretion in media containing 0, 0·6 or 3·0 mg glucose/ml. Theophylline and dibutyryl 3′,5′-cyclic adenosine monophosphate were effective in all experients and glucagon stimulated insulin release in four out of six experiments. At all ages studied, histological examination of the pancreas after each experiment revealed islets of Langerhans containing β cells. In most cases the islets were of the mantle type but occasionally bipolar islets were seen. Cellular normality, as judged by light microscopy, was preserved after periods of incubation for up to 5½ h. Glycogen was demonstrable in the pancreatic acinar tissue but not in the islets.

The results of these experiments indicate that, between the 16th and 24th week of foetal life, the human β cell is capable of releasing insulin in vitro when stimulated appropriately.

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R. D. G. MILNER
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M. A. ASHWORTH
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A. J. BARSON
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SUMMARY

Pieces of pancreas from human foetuses of 14–24 weeks gestational age and weighing between 50 and 625 g were incubated in vitro. Insulin release was studied under control conditions and in media supplemented with glucose (3 mg/ml), leucine (5 mmol/1) or arginine (5 mmol/1). Glucose never caused a significant rise in insulin release from the pancreas. The failure of mannoheptulose (3 mg/ml) and 2-deoxyglucose (3 mg/ml) to suppress basal insulin release in a glucose-free medium indicated that basal insulin release was not governed by the liberation of glucose from glycogen in the exocrine pancreas. Arginine stimulated insulin release in four experiments using pancreas from foetuses weighing more than 200 g, but failed to cause insulin release from the pancreas of foetuses weighing less than 200 g in three experiments. Leucine consistently stimulated insulin release from the pancreas of foetuses of less than 200 g body weight but was only variably effective in causing insulin release from pancreas of foetuses weighing more than 200 g. The experimental results illustrate the development of different mechanisms for the release of insulin from the human foetal β cell.

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R.J. Ashworth
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J.M. Morrell
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A. Aitken
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Y. Patel
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S.M. Cockle
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ABSTRACT

A new TRH-like peptide pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2) has recently been purified and characterized from both the rabbit prostate complex and human semen. In this study, TRH-immunoreactive peptides were extracted from anterior pituitary, posterior pituitary and hypothalamus and subjected to gel exclusion chromatography. For each tissue, TRH was resolved from pGlu-Glu-ProNH2 by anion-exchange chromatography at pH 7.6 In the anterior pituitary, 63% of the TRH immunoreactivity was chromatographically identical to pGlu-Glu-ProNH2 whereas in the posterior pituitary the new peptide represented less than 5% of the total TRH immunoreactivity. Only trace levels of pGlu-Glu-ProNH2 were observed in hypothalamus, suggesting that the acidic TRH-related peptide found in the anterior pituitary may not be of hypothalamic origin. The new TRH-like peptide was purified from whole pituitaries by gel exclusion and ion-exchange chromatography, followed by high power liquid chromatography and was shown to have chromatographic properties identical to pGlu-Glu-ProNH2. Amino acid analysis of the purified peptide revealed glutamic acid and proline residues in the ratio Glx:2 Pro:1, which is the expected composition of pGlu-Glu-ProNH2 after acid hydrolysis.

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S. Harvey
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V. L. Trudeau
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R. J. Ashworth
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S. M. Cockle
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ABSTRACT

Pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2) is a tripeptide with structural and immunological similarities to thyrotrophin-releasing hormone (TRH; pGlu-His-ProNH2). Since TRH stimulates GH secretion in domestic fowl, the possibility that pGlu-Glu-ProNH2 may also provoke GH release was investigated. Unlike TRH, pGlu-Glu-ProNH2 alone had no effect on GH release from incubated chicken pituitary glands and did not down-regulate pituitary TRH receptors. However, pGlu-Glu-ProNH2 suppressed TRH-induced GH release from pituitary glands incubated in vitro and competitively displaced [3H]methyl3-histidine2-TRH from pituitary membranes. Systemic injections of pGlu-Glu-ProNH2 had no significant effect on basal GH concentrations in conscious birds, but promptly lowered circulating GH levels in sodiumpentobarbitone anaesthetized fowl. Submaximal GH responses of conscious and anaesthetized birds to systemic TRH challenge were, however, potentiated by prior or concomitant administration of pGlu-Glu-ProNH2. These results demonstrate, for the first time, that pGlu-Glu-ProNH2 has biological activity, with inhibitory and stimulatory actions within the avian hypothalamo-pituitary axis. These results indicate that pGlu-Glu-ProNH2 may act as a TRH receptor antagonist within this axis.

Journal of Endocrinology (1993) 138, 137–147

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F. N. LEACH
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M. A. ASHWORTH
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A. J. BARSON
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R. D. G. MILNER
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SUMMARY

Various methods of tissue culture were studied in an attempt to grow human foetal pancreas under conditions favourable for insulin release. Simple dicing of pancreas was superior to plasma clot adhesion or collagenase digestion for the preparation of tissue for culture. The culture medium described by Kahri (1966) (containing 25% heated postnatal calf serum) was the most suitable of four tested for the study of insulin release from the tissue culture. In this medium insulin could be measured quantitatively by radioimmunoassay and insulin degradation occurred at the rate of 20–25%/24 h. In other media the pancreas grew less well or insulin degradation was much greater.

Human foetal pancreas grown under optimal conditions released insulin for up to 34 days. Insulin released into the culture medium did not appear to inhibit the further release of insulin. In some experiments the total amount of insulin released into the culture medium was several-fold greater than that in the pancreas originally seeded. In acute incubation experiments barium and theophylline stimulated insulin release from pancreas cultures. It proved impossible to identify the cells from which insulin was released but they did not appear to be in the monolayer which was composed of fibroblasts.

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