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M Kapuscinski and A Shulkes


Gastrin is a regulator of both gastric acidity and gastrointestinal growth and is expressed transiendy in the neonatal ovine and human pancreas. C-terminal amidation of glycine extended gastrin (G-gly) to gastrin amide (G-amide) by peptidylglycine α-amidating mono-oxygenase (PAM) is the final processing step. To investigate the relationship between PAM and gastrin synthesis in the developing pancreas, we measured PAM activity and the concentrations of gastrins in ovine pancreatic extracts from 95 days of gestation onwards.

Pancreatic PAM activity was highest in the 95-day-old fetus (138 ±29 pmol/h/mg protein, mean ± s.e.m.) and decreased to 9·5 ±3·7 pmol/h/mg protein in the adult. The circulating enzyme was also highest in the youngest fetus (1840 ± 165 pmol/h/ml plasma) decreasing to approximately 50% in the 135-day-old fetus, with no further significant changes. The concentration of bioactive G-amide in the pancreas was 2·0 ±0·6 pmol/g at 95 days of gestation, 3·4 ± 0·9 pmol/g at 135 days and decreased to 0·7±0·1 pmol/g in the adult. The precursor G-gly followed a similar pattern but its concentration was less than 10% of G-amide. These results show that: (a) there are high levels of PAM activity in the ovine fetal pancreas and in the fetal circulation, (b) PAM activity is apparently not rate-limiting in the bioactivation of pancreatic gastrin and (c) the dual expression of both PAM and gastrin in the fetal pancreas is similar to that observed in peptide-secreting tumours of the adult.

Journal of Endocrinology (1995) 145, 137–142

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JC Whitley, C Moore, AS Giraud, and A Shulkes

High concentrations of a peptide related to gastrin-releasing peptide (GRP) are produced in the utero-placental unit of the human and sheep and secreted into the general circulation. This suggests an endocrine role in addition to its role as a neurotransmitter/neuromodulator. The GRP is larger than the previously described form GRP(1-27) but it is not known whether the larger form is the product of a related GRP-like gene or differences in post-translational processing. We have therefore cloned the gene for the sheep homologue of the GRP gene and determined its distribution. Only a single GRP gene was found in the sheep. This had a similar organisation to the human GRP gene with three exons and two introns. The larger form of GRP in the pregnant endometrium therefore appears to be the result of an alteration in processing of the GRP prohormone. The expression of GRP mRNA in the pregnant uterus was extraordinarily high comprising one-third of all mRNA synthesised by the pregnant endometrium. As the endometrial GRP mRNA arises solely from the glandular epithelium, the localised synthesis of GRP mRNA would be far higher. GRP mRNA was expressed in a wide variety of fetal tissues (fundus, colon, jejunum, ileum, duodenum, kidney, adrenal, lung, heart and pancreas) with a corresponding presence of GRP immunoreactivity. The expression of GRP in the fetal lung was biphasic with peaks at mid-term and near parturition but none in the adult supporting the concept of a specific developmental role of GRP in the lung.

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JC Whitley, AS Giraud, AO Mahoney, IJ Clarke, and A Shulkes

In the ovine endometrium, dramatic increases in gastrin-releasing peptide (GRP) mRNA and immunoreactivity are observed during the luteal regression phase of the oestrous cycle (24-fold) and during pregnancy (at least 150-fold). This study sought to determine whether oestrogen and/or progesterone were responsible for the temporal regulation of GRP observed in the uterus. Ovariectomized sheep were divided into four groups (n=4), as follows: 1, untreated; 2, given subcutaneous and intravaginal progesterone implants; 3, given subcutaneous oestrogen implants; and 4, treated with both oestrogen and progesterone. After 10 days, the animals were sacrificed and plasma, pituitary and endometrium were obtained. A fifth group of sheep with intact ovaries was included. Analysis of endometrial GRP-immunoreactivity (GRP-ir) revealed a twofold drop for groups treated with oestrogen, progesterone or both hormones. A dramatic reduction in endometrial GRP mRNA was o! bserved in the group treated with both hormones. GRP-ir was measured in whole pituitaries and found to vary greatly (1.7-53.7 pmol/g tissue) within all groups of ovariectomized animals. There were no significant differences between any of the five groups. A significant reduction in circulating GRP-ir was observed after 10 days of treatment with either oestrogen or progesterone. These studies demonstrate that, in sheep, the synthesis, storage and secretion of GRP are differentially affected by oestrogen and progesterone. Regulation appears to be tissue specific since GRP content in the pituitary is unchanged by oestrogen or progesterone whereas GRP expression in the endometrium is inhibited. Changes in GRP mRNA expression did not correlate with changes in endometrial expression of mRNA for oestrogen receptor alpha, oestrogen receptor beta and the progesterone receptor. This study is the first reported demonstration that expression of the GRP gene can be influenced by the presence of ovarian steroids, with the conclusion that oestrogen and/or progesterone act as negative regulators of endometrial GRP expression.

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J Pannequin, JP Tantiongco, S Kovac, A Shulkes, and GS Baldwin

Amidated forms of the peptide hormone gastrin act via the cholecystokinin-2 receptor to stimulate gastric acid secretion, whereas non-amidated forms stimulate colonic mucosal proliferation via a novel, as yet uncharacterised, receptor. Nuclear magnetic resonance (NMR) and fluorescence spectroscopic studies have revealed that glycine-extended gastrin17 bound two ferric ions, and that ferric ion binding was essential for biological activity. We have therefore investigated the role of ferric ions in the biological activity of amidated gastrin17. As with glycine-extended gastrin17, fluorescence quenching experiments indicated that Glu7 Ala and Glu8,9 Ala mutants of amidated gastrin17 each bound only one ferric ion. The affinity of the mutant peptides for the cholecystokinin-2 receptor on transfected COS-7 cells or on Tlymphoblastoid Jurkat cells, and their potency in stimulation of proliferation in Jurkat cells and inositol phosphate production in transfected COS-7 cells, were similar to the values obtained for amidated gastrin17. In addition, the iron chelator desferrioxamine did not significantly inhibit either binding of amidated gastrin17 to the cholecystokinin-2 receptor, or stimulation of inositol phosphate production by amidated gastrin17 in transfected COS-7 cells. We conclude that, in contrast to glycine-extended gastrin17, binding of ferric ions is not essential for the biological activity of amidated gastrin17. Our results support the concept of distinct modes of action for amidated and non-amidated gastrins, and raise the possibility of developing selective antagonists of the actions of non-amidated and amidated gastrins.

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JC Whitley, A Shulkes, LA Salamonsen, D Vogiagis, M Familari, and AS Giraud

Synthesis of both mRNA and peptide for gastrin-releasing peptide (GRP) has been demonstrated in the pregnant endometrium of sheep and women. However, it is not known whether GRP is synthesized in the sheep uterus during the oestrous cycle. Furthermore the cellular site of GRP mRNA synthesis in the uterus has not been determined. Therefore we examined the synthesis of GRP and determined the cellular location of GRP peptide and mRNA in sheep uterus taken at different times during the oestrous cycle (duration 17 days) and pregnancy (duration 145 days). Northern blot analysis of RNA isolated from ovine endometrium revealed low or no GRP mRNA at days 4, 10, 12 and 14 of the oestrous cycle and a 24-fold rise in GRP mRNA (normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA) between days 14 and 16. A similar pattern was observed during early pregnancy, with a 12-fold rise in GRP mRNA:GAPDH mRNA between days 17 and 20 of pregnancy. Levels of GRP peptide were determined by RIA and found to be low in endometrium isolated at days 4, 10, 12 and 14 of the oestrous cycle (1.0-1.6 pmol/g) and 4 to 5-fold higher at day 16. In situ hybridization localized GRP synthesis to the epithelial cells of the uterine glands at day 16 of the oestrous cycle and at days 17, 20, 40 and 50 of pregnancy. At day 140 of pregnancy diffuse hybridization to cells of the myometrium was also observed. Immunohistochemistry localized GRP peptide to the apical cytoplasm of uterine glandular epithelial cells at day 16 of the oestrous cycle. For samples obtained at day 20 of pregnancy, the area surrounding the glands also showed moderately strong staining. Further staining in the glandular lumen and the stromal tissue surrounding the glands was apparent at day 140 of pregnancy. No GRP immunoreactivity could be detected in the peripheral plasma during the oestrous cycle or the first 20 days of pregnancy. Sizing chromatography of GRP immunoreactivity extracted from endometrial tissue taken at day 10 of the oestrous cycle revealed two peaks that co-eluted with GRP(1-27) and GRP(18-27). However, during luteolysis and oestrus the major peak of GRP immunoreactivity extracted from endometrial tissue was larger than GRP(1-27) and similar to that seen previously in the gravid ovine endometrium. These studies demonstrate that a peptide similar to, but larger than, GRP is a major product of the glandular epithelium of the ovine uterus during the luteal regression phase of the oestrous cycle and post-blastocyst implantation in pregnancy and provide further evidence that GRP-related peptides have important regulatory roles in uterine function.

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K. G. Braslis, A. Shulkes, D. R. Fletcher, and K. J. Hardy


Calcitonin gene-related peptide (CGRP) is a product of the calcitonin gene with a widespread distribution in neural tissue of the brain, gut and perivascular nerves. Infusion of CGRP produces multiple biological effects, but the physiological significance of these findings will be influenced by the sites and rates of CGRP metabolism.

The metabolic clearance rate and half-life of disappearance of human CGRP were estimated in conscious sheep after infusing CGRP at 1 or 5 pmol/kg per min to steady-state conditions. The particular organs involved in the clearance of CGRP were assessed by measuring the inflow and outflow concentrations across the liver, gut, kidney, lung and brain.

The metabolic clearance rate at steady state was 22·6 ± 2·1 (s.e.m.) and 15·0±1·7 ml/kg per min for the 1 and 5 pmol/kg per min doses respectively. The half-life of disappearance was bi-exponential: 3·6±0·3 min for the first phase and 13·6±1·0 min for the second phase. High-pressure liquid chromatography of plasma at equilibrium revealed only a single peak coeluting with CGRP(1–37): no immunoreactive metabolites were detected. These pharmacokinetic values are intermediate between that of a neurotransmitter and a hormone and are therefore consistent for a peptide with both circulatory and neurotransmitter modes of action. The kidney, with an arterial–renal vein gradient of 14%, and the liver, with a portal– hepatic vein gradient of 25%, were the major organs involved in the clearance of CGRP. The specific organ clearance, however, accounted for only one-third of the whole body metabolic clearance rate of CGRP, suggesting that other more generalized degradative systems are involved, such as endothelial-bound enzymes of blood vessels. This information on clearance and organ-specific metabolism should form a basis for evaluating the physiological roles and modes of action of CGRP.

J. Endocr. (1988) 118,25–31