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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.

The bombesin receptor subtype 3 (BRS-3) is considered an orphan receptor as it has a low affinity for bombesin-like peptides and no identified natural ligand. We have reported a novel form of gastrin-releasing peptide (GRP) present in high abundance in the pregnant uterus of women and sheep. As BRS-3 was originally cloned from guinea pig uterus, we postulated that the uterine GRP-like peptide may be its natural ligand. We have therefore cloned the gene for the sheep homologue of BRS-3 and determined its distribution. The sheep BRS-3 gene spans 4 kbp and comprises three exons with intron-exon borders at positions similar to those observed for the human and mouse BRS-3 genes. The predicted amino acid sequence of ovine BRS-3 has approximately 85% identity with the human, mouse and guinea pig receptors. Highly conserved amino acids important in mediating receptor G-protein coupling to second messengers and important in ligand binding were found to be conserved in ovine BRS-3. One potentially important deviation was noted: ovine BRS-3 possesses an arginine residue at position 294 instead of a histidine residue as found in all other BRS-3. His(294) was previously identified as important in ligand-receptor interactions while Arg(294) was implicated in high ligand affinity. Thus ovine BRS-3 may have binding characteristics different from those of the human, mouse and guinea pig BRS-3 receptors. In the ewe, BRS-3 mRNA expression was detected in pituitary and hypothalamus but not in tissues of the pregnant uterus (endometrium, myometrium, chorioallantois or amnion). Nor was BRS-3 expression detected in the non-pregnant uterus or in testis. This pattern of BRS-3 expression is similar to that observed in the mouse but different from that observed in the human, rat and guinea pig. We conclude that there is no local interaction between uterine GRP-like peptide and BRS-3. However, the high expression of BRS-3 in the pituitary coupled with elevated circulating levels of this GRP-like peptide during pregnancy suggests an alternate pathway. Cloning of the ovine BRS-3 gene will permit a detailed functional analysis of this receptor in the sheep and its role in the mediation of action of uterine GRP.

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.

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

In the sheep fetus, plasma levels of gastrin are raised above adult levels from 2 weeks before birth. This observation initiated the present study on the maternal and fetal secretion rate, metabolism and placental transfer of gastrin. The experiments were performed on conscious pregnant ewes with chronically cannulated fetuses and on newborn lambs. Metabolic clearance rate (MCR), production rate (PR) and placental transfer of gastrin were measured by alternate steady-state infusion of gastrin into the mother and fetus. Plasma levels of gastrin were measured by radioimmunoassay. Metabolic clearance rate was similar in the pregnant and non-pregnant ewe (8·4± 1·1 (s.e.m.) and 9·0±1·4 ml/min per kg) respectively. However, fetal MCR was significantly increased. Term was 145 days. Metabolic clearance rate was 15·5 ± 1·7 at 110–125 days of gestation, 25·6 ± 2·9 at 126–135 days, 29·7 ± 4·9 at 136–145 days and remained raised in the first 2 weeks post partum. Gastrin did not cross the placenta in either direction. Placental destruction of gastrin was not responsible for the increased fetal MCR as umbilical artery and umbilical vein levels were not significantly different during fetal gastrin infusion. Furthermore, MCR remained raised in the newborn lambs. Gastrin PR was significantly increased at all ages. The results showed that the previously reported fetal hypergastrinaemia is from fetal sources and is not a result of immaturity of clearance mechanisms. In fact, fetal MCR was significantly increased. The increased fetal plasma gastrin levels are due to an increased rate of production from the fetus.

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