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Gastrin releasing peptide-like immunoreactivity (GRPLI) in human pregnancy
Abstracts from the l lth International Symposium on Regulatory Peptides
Gastrin Releasing Peptide-like Immunoreactivity (GRPLI) in Human Pregnancy. Q. Xiao, J.R.G. Challis, X. Han, E.R. Spindel, C. Prasad, D. Hill, and T.J. McDonald, Department of Medicine, University of Western Ontario, London, Ontario; Department of Physiology, University of Toronto, Toronto, Ontario, Oregon Regional Primate Center, Beaverton, Or. Having recently reported the presence of a novel larger molecular weight form of GRPLI circulating in ovine pregnancy (Endocrinol. 131:2033-5, 1992, and 135:2440-5, 1994), we hypothesized that GRP might similarly be present in human pregnancy. Extracts of normal term amnion, placenta and chorion/decidual tissue (n=5) contained GRPLI in amounts of 4.7 ± 2.9 (pmol.g -1 wet weight; mean ± SEM), 3.6 ± I.i and 2.9 ± 1.5, respectively, using C-terminally directed antisera, GRPLI in all tissues eluted at the positions of GRPI_27 and GRP18_27 on gel filtration. On reverse-phase HPLC, GRPLI occurred at the retention times of GRP1_27 and GRP18_27 but there were two larger GRPLI peaks which eluted at unique retention times, not corresponding to any known GRP form or oxidation product; these novel GRP-like peptide forms have a molecular size near that of GRP18_27. In contrast, human fetal lung extracts contained only GRPI_27, GRP14_27 and GRP18_27. The products of RT-PCR of RNA extracted from 6 weeks, 17 weeks and term human-fetal membranes, placental villi, and decidua, hybridized with a specific GRP oligonucleotide probe. Positive i~unohistochemical staining for GRP occurred in tissues obtained in the first (9.5 weeks) and second (22 weeks) trimesters and at term. GRPLI was seen in cytotrophoblasts, the syncytiotrophoblast, in amniotic epithelial cells and in extravillous trophoblasts present in decidual septa and fetal membranes. Hence, human GRP and apparently novel GRPLI entities are present in specific cells of the human placenta. The GRP gene is activated early in gestation and remains active throughout pregnancy. Given the proven trophic nature of GRP, GRP and GRP-like peptides may play important roles in maternal, placental and fetal development and maintenance.
Influence of Prandial State on the Gene Expression of Gastrin, Cholecystokinin, Somatostatin, Acltn, GAPDH, 18S and 28S rRNA in the Pancreas and Upper Digestive Tract of Rats. Yamada H l, Nylander A-G I, Chen D 1, Kimura K 1 and Monstein H-J2. 1Department of Pharmacology, University of Lund, Land, Sweden. 2Department of Clinical Microbiology, Molecular Biology Laboratory, University Hospital, Linkgping, Sweden.
The gene expression of gastrin, cholecystokinin (CCK) and somatostatin in the pancreas and upper digestive tract is probably regulated by food intake. The expression of bouse-keeping genes, e.g. the enzyme glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) or structural proteins, such as actin, 18S and 28S ribosomal RNA (rRNA), should not be affected by the prandial state if they are to be used as standard markers. The aim of this study was to determine the effect of fasting on the expression of gastrin, CCK and somatostatin as well as of various standard marker genes. Methods Total RNA was isolated from fundi¢ and antral mucosa in stomach, duodenum and pancreas in freely fed rats or rats fasted for 48 hours and analysed by Northern blot analysis, using the same amount of total RNA in each slot as measured by OD260 (10-20 ~tg). Gastrin, CCK and somatostatin mRNA expressions were analyzed by hybridization with complementary DIG-labelled RNA probes. Probes for GAPDH, actin, 18S and 28S rRNA were also used in hybridization studies. mRNA levels in tissues from fasted and fed rats were compared after hybridization by means of measured OD-units obtained from X-ray films. Results Fasting reduced antral gastrin mRNA and duodenal CCK mRNA levels. GAPDH and actin mRNA levels were markedly reduced in fundus and antrum, but not in duodenum. Somatostatin mRNA,18S and 28S rRNA levels were not affected by fasting in any of the tissues examined. Fasting reduced not only the mRNA expression of gastrin and CCK, but also the expression of GAPDH and actin, suggesting that the choice of "house-keeping" gene for internal standard in the Northem blot analysis will greatly influence the quantitative assessment of the peptide hormone mRNA level.
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Gastrin Releasing Peptide-like Immunoreactivity (GRPLI) in Human Pregnancy. Q. Xiao, J.R.G. Challis, X. Han, E.R. Spindel, C. Prasad, D. Hill, and T.J. McDonald, Department of Medicine, University of Western Ontario, London, Ontario; Department of Physiology, University of Toronto, Toronto, Ontario, Oregon Regional Primate Center, Beaverton, Or. Having recently reported the presence of a novel larger molecular weight form of GRPLI circulating in ovine pregnancy (Endocrinol. 131:2033-5, 1992, and 135:2440-5, 1994), we hypothesized that GRP might similarly be present in human pregnancy. Extracts of normal term amnion, placenta and chorion/decidual tissue (n=5) contained GRPLI in amounts of 4.7 ± 2.9 (pmol.g -1 wet weight; mean ± SEM), 3.6 ± I.i and 2.9 ± 1.5, respectively, using C-terminally directed antisera, GRPLI in all tissues eluted at the positions of GRPI_27 and GRP18_27 on gel filtration. On reverse-phase HPLC, GRPLI occurred at the retention times of GRP1_27 and GRP18_27 but there were two larger GRPLI peaks which eluted at unique retention times, not corresponding to any known GRP form or oxidation product; these novel GRP-like peptide forms have a molecular size near that of GRP18_27. In contrast, human fetal lung extracts contained only GRPI_27, GRP14_27 and GRP18_27. The products of RT-PCR of RNA extracted from 6 weeks, 17 weeks and term human-fetal membranes, placental villi, and decidua, hybridized with a specific GRP oligonucleotide probe. Positive i~unohistochemical staining for GRP occurred in tissues obtained in the first (9.5 weeks) and second (22 weeks) trimesters and at term. GRPLI was seen in cytotrophoblasts, the syncytiotrophoblast, in amniotic epithelial cells and in extravillous trophoblasts present in decidual septa and fetal membranes. Hence, human GRP and apparently novel GRPLI entities are present in specific cells of the human placenta. The GRP gene is activated early in gestation and remains active throughout pregnancy. Given the proven trophic nature of GRP, GRP and GRP-like peptides may play important roles in maternal, placental and fetal development and maintenance.
Influence of Prandial State on the Gene Expression of Gastrin, Cholecystokinin, Somatostatin, Acltn, GAPDH, 18S and 28S rRNA in the Pancreas and Upper Digestive Tract of Rats. Yamada H l, Nylander A-G I, Chen D 1, Kimura K 1 and Monstein H-J2. 1Department of Pharmacology, University of Lund, Land, Sweden. 2Department of Clinical Microbiology, Molecular Biology Laboratory, University Hospital, Linkgping, Sweden.
The gene expression of gastrin, cholecystokinin (CCK) and somatostatin in the pancreas and upper digestive tract is probably regulated by food intake. The expression of bouse-keeping genes, e.g. the enzyme glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) or structural proteins, such as actin, 18S and 28S ribosomal RNA (rRNA), should not be affected by the prandial state if they are to be used as standard markers. The aim of this study was to determine the effect of fasting on the expression of gastrin, CCK and somatostatin as well as of various standard marker genes. Methods Total RNA was isolated from fundi¢ and antral mucosa in stomach, duodenum and pancreas in freely fed rats or rats fasted for 48 hours and analysed by Northern blot analysis, using the same amount of total RNA in each slot as measured by OD260 (10-20 ~tg). Gastrin, CCK and somatostatin mRNA expressions were analyzed by hybridization with complementary DIG-labelled RNA probes. Probes for GAPDH, actin, 18S and 28S rRNA were also used in hybridization studies. mRNA levels in tissues from fasted and fed rats were compared after hybridization by means of measured OD-units obtained from X-ray films. Results Fasting reduced antral gastrin mRNA and duodenal CCK mRNA levels. GAPDH and actin mRNA levels were markedly reduced in fundus and antrum, but not in duodenum. Somatostatin mRNA,18S and 28S rRNA levels were not affected by fasting in any of the tissues examined. Fasting reduced not only the mRNA expression of gastrin and CCK, but also the expression of GAPDH and actin, suggesting that the choice of "house-keeping" gene for internal standard in the Northem blot analysis will greatly influence the quantitative assessment of the peptide hormone mRNA level.
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