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Identity and Regulation of Stored and Secreted Progastrin-Derived Peptides in Sheep

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Identity and Regulation of Stored and Secreted Progastrin-Derived Peptides in Sheep 0013-7227/04/$15.00/0 Endocrinology 145(11):5129–5140 Printed in U.S.A. Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2004-0912 Identity and Regulation of Stored and Secreted Progastrin-Derived Peptides in Sheep ADRIENNE C. PATERSON, SHARON M. LOCKHART, JOSEPHINE BAKER, GREG NEUMANN, GRAHAM S. BALDWIN, AND ARTHUR SHULKES Department of Surgery, University of Melbourne, Austin Health, Melbourne, Victoria 3084, Australia Downloaded from https://academic.oup.com/endo/article/145/11/5129/2500504 by guest on 25 September 2021 (Amidated and nonamidated progastrin-derived peptides have antral (CTFP, 710 ؎ 62 pmol/liter; Gamide, 211 ؎ 35 pmol/liter distinct biological activities that are mediated by a range of and jugular (CTFP, 308 ؎ 16 pmol/liter; gastrin amide, 32 ؎ 3 receptor subtypes. The objective was to determine the nature pmol/liter) veins. Alteration of gastric acidity in sheep by iv of the stored and secreted progastrin-derived peptides and to infusion of a H/K-adenosine triphosphatase inhibitor or so- investigate whether progastrin release is regulated by gastric matostatin or by intragastric infusion of HCl demonstrated acidity. Using an antiserum directed to the C terminus of that the CTFP concentrations changed, although to a lesser progastrin for identification and to monitor purification, C- extent than the changes in circulating gastrin amide. We con- terminal flanking peptides (CTFP) of progastrin (prog76–83, clude that the CTFP of progastrin is the major stored and prog77–83, and prog78–83 in approximately equivalent amounts) circulating species of the gastrin gene, and that it is secreted were isolated and identified from extracts of sheep antrum in a regulated fashion rather than constitutively. Because using ion exchange, HPLC, and mass spectrometry. Only trace full-length progastrin is bioactive, but is only a minor antral amounts of full-length progastrin were present. Progastrin and secreted form, determination of the biological activity of CTFP was the predominant progastrin-derived peptide in the the C-terminal flanking peptides will be important for a com- Sim- plete understanding of gastrin endocrinology. (Endocrinology .[3 ؍ (antrum [progastrin CTFP/gastrin amide (Gamide ilarly, progastrin CTFP was the major circulating form in the 145: 5129–5140, 2004) ASTRIN, A CLASSICAL gastrointestinal hormone pro- and colorectal cancers in vitro and in vivo (15–18). The re- G duced by antral G cells, is the principal stimulant of ceptors mediating the effects of Ggly and progastrin have not gastric acid secretion (1, 2). Like many other peptide hor- yet been characterized, but, based on responses to specific mones, gastrin is synthesized as a large precursor molecule receptor antagonists, are distinct from the CCK-2 and CCK-1 (101 amino acids in man and 104 amino acids in most other receptors that interact with amidated gastrin and CCK (6). species) that is converted to progastrin (80 and 83 amino Depending on the extent of posttranslational processing acids in man and sheep, respectively) by cleavage of the and the source (antrum, duodenum, or tumor), the tissues NH2-terminal signal peptide. Subsequent processing results can contain variable amounts of progastrin, progastrin- in the generation of glycine-extended gastrins (Ggly) (G34gly processing intermediates, Ggly, and Gamide (19). Colorectal and G17gly), with the final step being amidation to gastrin carcinomas express progastrin, but processing is attenuated, amide (Gamide; G34amide and G17amide; Fig. 1) (3, 4). so that much of the synthesized progastrin is apparently not Until recently, amidated gastrin was thought to be the only fully processed to the amidated end product (20–22). biologically active form of gastrin. A major reassessment of In humans and other species, Gamide is generally ac- our understanding of gastrin biology occurred with the rec- cepted to be the predominant gastrin form found in the ognition that the precursor forms and the amidated end antrum, whereas full-length progastrin and Ggly are less product have distinct biological activities mediated by dif- than 5% as abundant as Gamide (13, 23–25). However, an- ferent receptors (5, 6). Gamide, acting through the cholecys- tisera to the carboxyl-terminal sequence of progastrin have tokinin-2 (CCK-2) receptor (previously known as the gas- shown high concentrations of C-terminal flanking peptides trin/CCK-B receptor) is the major hormonal regulator of (CTFP) in approximately equal concentration to Gamide (26, gastric acid secretion (1) and is a mitogen for normal gastric 27). This is true for the human and a number of species, epithelium and some gastric cancers in vitro and in vivo (5–8). including the antrum of pig, dog, ferret, and rat (13, 28). In In contrast, the precursor forms and intermediates, such as the human, the predominant form was progastrin75–80;in Ggly, have little direct effect on gastric function, but may other species, it was progastrin75–83 (13, 27). Serine 75 was potentiate the effects of Gamide (9–14). Ggly and progastrin phosphorylated to varying extents in the different species independently stimulate proliferation of the colonic mucosa (13, 27, 28). Fragments lacking serine 75 are present in the antrum of pig and dog, but at much lower concentrations than the nonapeptide progastrin75–83 (13). Abbreviations: CCK, Cholecystokinin; CTFP, C-terminal flanking There are few studies of the regulation of secretion of peptide; CTI, C-terminal immunoreactivity; Gamide, gastrin amide; Ggly, glycine-extended gastrin; TFA, trifluoroacetic acid. Gamide precursors. The release of Ggly from the antrum Endocrinology is published monthly by The Endocrine Society (http:// appears to be controlled in a similar fashion to Gamide (29, www.endo-society.org), the foremost professional society serving the 30). However, the ratio of Ggly to Gamide in the circulation endocrine community. is higher than that in the antrum, suggesting a differential 5129 5130 Endocrinology, November 2004, 145(11):5129–5140 Paterson et al. • Identity and Regulation of Progastrin-Derived Peptides Downloaded from https://academic.oup.com/endo/article/145/11/5129/2500504 by guest on 25 September 2021 FIG. 1. Structure of sheep progastrin and progastrin-derived peptides and specificity of antibodies. Preprogastrin (104 amino acids) is converted to progastrin (83 amino acids) by removal of the signal peptide. The sequential action of prohormone convertases (endopeptidases and carboxypeptidase B-like enzymes) converts progastrin to glycine-extended forms and CTFP by cleavage at paired basic residues. The C terminus of Ggly is then amidated by peptidyl ␣-amidating monooxygenase. For simplicity, only the 17-amino acid forms of Ggly and Gamide are depicted. The epitopes for the different antisera are shown. secretion (24, 31, 32). This may relate to the heterogeneity of a purse string suture into the antral region of the abomasum. Gastric the secretory granules, which contain different proportions juice was collected by first clearing the cannula of any contents, then of progastrin and its processing products (6, 33). The pro- aspirating approximately 5 ml gastric secretion. Cannulas were also inserted into the jugular veins, as described for the conscious sheep cessing of progastrin occurs within the secretory granules studies. Basal samples were not collected until 30 min after the surgery and involves cleavage, phosphorylation, sulfation, and ami- was completed. dation. The extent of processing is dependent on residence times and the strength and duration of the stimulation (34). Studies in conscious sheep However, the regulated secretion of progastrin has not been examined in vivo. The studies were performed in Merino-Corriedale-cross sheep, weighing 35–45 kg. After a local anesthetic (2 ml 1% lignocaine) had been Progastrin has recently been demonstrated to have administered, a hypodermic needle was inserted into the jugular vein, growth-promoting effects on normal colonic mucosa and directed toward the head (up). A polyvinyl cannula (inside diameter, colorectal cancers (15, 17, 18). These studies were performed 0.76 mm; outside diameter, 1.65 mm) was then passed through the using full-length progastrin, either as a recombinant peptide needle into the vein. The same procedure was followed for the con- tralateral jugular vein, except that the cannula was directed toward the or overexpressed as a transgene in the liver and secreted heart (down). Agonists and antagonists were infused through the can- constitutively. Although recombinant progastrin is relatively nula directed downward, and blood was collected from the cannula stable in the circulation (35), a comparison of the antral and directed upward. A 24-h recovery period was allowed, and animals were secreted forms of progastrin has not been reported. We, housed in metabolism cages for the duration of the experiments. therefore, determined the structure of the stored and secreted forms of ovine progastrin and compared the regulation of Studies in anesthetized sheep antral secretion of progastrin to that of Gamide. Protocol 1: effect of iv omeprazole and intragastric HCl infusions. After a 30-min basal period, omeprazole was administered via the jugular can- Materials and Methods nula pointing toward the heart as a bolus (0.65 mg/kg), followed im- Animal preparation mediately by an infusion of 0.90 mg/kg⅐h at 20 ml/h for 180 min. During the last hour of omeprazole infusion, 0.1 m HCl was administered via All experiments conformed with the National Health and Medical the gastric cannula to return the pH to between 1.5 and 2.5. This required Research Council of Australia code of practice for the care and use of an average of 120 ml over 30 min. Blood samples were obtained from animals for scientific purposes and were approved by the Austin health the jugular, antral, and fundic cannulas at regular intervals before, animal ethics committee. during, and after the infusion. Gastric pH was also measured at these times. Studies in anesthetized sheep The model for collecting antral and fundic blood has been reported Studies in conscious sheep previously (36).
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