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Endocrinology, doi:10.1210/en.2004-0912
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Endocrinology Vol. 145, No. 11 5129-5140
Copyright © 2004 by The Endocrine Society

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

Address all correspondence and requests for reprints to: Dr. Arthur Shulkes, Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia. E-mail: aas{at}unimelb.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Statistical analyses
 Results
 Discussion
 References
 
Amidated and nonamidated progastrin-derived peptides have distinct biological activities that are mediated by a range of receptor subtypes. The objective was to determine the nature of the stored and secreted progastrin-derived peptides and to investigate whether progastrin release is regulated by gastric acidity. Using an antiserum directed to the C terminus of progastrin for identification and to monitor purification, C-terminal flanking peptides (CTFP) of progastrin (prog76–83, prog77–83, and prog78–83 in approximately equivalent amounts) were isolated and identified from extracts of sheep antrum using ion exchange, HPLC, and mass spectrometry. Only trace amounts of full-length progastrin were present. Progastrin CTFP was the predominant progastrin-derived peptide in the antrum [progastrin CTFP/gastrin amide (Gamide) = 3]. Similarly, progastrin CTFP was the major circulating form in the antral (CTFP, 710 ± 62 pmol/liter; Gamide, 211 ± 35 pmol/liter) and jugular (CTFP, 308 ± 16 pmol/liter; gastrin amide, 32 ± 3 pmol/liter) veins. Alteration of gastric acidity in sheep by iv infusion of a H/K-adenosine triphosphatase inhibitor or somatostatin or by intragastric infusion of HCl demonstrated that the CTFP concentrations changed, although to a lesser extent than the changes in circulating gastrin amide. We conclude that the CTFP of progastrin is the major stored and circulating species of the gastrin gene, and that it is secreted in a regulated fashion rather than constitutively. Because full-length progastrin is bioactive, but is only a minor antral and secreted form, determination of the biological activity of the C-terminal flanking peptides will be important for a complete understanding of gastrin endocrinology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Statistical analyses
 Results
 Discussion
 References
 
GASTRIN, A CLASSICAL gastrointestinal hormone produced by antral G cells, is the principal stimulant of gastric acid secretion (1, 2). Like many other peptide hormones, gastrin is synthesized as a large precursor molecule (101 amino acids in man and 104 amino acids in most other species) that is converted to progastrin (80 and 83 amino acids in man and sheep, respectively) by cleavage of the NH2-terminal signal peptide. Subsequent processing results in the generation of glycine-extended gastrins (Ggly) (G34gly and G17gly), with the final step being amidation to gastrin amide (Gamide; G34amide and G17amide; Fig. 1Go) (3, 4).



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  		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 {alpha}-amidating monooxygenase. For simplicity, only the 17-amino acid forms of Ggly and Gamide are depicted. The epitopes for the different antisera are shown.

 
Until recently, amidated gastrin was thought to be the only biologically active form of gastrin. A major reassessment of our understanding of gastrin biology occurred with the recognition that the precursor forms and the amidated end product have distinct biological activities mediated by different receptors (5, 6). Gamide, acting through the cholecystokinin-2 (CCK-2) receptor (previously known as the gastrin/CCK-B receptor) is the major hormonal regulator of gastric acid secretion (1) and is a mitogen for normal gastric epithelium and some gastric cancers in vitro and in vivo (5, 6, 7, 8). In contrast, the precursor forms and intermediates, such as Ggly, have little direct effect on gastric function, but may potentiate the effects of Gamide (9, 10, 11, 12, 13, 14). Ggly and progastrin independently stimulate proliferation of the colonic mucosa and colorectal cancers in vitro and in vivo (15, 16, 17, 18). The receptors mediating the effects of Ggly and progastrin have not yet been characterized, but, based on responses to specific receptor antagonists, are distinct from the CCK-2 and CCK-1 receptors that interact with amidated gastrin and CCK (6).

Depending on the extent of posttranslational processing and the source (antrum, duodenum, or tumor), the tissues can contain variable amounts of progastrin, progastrin-processing intermediates, Ggly, and Gamide (19). Colorectal carcinomas express progastrin, but processing is attenuated, so that much of the synthesized progastrin is apparently not fully processed to the amidated end product (20, 21, 22).

In humans and other species, Gamide is generally accepted to be the predominant gastrin form found in the antrum, whereas full-length progastrin and Ggly are less than 5% as abundant as Gamide (13, 23, 24, 25). However, antisera to the carboxyl-terminal sequence of progastrin have shown high concentrations of C-terminal flanking peptides (CTFP) in approximately equal concentration to Gamide (26, 27). This is true for the human and a number of species, including the antrum of pig, dog, ferret, and rat (13, 28). In the human, the predominant form was progastrin75–80; in other species, it was progastrin75–83 (13, 27). Serine 75 was phosphorylated to varying extents in the different species (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).

There are few studies of the regulation of secretion of Gamide precursors. The release of Ggly from the antrum appears to be controlled in a similar fashion to Gamide (29, 30). However, the ratio of Ggly to Gamide in the circulation is higher than that in the antrum, suggesting a differential secretion (24, 31, 32). This may relate to the heterogeneity of the secretory granules, which contain different proportions of progastrin and its processing products (6, 33). The processing of progastrin occurs within the secretory granules and involves cleavage, phosphorylation, sulfation, and amidation. The extent of processing is dependent on residence times and the strength and duration of the stimulation (34). However, the regulated secretion of progastrin has not been examined in vivo.

Progastrin has recently been demonstrated to have growth-promoting effects on normal colonic mucosa and colorectal cancers (15, 17, 18). These studies were performed using full-length progastrin, either as a recombinant peptide or overexpressed as a transgene in the liver and secreted constitutively. Although recombinant progastrin is relatively stable in the circulation (35), a comparison of the antral and secreted forms of progastrin has not been reported. We, therefore, determined the structure of the stored and secreted forms of ovine progastrin and compared the regulation of antral secretion of progastrin to that of Gamide.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Statistical analyses
 Results
 Discussion
 References
 
Animal preparation
All experiments conformed with the National Health and Medical Research Council of Australia code of practice for the care and use of animals for scientific purposes and were approved by the Austin health animal ethics committee.

Studies in anesthetized sheep
The model for collecting antral and fundic blood has been reported previously (36). Merino-Corriedale-cross sheep. weighing 35–45 kg, were used. Anesthesia was induced by injection of pentothal into the jugular vein and was maintained throughout the experiment with a 1–2% fluothane-oxygen mixture (ICI Australia Operations Pty. Ltd., Heidelberg, Australia). Using a midline incision, cannulas (inside diameter, 0.76 mm; outside diameter, 1.65 mm; Dow Corning Corp., Midland, MI) were inserted into the gastric vein, draining blood from the fundus, and the gastroepiploic vein, draining blood from the antrum. The vein connecting the antrum to the fundus was ligated to prevent contamination of the samples by blood flow from the fundic to the antral vein. The stomach was cannulated by the insertion of a catheter through a purse string suture into the antral region of the abomasum. Gastric juice was collected by first clearing the cannula of any contents, then aspirating approximately 5 ml gastric secretion. Cannulas were also inserted into the jugular veins, as described for the conscious sheep studies. Basal samples were not collected until 30 min after the surgery was completed.

Studies in conscious sheep
The studies were performed in Merino-Corriedale-cross sheep, weighing 35–45 kg. After a local anesthetic (2 ml 1% lignocaine) had been administered, a hypodermic needle was inserted into the jugular vein, directed toward the head (up). A polyvinyl cannula (inside diameter, 0.76 mm; outside diameter, 1.65 mm) was then passed through the needle into the vein. The same procedure was followed for the contralateral jugular vein, except that the cannula was directed toward the heart (down). Agonists and antagonists were infused through the cannula directed downward, and blood was collected from the cannula directed upward. A 24-h recovery period was allowed, and animals were housed in metabolism cages for the duration of the experiments.

Studies in anesthetized sheep
Protocol 1: effect of iv omeprazole and intragastric HCl infusions.
After a 30-min basal period, omeprazole was administered via the jugular cannula pointing toward the heart as a bolus (0.65 mg/kg), followed immediately by an infusion of 0.90 mg/kg·h at 20 ml/h for 180 min. During the last hour of omeprazole infusion, 0.1 M HCl was administered via the gastric cannula to return the pH to between 1.5 and 2.5. This required an average of 120 ml over 30 min. Blood samples were obtained from the jugular, antral, and fundic cannulas at regular intervals before, during, and after the infusion. Gastric pH was also measured at these times.

Studies in conscious sheep
Protocol 2: effect of omeprazole infusion.
After a 30-min basal period, omeprazole was administered via the jugular cannula pointing toward the heart as a bolus (0.65 mg/kg), followed immediately by an infusion of 0.90 mg/kg·h at 20 ml/h for 120 min. Blood samples were obtained from the upward-pointing cannula at regular intervals before, during, and after the infusion.

Protocol 3: effect of somatostatin infusion.
After a 30-min basal period, somatostatin-14 (5 µg/kg·h; Bachem, Bubendorf, Switzerland) was infused via the jugular cannula pointing toward the heart at 20 ml/h for 120 min. Blood samples were obtained from the upward-pointing cannula at regular intervals before, during, and after the infusion.

Laboratory methods
Tissue extracts.
Antral tissue was collected immediately after a lethal dose of barbiturate, washed with ice-cold 0.9% saline, snap-frozen in liquid nitrogen, and stored at –70 C until extraction. Antral mucosae from four sheep were extracted for gastrin peptides using either a two-step boiling water/acetic acid extraction or an organic solvent extraction. The water/acid procedure involved boiling the specimen in 5 vol water for 5 min, followed by mechanical homogenization (Polytron, Brinkmann Instruments, Lucerne, Switzerland). The sample was boiled for an additional 5 min, then microfuged at 10,000 rpm for 15 min at 4 C. The supernatant (water extract) was collected and stored at –20 C until assay. The pellet was resuspended in 5 vol boiling 3% acetic acid, boiled for 10 min, and centrifuged as before. The supernatant (acid extract) was collected and stored at –20 C until assay. The organic solvent extract was obtained by pulverizing frozen tissue with a mortar and pestle on dry ice and homogenizing (Polytron) in 10 vol 80% acetonitrile. Cellular debris was pelleted by centrifugation (20,400 x g, 4 C, 10 min). The supernatant was collected, and organic solvent was removed by air stream. Particulates were removed by centrifugation (3,000 x g, 4 C, 10 min), and the supernatant was stored at –20 C.

Concentrating plasma.
Antral plasma samples from seven sheep infused with omeprazole and HCl (protocol 1) were pooled and applied to a reverse phase C18 Sep-Pak (Waters Corp., Milford, MA) according to the manufacturer’s instructions. Briefly, 2 ml plasma were mixed with 4 vol 0.05% trifluoroacetic acid (TFA) and 60 µl 10% TFA (final pH ~3), and passed through an activated reverse phase C18 Sep-Pak three times. Unbound material was removed by washing with 10 ml 0.05% TFA, and peptide was eluted with 6 ml 80% acetonitrile. Eluates from five Sep-Paks were combined and lyophilized by air stream. Samples were stored at –20 C until RIA and chromatography.

RIAs.
The epitopes for the different antisera are shown in Fig. 1Go. A new antiserum was generated to measure the C terminus of ovine progastrin. The decapeptide (TyrSerAlaGluGluGlyAspGlnHisPro), which corresponds to the ovine C-terminal-flanking peptide progastrin75–83 (CTFP) with an additional N-terminal tyrosine (37), was custom-synthesized by Chiron (Melbourne, Australia), with sequence and purity (>95%) determined by mass spectrometry and HPLC. This peptide was used for conjugation and radiolabeling. For antibody production, the N terminus of the peptide was conjugated with glutaraldehyde to keyhole limpet hemocyanin in a molar ratio of 600:1:6000 (peptide/keyhole limpet hemocyanin/glutaraldehyde). The conjugate was emulsified in Freund’s complete adjuvant (Sigma-Aldrich Corp., Castle Hill, Australia) in a 1:1 ratio (vol/vol), and 2 ml emulsified conjugate (which contained about 100 nmol CTFP) were injected sc into each of four New Zealand White Cross rabbits. For subsequent booster injections (at 6- to 8-wk intervals), the conjugate was emulsified using Freund’s incomplete adjuvant (Sigma-Aldrich Corp.) in a 1:1 ratio (vol/vol). After several booster injections, one rabbit (no. 152) generated high titer, high affinity antibodies against the CTFP. There was insignificant (<0.001%) cross-reactivity with Ggly, Gamide, or human Tyr-progastrin71–80 even at relatively high concentrations (10 µM). Because this antiserum detects both intact progastrin and the CTFP of progastrin, the results are expressed as progastrin C-terminal immunoreactivity (progastrin CTI) for clarity. Assay incubations were made in veronal buffer (1 ml) containing 0.1% BSA, pH 8.7. The antibody was used at a final dilution of 1:40,000 with [125I]Tyr-progastrin75–83, prepared by the chloramine-T method, as the label. The calculated 50% inhibitory dose was 12.2 fmol/tube, and the intra- and interassay variations were less than 6% and less than 9% respectively. Progastrin CTI was measured in tissue extracts (50–100 µl of a 1:1000 dilution), plasma (50 µl), and chromatography fractions (50–800 µl) against a Tyr-progastrin75–83 standard curve. Solutions of the decapeptide were prepared gravimetrically using a microbalance, and their concentrations were checked by absorbance at 280 nm before use as the standard for the RIA. RIA of various dilutions of antral extracts and plasma samples showed the samples diluted in parallel to the standard curve.

Amidated gastrin.
Tissue extracts, plasma, and chromatography fractions were measured with antiserum 1296 as previously reported (38). This antiserum detects all amidated C-terminal fragments greater than the pentapeptide and does not cross-react with CCK or Ggly.

Glycine-extended gastrin.
The antral tissue extract and sizing chromatography fractions were assayed with antiserum 109-2 using a previously described RIA (32). This antiserum detects Ggly forms and CCK-glycine, but does not cross-react with Gamide (29).

Somatostatin.
Plasma somatostatin was measured in ethanol-extracted plasma, as described previously (39). Extracted plasma was assayed against a somatostatin-14 standard curve with antiserum 8402, which detects somatostatin-14 and somatostatin-28 to an equal extent. [125I]Tyr-somatostatin-14 was prepared using the chloramine-T method and purified by reverse phase HPLC. The 50% inhibitory dose was 8 fmol/ml, and the inter- and intraassay coefficients of variation were less than 5% and 12%, respectively.

Sizing chromatography.
Antral plasma and antral tissue extracts were chromatographed on a calibrated Sephadex G-50 superfine column (10 x 1200 mm; Pharmacia Biotech, Uppsala, Sweden) eluted with 0.02 M veronal, 0.05% BSA, and 0.005% sodium azide, pH 8.7, at 4 C at a flow rate of 6.3 ml/h in 1-ml fractions. Blue dextran (Pharmacia Biotech) and Na-125I (ICN, Sydney, Australia) were used to determine void and total volumes, respectively.

Ion exchange chromatography.
Antral extract (16 ml of an organic solvent extract, containing ~5.1 nmol progastrin CTI, in a final concentration of 0.02 M triethanolamine, pH 8) was filtered, loaded onto a Pharmacia Mono Q HR 5/5 column equilibrated with 0.02 M triethanolamine, pH 8, and eluted with a gradient from 0.02 M triethanolamine, pH 8 to 8% acetic acid in 0.02 M triethanolamine, pH 2 over 40 min.

Reverse phase HPLC.
A portion of the pooled immunoreactive peaks from ion exchange chromatography was applied to a C18 µBondapak Radial Pak cartridge with a gradient from 0–30% acetonitrile in 0.05% TFA over 45 min at 1 ml/min. A single immunoreactive peak eluting at 9.3% acetonitrile was recovered and used for mass spectrometry.

To compare stored and circulating forms of ovine progastrin CTI, organic antral extract containing 77 pmol progastrin CTI was subjected to reverse phase HPLC as described above. Concentrated plasma was reconstituted in 0.05% TFA, filtered (0.45 µm), and subjected to reverse phase HPLC as described above immediately after the antral extract.

Mass spectrometry.
Electrospray ionization mass spectrometry was performed on a Sciex API-300 triple quadrupole mass spectrometer (PerkinElmer Life Sciences, Melbourne, Australia) fitted with a micro-ion spray ion source (flow rate, 0.2 µl/min), previously calibrated to an accuracy of ± 0.01% using singly charged (polypropylene glycol) reference ions. Portions from the reverse phase HPLC fractions were concentrated 6-fold by freeze-drying. Three to 5 µl of the concentrated fractions were mixed with acetonitrile/0.2% formic acid (1:1) before analysis. MS/MS spectra (Q3 scans) were obtained using nitrogen collision gas (4 millitorr pressure, 20.7 cm cell length) and optimized collision energies of 32–64 eV. Signal-averaged raw mass spectra were analyzed manually and transformed to a true mass scale using the PE-Sciex BioMultiview program Biospec Reconstruct (PerkinElmer Life Sciences). Peptide sequences inferred from observed masses were confirmed by analysis of MS/MS spectra manually and using the BioMultiview programs Predict Sequence and Peptide Fragments (PerkinElmer Life Sciences).


   Statistical analyses
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analyses
Results
 Discussion
 References
 
Results are expressed as the mean ± SE. The integrated gastrin/progastrin CTI response was calculated as the area under the concentration-time curves as reported previously (36). Statistical comparison between more than two groups was made by one-way ANOVA, followed by Dunnett’s test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analyses
 Results
Discussion
 References
 
Antral tissue concentrations
Progastrin CTI, as measured with antiserum 152, was the major antral form of progastrin-derived peptide with a mean concentration of 1961 ± 806 pmol/g. The concentration of Gamide (antiserum 1296) was about 3-fold less at 735 ± 157 pmol/g, whereas the Ggly concentration (antiserum 109-2) was 74 ± 18 pmol/g, an order of magnitude less than Gamide.

Sizing chromatography of antral progastrin-derived peptides
Antral extracts from several sheep were examined by size exclusion chromatography, and a representative chromatograph of a water extract is shown in Fig. 2Go. Comparison of the peak area of progastrin CTI (Fig. 2AGo) and Gamide (Fig. 2BGo) confirms that progastrin CTI is the predominant immunoreactive form in the antrum. The elution profile indicated that ovine progastrin CTI was considerably smaller than the recombinant human progastrin marker. Interestingly, ovine progastrin CTI eluted in a similar position to G17amide (2098 Da; Fig. 2Go). However, this appears to be the result of an interaction with the separation medium, because synthetic ovine C-terminal flanking peptide (SAEEGDQHP, prog75–83; molecular mass, 1132 Da) also eluted in the same position as G17amide. Sizing chromatography anomalies such as these have also been encountered in previous studies characterizing progastrin forms (25, 26, 40). The elution profiles from both the water and organic solvent extracts were identical (data not shown).



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  		FIG. 2. Sizing chromatography of water extract from ovine antrum. Fractions were assayed with antiserum 152 for progastrin CTI (A) and antiserum 1296 for Gamide (B). A similar profile was observed with an organic solvent extract (data not shown). The elution positions of human progastrin6–80 (hPG), G17amide (G17), and human and ovine C-terminal flanking peptides (hCTFP and oCTFP), run on separate occasions, are indicated by arrows.

 
Peptide purification
The organic solvent antral extract was fractionated on an ion exchange column. Two immunoreactive peaks were identified (>90% of amount recovered; Fig. 3AGo). The fractions encompassing these two peaks were pooled and rechromatographed by reverse phase HPLC (Fig. 3BGo). A single major immunoreactive peak eluting at 9.3% acetonitrile was obtained and subjected to mass spectrometry for molecular weight determination and sequence analysis.



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  		FIG. 3. Purification of ovine progastrin CTI. Organic solvent extract from ovine antrum was subjected to ion exchange chromatography (A). The peak of progastrin CTI (fractions 35–39) was additionally purified by reverse phase HPLC (B), as described in Materials and Methods. The peak from the latter chromatography (fractions 18–20) was used for molecular mass and sequence determination by mass spectrometry. Dashed and dotted lines represent eluant gradient and optical absorbance at 214 nm, respectively.

 
Mass spectrometry
The immunoreactive fractions from the reverse phase HPLC were concentrated by freeze-drying. Electrospray ionization mass spectrometry identified three peaks with molecular masses of 681.3, 810.3, and 881.4 Da in approximately equal amounts (Table 1Go). These molecular masses corresponded precisely with those expected for three peptides (EGDQHP, EEGDQHP, and AEEGDQHP, respectively) derived from the known ovine progastrin sequence (Table 1Go). In the case of the largest peptide, AEEGDQHP, the sequence was confirmed by MS/MS collisional fragmentation. Similar mass spectrometric results were obtained when antral extracts were purified by chromatography on a C18 µBondapak Radial Pak cartridge (as described in Materials and Methods), followed by purification of the immunoreactive peak on a C18 microbore column (1 mm x 250 m; 40 µl/min flow rate; linear applied gradient of 0–50% acetonitrile/0.1% TFA over 150 min).


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  		TABLE 1. Mass spectrometry of antral progastrin CTI

 
Basal concentrations
In the anesthetized animals, Gamide concentrations in the antral vein were significantly higher than those in the fundic and peripheral circulations (Table 2Go). Progastrin CTI was also higher in the antral vein than in the peripheral circulation and was by far the major circulating form (Table 2Go). The progastrin CTI to Gamide ratio increased from 3.4 in the antral circulation to 9.6 in the peripheral circulation, suggesting that Gamide was being cleared more rapidly than progastrin CTI. Similar results were seen in the peripheral circulation of the nonanesthetized animals (Table 2Go). The relative amounts of circulating progastrin-derived peptides were similar to the stored antral concentrations, with progastrin CTI being the major form.


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  		TABLE 2. Basal plasma concentrations of progastrin CTI and Gamide (picomoles per liter)

 
Studies in anesthetized sheep
Protocol 1: effect of iv omeprazole and intragastric HCl infusion.
Gamide concentrations in the antral and peripheral circulations increased by 30% after 2 h of omeprazole infusion and reached maximums of 291 ± 73 and 48 ± 11 pmol/liter for antral and jugular concentrations, respectively (Fig. 4Go). Intragastric HCl rapidly reversed this increase to below basal levels, reaching minimums at 180 min of 184 ± 62 and 27 ± 5 pmol/liter for antral and jugular concentrations, respectively. On cessation of both omeprazole and intragastric HCl, there was a large rebound increase in plasma Gamide (Fig. 4Go). Progastrin CTI concentrations also changed in response to the alterations in gastric pH, although not to the same extent or as rapidly as plasma Gamide. Thus, as shown in Fig. 5Go, the omeprazole infusion caused a progressive increase in antral and peripheral progastrin CTI concentrations that was moderated by the administration of intragastric HCl. The extent of these changes for Gamide and progastrin CTI are demonstrated with the integrated outputs, which measure the net changes compared with control, rather than the absolute changes. These are shown for the antrum only (Fig. 6Go). Antral Gamide output in response to the omeprazole-induced increase in gastric pH was 0.87 ± 0.29 pmol/ml·min, reaching a nadir of –0.56 ± 0.71 pmol/liter after 1 h of intragastric HCl, with a rebound increase after cessation of acid administration. Antral progastrin CTI output was higher than antral Gamide, with a maximum output of 1.99 ± 0.57 pmol/ml·min after 2-h omeprazole infusion. The progastrin CTI output remained at or below this level during intragastric acid administration, with a rebound increase to 3.89 ± 1.41 pmol/ml·min in the 30 min after cessation of acid administration (Fig. 6Go).



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  		FIG. 4. Effect of iv infusion of omeprazole (0–120 min) and then of omeprazole plus intragastric HCl on Gamide in the antral (A) and jugular (B) veins of anesthetized sheep (n = 7).

 


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  		FIG. 5. Effect of iv infusion of omeprazole (0–120 min) and then of omeprazole plus intragastric HCl on plasma progastrin CTI in the antral (A) and jugular (B) veins of anesthetized sheep (n = 7).

 


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  		FIG. 6. Antral integrated outputs for Gamide (A) and progastrin CTI (B) in response to omeprazole and omeprazole plus intragastric HCl administered to anesthetized sheep (n = 7). *, P < 0.05 compared with basal output (–30 to 0 min).

 
Basal somatostatin was highest in the fundic vein (327 ± 62 pmol/liter), followed by levels in the antral vein (186 ± 25 pmol/liter) and peripheral circulation (79 ± 5 pmol/liter). Omeprazole with or without concurrent intragastric HCl had no effect on jugular or fundic somatostatin concentrations (data not shown). Omeprazole alone had no effect on antral vein somatostatin concentrations. However, omeprazole plus intragastric HCl resulted in a rapid and large increase in antral vein somatostatin from 170 ± 44 to a maximum of 519 ± 114 pmol/liter (Fig. 7Go).



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  		FIG. 7. Effect of iv infusion of omeprazole (0–120 min) and then of omeprazole plus intragastric HCl on plasma somatostatin in the antral vein of anesthetized sheep (n = 7).

 
Studies in conscious sheep
Protocol 2: effect of iv omeprazole.
As in the anesthetized animals, omeprazole infusion resulted in a sustained increase in circulating Gamide and progastrin CTI. Although, as noted previously, basal levels of Gamide and progastrin CTI were quite different, the net increase was similar for both peptides, with a maximum increase of about 0.5 pmol/liter·min (Fig. 8Go).



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  		FIG. 8. Peripheral integrated outputs for Gamide (A) and progastrin CTI (B) in response to omeprazole infusion to the conscious sheep. *, P < 0.05 compared with basal output (–30 to 0 min).

 
Protocol 3: effect of somatostatin infusion.
Somatostatin decreased Gamide concentrations, and the decrease was followed by a rebound increase after the cessation of the infusion (Fig. 9AGo). Somatostatin also appeared to inhibit progastrin CTI secretion, although the change was not statistically significant. After the somatostatin infusion was stopped, there was a significant rebound increase in plasma progastrin CTI (Fig. 9BGo).



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  		FIG. 9. Peripheral integrated outputs for Gamide (A) and progastrin CTI (B) in response to somatostatin infusion to the conscious sheep. *, P < 0.05 compared with basal output (–30 to 0 min); #, P < 0.05 compared with output at 90–120 min.

 
Comparison of stored and circulating forms of ovine progastrin CTI
Size exclusion chromatography demonstrated that antral and plasma progastrin CTI eluted with a similar Kav of approximately 0.65 (Fig. 10Go, A and B). Amidated gastrin from both antrum and plasma eluted in the same position for reverse phase HPLC (Fig. 10Go, C and D, dotted line), indicating that conditions were the same between consecutive HPLC runs. However, the plasma progastrin CTI had several peaks, two eluting slightly earlier than the stored form (17 and 20 min compared with 22 min) and two eluting later (56 and 66 min; Fig. 10Go, C and D). Trace amounts of these latter peaks were also detected in the antral extracts (Fig. 10CGo).



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  		FIG. 10. Comparison of antral and circulating forms of progastrin CTI by reverse phase HPLC. Organic solvent antral extract (A and C) and concentrated plasma (B and D) were subjected to sizing chromatography (A and B) and reverse phase HPLC (C and D). Fractions were assayed with antiserum 152 for progastrin CTI (solid line) and antiserum 1296 for Gamide (dotted line). The elution positions for sizing chromatography of human progastrin6–80 (hPG), G17amide (G17), and human and ovine C-terminal flanking peptides (hCTFP and oCTFP) are indicated by arrows.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analyses
 Results
 Discussion
References
 
The present report describes the isolation and characterization of the predominant progastrin-derived peptides in sheep antrum. The molecular masses and sequence correspond to peptides derived from the progastrin C terminus, namely progastrin76–83, progastrin77–83, and progastrin78–83. The three peptides were present in approximately equivalent amounts. A peptide of similar chromatographic characteristics was the major secreted form, and its release was regulated by gastric pH and somatostatin, suggesting that the CTFP was stored in secretory granules. Indeed, the dibasic residues adjacent to the CTFP appear to be essential for sorting of progastrin into secretory granules and to the regulated secretory pathway (41).

Nonamidated forms of gastrin, such as progastrin and Ggly, are now known to be biologically active. Studies with transgenic animals overexpressing progastrin or Ggly or after infusion of synthetic peptides have shown that nonamidated gastrins can stimulate colonic proliferation, accelerate the development of colon cancer, and potentiate gastric acid secretion (9, 15, 16, 42, 43). To render these findings informative, it is essential that relevant molecular forms of progastrin are examined. However, the studies described above used full-length progastrin or Ggly. Depending on the assay system, full-length progastrin comprises only 1–10% of gastrin immunoreactivity in humans (19, 20, 26, 27). As found in the present study in sheep, the pig, ferret, and dog have only trace amounts of full-length progastrin (13, 25). An increased proportion of intact progastrin is found in gastrinomas (~40%) (26) and in colorectal cancers (~30%) (22), consistent with attenuated processing in the hypersecretory gastrinoma cell and the nonendocrine gastrin-containing cell in colorectal cancer (5, 19). Nevertheless, even in these circumstances, the most abundant progastrin-derived peptide is the CTFP, not intact progastrin (20, 22, 26). C-Terminal flanking peptides of the related peptide, CCK, which also start with the sequence Ser-Ala-Glu, have been isolated from pig (44) and rat (45, 46) brain in similar concentrations to amidated CCK. These peptides were not active in bioassay systems that respond to amidated CCK (44, 45).

Previous studies of a number of species, including pig, dog, ferret, and rat (13, 28, 47), have reported that the sum of Gamide and its immediate precursor, Ggly, is approximately equal to the CTFP concentration. Because all three peptides are derived from the same precursor, this stoichiometry is expected. However, in the human antrum the CTFP concentration is about 40% higher than the Gamide concentration, and in gastrinomas the proportion ranges from 0.5- to 18-fold (26). In the current study we found that the CTFP concentration was about 2.5-fold higher than the sum of the concentrations of Gamide and Ggly. The excess of CTFP in man and sheep is not the result of reduced secretion, because the antral vein concentration of CTFP was more than 3-fold higher than Gamide. The presence of nonimmunoreactive metabolites offers a possible explanation for the apparent excess of CTFP, because variable amounts of N-terminal fragments of G17, such as G13, or of G17 extended by Gly-Arg or Gly-Arg-Arg at the C terminus have been reported in the antrum and in gastrinomas (23, 24, 48, 49). Alternative explanations that require testing include the presence of nonimmunoreactive breakdown products of Gamide or Ggly, heterogeneity of the secretory granules, differences in processing within secretory granules, the influence of phosphorylation on precursor cleavage, and preferential sorting of CTFP into secretory granules (6, 33, 41).

Our findings in the sheep agree with previous reports on the presence of substantial amounts of CTFP in the antrum of other species, but differ on the lengths and relative amounts of the CTFPs present. Processing of progastrin involves cleavage after three dibasic sites (Arg36Arg37, Lys53Lys54, and Arg73Arg74). Gly72 then provides the donor for the C-terminal amidation of gastrin (4, 6). In human, dog, pig, and ferret, the major CTFP commences with Ser75 and is present in both phosphorylated and nonphosphorylated forms (13, 26, 28, 47). Smaller amounts of nonserine-containing peptides are also detected. We found three different CTFP peptides: AEEGDQHP (progastrin76–83), EEGDQHP (progastrin77–83), and EGDQHP (progastrin78–83), none of which contained Ser75. This appears to be a true species difference and not an artifact of extraction, because the tissues were rapidly removed and frozen, and two different extraction procedures, namely, boiling water, as used previously (13), and cold organic solvent extraction, were used with the same result. Furthermore, two different purification procedures were used before mass spectroscopy for molecular mass and sequence determination.

The present study is the first to report that very high concentrations of CTFP are present in the circulation, and that CTFP release is regulated by similar factors that control amidated gastrin. Sizing and reverse phase chromatography of plasma confirmed that the progastrin C-terminal immunoreactivity was not intact progastrin, but a CTFP of similar chromatographic characteristics to the antral forms. The CTFP was the predominant circulating progastrin-derived peptide. The CTFP to Gamide ratio in the antral vein was about 3:1, whereas in the peripheral circulation it was 10:1, demonstrating that not only was CTFP being secreted at a higher rate, but apparently it was also more stable than Gamide after secretion.

Our studies in anesthetized sheep used a previously reported model that allows antral vein concentrations (secretion) and peripheral concentrations (equilibrium values) to be measured simultaneously (36). An increase in gastric pH by inhibition of the H/K-adenosine triphosphatase with omeprazole increased circulating CTFP, although the response was delayed compared with Gamide. pH reversal by intragastric HCl prevented any additional increase in CTFP, but reversed the increase in Gamide. These findings demonstrate that CTFP was regulated by gastric pH, but to a lesser extent than Gamide, and suggest that different populations of secretory granules were involved, with secretion from the Gamide-containing granules being more tightly regulated. Omeprazole also increased the CTFP concentration in the conscious animal, showing that the changes were not related to the anesthetic. The final demonstration of differential regulation of CTFP secretion was that somatostatin infusion into conscious sheep reduced CTFP secretion to a lesser extent than Gamide secretion. After cessation of the somatostatin infusion, there was a rebound increase above basal concentrations for both gastrin forms resulting from the removal of the direct inhibitory effect on the G cell coupled with the still elevated pH (50). Taken together with the pH effects on CTFP secretion, these findings indicate that CTFP is regulated, but in a different way from Gamide.

Although there have been no previous reports on the quantity and regulation of circulating CTFP, antral CTFP is known to be influenced by alterations in the gastric luminal environment. Fasting of rats for 48 h caused a 60% decrease in CTFP content and decreased the proportion of Ser75 phosphorylation (28). In dogs with surgically excluded antra, the concentration of CTFP increased 4-fold, and the concentration of Ggly increased 8-fold, whereas Gamide increased only 3-fold compared with control antrum. Intact progastrin remained about 1% of the total immunoreactivity, indicating that endopeptidase cleavage of progastrin was normal, whereas conversion to Gamide was suppressed (40). The available data imply that the processing of progastrin is regulated, but the initial endopeptidase cleavage of progastrin is virtually complete and is not a rate-limiting step.

There are few reports on the biological activity of the CTFP. Short-term (5-min) CTFP infusions did not alter acid secretion or modify the acid response to Gamide (13). CTFP stimulated histamine release, but at 1% the potency of Gamide (14). However, by analogy with Ggly, long-term administration may be required to demonstrate a biologically relevant effect (9, 10, 12, 51). The long-term effects of CTFP on gastric acidity and gastrointestinal mucosal proliferation, either alone or in combination with other gastrin peptides, have not been reported. Because CTFP is the major stored and secreted form of progastrin-derived peptide, and nonamidated forms of gastrin have been implicated in the acceleration of gastric and colorectal cancer development (43, 52), studies of the biological activity of CTFP will be of great interest.


   Acknowledgments
 
We thank Ms. Lisa Hogan for expert technical assistance. Antibody 1296, raised against gastrin amide, 109–2 (gastrin gly), and 8402 (somatostatin), was provided by CURE/Digestive Diseases Research Center, Antibody/RIA Core.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia, the Austin Hospital Medical Research Foundation, and National Institutes of Health Grant DK-41301.

Abbreviations: CCK, Cholecystokinin; CTFP, C-terminal flanking peptide; CTI, C-terminal immunoreactivity; Gamide, gastrin amide; Ggly, glycine-extended gastrin; TFA, trifluoroacetic acid.

Received July 15, 2004.

Accepted for publication August 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analyses
 Results
 Discussion
 References
 

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