Identification of a gastrin binding protein in porcine gastric mucosal membranes by covalent cross-linking with iodinated gastrin

A gastrin binding protein (GBP) has been identified in detergent extracts of porcine gastric mucosal membranes by covalent cross-linking to 125I-[Nle15]gastrin with disuccinimidyl suberate. The apparent molecular weight of the cross-linked complex (80,000) is uneffected by reduction suggesting that the GBP is not composed of disulfide-bonded subunits. Subtraction of the molecular weight of 125I-gastrin indicates that the molecular weight of the GBP is 78,000. A similar molecular weight has been observed previously for the gastrin receptor (74,000) on intact canine parietal cells and plasma membranes therefrom, and for the receptor for the related hormone cholecystokinin (76,000-85,000) on pancreatic acinar membranes under reducing conditions. The similarity in molecular weight between the gastrin receptor and the solubilized GBP suggests that the latter protein is probably the gastrin receptor. However, the concentration (2 microM) of [Nle15]gastrin required for 50% inhibition of cross-linking of gastrin to the GBP solubilized in 0.1% Triton X-100 is 200-fold greater than the value (10 nM) observed for the gastrin receptor on isolated canine gastric parietal cells. A lower concentration (0.3 microM) of [Nle15]gastrin was required to inhibit cross-linking in a milder detergent (0.4% digitonin, 0.08% cholate). Thus, the reduced affinity for gastrin of the putative solubilized form of the gastrin receptor appears to be a result of detergent extraction.     

Metal ion-dependent binding of gastrin to albumin

Binding of 125I-[Nle15]gastrin to albumin purified from porcine serum, from porcine gastric mucosal cytosol, and from bovine serum has been demonstrated by covalent cross-linking and ultracentrifugation. Binding was enhanced in the presence of Zn2+, Ni2+, Cu2+, Co2+, and Cd2+, but not Ca2+, Mg2+, or Mn2+. The best fit to the binding data for bovine serum albumin was obtained with a model assuming two nonequivalent binding sites. The affinity of both sites for gastrin was increased in the presence of 100 microM Zn2+ or Ni2+ ions. The highest association constant observed was 2.3 X 10(5) M-1 in the presence of 100 microM Zn2+ ions. The similarity of the Zn(2+)-dependence of binding for bovine and porcine serum albumins, despite the replacement of His3 by Tyr, suggested that the N-terminal metal ion-binding site was not involved. Although all gastrin affinities were reduced by 50% in the presence of 150 mM NaCl, the Zn(2+)-dependence of binding was retained. We therefore propose that the ternary complex of gastrin, Zn2+ ions, and albumin may play a physiological role in the serum transport of Zn2+ ions and in the uptake of Zn2+ ions from the lumen of the gastrointestinal tract.     

Isolation of transferrin from porcine gastric mucosa: comparison with porcine serum transferrin

1. An iron-binding glycoprotein has been purified to homogeneity from porcine gastric mucosa. 2. The molecular weight (80,000), amino acid composition, carbohydrate content, N-terminal amino acid sequence, tryptic map, stoichiometry of iron binding (2 mol/mol), visible absorption spectrum of the ferric complex and chromatographic behaviour of the gastric protein are all strikingly similar to the corresponding properties of porcine serum transferrin. 3. The quantity of the gastric protein (1.3 mg/g wet weight) present in the gastric mucosa suggests that it is not serum transferrin (plasma concentration 1.8 mg/ml) contaminating the tissue. 4. A role for transferrin in the uptake of dietary iron by the gastrointestinal tract is proposed.     

A qualitative comparison of canine plasma gastroenteropancreatic hormone responses to bombesin and the porcine gastrin-releasing peptide (GRP)

The effect on plasma gastroenteropancreatic hormone levels on infusing the porcine gastrin-releasing peptide and bombesin into dogs demonstrated no qualitative difference in the spectrum of activity of the two peptides. Sustained elevations in plasma immunoreactive gastrin, pancreatic polypeptide, enteroglucagon, gastric inhibitory polypeptide, pancreatic glucagon and transient elevations in plasma insulin were seen during infusions of both peptides. The similar spectrum of activities and the structural homology between the two peptides suggests that the porcine gastrin releasing peptide is the porcine counterpart of the amphibian peptide bombesin.     

Binding of pepsinogen to the 78 kDa gastrin binding protein.

An endogenous ligand of the 78 kDa gastrin-binding protein (GBP) has been purified from detergent extracts of porcine gastric mucosal membranes by ion exchange chromatography and preparative gel electrophoresis. The ligand bound to the GBP with high affinity (mean IC50 value of 0.31+/-0.09 microgram/ml, or 8 nM), as assessed by inhibition of cross-linking of iodinated gastrin2,17 to the GBP. Both the N- and C-terminal halves of the GBP, which had been expressed individually as glutathione-S-transferase fusion proteins in Escherichia coli, and purified on glutathione-agarose beads, bound the ligand. Two peptides derived from the ligand were purified by reversed-phase high-performance liquid chromatography (HPLC), and characterised by mass spectrometry and Edman sequencing. The peptides were 97% and 100% identical, respectively, to amino acids 119-157 and 199-219 of porcine pepsinogen A. Commercial samples of pepsinogen also bound to the GBP, with a mean IC50 value of 3.9+/-1. 2 micrograms/ml (100 nM). We conclude that the ligand is closely related, but not identical, to pepsinogen A.     

Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue.

A heptacosapeptide with potent gastrin releasing activity has been isolated from porcine non-antral gastric and intestinal tissue. The amino acid sequence suggested from a preliminary study on the gastric peptide is: Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH₂. Striking homology in the C-terminal region is seen with bombesin, accounting for the similar bioactivities of the two peptides. Some structural resemblance with porcine cholecystokinin in the N-terminal region is noted.     

Gastrins, iron homeostasis and colorectal cancer

The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron deficiency anemia was recognized as a long-term consequence of partial gastrectomy. This review summarizes the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyze the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract.     

Effect of porcine gastrin releasing peptide on anterior pituitary hormone release

The effect of porcine gastrin releasing peptide (GRP), a heptacosapeptide with potent gastrin releasing activity which has recently been isolated from porcine non-antral gastric tissue, on pituitary function was investigated in the rat. Graded doses of synthetic porcine GRP were injected intravenously and the animals were killed at various intervals after injection. Growth hormones, LH, FSH, and TSH were measured in serum by specific radioimmunoassays. GRP had no significant effect on growth hormone or FSH serum concentrations at any dose or sampling time studied. In contrast, the heptacosapeptide significantly stimulated LH and suppressed TSH secretion in a dose-related fashion. Since there are striking structural similarities between GRP and bombesin, a tetradecapeptide from amphibian skin which shows amino acid homology with the C-terminal region of GRP, GRP may be the mammalian counterpart of bombesin.     

Immunofluorescent localization of the gastrin-secreting G cells in the pyloric antrum of the pig

Summary An indirect immunofluorescence technique, using anti-human gastrin serum, was applied to formalin-fixed, paraffin-embedded material from antral tissues of porcine stomach. After photographing the result, post-fixation in formalin was followed by the Grimelius silver technique. The argyrophil G cells, situated predominantly in the middle zone of the gastric glands, all showed positive gastrin immunofluorescence. Some weakly fluorescent cells failed to stain by the silver technique, suggesting that the latter is less sensitive than immunofluorescence in that it depends on the presence of the granular storage product.     

Characterization of porcine gastric galanin.

The presence of a peptide capable of producing powerful contractions of rat small intestinal smooth muscle was detected in chromatographic fractions derived from porcine gastric corpus extracts. The pharmacological characteristics of this entity suggested that it might be galanin and on its purification to homogeneity, amino acid composition and sequence analysis demonstrated the identify of the gastric and intestinal forms of galanin. The presence of galanin in the gastric corpus tissue and its ability to affect gastric smooth muscle activity, gastrin release, and gastric acid secretion suggest potential important physiological roles for galanin in the stomach.     

Dose-related inhibition of gastric secretion by intraduodenal trypsinogen in dogs

It has been shown previously that trypsinogen and its activation peptide but not trypsin decreased gastric secretion. The purpose of this work was to study the dose-action relation between the intraduodenal infusion of trypsinogen and gastric secretion. Three dogs provided with gastric and duodenal Thomas fistulae were stimulated by continuous i.v. perfusion of porcine gastrin I-II (6 microgram kg-1 h-1). Pancreatic juice was diverted to the exterior and gastric secretion was collected. Upon reaching a gastric secretory plateau, porcine trypsinogen was infused intraduodenally at doses of 5, 10, 20, 40, 80 and 160 mg. Each test was continued for a further 60 min. Control was made with isotonic saline. There was a dose-related inhibition of the gastrin-stimulated gastric acid output. This inhibition reached a maximum of 50% with 40 mg of intraduodenal trypsinogen, showing no increase with higher doses.     

Interrelationships between circulating gastrin and iron status in mice and humans

The observations that the peptide hormone gastrin interacts with transferrin in vitro and that circulating gastrin concentrations are increased in the iron-loading disorder hemochromatosis suggest a possible link between gastrin and iron homeostasis. This study tested the hypothesis that gastrin and iron status are interrelated by measurement of iron homeostasis in mice and humans with abnormal circulating gastrin concentrations. Intestinal iron absorption was determined by (59)Fe uptake following oral gavage, and concentrations of duodenal divalent metal transporter-1 (DMT-1) and hepatic hepcidin mRNAs were determined by quantitative real-time PCR in agastrinemic (GasKO), hypergastrinemic cholecystokinin 2 receptor-deficient (CCK2RKO), or wild-type mice. Iron status was measured by standard methods in the same mice and in hypergastrinemic humans with multiple endocrine neoplasia type 1 (MEN-1). Iron absorption was increased sixfold and DMT-1 mRNA concentration fourfold, and transferrin saturation was reduced 0.8-fold and hepcidin mRNA expression 0.5-fold in juvenile GasKO mice compared with age-matched wild-type mice. In mature mice, few differences were observed between the strains. Juvenile CCK2RKO mice were hypergastrinemic and had a 5.4-fold higher DMT-1 mRNA concentration than wild-type mice without any increase in iron absorption. In contrast to juvenile GasKO mice, juvenile CCK2RKO mice had a 1.5-fold greater transferrin saturation, which was reflected in a twofold increase in liver iron deposition at maturity compared with wild-type mice. The correlation between transferrin saturation and circulating gastrin concentration observed in mutant mice was also observed in human patients with MEN, in whom hypergastrinemia correlated positively (P = 0.004) with an increased transferrin saturation. Our data indicate that, in juvenile animals when iron demand is high, circulating gastrin concentrations may alter iron status by a CCK2R-independent mechanism.     

Interaction of gastrin with transferrin: effects of ferric ions

The sedimentation behavior of 125I-labeled gastrin has been studied as a function of Fe3+ ion concentration and pH. Both sedimentation velocity and sedimentation equilibrium experiments indicated that high-molecular-weight Fe3+-gastrin complexes were formed at pH 5.0 and pH 7.4. Self-association of gastrin alone was observed at pH values below 5.0. 125I-labeled gastrin bound to human serum apotransferrin at pH 7.4. Scatchard analysis of the gastrin-apotransferrin complex gave a Kd of approximately 6.4 microM at 37 degrees C, with two binding sites per molecule of apotransferrin. No significant binding of gastrin to diferric transferrin was observed under the same conditions. The binding of gastrin to apotransferrin was inhibited by NaCl. The results are consistent with the hypothesis that gastrin and transferrin act synergistically in the uptake of dietary iron by the gastrointestinal tract.     

Effect of cortisone acetate on acid secretion induced by histamine, pentagastrin, gastrin and food in dogs]

The effects of long term treatment with cortisone on the gastric secretion induced by histamine, pentagastrin, porcine gastrin and a meal have been investigated in four dogs with both gastric fistula and Heidenhain pouch. Cortisone increased the postcibal acid output and the observed maximal acid response from the pouch to all three exogenous stimuli. The ED 50'S remained unchanged. The same effects although less marked were observed in the innervated stomach. These data indicate that the increased acid secretion observed after long term treatment with cortisone is largely due to an increased secretory capacity of the gastric mucosa. This latter could result from an increase in the number of secretory units or to partial removal of a non competitive inhibitor of gastric secretion.     

Catabolism of gastrin releasing peptide and substance P by gastric membrane-bound peptidases

The catabolism of two gastric neuropeptides, the C-terminal decapeptide of gastrin releasing peptide-27 (GRP10) and substance P (SP), by membrane-bound peptidases of the porcine gastric corpus and by porcine endopeptidase-24.11 ("enkephalinase") has been investigated. GRP10 was catabolized by gastric muscle peptidases (specific activity 1.8 nmol min-1 mg-1 protein) by hydrolysis of the His8-Leu9 bond and catabolism was inhibited by phosphoramidon (I50 approx. 10(-8) M), a specific inhibitor of endopeptidase-24.11. The same bond in GRP10 was cleaved by purified endopeptidase-24.11, and hydrolysis was equally sensitive to inhibition by phosphoramidon. SP was catabolized by gastric muscle peptidases (specific activity 1.7 nmol min-1 mg-1 protein) by hydrolysis of the Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10 bonds, which is identical to the cleavage of SP by purified endopeptidase-24.11. The C-terminal cleavage of GRP10 and SP would inactivate the peptides. It is concluded that a membrane-bound peptidase in the stomach wall catabolizes and inactivates GRP10 and SP and that, in its specificity and sensitivity to phosphoramidon, this peptidase resembles endopeptidase-24.11.     

Effects of morphine, enkephalins and naloxone on postprandial gastric acid secretion, gastric emptying and gastrin release in dogs

The effects of intravenous infusions of morphine, met-enkephalin and leu-enkephalin on gastric acid secretion, gastrin release and gastric emptying were investigated in four dogs with gastric cannulas stimulated by a liquid peptone meal. The actions of a potent opiate antagonist, naloxone, used alone or combined with opiates were also studied. Morphine, met-and leu-enkephalin decreased the fractional gastric emptying rate. Acid secretion was decreased by enkephalins and increased by high doses of morphine. Enkephalins and to a lesser degree morphine inhibited gastrin release during the first hour following the administration of the meal. Only leu-enkephalin decreases significantly the integrated gastrin response. Naloxone at the doses used antagonized partly or totally the effects of opiates on gastric emptying but not those on gastric secretion or gastrin release. Naloxone infused alone had no significant effect on the gastric functions tested. These studies indicate that in dogs stimulated by a liquid test meal, enkephalins inhibit gastric emptying, acid secretion and gastrin release. Morphine inhibits gastric emptying and gastrin release and enhances acid secretion.     

Isolation and identification of a putative porcine transferrin receptor from Actinobacillus pleuropneumoniae biotype 1.

Each of two affinity isolation methods, the first based on biotinylated porcine transferrin plus streptavidin-agarose, and the second on Sepharose-coupled porcine transferrin, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine-transferrin-binding polypeptides (approximately 64 kDa and 99 kDa) from total membranes of Actinobacillus pleuropneumoniae grown under iron-restricted conditions. Both polypeptides were iron-repressible and were identified as potential receptor candidates as they were not isolated when biotinylated human transferrin was used instead of biotinylated porcine transferrin. The 64 kDa polypeptide was the more easily removed from Sepharose-coupled porcine transferrin and only the 99 kDa polypeptide appeared to be an outer-membrane protein. While these results suggest that the 99 kDa polypeptide represents the porcine transferrin receptor of A. pleuropneumoniae, and that the 64 kDa polypeptide represents an associated protein serving an accessory role, other interpretations are also possible.     

Differentiation of the gastric mucosa. II. Role of gastrin in gastric epithelial cell proliferation and maturation

Gastrin is the principal hormonal inducer of gastric acid secretion. The cellular targets for gastrin in the stomach are the acid-secreting parietal cell and histamine-producing enterochromaffin-like (ECL) cell. Gastrin is also a growth factor, with hypergastrinemia resulting in increased proliferation of gastric progenitor cells and a thickened mucosa. This review presents insights into gastrin function revealed by genetically engineered mouse models, demonstrating a new role for gastrin in the maturation of parietal and ECL cells. Thus, gastrin regulates many aspects of gastric physiology, with tight regulation of gastrin levels required to maintain balanced growth and function of gastric epithelial cells.