Identification of progastrin in gastrinomas, antrum, and duodenum by a novel radioimmunoassay.

Recent studies on the gene sequence encoding the human pyloric antral hormone, gastrin, indicate a precursor of 101 residues. We have now raised antibodies to a synthetic analogue corresponding to (Tyr)-human progastrin COOH-terminal pentapeptide. The antibodies could be used in radioimmunoassay to measure this peptide, but they did not react with corresponding fragments of procholecystokinin, porcine progastrin, or other human progastrin-derived peptides, notably heptadecapeptide gastrin (G17), and 34-residue gastrin (G34). Radioimmunoassay of human antral and duodenal extracts revealed a major peak of activity that corresponded to the native COOH-terminal fragment of progastrin, and occurred in approximately equimolar amounts with COOH-terminal G17 immunoreactivity. In addition, there was a minor peak of apparently higher molecular weight material. In some gastrinomas the latter material was the predominant immunoreactive form, and it occurred in higher molar concentrations than any other form of gastrin. Digestion of this material with trypsin liberated peptides that reacted with antibodies specific for the NH2-terminus of G34, and G17. On this basis the high molecular weight component was identified as a form of gastrin that extended from the COOH-terminus of the precursor to a point at least beyond the NH2-terminus of G34, and probably included the entire progastrin sequence. The results suggest differences in posttranslational processing pathways of progastrin in antrum, duodenum, and gastrinomas. They also indicate that the present experimental approach allows the identification of progastrin-like substances, which should open the way to studying the mechanisms of gastrin biosynthesis.

Identical biological effects of pancreatic glucagon and a purified moiety of canine gastric immunoreactive glucagon.

Because in the dog, the gastric fundus contains the largest amount of glucagon immunoreactivity (IRG), the IRG of mucosal scrapes of 105 canine stomachs was extracted by acid-ethanol and then precipitated by ether-ethanol. The IRG recovered was measured by antisera 30K, specific for glucagon and K-4023, which cross-reacts with glucagon-like immunoreactivity. Extracts of mucosa of stomach fundus were further purified by gel filtration on Bio-Gel P-30 in 3M acetic acid. One pooled fraction corresponding to marker pancreatic glucagon in its elution volume was then gel-filtered on Bio-Gel P-30 in 0.05 M NH(4)HCO(3) and yielded one IRG peak, which, however, showed three immunoreactive components on polyacrylamide disc gel electrophoresis in urea. In addition, antiserum K-4023 reacted more strongly with that peak than antiserum 30K indicating the presence of glucagon-like immunoreactivity in this fraction. Subsequent ion-exchange column chromatography on DEAE-Sephadex A-25 and then CM-Bio-Gel A allowed purification to a single protein band on disc gel electrophoresis reacting equally to both antisera 30K and K-4023. 1.5 mug of purified gastric glucagon was obtained and its biological effects were compared to those of pancreatic glucagon in isolated rat hepatocytes. When immuno-equivalent amounts (300-2,500 pg/ml) of either type of glucagon were used, the same biological responses with respect to glycogenolysis and gluconeogenesis as well as urea, lactate, and pyruvate production were observed. Liver cyclic AMP was also raised to the same extent by either one of these hormones. We conclude that this moiety of gastric IRG is apparently identical to pancreatic glucagon because (a) their molecular weights, elution properties in ion exchange chromatography, and their electrophoretic mobility are indistinguishable and (b) both hormones elicited identical biological effects in isolated rat hepatocytes.

Mutagens in extracts of human gastric mucosa.

Extracts of mucosa from 53 stomachs were tested for mutagenicity with the use of Salmonella typhimurium strains TA98 and TA100. Mutagenic activity was observed in nonneoplastic mucosa from seven stomachs with S. typhimurium strain TA98 without the S-. mix (a rat liver microsomal enzyme preparation) and in three stomachs with TA100 without S-. mix. All of the TA98 mutagenic samples were derived from stomachs showing evidence of intestinal metaplasia. The exception was an extract of corpus mucosa that was mutagenic with strain TA100. "Transnitrosoase" activity was found in 12 of 53 samples, all of which showed TA98 mutagenicity. Whether the presence of mutagens and transnitrosoase activity causes intestinal metaplasia or results from it is uncertain.

Enhanced expression of c-Ha-ras p21 in human stomach adenocarcinomas defined by immunoassays using monoclonal antibodies and in situ hybridization.

Using c-Ha-, c-Ki-, and c-N-ras-specific probes in a RNA-RNA hybridization assay we found enhanced expression of c-Ha-ras protooncogene in stomach adenocarcinomas relative to nonneoplastic epithelium, whereas little or no transcription of either c-Ki- or c-N-ras was detected. Enhanced levels of c-Ha-ras RNA expression were detected in all of the adenocarcinomas examined. Hybridization with c-Ha-ras was also detected in nonneoplastic gastric epithelium adjacent to carcinoma, although the labeling was less intense than that of carcinoma cells. More extensive analysis of the c-Ha-ras p21 expression was then carried out in formalin-fixed, paraffin-embedded tissue sections and extracts from surgically resected stomach tissues using monoclonal antibodies (MAbs) RAP-5 and Y13-259. The data obtained from the immunohistochemical studies were consistent with the results of in situ hybridization assay. Adenocarcinomas were much more reactive with MAb RAP-5 than benign and normal tissues, and the majority of carcinomas demonstrated increased expression of c-Ha-ras p21. Quantitative liquid competition radioimmunoassays using MAb Y13-259 also demonstrated significantly higher levels of c-Ha-ras p21 in extracts from stomach adenocarcinomas than those from normal mucosae. No strict correlation was found between ras p21 expression and the degree of tumor differentiation or histological type. Although advanced carcinomas generally demonstrated higher levels of ras p21 than early carcinomas, no correlation among advanced carcinomas and ras p21 levels was observed in relation to depth of tumor invasion to the muscularis propria, subserosa, or serosa. Benign lesions, in comparison, were much less reactive with MAb RAP-5 than carcinomas. Among the benign lesions tested, dysplastic lesions were more reactive than nondysplastic lesions. Normal stomach mucosa was generally nonreactive with the exception of parietal cells. Our results indicate that transformation of the stomach mucosa from benign to malignant phenotype is associated with an increase in c-Ha-ras p21 expression.

Plasma gastrin responses to bombesin and antral gastrin concentrations in patients with the intestinal type of gastric cancer.

Mucosal atrophy of the gastric antrum (type B atrophic gastritis) is generally accepted as predisposing to the development of the intestinal type of gastric cancer. Since bombesin stimulates gastrin release selectively from the antral mucosa, the response can be used as a marker for antral mucosal atrophy. In this study we have investigated bombesin-stimulated plasma gastrin responses in 21 patients with the intestinal type of gastric cancer and we have compared the results with 12 patients with the diffuse type of gastric cancer, 17 patients with benign gastric ulcer, and 30 dyspeptic patients without endoscopical or histological abnormalities. Gastrin concentrations were also measured in extracts of antral biopsies. Basal plasma gastrin concentrations were not significantly different. In contrast, patients with the intestinal type of gastric cancer had a significantly lower plasma gastrin response to bombesin than did the normal subjects (P less than 0.01) and patients with the diffuse type of gastric cancer (P less than 0.05), but the result was not significantly different from that of the gastric ulcer patients. The antral gastrin content of the patients with the intestinal type of gastric cancer was significantly lower than in controls (P less than 0.005), the patients with the diffuse type of gastric cancer (P less than 0.05), and those with gastric ulcer (P less than 0.05). It is concluded that patients with the intestinal type of gastric cancer have, in contrast to those with the diffuse type of gastric cancer, an abnormally low plasma gastrin response to bombesin. This low response is due to a reduced gastrin content of the antral mucosa.

Expression of growth factor and epidermal growth factor receptor encoded transcripts in human gastric tissues.

Expression of mRNA-encoding transforming growth factors alpha and beta (TGF alpha and beta), epidermal growth factor receptor (EGFR), and platelet-derived growth factors (PDGF) A and B chains was examined in 63 human gastric biopsies. Despite considerable individual variation, transcript levels were generally higher in 16 paired gastric tumors compared with surrounding epithelium. Marked increases were observed for the TGFs and c-sis, whereas EGFR mRNA was poorly expressed; there was no correlation with pathological staging of the cancers. In the nonneoplastic tissues, 14 had normal histology and 27 displayed superficial (SG) or atrophic gastritis (AG). Transcript levels greater than or equal to + were similar between these categories for all the growth factors, but were about 50% higher for EGFR in the tissues with gastritis. Concurrent expression of TGF alpha and EGFR (greater than or equal to + level) was more frequent in the paired tumors (38%) than in adjacent nonmalignant tissue (6%) and was seen in only one of 14 (7%) normal samples, in three of 19 (16%) of those with AG, and none of eight of those displaying SG. High levels of TGF beta and PDGFA mRNA were expressed in gastric ulcers, with little or no TGF alpha and EGFR transcripts; in contrast both TGFs and EGFR message were found in normal oesophagus. Stomach tissues are thus capable of synthesizing a variety of growth factors. These may be associated with nonneoplastic hyperplasia and/or malignant proliferation. Coexpression of TGF alpha/EGFR supports the possibility of an autocrine loop sustaining tumor growth which is different from the mechanisms responsible for normal cellular proliferation.

Promotion by bombesin of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effects of bombesin on the incidence, number, histological type, and depth of involvement of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in male Wistar rats. Rats received alternate-day s.c. administration of 20 or 40 micrograms/kg body weight of bombesin in depot form after p.o. treatment with the carcinogen for 25 weeks. Prolonged administration of bombesin at 40 micrograms/kg led to a significant increase in the incidence and number per rat of gastric cancers of the glandular stomach at Week 52. In rats that had received alternate-day injections of 20 micrograms/kg of bombesin, the number of gastric cancers per rat, but not the incidence of cancer, was significantly more than in untreated rats. However, bombesin at both dosages did not affect the histological appearance of the lesions or their depth of involvement. At Weeks 30 and 52, norepinephrine concentrations in the fundic and antral portion of the gastric walls and labeling indices in the antral and fundic mucosae were significantly higher in rats treated with bombesin at both dosages than in untreated rats. These findings indicate that bombesin enhances gastric carcinogenesis after administration of N-methyl-N'-nitro-N-nitrosoguanidine is stopped and that this effect may be related to its effects in increasing tissue norepinephrine concentrations in the stomach wall and increasing cell proliferation in the gastric mucosa.

Enhancement of experimental gastric carcinogenesis induced in spontaneously hypertensive rats by N-methyl-N'-nitro-N-nitrosoguanidine.

The incidence, number, and histological types of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine and the tissue norepinephrine concentration of the gastric wall were investigated in spontaneously hypertensive rats and in control Wistar Kyoto rats and Wistar rats. All rats were given drinking water containing 25 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine for 25 weeks. During Week 52, the incidence and number per rat of gastric cancers were significantly greater in spontaneously hypertensive rats than in Wistar Kyoto and Wistar rats. All tumors induced in the glandular stomach were adenocarcinomas, but no significant difference was found in the histological types of adenocarcinoma in the three strains of rats. At Weeks 15, 30, and 52, norepinephrine concentrations in the fundic and antral portion of the gastric walls and labeling indices in the antral and fundic mucosa were significantly higher in spontaneously hypertensive rats than in Wistar Kyoto and/or Wistar rats. These findings indicate that increased sympathetic nervous system activity enhances the development of gastric cancers, but immunological dysfunction in spontaneously hypertensive rats may contribute to the increased susceptibility to gastric cancer.

Molecular species of phosphatidylcholine from rat gastric mucosa.

Phosphatidylcholine of rat gastric mucosa were found to constitute about half of the total phospholipids. The composition of 20 molecular species accounting for approx. 90% of the total phosphatidylcholine was determined by specific enzymic hydrolyses and AgNO3 thin-layer and gas-liquid chromatography. Disaturated (dipalmitoyl) phosphatidylcholine made up about 31% of the total phosphatidylcholines. Other species which occurred in significant concentrations included 16:0/18:1, 18:0/18:1, 16:0/18:2, 18:0/18:2, 16:0/18:3, 18:0/18:3, 16:0/20:4, and 18:0/20:4. These results indicate that rat gastric mucosa is similar to lung in that both contain elevated amounts of dipalmitoyl phosphatidylcholine. Other similarities between these two tissues are discussed.

Common or distinct receptors for gastrin and cholecystokinin in gastric mucosa?

The differentiation between gastrin (HG) and cholecystokinin (CCK) receptors in gastric mucosa was examined on isolated parietal (F3) and non-parietal (F1) cells from rabbit fundic mucosa separated by elutriation. Direct binding assays on enriched cell populations were performed using 125I-labeled HG-17, 125I-labeled CCK-8 and 125I-labeled CCK-39 as probes. (1) On F1 cells, the dissociation constants (Kd) for the two labeled CCKs were nearly the same (62 pM for CCK-8 and 74 pM for CCK-39) but the binding capacity for CCK-8 was 2-times higher than for CCK-39. HG-17 also bound to this cell population, but its Kd value as about 2-times higher (110 pM) than that of CCK. The presence of two distinct classes of sites on F1 cells can be suggested from competition studies: one more specific for CCK, which bound CCK-8 and CCK-39 with the same affinity, and another class more specific for gastrin, which bound CCK-8 and HG-17 with the same affinity and CCK-39 with a low affinity. (2) On F3 cells, CCK-8 and HG-17 bound with similar affinities (Kd values 81 pM for CCK-8 and 87 pM for HG-17), but CCK-39 did not specifically bind to this cell population. The presence of a binding site more specific for HG than for CCK on F3 cells was confirmed by competition studies in which CCK-33 competed for binding with labeled HG-17 and labeled CCK-8 with a 50-times lower affinity than the other peptides.

The presence of blood group A-active glycolipids in cancer tissues from blood group O patients.

Glycolipids were isolated from human gastric cancer tissues and normal mucosae. Sulfogalactosylceramide, ganglioside and neutral glycolipid fractions were separated by DEAE-Sephadex and silica gel column chromatography. Sulfogalactosylceramide contents were higher in the cancer tissues than in the normal mucosae. Ganglioside contents showed considerable variations but in the cancer tissues in mole percentage of ganglioside GM3 was higher than in the normal mucosae. The cancer tissues contained more neutral glycolipids than normal tissues. Glycolipids of lacto-series, including fucolipids, were markedly increased in the cancer tissues. Blood group A-active glycolipids were found in the cancer tissues from two patients with blood group O but not found in the uninvolved tissue associated with the cancer tissue.

Glyceroglucolipids of the mucous barrier of dog stomach.

Distribution of glyceroglucolipids in the mucous barrier of dog stomach fundus, body and antrum was investigated. Surface mucus lining and preformed intracellular mucus were obtained by instillation of the ligated stomach compartments with 2 M NaCl. Lipids were extracted from the dialyzed and lyophilized samples, and the glycolipids were separated into neutral and acidic fractions. The glyceroglucolipids contained in each fraction were purified into individual components by thin-layer chromatography and were quantified. The content of glyceroglucolipids (mumol glucose/g protein) in the antral portion of the stomach was 2.1 times greater than that of the fundus and 3.4 times greater than that of the body. All three areas of the stomach contained neutral and sulfated glyceroglucolipids. However, the level of sulfated glycerolglucolipids (mumol glucose/g protein) was three times higher in the antrum as compared to the fundus and four times higher as compared to the body. The neutral and sulfated glyceroglucolipids were present in a molar ratio of 1.0 : 0.7 in the fundus, 1.0 : 1.1 in the body and 1.0 : 1.5 in the antrum.

Heterogeneity in gastrointestinal mucins.

Pig digestive tract mucins have often been used as model mucins for studying mucin structure, function and metabolism. In the present study pig gastric mucin and pig colonic mucin in the subunit form have been characterised and compared. Following Sepharose 4B or 2B-CL gel chromatography, the mucin eluant fractions were assayed colorimetrically by both the periodic acid-Schiff and the Alcian blue binding assays. Subunit colonic mucin eluted as a single unimodel peak that was easily detected by both assays. In contrast, subunit gastric mucin gave a peak primarily detected by periodic acid-Schiff that was overlapped by, but partially separated from, another peak primarily detected by Alcian blue. Subunit gastric mucin was separated into two periodic acid-Schiff staining spots when electrophoresed on cellulose acetate. Cetylpyridinium chloride (CPC) was able to precipitate only about half the subunit gastric mucin. The CPC-precipitable subunit gastric mucin corresponded to the faster running spot on electrophoresis, and the subunit gastric mucin in the CPC supernatant (which may have more than one subunit mucin type) to the slower spot(s). The former had a higher sulphate content and stained with Alcian blue. The latter had a lower sulphate content and showed very little Alcian blue reactivity. These results indicate that subunit pig gastric mucin is heterogeneous with respect to both size and charge. The differences between the types may be important in biological and physiochemical behaviour of gastric mucin. It seems likely that different laboratories may have worked on one or other of the pig gastric mucin types or a mixture, depending on the preparation method.

Isolation and partial characterization of rat gastric mucous glycoprotein.

Mucus glycoproteins from the rat stomach were characterized after their isolation from homogenates of the superficial gastric mucosa by equilibrium centrifugation in CsCl density gradients. Water-soluble as well as water-insoluble glycoproteins were studied. The latter were solubilized by 2-mercaptoethanol reduction of the homogenate. From both homogenate fractions the sames two glycoproteins 1 and 2 were purified, glycoprotein 1 being present in considerably higher amount than glycoprotein 2. Their respective buoyant densities in a CsCl gradient were 1.47--1.50 g/ml and 1.56--1.58 g/ml. The two glycoproteins expressed slight differences in gel electrophoresis and gel filtration. The results from column chromatographic comparisons between reduced and unreduced glycoproteins indicated strongly that both glycoproteins 1 and 2 were built from subunits kept together by S-S bonds. The s20,w values of the reduced glycoproteins 1 and 2 were 15.7 S and 11.6 S. Glycoprotein 1 contained 5% protein, 70% carbohydrate and 1--2% sulphate, whereas these percentages for glycoprotein 2 were 10% protein, 65% carbohydrate and 10% sulphate. The molar proportions of the main sugar components galactose, fucose, glucosamine and galactosamine were 4 :2 : 4 : 1 (glycoprotein 1) and 3 : 2 : 3 : 1 (glycoprotein 2). Blood-group activity A was expressed by glycoprotein 1, whereas glycoprotein 2 showed mainly blood-group activity Leb, some B activity and also some A activity, but to a lesser extent than glycoprotein 1.

Glycosphingolipids of guinea pig gastric mucosa.

Glycosphingolipids have beenn isolated from guinea pig gastric mucosa and their composition and content determined. The neutral glycospingolipids were found to consist of mono-, di-, tri- and pentaglycosylceramide. The acidic glycosphingolipids wee represented by galactosyl and lactosyl sulfatides, and GM4, GM3 and GD3 gangliosides. None of the analyzed glycolipids contained N-acetylglucosamine and fucose.

Characterization of gastric mucosal membranes. II. Lipid compositions of purified gastric mitochondria from pig, rabbit and frog.

1. The lipid composition of purified mitochondrial fractions from the fundic mucosa of pig, rabbit and frog were determined. 2. The total lipids expressed as mg lipid per 100 mg mitochondrial protein were approx. the same in pig and rabbit (13.4 and 15.5, respectively) and much higher than in frog (8.5). 3. The levels of phospholipids were about the same in pig and frog (approx. 61% of the total lipid) and lower than rabbit (78%). However, the levels of cholesterol were significantly different in the three species and constituted 22, 9 and 18.2% of the total lipids in pig, rabbit and frog mitochondria, respectively. 4. The glycolipid content in the mitochondrial lipids from pig, rabbit and frog were 7, 5.6 and 10.5%, respectively. 5. Cardiolipin contributed from 5.6 to 7.5% of the total phospholipids in the various species. Phosphatidylethanolamine and phosphatidylcholine together accounted for 80 90% of the total phospholipids in the various species; the contribution of phosphatidylcholine being always higher than that of phosphatidylethanolamine. Small but significant amounts of phosphatidylinositol were present in all species. 6. Generally, the predominant saturated fatty acid in the phospholipids was 16:0 from all species (except in phosphatidylethanolamine from pig and frog), and 18:1 and 18:2 were the predominant unsaturated fatty acids from all species. Sphingomyelin contained the highest amount of saturated fatty acids (over 80%) in both the species (pig and rabbit) studied.

Blood group A active difucosyl glycolipid from hog gastric mucosa.

A new difucosyl glycolipid exhibiting blood group A activity was isolated from water-soluble glycolipid fraction of hog gastric mucosa. The structure of this glycolipid was identified by partial acid hydrolysis, sequential degradation with specific glycosidases and methylation analysis, as: "see article".

[Purification of human gastric glycoprotein and a study of its carbohydrate components (author's transl)]. / Purification d'une glycoprotéine gastrique humaine et étude de ses composants glucidiques.

Mucus was extracted from human gastric mucosa by homogenization in distilled water. The crude extract was purified from plasma, salivary and tissue contaminants by different steps involving chromatography on Sepharose 6B, Sepharose 2B and immunosorbents. The native glycoprotein so prepared was found to be immunologically pure; it migrated as a single band in acrylamide agarose gel electrophoresis at pH 8.6 and 3.5. The molecule exhibits blood group acitivity, contains 0.71 per cent sialic acid but no sulphate. The carbohydrate moiety contains glucosamine, galactosamine, fucose, galatose and mannose in the molar proportions 3: 1.95: 2.92:4.53:0.05.

Distribution and molecular forms of peptides containing somatostatin immunodeterminants in extracts from the entire gastrointestinal tract of man and pig.

Specimens from human porcine mucosal and muscular tissue from the entire gastrointestinal tract were extracted in acid ethanol, subjected to chromatography and analysed for somatostatin-like immunoreactivity by region-specific radioimmunoassays. The concentration of somatostatin-like immunoreactivity from man and pig ranged from 1.13 +/- 0.37 to 101.15 +/- 33.93 pmol/g wet weight, and from 7.64 to 159.48 +/- 23.79 pmol/g wet weight, respectively. In both species the highest concentrations were found in the jejunum. The immunoreactivity in intestinal mucosal extracts was distributed among four major peaks, two of which were identified by HPLC as somatostatin 1-28 and somatostatin 1-14, respectively. A peak of approx. 10 kDa was resolved by ion exchange plus HPLC into three components, two containing at least part of the somatostatin 1-14 sequence as well as (part of) the somatostatin 1-28(1-14) sequence (but differing in charge), the third containing only the 1-28(1-14) sequence. These peptides probably represent uncleaved and partially cleaved prosomatostatin. The fourth component to be identified by gel filtration was slightly larger than somatostatin 1-14. Extracts from the antrum, the pancreas and from muscular tissues contained almost exclusively somatostatin 1-14, and very little somatostatin 1-28, indicating that the somatostatin precursor is processed differently at these sites. Furthermore, extracts of porcine gastric antrum, analysed for somatostatin 1-28(1-14) immunoreactivity, showed two immunoreactive forms in the mucosa and three major forms in the muscular layers.

Characterization of peptic inhibitory activity associated with sulfated glycoprotein isolated from gastric mucosa.

Sulfated glycoproteins were extracted and purified from porcine stomach mucous scraping. Four sulfated glycoprotein fractions were separated and subsequently purified. These compounds always accompanied the apparent peptic inhibitory activity and consisted of 15-18% (w/w) protein. The carbohydrate portions contained an equimolar ratio of galactose and hexosamine (mainly glucosamine), together with lesser amounts of fucose and sialic acid. The sulfate content of the above fractions was 2-9% (w/w) of the total sulfated glycoprotein. The mode of inhibition of the sulfated glycoproteins to peptic activity was investigated and suggested that there was binding of the sulfated glycoproteins to the substrate of pepsin, making the substrate resistant to peptic activity. The sulfated glycoproteins neither bound pepsin at pH 1.8 nor inhibited the hydrolysis of a synthetic dipeptide substrate of pepsin. Desulfation of the sulfated glycoproteins resulted in the loss of both the inhibitory activity and the precipitate formation. The precipitation curve for sulfated glycoprotein and porcine serum albumin showed that both bound in varying proportions and suggests that both components are multivalent in this precipitate formation.