Xenopsin immunoreactivity in antral G-cells may reside in the N-terminus of gastrin 17

Summary The nature of xenopsin immunoreactivity in mammalian antral G-cells has been reassessed. Xenopsin immunostaining was most intense in human antral G-cells, present in those of the dog and pig and not detected in guinea pig or rat tissues. Rigorous specificity controls for ionic binding of immunoglobulins to antral G-cell granules indicated that this mechanism was not responsible for xenopsin immunostaining. Preincubation of the xenopsin antiserum with xenopsin, human gastrin 1–13 and gastrin 2–17 completely abolished immunostaining at similar molar concentrations. Gastrin 34 was ineffective at much higher concentrations. These results infer that xenopsin-immunoreactivity in antral G-cells resides in the N-terminal region of gastrin 17. Examination of the primary structures of xenopsin and the N-terminal regions of some mammalian gastrins reveals a hitherto unrecognized homology.     

Co-localization of xenopsin and gastrin immunoreactivity in gastric antral G-cells

Studies indicating evidence for the presence of the amphibian octapeptide xenopsin in gastric mucosa of mammals prompted us to investigate the cellular localization of this peptide. Using the peroxidase-antiperoxidase method and a specific antiserum to xenopsin (Xen-7) on paraffin and adjacent semithin sections of gastric antral mucosa from man, dog, and Tupaia belangeri, we found numerous epithelial cells showing a specific positive immunoreaction. These cells were of typical pyramidal shape and could be classified as of the "open" type. Cell quantification in serial sections processed for xenopsin and gastrin immunoreactivity, respectively, revealed an identical number of cells per section and an identical distribution of these cells in the middle zone of the antral mucosa. Furthermore, adjacent semithin sections demonstrated the colocalization of xenopsin and gastrin immunoreactivity within the same G-cell. The xenopsin antiserum could be completely absorbed with synthetic xenopsin but not with gastrin. Preabsorption tests with neurotensin, a xenopsin related peptide, or with somatostatin, glucagon, and enkephalins gave no evidence for crossreactivity of the xenopsin antiserum with these peptides. It is concluded that gastric antral G-cells in addition to gastrin also contain the amphibian peptide xenopsin.     

Co-localization of xenopsin and gastrin immunoreactivity in gastric antral G-cells

Summary Studies indicating evidence for the presence of the amphibian octapeptide xenopsin in gastric mucosa of mammals prompted us to investigate the cellular localization of this peptide. Using the peroxidase-antiperoxidase method and a specific antiserum to xenopsin (Xen-7) on paraffin and adjacent semithin sections of gastric antral mucosa from man, dog, and Tupaia belangeri, we found numerous epithelial cells showing a specific positive immunoreaction. These cells were of a typical pyramidal shape and could be classified as of the open type. Cell quantification in serial sections processed for xenopsin and gastrin immunoreactivity, respectively, revealed an identical number of cells per section and an identical distribution of these cells in the middle zone of the antral mucosa. Furthermore, adjacent semithin sections demonstrated the colocalization of xenopsin and gastrin immunoreactivity within the same G-cell. The xenopsin antiserum could be completely absorbed with synthetic xenopsin but not with gastrin. Preabsorption tests with neurotensin, a xenopsin related peptide, or with somatostatin, glucagon, and enkephalins gave no evidence for crossreactivity of the xenopsin antiserum with these peptides.It is concluded that gastric antral G-cells in addition to gastrin also contain the amphibian peptide xenopsin.     

Antral G-cell in gastrin and gastrin-cholecystokinin knockout animals

The antral hormone gastrin is the key regulator of gastric acid secretion, mucosal growth and differentiation. Gastrin is synthesized in the endocrine G-cells in the antroduodenal mucosa. We have now examined the way in which the loss of gastrin alone or gastrin plus cholecystokinin (CCK) affects the antral G-cell. Immunohistochemistry, radioimmunoassay and quantitative real-time polymerase chain reaction techniques were employed to examine the expression of genes belonging to the G-cell secretory pathway in gastrin and gastrin-CCK knockout mice. Transmission electron microscopy was used to examine the ultrastructure of the G-cells. The number of G-cells increased but the secretory granules were few and abnormally small in the G-cells of both mouse models compared with wildtypes. Thus, gastrin is not necessary for the formation of G-cells as such but the lack of gastrin reduces the number and size of their secretory granules suggesting that gastrin is vital for the formation and/or maintenance of secretory granules in G-cells. This work was supported by the Novo Nordic Foundation (L.F.-H.) and Swedish Research Council (grant no. 4499; F.S. and N.W.).     

Interleukin-1beta up-regulates RGS4 through the canonical IKK2/IkappaBalpha/NF-kappaB pathway in rabbit colonic smooth muscle

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and alpha-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg(36)Arg(37) and Arg(73)Arg(74) sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, alpha-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys(53)Lys(54)), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.     

Occurrence of met-enkephalin,met-enkephalin-Arg6-Phe7 and met-enkephalin-Arg6-Gly7-Leu8 in gastrin cells of hog antral mucosa

Summary Region-specific antisera to three enkephalins: met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸, together with four region specific antisera to progastrin: C-terminal G17 specific, N-terminal G34 specific, cryptic peptides A- and B-specific, were used in immunohistochemical studies of hog antral mucosa. A sub-population (6–10%) of the gastrin-containing endocrine cells (G-cells) was found to react with antisera to met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸. About 30% of all the enkephalin-containing cells were identified as G-cells. The results indicate that a fraction of G-cells produces both enkephalin-like peptides and gastrin.     

The G-cells in the dog: a light and electron microscope immunocytochemical study

Summary An immunohistochemical study has been performed to analyse the distribution of gastrin cells in the gastrointestinal tract of the dog. This study revealed that G-cells immunoreactive for gastrin were almost exclusively present in the pyloric antral mucosa, mainly in the middle third of the pyloric mucosa. The calculated number of G-cells per surface unit area was 8.5×10³–1.2×10⁴ cells cm^–2. Some gastrin-immunopositive cells were found in the first 10 mm of the proximal duodenum, mainly in the villous region. The fundic area of the dog stomach, the oesophagus, small intestine, caecum, colon, rectum, salivary glands, liver and pancreas were all immunonegative for gastrin. At the ultrastructural level, three different types of granules (150–400 nm) were evident in G-cells: electron-dense, electron-lucent and intermediate forms. Most of them were located in the subnuclear region of the cell. The effect of fixation of the antral mucosa at different pH levels was studied. In samples fixed with acid solutions, most of the G-cell granules were of the electron-dense type and were stronly immunopositive for gastrin. Fixation of samples at a basic pH resulted in most of the gastrin granules losing their contents into the cytoplasm, and the positive reaction to gastrin was then located in the cytoplasm and at the periphery of the electron-lucent granules.     

Identification and characterization of glycine-extended post-translational processing intermediates of progastrin in porcine stomach

We developed a radioimmunoassay specific for glycine-extended progastrin processing intermediates (G-Gly) using antisera generated against the synthetic peptide Tyr-Gly-Trp-Met-Asp-Phe-Gly. Distribution of immunoreactivity in the porcine gastrointestinal tract obtained with this antibody paralleled that of gastrin with the mucosa containing the highest quantity, 116 +/- 22 pmol/g, wet weight (mean +/- S.E., n = 5), or roughly 4% of gastrin concentration. This immunoreactivity was localized specifically to antral mucosal G-cells by immunohistochemistry. On Sephadex G-50 column chromatography of porcine antral mucosal extracts glycine-extended progastrin processing intermediates were separated into three principal molecular forms, each corresponding to known molecular forms of gastrin, component I, tetratriacontagastrin (G34) and heptadecagastrin (G17). Following purification by antibody-coupled affinity chromatography, one molecular form corresponding to G17 in size was shown to have an amino terminus identical to that of G17. Another molecular form corresponding to G34 in size could be converted to the molecular form corresponding to G17 by tryptic digestion. Our findings indicate that glycine-extended progastrin processing intermediates may serve as immediate precursors for each molecular form of gastrin, thus suggesting an alternative pathway for gastrin biosynthesis more complex than that previously conceived.     

The antrat gastrin-producing G-cell: Biochemical and ultrastructural responses to feeding

Summary The effect of feeding on serum and antral immunoreactive gastrin (IRG) concentrations and on the ultrastructural appearance of antral G-cell granules has been examined. Serum and tissue IRG concentrations were dependent upon the length of time (12 or 48 h) the rats had been fasted before receiving food; IRG release was biphasic; the first peak was more pronounced in rats fasted 12h. Antral tissue IRG content increased significantly postprandially. An initial depletion of antral IRG was seen in rats fasted 48 h. Examination of the subcellular distribution of antral IRG revealed more of the 5–15 min postprandal total IRG in the cytoplasm and less in the secretory granules.Ultrastructurally, G-cells from fasting rats contained mainly electron-dense granules. Five minutes postprandially numerous electron-lucent granules were observed. More electron dense granules were apparent 60 and 120 min postprandially. Fasting rats had the highest G-cell granule density index; a significantly lower index was observed 5 min postprandially. Indices at 60 and 120 min postprandially increased but were still lower than the fasting index. These studies indicate that gastrin biosynthesis is necessary for food stimulated gastrin release and that the electron density of the G-cells' granules is not an accurate reflection of the G-cell gastrin content.The authors thank Elisabeth Bothe, Heidi Dörler and Heide Karl for technical assistance and the Deutsche Forschungsgemeinschaft (Bonn-Bad Godesberg, Grant Cr 20/7), the Atkinson Charitable Foundation and the Canadian MRC for financial support     

Expression and post-translational processing of gastrin in heterologous endocrine cells

The biosynthesis of gastrin involves a complex series of post-translational processing reactions that result in the formation of a biologically active secretory product. To study the mechanisms for two specific reactions in gastrin processing, namely dibasic cleavage and amidation, we infected AtT-20, GH3, and Rin5-f cells with the retroviral expression vector, pZip-NeoSV(X), containing human gastrin cDNA. We detected gastrin and its glycine extended post-translational processing intermediates (G-gly) in the media and cell extracts of successfully infected cells. Characterization of the molecular forms of gastrin in these cell lines revealed that GH3 and Rin5-f processed gastrin in a manner similar to antral G-cells but the cleavage of the Lys74-Lys75 bond that converts G34 to G17 appeared to be suppressed in AtT-20 cells. Even after conversion of this site to Arg74-Arg75 via site-directed mutagenesis, the At-20 cells synthesized G34 predominantly. All of the infected cells amidated gastrin but the gastrin/G-gly ratio, a reflection of amidation within the cells, was enhanced in GH3 and Rin5-f cells but diminished in AtT-20 cells upon treatment with dexamethasone (10(-4) M) for 3 days. The dibasic cleavage of gastrin was uneffected by dexamethasone. Our data suggest that the activities of post-translational processing reactions responsible for the synthesis of biologically active gastrin exhibit considerable tissue and substrate specificity.     

Effects of omeprazole on the number of immunoreactive gastrin- and somatostatin-cells in the rat gastric mucosa.

The effects of omeprazole--an inhibitor of gastric acid secretion--on gastrin (G)- and somatostatin (D)-cell density in the gastric antral mucosa epithelium in rats were examined, following a 5-day treatment. It was found that omeprazole increased the density of G-cells, whereas it decreased the density of D-cells. That effect was probably independent of hypergastrinaemia, since it could not be blocked by a simultaneous treatment with proglumide--a gastrin receptor blocker. It is concluded that the observed phenomenon is a direct result of a lower gastric acidity, as a consequence of omeprazole treatment.     

Cellular origins of different forms of gastrin. The specific immunocytochemical localization of related peptides.

We have localized the antigenic determinants for the main forms of gastrin (big gastrin, G34, and little gastrin, G17) in hog antral mucosa using sequence specific antibodies and an indirect immunofluorescence technique. Populations of monospecific antibodies were obtained after affinity immunoadsorption to remove populations of unwanted specificity. The specificity of the purified antisera was established by direct binding of 125I labeled peptides to antisera at the same dilutions as those used in immunocytochemistry. The results indicate that in hog antral mucosa there is a single population of cells with the antigenic determinants of the C-terminal region of G17 and G34, the N-terminal region of G17, the N-terminal region of G34, and the intact G17 molecule. In duodenum there are cells with only C-terminal reactivity; since gastrin and CCK share a common C-terminal sequence it is concluded that this cell type contains CCK-like peptides rather than gastrin.     

Quantitative electron microscopic studies on the kinetics of secretory granules in G-cells

Summary Ultrastructural studies of secretory granules of rat antral G-cells and measurement of serum gastrin level were performed under the condition of fasting and administration of alkaline solution into the stomach. On electron micrographs, no qualitative difference was observed among those experimental groups. However, morphometrical analysis revealed significant quantitative differences. The population density of secretory granules of the rats treated once with alkali first increased and then decreased reaching that of the fasted group, while that of the repeatedly treated group remained nearly equal to the maximum value. The average sectioned surface area of secretory granules tended to decrease for 1.5h after the stimulation but the difference was not significant among those groups.From the results obtained at present, responding to chemical stimulation such as pH changes in the antrum, it seems probable that not only exocytosis but also migration of secretory granules from supra- and/or para-nuclear portion to the basal portion of the cell occurs rapidly in G-cells and that both these processes are inhibited immediately by antral acidification. Moreover, the present results apparently indicate that under the condition of no antral acidification G-cells have a capacity of secreting gastrin for a fairly long time, such as 4–8 h, responding to adequate stimulus. These findings are strongly suggestive of the existence of a capacious pool of granules in the supra- and/or para-nuclear cytoplasm or of fairly speedy production of secretory granules in the Golgi area.The author wishes to express thanks to Prof. R. Furihata, Department of Surgery, and Prof. T. Nagata, Department of Anatomy, Shinshu University School of Medicine, for their constant interest and guidance, and to Dr. F. Iida, Department of Surgery, who has followed the course of this work throughout     

Diminished prohormone convertase 3 expression (PC1/PC3) inhibits progastrin post-translational processing

Gastrin is initially synthesized as a large precursor that requires endoproteolytic cleavage by a prohormone convertase (PC) for bioactivation. Gastric antral G-cells process progastrin at Arg(94)Arg(95) and Lys(74)Lys(75) residues generating gastrin heptadecapeptide (G17-NH(2)). Conversely, duodenal G-cells process progastrin to gastrin tetratriacontapeptide (G34-NH(2)) with little processing at Lys(74)Lys(75). Both tissues express PC1/PC3 and PC2. Previously, we demonstrated that heterologous expression of progastrin in an endocrine cell line that expresses PC1/PC3 and little PC2 (AtT-20) resulted in the formation of G34-NH(2). To confirm that PC1/PC3 was responsible for progastrin processing in AtT-20 cells and capable of processing progastrin in vivo we coexpressed either human wild-type (Lys(74)Lys(75)) or mutant (Arg(74)Arg(75), Lys(74)Arg(75), and Arg(74)Lys(75)) progastrins in AtT-20 cells with two different antisense PC1/PC3 constructs. Coexpression of either antisense construct resulted in a consistent decrease in G34-NH(2) formation. Gastrin mRNA expression and progastrin synthesis were equivalent in each cell line. Although mutation of the Lys(74)Lys(75) site within G34-NH(2) to Lys(74)Arg(75) resulted in the production of primarily G17-NH(2) rather than G34-NH(2), inhibition of PC1/PC3 did not significantly inhibit processing at the Lys(74)Arg(75) site. We conclude that PC1/PC3 is a progastrin processing enzyme, suggesting a role for PC1/PC3 progastrin processing in G-cells.     

A novel gastrin-processing pathway in mammalian antrum

An antiserum, L221, has been developed that is specific for the C-terminal region of the N-terminal tridecapeptide (i.e., 1-13) fragment of the acid-stimulating hormone, G17. In contrast to N-terminal G17 antisera previously used to estimate 1-13 G17, L221 does not cross-react with other N-terminal gastrin fragments or with C-terminal extensions of G17. Using L221 in conjunction with conventional gastrin antisera, and reversed-phase HPLC, it has been possible to identify in addition to 1-13 G17 a further, formerly unrecognised gastrin fragment, 1-11 G17, in stomach extracts. The production of 1-13 G17, 1-11 G17 and other gastrin forms such as the biologically active hexapeptide G6 which is known to occur naturally cannot be explained by tryptic cleavage of progastrin. Instead, their biosynthesis could be explained by the actions of an enzyme with an endopeptidase 24.11-like specificity. In porcine antrum, unsulphated and sulphated G17 are present in similar amounts, but unsulphated 1-13 G17 was about twice as abundant as sulphate 1-13 G17. This is consistent with previous in vitro findings that endopeptidase 24.11 has a higher affinity for the Ala-11-Tyr-12 and Gly-13-Trp-14 bonds in unsulphated G17, than in sulphated G17. The results suggest a novel albeit minor, processing pathway for gastrin biosynthesis in pig antrum involving an enzyme resembling endopeptidase 24.11.     

Identification by specific radioimmunoassay of two novel peptides derived from the C-terminus of porcine preprogastrin

A radioimmunoassay has been developed using antibodies to a synthetic analogue of the C-terminal hexapeptide sequence of the porcine gastrin precursor. Boiling water extracts of porcine antral mucosa contained immunoreactive material that diluted in parallel with standard peptide. Concentrations of immunoreactivity were 5.5 +/- 0.8 nmol X g-1 (mean +/- S.E.M.) in antral mucosa and were closely similar to those of C-terminal heptadecapeptide gastrin immunoreactivity (5.0 +/- 0.6 nmol X g-1). Approximately 30-fold lower concentrations were found in porcine duodenum. A similar distribution was found in ferret, but human, rat and chicken antrum did not contain significant quantities of immunoreactivity. Gel filtration of porcine antral extracts on Sephadex G-50 revealed a single peak of immunoreactivity eluting in a similar position to G17, but on anion-exchange chromatography two peaks of immunoreactive material were separated. These also differed in their retention time on reverse phase HPLC. Both peptides are probably derived by tryptic cleavage at the C-terminus of porcine preprogastrin. No evidence was found to suggest that there are significant quantities of unprocessed preprogastrin in hog antral mucosa. The precise chemical difference between the two immunoreactive peptides identified here remains to be established; together, however, they provide specific markers for progastrin synthesis.     

Sequences of gastrins purified from a single antrum of dog and of goat

Heptadecapeptide gastrins (G17) have been purified and sequenced from a variety of species. However, progastrin (G34) sequences have been determined only for pig and human from purified peptides and for rat from cDNA. Since G34 in most species accounts for only approximately 5% of total antral gastrin, micropurification techniques must be employed to avoid the need for large quantities of antral tissue. Efficient purification methodology yielded 1.5 and 1.3 nmol of G34 from the antrum of a single goat and of a single dog, respectively. The N-terminal pyroglutamyl residues were enzymatically removed and the peptides were sequenced through to the proximity of their COOH-termini. The COOH-terminal sequences of goat and dog G34 were confirmed by sequencing the corresponding deblocked G17 from each animal. The previously published dog G17 sequence was shown to be incorrect. The sequences for dog and goat G34 are: Dog less than ELGLQGPPQLVADLSKKQGPWMEEEEAAYGWMDF# Goat less than ELGLQDPPHMVADLSKKQGPWVEEEEAAYGWMDF# Dog and goat gastrins differ in 3 sites in the 17 amino acid NH2-terminus and only a single site in G17 (the sites of differences are underlined). The ratio for sulfated to non-sulfated antral G17 is 9:1 for the goat and 1:9 for the dog.     

Progastrin maturation during ontogenesis. Accumulation of glycine-extended gastrins in rat antrum at weaning.

The post-translational maturation of antral progastrin was studied in the developing rat. While N-terminal proteolysis remained unchanged and tyrosine O-sulphation varied only slightly during ontogenesis, major changes were observed in the degree of alpha-carboxyamidation. In the third week of life the immediate precursor of amidated gastrin, glycine-extended gastrin, accumulated, and at weaning (day 21) the concentrations exceeded those of amidated gastrin. Our results confirm that weaning is accompanied by an increased synthesis of gastrin and imply that alpha-carboxyamidation is the rate-limiting step during the biosynthetic maturation of gastrin.     

Gastrin release in obese Zucker rats

In this study, gastrin release in the obese Zucker rat was investigated by in vivo and in vitro experiments. Obese rats exhibited normal plasma gastrin levels at 3 weeks (preobese), were moderately hypergastrinemic at 3 months and severely hypergastrinemic at 5 months, compared to lean littermates. Following oral peptone, plasma gastrin levels doubled in both lean and obese rats. Basal and vagally stimulated gastrin release from perfused stomachs was greater in obese compared to lean rats and atropine had no effect on basal gastrin release in either group. Basal somatostatin release from the perfused stomach was found not to differ in both groups of animals. Morphological studies revealed an increase in the number of gastrin-containing G-cells in adult obese rats compared to lean littermates, but not in 3-week-old pups compared to lean littermates, indicating a strong correlation between cell number and plasma gastrin levels. These data indicate that the obese Zucker rat exhibits fasting hypergastrinemia in vivo, a condition which appears after weaning and increases in severity with age. Gastrin hypersecretion persists from the vascularly perfused stomach preparation. The basal hypergastrinemia of the obese Zucker rat is independent of a hyperactive postganglionic cholinergic drive but is associated with and probably causally related to an increase in antral G-cell numbers.