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.     

Identification of xenin, a xenopsin-related peptide, in the human gastric mucosa and its effect on exocrine pancreatic secretion.

One of the peptides previously discovered in amphibians is the octapeptide xenopsin. As immunohistochemistry has also indicated the presence of xenopsin immunoreactivity in man, we extracted in the present investigation xenopsin-immunoreactive material from human gastric mucosa and purified it to homogeneity with several high performance liquid chromatography (HPLC) reverse phase and ion exchange chromatographic steps. The eluates were monitored with a radioimmunoassay for amphibian xenopsin. Determination of the amino acid sequence revealed a 25-amino acid peptide having 6 C-terminal amino acids in common with amphibian xenopsin. The sequence of this peptide, termed xenin 25, is M-L-T-K-F-E-T-K-S-A-R-V-K-G-L-S-F-H-P-K-R-P-W-I-L. The peptide was custom-synthesized. Mass spectrometry of the synthetic and the extracted peptide revealed identical molecular mass. Purification of 250 ml of human postprandial plasma with Sep-Pak C18 cartridges, reverse phase HPLC, and ion exchange chromatography demonstrated circulating xenin immunoreactivity at a retention time identical to xenin 25. The amount of xenin immunoreactivity at the position of xenin 25 on C18-HPLC increased significantly after a meal. A radioimmunoassay utilizing antibodies to xenin 25 and a 125I-labeled analogue of xenin 25 was used to measure immunoreactive xenin in the plasma of 10 volunteers. There was a significant rise of xenin immunoreactivity in the plasma after a meal. Intravenous infusion of the synthetic peptide in dogs stimulated exocrine pancreatic secretion beginning at a dose of 4 pmol/kg/min. The maximal effect was seen with 64 pmol/kg/min. We have detected, therefore, a new peptide, xenin 25, in human gastric mucosa; we have provided evidence for the presence of this peptide in the human circulation, and have shown a rise of plasma xenin concentrations after a meal. This peptide stimulates exocrine pancreatic secretion. Its physiologic role deserves further investigation.     

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.     

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

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.     

Distribution of ACTH-like immunoreactivity in rat brain and gastrointestinal tract

Summary Immunocytochemistry reveals ACTH-like immunoreactivity to reside not only in the pituitary but also in central nerves and in antral gastrin cells. In all probability, the central nerves store a peptide identical with or closely resembling true ACTH. Their pattern of distribution is, in some regions, similar to that of enkephalin-immunoreactive nerves. The antiserum demonstrating ACTH-like immunoreactivity in central nerves and in antral gastrin cells is directed towards the COOH-terminal part of the hormone. A peptide corresponding to this part, the corticotrophin-like intermediate peptide (CLIP) is manufactured by the pars intermedia of the pituitary. CLIP is devoid of adrenocortical activity but has recently been shown to possess insulin-releasing activity. The occurrence of CLIP-like peptides in antral gastrin cells may indicate a role for such peptides in the gastrointestinal regulation of insulin release. The simultaneous occurrence of enkephalin-like and ACTH-like immunoreactivity in the antral gastrin cells is of particular interest since a large precursor molecule, containing both the enkephalin and the ACTH sequence has recently been identified.     

Distribution of ACTH-like immunoreactivity in rat brain and gastrointestinal tract.

Immunocytochemistry reveals ACTH-like immunoreactivity to reside not only in the pituitary but also in central nerves and in central nerves and in antral gastrin cells. In all probability, the central nerves store a peptide identical with or closely resembling true ACTH. Their pattern of distribution is, in some regions, similar to that of enkephalin-immunoreactive nerves. The antiserum demonstrating ACTH-like immunoreactivity in central nerves and in antral gastrin cells is directed towards the COOH-terminal part of the hormone. A peptide corresponding to this part, the corticotrophin-like intermediate peptide (CLIP) is manufactured by the pars intermedia of the pituitary. CLIP is devoid of adrenocortical activity, but has recently been shown to possess insulin-releasing activity. The occurrence of CLIP-like peptides in antral gastrin cells may indicate a role for such peptides in the gastrointestinal regulation of insulin release. The simultaneous occurrence of enkephalin-like and ACTH-like immunoreactivity in the antral gastrin cells is of particular interest since a large precursor molecule, containing both the enkephalin and the ACTH sequence has recently been identified.     

Functional and anatomical relationships between antral gastrin cells and gastrin-releasing peptide neurons

The present studies were directed to examine the effect of gastrin-releasing peptide (GRP) on beta-adrenergic stimulated gastrin release by cultured rat antral mucosa and to assess the anatomical relationship between gastrin cells and GRP nerves in rat and human antrum. Peptide-containing cells were identified by application of an avidin-biotin-peroxidase immunocytochemical double staining method utilizing antibodies to GRP and gastrin prepared in rabbits. Rat antral mucosa was cultured for 60 min and gastrin released into the culture medium was measured by radioimmunoassay. Inclusion of antibodies to GRP in culture medium did not affect carbachol-stimulated gastrin release, whereas isoproterenol-stimulated gastrin release into the medium was inhibited significantly by addition of GRP antiserum to the culture medium. GRP-containing neurons and axonal fibers were stained immunocytochemically with diaminobenzidine (reddish-brown specific staining) and were located in the lamina propria adjacent to and surrounding the main lobules of antral glands. After double staining utilizing 4-Cl-1-Naphthol as substrate, blue stained gastrin-containing cells were identified in the middle and deeper regions of antral glands in close proximity to GRP neuronal elements. These studies suggest that beta-adrenergic, but not cholinergic, stimulation of gastrin release is mediated, at least in part, through GRP. They also demonstrate intimate anatomical, as well as functional, relationships between gastrin cells and GRP-containing neurons.     

Characterisation of xenopsin immunoreactivity derived from pepsinised human skin and possible mechanism of in vivo generation

Human skin was subjected to a variety of extraction and enzymatic digestion procedures. Extracts and digests were subjected to neurotensin and xenopsin radioimmunoassays of known specificity. No neurotensin immunoreactivity was detected in any preparation with any region-specific antiserum. C-terminal xenopsin immunoreactivity was present in skin homogenates following incubation with both soluble and solid-phase pepsin and in those incubated with a leucocyte lysate or purified cathepsin D. The generation of xenopsin immunoreactivity was dependent on low pH and enzymes of pepsin-type specificity acting on a tissue precursor of approximately 30 kDa. Gel permeation chromatography of skin-derived xenopsin immunoreactivity identified a single molecular species larger than synthetic xenopsin which was resolved into two components by reverse-phase HPLC with retention times similar to synthetic xenopsin and kinetensin. Human skin thus contains a high-molecular-weight precursor protein and an endogenous acid protease, cathepsin D, capable of generating a peptide of similar size and C-terminal structure to amphibian xenopsin under acidic conditions such as might occur locally in wounds or at sites of inflammation.     

FMRF-amide immunoreactivity in the mammalian gastroenteropancreatic neuroendocrine system

The presence of FMRF-amide, a cardioactive tetrapeptide, was studied by immunocytochemistry in human and rat gastric antrum and pancreas, and in the ovine, bovine, canine and rabbit pancreas. In human and rat gastric antrum, numerous cells contained FMRF-amide immunoreactive material. By staining of serial sections and by double staining, colocalization of immunoreactivity for gastrin and FMRF-amide was observed in part of the gastrin cells. In the pancreas of these and the other species, immunoreactivity for FMRF-amide was located both in acinar and islet endocrine cells. Colocalization of FMRF-amide and pancreatic polypeptide was found in a proportion of pancreatic polypeptide cells in the pancreas. FMRF-amide immunoreactivity never colocalized with the other neurohormonal peptides which occur in the gastric antrum and the pancreas. Our observations show that neuroendocrine cells occur in the gastric antrum and pancreas which are exclusively immunoreactive or gastrin and for pancreatic polypeptide respectively. In addition cells occur which show immunoreactivity for FMRF-amide as well as for gastrin in the gastric antrum and with antiserum to FMRF-amide as well as for pancreatic polypeptide in the pancreas. It is concluded that FMRF-amide antibodies probably recognize a substance in G and PP cells which is not identical but may be structurally related to gastrin and pancreatic polypeptide.     

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.     

Histamine and histidine decarboxylase are hallmark features of ECL cells but not G cells in rat stomach

The oxyntic mucosa of the rat stomach is rich in ECL cells which produce and secrete histamine in response to gastrin. Histamine and the histamine-forming enzyme histidine decarboxylase (HDC) have been claimed to occur also in the gastrin-secreting G cells in the antrum. In the present study, we used a panel of five HDC antisera and one histamine antiserum to investigate whether histamine and HDC are exclusive to the ECL cells. By immunocytochemistry, we could show that the ECL cells were stained with the histamine antiserum and all five HDC antisera. The G cells, however, were not stained with the histamine antiserum, but with three of the five HDC antisera. Thus, histamine and HDC coexist in the ECL cells (oxyntic mucosa) but not in G cells (antral mucosa). Western blot analysis revealed a typical pattern of HDC-immunoreactive bands (74, 63 and 54 kDa) in oxyntic mucosa extracts with all five antisera. In antral extracts, immunoreactive bands were detected with three of the five HDC antisera (same as above); the pattern of immunoreactivity differed from that in oxyntic mucosa. Food intake of fasted rats or treatment with the proton pump inhibitor omeprazole raised the HDC activity and the HDC protein content of the oxyntic mucosa but not of the antral mucosa; the HDC activity in the antrum was barely detectable. We suggest that the HDC-like immunoreactivity in the antrum represents a cross-reaction with non-HDC proteins and conclude that histamine and HDC are hallmark features of ECL cells but not of G cells.     

Localization by immunoelectron microscopy of somatostatin-containing cells in the gastric mucosa of a teleost fish, Perca fluviatilis

Somatostatin-immunoreactive cells were localized on semithin and ultrathin sections of Epon-embedded samples of perch gastric mucosa, classically fixed with aldehydes and osmium tetroxide. On semithin sections, somatostatin cells were identified by using the immunoperoxidase method. The ultrastructural localization of somatostatin immunoreactivity was achieved using the colloidal gold method. Cells showing somatostatin immunoreactivity are found to be scattered among the surface mucous cells and the mucous neck cells. Somatostatin appears to be localized in cytoplasmic granules. Somatostatin-containing cells are identified as the type I cells which were described in a previous ultrastructural study. The present report also points out that tissue samples which have been classically processed for ultrastructural study could be in some cases suitable for immunocytochemical investigations.     

FMRF-amide immunoreactivity in the mammalian gastroenteropancreatic neuroendocrine system

Summary The presence of FMRF-amide, a cardioactiv tetrapeptide, was studied by immunocytochemistry in human and rat gastric antrum and pancreas, and in the ovine, bovine, canine and rabbit pancreas. In human and rat gastric antrum, numerous cells contained FMRF-amide immunoreactive material. By staining of serial sections and by double staining, colocalization of immunoreactivity for gastrin and FMRF-amide was observed in part of the gastrin cells. In the pancreas of these and the other species, immunoreactivity for FMRF-amide was located both in acinar and islet endocrine cells. Colocalization of FMRF-amide and pancreatic polypeptide was found in a proportion of pancreatic polypeptide cells in the pancreas. FMRF-amide immunoreactivity never colocalized with the other neurohormonal peptides which occur in the gastric antrum and the pancreas.Our observations show that neuroendocrine cells occur in the gastric antrum and pancreas which are exclusively immunoreactive or gastrin and for pancreatic polypeptide respectively. In addition cells occur which show immunoreactivity for FMRF-amide as well as for gastrin in the gastric antrum and with antiserum to FMRF-amide as well as for pancreatic polypeptide in the pancreas. It is concluded that FMRF-amide antibodies probably recognize a substance in G and PP cells which is not identical but may be structurally related to gastrin and pancreatic polypeptide.In honour of Prof. P. van Duijn     

Nutrient sensing receptors in gastric endocrine cells

Sensing protein breakdown products in the luminal content is of particular importance for the regulation of digestive activities in the stomach which are mainly governed by gastric hormones. The molecular basis for tuning the release of hormones according to the protein content is still elusive. In this study we have analysed the murine stomach for candidate nutrient receptors. As a promising candidate we have concentrated on the broadly tuned amino acid receptor GPRC6A. Expression of GPRC6A could be demonstrated in different regions of the murine stomach; especially in the gastric antrum. Using immunohistochemical approaches, a large cell population of GPRC6A-positive cells was visualized in the basal half of the antral gastric mucosa. Molecular phenotyping of GPRC6A-immunoreactive cells revealed that most of them contained the peptide hormone gastrin. A small population turned out to be immunoreactive for somatostatin. In search for additional amino acid receptors in antral gastric mucosa, we obtained evidence for expression of the gustatory amino acid receptor subunit T1R3 and the calcium-sensing receptor CaSR. Many CaSR-cells were found in the gastric antrum and most of them also contained gastrin; very similar to GPRC6A-cells. In contrast, T1R3 was found only in a small population of gastrin-negative cells. The finding that GPRC6A-and CaSR-receptors are both expressed in many if not all gastrin cells strongly suggests that both receptor types are co-expressed in the same cells, where they could form heterodimers providing a unique response spectrum of these cells.     

Characterization of the carboxyl terminal flanking peptide of rat progastrin

A peptide identical in structure to the carboxyl-terminal flanking nonapeptide of rat progastrin, predicted by cDNA sequence, was synthesized. The synthetic peptide was used for production of a rabbit antiserum. This antiserum was used to develop a radioimmunoassay specific for rat carboxyl terminal flanking peptide. This assay was used to monitor the purification of immunoreactivity from rat antral extracts. Gel permeation, anion exchange and reverse phase chromatography steps resulted in a single absorbance peak associated with the carboxyl terminal flanking peptide immunoreactivity. The purified peptide eluted in the same position as the synthetic peptide during all three types of chromatography. This material was shown to be identical in mass to Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn, the predicted sequence of the carboxyl terminal nonapeptide of rat progastrin.     

Endocrine profile in gastric carcinomas. An immunohistochemical study.

22 gastric carcinomas (13 intestinal type and nine diffuse type) were immunostained for neuron specific enolase, chromogranin, Leu-7 and a panel of fifteen different peptide hormones. Five out of the 13 tumours of intestinal type and four out of the nine diffuse carcinomas expressed immunoreactivity for one or more of the pan endocrine markers. Seven out of the 13 tumours of intestinal type and five out of the nine diffuse carcinomas also expressed immunoreactivity for gastrin (3), ACTH (3), serotonin (7) and calcitonin (7). Immunoreactivity for somatostatin (1) and substance P (1) were also seen in two tumours of intestinal type. Seven out of 18 cases with benign mucosa adjacent to the tumours expressed a focal immunoreactivity for chromogranin (6), serotonin (6), gastrin (5) and calcitonin (1). All hormone-producing tumours also expressed immunoreactivity for carcino-embryonic antigen. Our results confirm that a high proportion of gastric carcinomas are hormone producing.     

Isolation from porcine antral mucosa of a hexapeptide corresponding to the C-terminal sequence of gastrin

The present studies were undertaken to confirm reports of high concentrations of the C-terminal tetrapeptide of gastrin in hog antral mucosa. A method was developed whereby synthetic tetrapeptide added to boiling water extracts of hog antral mucosa could be purified to homogeneity by adsorption to Amberlite XAD2 resin, ion exchange chromatography on DEAE cellulose, and reverse phase HPLC. The product had the amino acid composition of gastrin tetrapeptide. When the same method was used on antral mucosa without prior addition of synthetic G4, several small peaks of material with C-terminal immunoreactivity could be found in DEAE column eluates but none could be unequivocally identified as the tetrapeptide. In the same column runs there was a relatively large peak of immunoreactivity eluting later than the tetrapeptide. This material was purified to homogeneity by HPLC and on the basis of its amino acid composition and sequence was identified as the C-terminal hexapeptide of gastrin.     

Ultrastructural studies of endocrine cell populations showing an argentaffin reaction and/or serotonin immunoreactivity in the rat antral mucosa

Summary On the basis of staining results in closely related semi-thin sections from rat antral mucosa immunostained with polyclonal serotonin antibodies and silver-stained for the argentaffin reaction, respectively, three different cell populations could be distinguished. One of these cell populations showed both serotonin immunoreactivity and an argentaffin reaction, a second one serotonin immunoreactivity alone, and a third one only an argentaffin reaction.These cell populations were studied electron microscopically in ultra-thin sections located between the stained semi-thin sections. The cell population displaying an agentaffin reaction and serotonin immunoreactivity showed secretory granules of the enterochromaffin cell type. A similar granular appearance was observed in cells which only exhibited an argentaffin reaction. Serotonin immunoreactivity in the absence of an argentaffin reaction was evident in some G (gastrin) cells. and in some D₁ and possibly also some D (somatostatin) cells; but not all the endocrine cells of the non-enterochromaffin type displayed serotonin immunoreactivity. The significance of the different reactions in the three cell populations is discussed.