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.

Molecular forms of glucagon-like immunoreactivity in porcine intestine and pancreas.

Glucagon-related polypeptides in porcine pancreas and intestine were analysed by gel-permeation chromatography and RIA. Three assays were employed: a nonspecific glucagon assay (R59) of 94% cross-reactivity with glicentin; a pancreatic glucagon assay (RCS5) directed against the C-terminal region of glucagon and of less than 0.01% cross-reactivity with glicentin; and a glicentin assay (R64) of less than 0.01% cross-reactivity with glucagon. For extracts of porcine pancreas all three assays gave similar molar concentrations of immunoreactivity. In porcine intestinal extracts immunoreactivity was detected in significant amounts only by the nonspecific glucagon (R59) and the glicentin (R64) assays, again in similar molar concentrations. The immunoreactivities present in pancreas and intestine were chromatographically and immunologically separable into six main peaks, peaks I, II, III, V, and VI being present in the pancreas, and peaks I, II, and IV in the intestine. The different immunoreactivities of the peaks allowed probable identities to be assigned to their main components. Apart from peak I, which consists of void-volume material that may interfere nonspecifically with the assays, the main components of the peaks can be interpreted as glicentin (in peak II) or fragments derived from glicentin. Peak III contains the N-terminal portion of glicentin (glicentin-related pancreatic peptide), peak IV probably contains glucagon with its 8 amino-acid C-terminal extension, peak V is pancreatic glucagon and peak VI contains smaller N-terminal glicentin fragments. These findings fit with the proposition that glicentin fulfills the role of proglucagon in the pancreas, and is the major component of enteroglucagon in the intestine.

The distribution of polypeptide YY-like immunoreactivity in rat tissues.

The distribution of peptide YY (PYY)-like immunoreactivity (IR) in rat tissues was determined by specific RIA after extraction with boiled 1 N acetic acid. The high concentration of PYY-IR was observed in the gastrointestinal tract, with concentrations gradually increasing from the duodenum to the end of colon. The concentration of PYY-IR in the colon was 298.7-449.5 pmol/g tissue (approximately 100-200 times more than that in the duodenum). Pituitary and pancreas contained measurable amounts of PYY-IR (6.8 and 6.3 pmol/g tissue). The concentration of PYY-IR in the mucosa was higher than that in the muscular layer in the small intestine, cecum, colon, and rectum. The ratio of the mucosal PYY-IR to the muscular PYY-IR was highest in the distal small intestine (4.7-6.8). Sephadex G-50 gel chromatography of the colon extracts revealed the one PYY-IR peak which corresponds to [125I]PYY. The gradual increase of PYY-IR from the duodenum to the end of the colon is different from the distribution of other known gut peptides.

Gastrointestinal amyloid deposition in familial amyloid polyneuropathy.

We found degeneration of enteric nerve plexuses in two patients with type I familial amyloid polyneuropathy. Amyloid deposition was more severe in the wall of the stomach than in the rectum. Hypomotility of the upper gastrointestinal tract, resulting from both amyloid deposition in the stomach and upper bowel and degeneration of the intrinsic autonomic nerves, may be responsible for anorexia, nausea, and vomiting. Diarrhea and constipation may be caused by degeneration of the enteric nerve plexuses. Gastric biopsy is valuable and safe in the diagnosis to type I familial amyloid polyneuropathy.

Histochemical study of rectal aminergic nerves in type I familial amyloid polyneuropathy.

We found degeneration of aminergic nerves in nine patients with type I familial amyloid polyneuropathy by histochemical study of rectal mucosa obtained by biopsy. There was prominent degeneration of aminergic nerves in four patients with uncontrollable alternating constipation and diarrhea, but aminergic nerves were relatively preserved in two patients with intermittent constipation or diarrhea. Sympathetic denervation of the gastrointestinal tract was probably important in causing bowel symptoms.

Localization of glucagon-like peptide (GLP) immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry.

The distribution of peptide immunoreactivities predicted from the sequence of the human preproglucagon gene in enteroglucagon (EG; glicentin-like immunoreactant-containing) cells of the human gut and A cells of the pancreas has been determined by light and electron microscopic immunocytochemistry. At light microscopy the application of peroxidase-antiperoxidase and immunogold-silver staining methods has revealed that glucagon-like peptide (GLP-1 and GLP-2) immunoreactivities coexist with a glicentin-related immunodeterminant in human colorectal EG cells and pancreatic A cells. Using single and double colloidal gold probe electron immunocytochemistry, we have been able to show the coexistence of glicentin, GLP-1, and GLP-2 immunoreactivities within single EG cell secretory granules. No morphologic segregation of the proglucagon immunoreactants was observed in EG cells of the colonic mucosa. In pancreatic A cells we have localized GLP-1, GLP-2, and glucagon-[16-29] immunoreactivities solely to the electron-dense core of the secretory granules, whereas glicentin-related immunoreactivity was restricted to the electron-lucent halo. The results obtained in the present study have shown that the peptide immunoreactivities predicted from cDNA sequencing of the human preproglucagon gene are indeed expressed in colorectal EG and pancreatic A cells. The topographical segregation of immunoreactivities in the A cell secretory granule shows that antigenic determinants derived from the C-terminal portion of proglucagon are stored with glucagon in the core of the secretory granule.

Ultrastructural localization of serotonin and polypeptide YY (PYY) in endocrine cells of the human rectum.

This study was performed with the aim of ultrastructurally localizing serotonin and polypeptide YY (PYY) in the endocrine cells of the human rectum. Existing basic methods for immunolocalization of antigenic sites in ultrathin sections were tested and modified to allow reproducible results with distinct localization of marker (colloidal gold probes coupled either to IgG or protein A). Probes signifying presence of serotonin were distinctly localized over all heteromorphous granules in argentaffin cells and, in addition, over some of the more monomorphous, rounded granules in a second cell type whose granules all were covered by probes showing localization of the PYY antigen. The results suggest that serotonin in endocrine cells of the gut is not confined to the enterochromaffin type but may also be present in trace amounts in non-enterochromaffin endocrine cells storing peptide hormones. Since probes marking sites of PYY were deposited over some heteromorphous granules in enterochromaffin cells, the evidence obtained also suggests that PYY may occur in low concentration in these cells. The distribution of probes in the sections indicated that antigenic sites were confined to granules in the cells.

Estimation and characterization of prostaglandins in the human gastrointestinal tract.

1 Prostaglandin-like material was extracted from muscle and mucosa of surgically removed human stomach, ileum and colon and assayed against prostaglandin E(2) on strips of rat gastric fundus. Superfused human isolated gastric mucosa released prostaglandin-like material and release was increased by stretching or clamping the tissue.2 The relative amounts of extracted biological activity were broadly as follows: gastric antral mucosa > colon muscle > gastric body mucosa approximately ileal mucosa > colon mucosa approximately gastric muscle approximately ileal muscle.3 Prostaglandin E and F were tentatively identified by chromatography and sensitivity to inactivation by alkali.4 Prostaglandin E apparently contributed most to the biological activity, possibly because the assay tissue is more sensitive to prostaglandin E than to F. Chromatography of gastric body mucosal extracts located material running with prostaglandin E(2) and a little with E(1). Colonic muscle and mucosal extracts contained material with R(F) values of prostaglandins E(1), E(2), E(3) and F(1a), whereas F(2a) and F(3a)-like substances were found only in the mucosa. The proportions of prostaglandin F varied between specimens.5 The amount of extracted prostaglandin-like activity was increased by adding cofactors and arachidonic acid, and lessened by homogenization with acid-ethanol.6 The type and amount of activity generated from arachidonic acid by partly purified colonic mucosal prostaglandin synthetase depended on the substrate concentration.7 The possible relationships of prostaglandins to mucus secretion and other physiological and pathological gut functions are discussed.

Glutathione and GSH-dependent enzymes in the tumorous and nontumorous mucosa of the human colon and rectum.

A high content of total glutathione and high activities of both GSH S-aryltransferase (CDNB) and GSH peroxidase were found in different segments of the human intestinal mucosa comparable to findings in human gastric mucosa. Intraindividual comparisons of tumorous and nontumorous tissue specimens in patients with adenocarcinomas of the colon and rectum revealed no marked differences in their glutathione content and enzyme activities except in the sigma, where we found significantly lower GSH concentrations and higher GSH S-aryltransferase activities in the carcinomatous tissue. gamma-Glutamyl-transpeptidase activity, a marker of neoplastic cell growth in experimental hepatocarcinogenesis, did not differ between tumorous and nontumorous tissue areas. The presence and high activity of the GSH-dependent enzyme system in different segments of the human intestinal mucosa may reflect its role in the defense against toxic and putative carcinogenic xenobiotics entering the body via the gastrointestinal tract.

Glycoprotein abnormalities in colonic carcinomata, adenomata, and hyperplastic polyps shown by lectin peroxidase histochemistry.

A technique for lectin-peroxidase histochemistry was adapted for the study of formalin fixed paraffin embedded colonic tissue. Ten lectins with differing carbohydrate binding specificity were tested against 20 normal rectal biopsy specimens and tissue from 19 colonic carcinomata, 19 tubular or tubulovillous adenomata, and 19 hyperplastic polyps. None of the normal rectal biopsy specimens bound the lectins peanut agglutinin (PNA), Griffonia simplicifolia II (GSII), and Ulex europaeus I (UEAI), whereas 18 carcinomata, 12 adenomata, and 18 hyperplastic polyps showed affinity for one or more of these lectins. Hyperplastic colonic polyps are shown to possess similar abnormalities in glycoprotein structure to malignant and adenomatous colonic tissue. This may simply indicate a non-specific reaction to changed rates of cell proliferation but might represent a more fundamental association between hyperplastic polyps and adenocarcinomas.

Distribution of fibronectin in the rectal mucosa.

Fibronectin is a glycoprotein of high molecular weight present in tissues, plasma, and tissue fluids. Its distribution in the rectal mucosa was studied by immunofluorescent and immunoperoxidase techniques using a monospecific antiserum. Immunofluorescent reactivity for fibronectin was present in the normal rectal mucosa of control subjects in epithelial cells, on basement membranes, and as a loose cribriform network of extracellular reactivity in the lamina propria that codistributed with histochemically demonstrable reticulin. Fibronectin was demonstrated immunoelectromicroscopically on collagen fibres, on smooth muscle cells and within and between columnar epithelial cells. In the rectal mucosa of patients with colitis with marked inflammatory changes, fibronectin appeared thickened and more prominent when present on basement membranes and as sparse strands between inflammatory cells infiltrating the lamina propria. In patients with longstanding colitis and less inflammatory cell infiltration there was a diffuse increase in fibronectin which was densely and uniformly present throughout the lamina propria. Fibronectin is a structural component of the rectal mucosa and changes in its distribution may form an important part of the local reaction to inflammatory bowel disease.

Immunohistochemical staining of colorectal tissues with monoclonal antibodies to ras oncogene p21 product and carbohydrate determinant antigen 19-9.

Two monoclonal antibodies were applied to benign, dysplastic, and malignant human colorectal tissues using immunohistochemical techniques on formalin fixed paraffin embedded material. RAP-5 antibody is directed against a synthetic peptide, reflecting an amino acid sequence of the ras oncogene p21 protein product. Despite using several different techniques and antibody dilutions differential staining between the various epithelial populations was not obtained. RAP-5 also showed other tissue components such as plasma cells, histiocytes, fibroblasts, smooth muscle and vascular endothelium. CA19-9 antibody recognizes an epithelial surface carbohydrate antigen originally derived from a human colorectal carcinoma cell line: it did not stain normal colorectal mucosa or adenomatous polyps, but showed focal expression of variable strength in regenerative, dysplastic, and cancerous mucosa in ulcerative colitis, and in non-colitic colorectal carcinoma. Neither antibody was found to be a reliable marker of the evolution of malignant mucosal changes, although CA19-9 may be of limited use in confirming adenocarcinoma of gastrointestinal origin.

Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity in the human gut.

The presence of the proenkephalin A-derived peptide Met5-enkephalin-Arg6-Gly7-Leu8 was demonstrated throughout the human gastrointestinal tract. Highest concentrations of Met5-enkephalin-Arg6-Gly7-Leu8, as assessed by radioimmunoassay, were measured in the separated muscularis externa, while lower levels were found in the submucosa and only small amounts in the mucosa. The results are consistent with a neuronal location of this peptide in the human gut. Over 65% of total immunoreactivity coeluted with the authentic peptide in both molecular exclusion chromatography and HPLC, while most of the remainder activity eluted earlier on gel filtration. The latter material probably represents N-terminally extended Met5-enkephalin-Arg6-Gly7-Leu8. Taken together with previous studies, our results appear to indicate that there are important species differences in post-translational processing of proenkephalin A in gut nerves.

The distribution of galanin message-associated peptide-like immunoreactivity in the pig.

Using a radioimmunoassay (RIA) developed to the N-terminal part of the predicted sequence of porcine galanin message-associated peptide (GMAP), we have confirmed the existence of GMAP-like immunoreactivity (-LI) in normal porcine tissues. GMAP-LI was found to parallel the distribution of galanin-immunoreactivity (-IR), although consistently the concentrations detected were, on a molar ratio, significantly less than those measured for galanin throughout the gastrointestinal tract, brain, spinal cord, adrenal and pituitary gland. As cleavage of the prohormone would be expected to produce galanin and GMAP on an equimolar basis, it is possible that the endogenous, intact GMAP peptide does not fully cross-react with the antibody raised to the N-terminal GMAP sequence. Gel chromatography of tissue extracts revealed a single molecular form of galanin-IR in the gut and four distinct molecular forms in the adrenal gland. GMAP-LI eluted as a single immunoreactive component in the gut, and in the adrenal gland there were two major molecular forms, one of which was apparently also detected by the galanin assay, and a small amount of N-terminal fragment. This molecular heterogeneity seems likely to be a result of the various possible prohormone cleavage products and/or posttranslational processing modifications. Further analysis of the galanin gene products needs to be undertaken in order to confirm this.

Co-existence of glicentin and peptide YY in colorectal L-cells in cat and man. An electron microscopic study.

Electron microscopic immunocytochemistry using protein A-gold labelling of ultrathin sections revealed immunoreactive glicentin (gut-type glucagon) and peptide YY (PYY) in virtually all secretory granules in a population of L-type endocrine cells in feline colon and human rectum. The granules of the human glicentin/PYY cells were considerably smaller in size than those in the cat. In both species the results indicate co-existence of glicentin and PYY in the same secretory granules, despite the probable derivation of the two peptides from two different precursors.

Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut.

Endocrine cells containing peptide YY (PYY) were numerous in the rectum, colon and ileum and few in the duodenum and jejunum of rat, pig and man. No immunoreactive cells could be detected in the pancreas and stomach. Coexistence of PYY and glicentin was revealed by sequential staining of the same section and by staining consecutive semi-thin sections. Since the PYY sequence is not contained in the glucagon/glicentin precursor molecule the results suggest that the PYY cell in the gut expresses two different genes coding for regulatory peptides of two different families.

Bombesin-like immunoreactive material in the gut, and the effect of bombesin on the stomach circulatory system of an elasmobranch fish, Squalus acanthias.

The distribution, nature and amount of bombesin-like immunoreactivity (IR) in the gastrointestinal canal and its afferent vessels was investigated in the spiny dogfish (Squalus acanthias) together with the in vitro effect of synthetic bombesin on perfusion flow through the vascularly perfused dogfish stomach. Nerve fibres showing bombesin-like IR frequently occurred in the walls of the anterior mesenteric and coeliac arteries and the intrinsic vessels of the gut. Chromatographic studies revealed that multiple peaks of bombesin-like IR material were present in extracts of the spiny dogfish gastrointestinal vessels. Bombesin-like IR was also present in muscle and mucosal layers of the gut with higher levels in muscle compared with mucosa, and higher levels in the stomach than in the intestine and the rectum. Exogenous bombesin increased the flow through the vasculary perfused spiny dogfish stomach in a dose-dependent manner. Studies with tetrodotoxin and atropine showed that bombesin probably exerts its effect directly on the vascular musculature. It is concluded from this study that bombesin-like material is present in nerves innervating the gut circulatory system of the spiny dogfish. Bombesin may affect the blood-flow to the gastrointestinal canal, possibly via a direct effect on vascular smooth muscle.

Intramural distribution of Met5-enkephalin-Arg6-Gly7-Leu8 in sphincter regions of the human gut.

The intramural distribution of Met5-enkephalin-Arg6-Gly7-Leu8 (MERGL) was studied in the oesophago-cardiac, pyloric, ileo-caecal and sigmoid-recto-anal regions of the human digestive tract. Serial samples encompassing each area were separated into mucosa, submucosa and muscularis externa and extracted for radioimmunoassay. Comparatively low levels of MERGL immunoreactivity were measured throughout the cardiac junction. Conversely, a remarkable peak of MERGL concentration was detected at the pyloric junction, in both submucosa and muscularis. A progressive decrease in tissue levels of the same peptide, most evident in the submucosa, was detected on the proximal side of the ileo-caecal region. In the distal sigmoid colon and rectum MERGL concentrations showed a rapid decline, down to very low levels in the anal canal. The results may suggest the involvement of an enkephalinergic mechanism in the control of the human pylorus.

Type III (chronic) GM1-gangliosidosis. Histochemical and ultrastructural studies of rectal biopsy.

Type III GM1-gangliosidosis is a rare hereditary storage disease caused by lack of lysosomal beta-galactosidase and characterized by a slowly progressive course, and extrapyramidal signs, but without prominent skeletal changes or visceromegaly. The storage substance was reported to be located only in the basal ganglia. There has been no detailed report on visceral lesions in type III GM1-gangliosidosis. In this report we describe a case of type III GM1-gangliosidosis, and the histochemical and ultrastructural findings from biopsied rectum. The patient was a 22-year-old female who exhibited dysarthria, gait disturbance, and generalized dystonia with rigidity. Beta-galactosidase activity in leukocytes was absent and sialidase activity in cultured fibroblasts was normal. Many histiocytes were found in biopsied rectal mucosa. Histochemical studies showed that the granules of histiocytes contained acidic glycoconjugates, beta-galactose, beta-N-acetylgalactosamine and sialic acid. Ultrastructural investigations revealed that ganglion cells of Meissner's plexus had many osmiophilic lamellar inclusions, similar to "membranous cytoplasmic bodies". These findings are crucial for the clinical diagnosis of type III GM1-gangliosidosis.

Subcutaneous fat biopsy in the diagnosis of amyloidosis secondary to chronic arthritis.

Subcutaneous fat biopsy was investigated for its sensitivity in giving a diagnosis in 44 consecutive patients with rheumatoid arthritis or ankylosing spondylitis suspected of systemic amyloidosis. In 26 of these patients amyloidosis could be demonstrated by fat or rectal biopsy or biopsies from organs suspected of amyloid deposition. Fourteen of the 26 (54%) fat biopsy specimens of the patients with amyloidosis were positive after staining with Congo red and 22 (85%) of the rectal biopsy specimens were positive. All 12 kidney biopsy specimens and 4 biopsy specimens from other organs of these 26 patients were positive for amyloidosis. In 2 patients with a negative rectal biopsy specimen, fat biopsy would have obviated the need for a more invasive biopsy. All patients experienced fat biopsy as less demanding compared to other biopsy procedures. These results imply that in patients with chronic arthritis subcutaneous fat biopsy is a useful screening procedure. In this patient group fat biopsy is less sensitive for the diagnosis of amyloidosis compared to rectal biopsy.