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Neuron-specific enolase as a marker for intestinal neurons. An immunocytochemical study of the human intestinal tract.

Surgical specimens from various parts of the human intestinal tract as well as suction biopsy specimens, including mucosa and submucosa of the rectum, were fixed in formalin and embedded in paraffin by routine procedures. The distribution of immunoreactive areas indicating the presence of neuron-specific enolase (NSE) was then determined by using a sheep anti-human-NSE antiserum prepared in our laboratory. The immunocytochemical method revealed, in distinct contrast to other tissue components, the cell bodies of ganglion cells in the submucosa (Meissner's plexus) and in the muscle layers (Auerbach's plexus). The nerve bundles of the submucosa, of the muscle layers, and of the subserosal connective tissue were also stained, whereas the thin nerve processes of the mucosa were identified only rarely. The smooth muscle cells were stained weakly, but this reaction did not interfere with the identification of the neurons and their processes. Immunocytochemical demonstration of NSE is obviously a valuable additional method for visualization of the intrinsic intestinal innervation. It might well be that this technique will be of advantage in the diagnosis of pathologic processes, such as those occurring in Hirschsprung's disease and allied conditions.

Adolescent↗

Effect of vasoactive intestinal peptide, somatostatin, neurotensin, cholecystokinin octapeptide, and secretin on intestinal absorption of amino acid in rat.

The effects of vasoactive intestinal peptide (VIP), somatostatin (SRIF), neurotensin (NT), cholecystokinin octapeptide (CCK-8), and secretin (SEC) on the intestinal absorption of amino acid were investigated. Six groups of Wistar rats were studied: (1) controls; (2) VIP treated; (3) SRIF treated; (4) NT treated; (5) CCK-8 treated; (6) SEC treated. [3H]Leucine was given intraluminally through a cannula at the ligament of Treitz, a number of blood samples were obtained through a superior mesenteric vein catheter 1-60 min after administration of [3H]leucine, and the radioactivity of plasma was measured to evaluate the absorption of [3H]leucine. It was shown that VIP and SRIF significantly inhibited the absorption of [3H]leucine (by 59.1% and 38.7%, respectively), whereas NT, CCK-8, and SEC significantly enhanced absorption (by 44.2%, 49.6%, and 39.1%, respectively). Radioimmunoassays of VIP, SRIF, and NT showed that at least some of the hormones or peptides exerted their effects on absorption of leucine at or near their physiological concentrations.

Animals↗

Neonatal intestinal perforation due to congenital defects in the intestinal muscularis.

Congenital defect of the muscular layer of the small intestine is a rare cause of spontaneous bowel perforation in premature infants. During the last 12 years we have observed four similar cases. We describe the most recent one, a premature infant who developed two abdominal events. On her 2nd day of life, spontaneous perforation of the distal ileum due to focal absence of the muscular layer occurred. Several weeks later she developed the typical clinical and histological picture of necrotizing enterocolitis. The clinical and histological characteristics of the two different conditions are compared, and the 24 cases reported in the literature are discussed. We conclude that focal absence of intestinal musculature may be not such a rare entity as is commonly believed.

Female↗

Megacystis-microcolon-intestinal hypoperistalsis syndrome: evidence of intestinal myopathy.

We investigated small- and large-bowel specimens of three newborn infants presenting with the clinical and radiological symptoms of megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS). Conventional histological staining revealed marked thinning of the longitudinal muscle layer. Electron-microscopic investigations showed typical "central core" vacuolic degeneration of smooth-muscle-cells combined with proliferation of col lagen fibres. The expression of alpha-smooth-muscle actin was absent or markedly reduced in the circular and longitudinal muscle layers and muscularis mucosae compared to the normal controls. These findings suggest that the intestinal obstruction in MMIHS is due to an abnormality of the smooth-muscle cells.

Abnormalities, Multiple↗

Intestinal absorption of dipeptides and beta-lactam antibiotics. II. Purification of the binding protein for dipeptides and beta-lactam antibiotics from rabbit small intestinal brush border membranes.

By photoaffinity labeling of brush border membrane vesicles from rabbit small intestine with photoreactive derivatives of beta-lactam antibiotics and dipeptides, a binding protein for dipeptides and beta-lactam antibiotics with an apparent molecular weight of 127,000 was labeled. The labeled 127 kDa polypeptide could be solubilized with the non-ionic detergents Triton X-100, n-octyl glucoside or CHAPS. If the vesicles were solubilized prior to photoaffinity labeling, no clear incorporation of radioactivity into the 127 kDa polypeptide occurred indicating a loss of binding ability upon solubilization. By affinity chromatography of solubilized brush border membrane proteins on an agarose wheat germ lectin column, the binding protein for dipeptides and beta-lactam antibiotics of Mr 127,000 was retained on the column. With N-acetyl-D-glucosamine the photolabeled binding protein for beta-lactam antibiotics and dipeptides was eluted together with the brush border membrane-bound enzyme aminopeptidase N. Separation from aminopeptidase N and final purification was achieved by anion-exchange chromatography on DEAE-sephacel. Polyclonal antibodies against the purified binding protein were raised in guinea pigs. The photolabeled 127 kDa protein could be precipitated from solubilized brush border membranes with these antibodies. Incubation of brush border membrane vesicles with antiserum prior to photoaffinity labeling significantly reduced the extent of labeling of the 127 kDa protein. Treatment of brush border membrane vesicles with antiserum significantly inhibited the efflux of the alpha-aminocephalosporin cephalexin from the brush border membrane vesicles compared to vesicles treated with preimmune serum. These studies indicate that the binding protein for dipeptides and beta-lactam antibiotics of apparent molecular weight 127,000 in the brush border membrane of rabbit small intestinal enterocytes is directly involved in the uptake process of small peptides and orally active beta-lactam antibiotics across the enterocyte brush border membrane.

Affinity Labels↗

Regional and cellular localization of glycosyltransferases in rat small intestine. Changes in enzymes with differentiation of intestinal epithelial cells.

Optimal assay conditions were determined for four glycosyltransferases in rat small intestinal mucosal homogenates and the regional distribution and cellular localization of these enzymes was studied. For each glycosyltransferase, similar levels of activity were found in duodenal, proximal jejunal and distal ileal segments; activities of the galactosyltransferases were lower in the distal jejunal-proximal ileal segment. Planar section studies indicated that the undifferentiated crypt cells had significantly higher levels of sialyltransferase activities in the jejunum and ileum than the mature villus cells. A similar crypt to villus gradient was found for a galactosyltransferase in the ileum. These data suggest that glycoprotein synthesis may be active in the undifferentiated crypt cells and that certain glycosyltransferases may serve as marker enzymes for cellular differentiation in the intestine.

Animals↗

Glycolipids of rat large intestine. Characterization of a novel blood group B-active tetraglycosylceramide absent from small intestine.

A novel blood group B-active tetraglycosylceramide has been isolated from rat large intestine. It is probably present only in the epithelial cells. Although the compound was not obtained pure, the structure was conclusively established by mass spectrometry, proton NMR spectroscopy and degradative studies to be Galp alpha 1 leads to 3Galp(2 comes from 1 alpha Fucp)beta 1 leads to 4Glcp beta 1 leads to 1Cer. The ceramide was composed of mainly trihydroxy long-chain base (18:0) and non-hydroxy fatty acids (16:0-24:0). The glycolipid was absent from small intestine.

Animals↗

Intestinal absorption and metabolism of 6-mercaptopurine in the rat small intestine.

The intestinal absorption of 6-mercaptopurine was examined in the rat by in vitro and in situ techniques for the purpose of establishing absorption characteristics which might explain the poor systemic oral availability of this drug. Experiments were designed to evaluate the significance of intestinal metabolism and active secretion processes. Since mucosal/serosal drug concentration gradients across in vitro segments were not significantly different (P greater than 0.05) for everted and noneverted preparations, with values of 1.21 +/- 0.27 and 0.91 +/- 0.12, respectively, it was concluded that active secretion or absorption mechanisms were absent. Varying the concentration of 6-mercaptopurine from 0.24 to 5.88 mM demonstrated saturability of the biotransformation of 6-mercaptopurine to 6-thiouric acid with a maximum rate of 1.6 X 10(-5) mmoles per min g for jejunal portions. Distal segments displayed 85% higher rates of biotransformation at concentrations of 1.47 mM. Inclusion of allpurinol (2 mM) completely inhibited biotransformation. With in situ loops perfused with 1.47 mM drug, collection of mesentery blood showed that absorption rates of 6-thiouric acid were 0.67 that of the parent drug.

Animals↗

Drug interactions in intestinal transport of folic acid and methotrexate. Further evidence for the heterogeneity of folate transport in the human small intestine.

The effect of sulfasalazine and olsalazine on the transport of [3H]folic acid and of [3H]methotrexate (MTX) was investigated in organ-cultured endoscopic biopsy specimens of small intestinal mucosa from normal subjects. Biopsy specimens obtained from patients undergoing routine diagnostic upper gastrointestinal endoscopy were organ-cultured at pH 5.5 and the effect of these two drugs on the initial rate of uptake of the two folates was determined. Both drugs inhibited the transport of [3H]folic acid with similar Ki values (1.38 and 1.32 mM for sulfasalazine and olsalazine, respectively). However, the uptake of [3H]MTX was only partially inhibited by sulfasalazine and was unaffected by olsalazine. Sulfasalazine inhibited 26.2% of the total flux of MTX, in close agreement with the fraction of MTX flux that has been shown previously to be inhibited by folic acid. These data corroborate previous findings of heterogeneity of transport of MTX in the mucosa of the human small intestine.

Aminosalicylic Acids↗

Immunoelectrophoretic studies on human small intestinal brush border proteins. A quantitative study of brush border enzymes from single small intestinal biopsies.

A method for measuring brush border membrane enzymes from small intestinal biopsies by crossed immunoelectrophoresis is presented. The use of a brush border specific antiserum made isolation of the brush border membrane before analysis unnecessary. This prevented loss of material which, together with inactivation of enzymes, was a limiting factor in previous studies of brush border enzymes from peroral biopsies. In 58 biopsies from patients without gastrointestinal disorders a close correlation between antigenic activity and corresponding enzymatic activity was shown for the following enzymes: sucrase-isomaltase (EC 3.2.1.48-EC 3.2.1.10), lactase-phlorizin hydrolase (EC 3.2.1.23-EC 3.2.1.62), microvillus aminopeptidase (microsomal, EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.X). The immunoelectrophoretic patterns of intestinal mucosa near the ligament of Treitz, and in jejunum and ileum were established. The method presented is thought to be of value in further studies of the molecular basis of brush border diseases.

Aminopeptidases↗

The effect of rhein and rhein anthrone on intestinal fluid transport and on large intestine transit in germ-free rats.

The effect of rhein and rhein anthrone on the transit and the transport of water and electrolytes in the large intestine was investigated in germ-free rats. After intracaecal administration, neither of the two compounds was found to accelerate the transit of a colour marker through the large intestine. Both drugs reduced the net absorption of sodium and chloride in the colon and enhanced net potassium secretion. Net water absorption was decreased by rhein and even reversed into net secretion by rhein anthrone. Our results show that the secretagogue activity of the compounds is not sufficient to induce laxation in germ-free rats. Furthermore rhein and rhein anthrone had no laxative properties under our experimental conditions.

Animals↗

Regional content of enteric substance P and vasoactive intestinal peptide during intestinal inflammation in the parasitized ferret.

Our aim was to characterize and quantitate changes in two key neuropeptides, substance P (SP) and vasoactive intestinal peptide (VIP), that are involved in governing neurally-mediated gastrointestinal (GI) reflex activity during enteric inflammation in the ferret. Neuropeptide content was determined by radioimmunoassay of extracts of jejunal, ileal and colonic muscularis externa from uninfected ferrets and ferrets infected with enteric stages of the parasitic nematode, Trichinella spiralis. Increased myeloperoxidase activity (MPO), an enzymatic marker of inflammation, occurred in all three gut regions. Histopathological changes were present only in the small intestine. Significant reductions were detected in both SP (72% decrease) and VIP (62% decrease) in the inflamed jejunum. Ileal concentrations of both SP (77% decrease) and VIP (46% decrease) were also decreased during T. spiralis infection compared to uninfected ferrets. Only SP (58% decrease) concentration showed a significant change in colonic tissues from infected ferrets; colonic VIP was unaltered. Parasite-induced inflammation caused significant changes in peptide-containing enteric neural pathways and might contribute to functional GI motor disturbances that occur during nematode infections in mammalian hosts.

Animals↗

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide and secretin from the ovine small intestine.

Vasoactive intestinal peptide (VIP), peptide histidine isoleucinamide (PHI) and secretin were separated and purified to homogeneity from ovine small intestine, using radioimmunoassay and radioreceptor assay for detection. An efficient and rapid purification sequence included acid extraction, concentration on a bulk C18 cartridge, filtration on a Fractogel column, ion-exchange chromatography on Mono-S and a maximum of three successive reverse-phase HPLC steps. The amounts of peptides obtained from 450 g wet weight tissue were 20 micrograms VIP, 15 micrograms PHI and 5 micrograms secretin. The as yet unknown amino acid sequences of the three peptides were found to be identical to those of the corresponding bovine peptides.

Amino Acid Sequence↗

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide (1-27) and secretin from the small intestine of guinea pig.

The neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucinamide (1-27) (PHI) and the hormone secretin were purified from the small intestine of guinea pig, being detected by radioimmunoassay and radioreceptor assay throughout six to seven chromatographic steps. After elution on a reverse-phase C18 column, the three peptides were separated on a Fractogel column. After cation-exchange chromatography of each peptide on Mono S, the final steps were performed using a reverse-phase RP8-e column. Guinea pig PHI differed from porcine PHI in having Tyr and Arg residues instead of Phe and Lys in, respectively, position 10 and 20. We confirmed the original sequence of guinea pig VIP previously documented (with Leu5, Thr9, Met19 and Val26). We also established the similarity of the primary structure of guinea pig secretin with that of porcine and bovine.

Amino Acid Sequence↗

Increased expression of c-fos and c-jun in the rat small intestinal epithelium after ischemia-reperfusion injury: a possible correlation with the proliferation or apoptosis of intestinal epithelial cells.

BACKGROUND AND PURPOSE: An increased expression of immediate early genes, such as the c-fos and c-jun, is observed in some organs after ischemia-reperfusion (I/R) injury. These factors have been revealed to potentially induce apoptosis and proliferation of the postischemic cells. The purpose of this study is to analyze the relationship between the expression patterns of such immediate early genes and the cellular responses in the intestinal epithelial cells (IECs) after I/R stress. METHODS: The rat small intestine was reperfused after 30 minutes ischemia. Semiquantitative reverse transcription-polymerase chain reaction was used to quantify c-fos and c-jun messenger RNAs. The proliferation and apoptosis of IECs were detected by immunohistochemistry and the in situ terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling method, respectively. RESULTS: The messenger RNA levels of the c-fos and c-jun showed characteristic patterns in the IECs after the I/R stress. The proliferation of the cells was initially observed after the I/R stress, followed by apoptosis of the cells. CONCLUSIONS: The sequential expression patterns of these factors are possibly related to the proliferation and apoptosis of the IECs.

Animals↗

Interleukin-11 enhances small intestine absorptive function and mucosal mass after intestinal adaptation.

BACKGROUND/PURPOSE: Interleukin-11 (IL-11) recently has been shown to enhance mucosal mass after massive small bowel resection (MSBR). However, enhanced mucosal mass may not correlate with increased substrate absorption. This study was designed to examine the effect of systemic administration of increasing doses of IL-11 on small intestine absorptive function and mucosal mass after MSBR. METHODS: Twenty-five Sprague-Dawley rats underwent an 80% small bowel resection and end-to-end jejunoileal anastomosis. Seven days after resection, all rats had placement of a jugular venous catheter connected to a subcutaneously placed osmotic pump. The rats were divided into 5 groups based on the content of the pump: group 1 (control, n = 5) received 0.1% bovine serum albumin (BSA) and groups 2 through 5 (n = 5 each) received IL-11 at 250, 500, 750, and 1,000 microg/kg/d, respectively. After a 14-day infusion period, [14C] galactose and [14C] glycine absorption was measured using an in vivo closed-recirculation technique. Mucosal DNA content also was determined for each group. Statistical analysis was performed by analysis of variance and expressed as mean +/-SEM. RESULTS: IL-11 administered at 250 microg/kg/d, a dose used in previous studies, did not significantly affect substrate absorption. However, compared with the control group, administration of higher doses of IL-11 produced a significant increase in substrate absorption and mucosal mass. The dose of IL-11 producing the overall optimal response based on the parameters measured (galactose absorption, 72% increase over control; glycine absorption, 112% increase over control; and DNA content, 98% increase over control) was 750 microg/kg/d. CONCLUSIONS: In addition to an increase in mucosal mass, these data show for the first time that IL-11 enhances absorptive function beyond the normal adaptive response after MSBR. Furthermore, the maximum effect of IL-11 on absorptive function was shown at 750 microg/kg/d, which is 3 times the dose used in previously reported studies. This study suggests that IL-11 may be useful clinically in patients with inadequate intestinal function.

Adaptation, Physiological↗