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Photoaffinity labelling of the vasoactive-intestinal-peptide-binding site on intact human colonic adenocarcinoma cell line HT29-D4. Synthesis and use of photosensitive vasoactive-intestinal-peptide derivatives.

N-Hydroxysuccinimidyl 4-azidobenzoate, a u.v.-sensitive heterobifunctional reagent, was used to synthesize photoreactive derivatives of the vasoactive intestinal peptide (VIP). Products of the reaction were purified by reverse-phase h.p.l.c. Three 4-azidobenzoyl-VIP (4-AB-VIP) derivatives were able to compete with monoiodinated 125I-VIP with an apparent KD of 2.5, 6.3 and 12.5 nM compared with 0.6 nM for native VIP. H.p.l.c.-purified mono[125I]iodinated VIP was used to synthesize 4-AB-125I-VIP derivatives. They were used to photoaffinity-label the VIP-binding site of HT29-D4 cells, a clone derived from the human colonic adenocarcinoma cell line HT29. Only one polypeptide, of Mr 70,000 +/- 5000 (mean +/- S.D.) was specifically labelled. The Mr of the component thus characterized was slightly higher than that of the major species (Mr 67,000) labelled after cross-linking experiments using 125I-VIP, conventional homobifunctional reagents and HT29 cells. Nevertheless, the specificity and extent of glycosylation of these two components were identical. These new photosensitive VIP derivatives should be useful tools with which to investigate further VIP-receptor structure and metabolism.

Affinity Labels↗

[Intestinal B-mode sonography in patients with endemic sprue. Intestinal sonography in endemic sprue].

AIM: The value of ultrasonography in the diagnosis, follow-up and for the detection of complications in patients with celiac sprue has not yet been sufficiently evaluated. A pronounced back and forth motility with echo-rich hump reflexes in a fluid-filled small bowel with a reduction of Kerckring's plicae circulares and with a loss of their density and uniformity was empirically defined as a diagnostic sign of celiac sprue. In the present study, the sonographic signs of celiac sprue were examined as an indicator of active sprue. METHOD: 50 patients with histologically proven celiac sprue were examined with real time ultrasonography (3.5-7 MHz). The detection or exclusion of the defined sonographic signs of celiac sprue with intensified motility and reduction of Kerckring's plicae circulares with a loss of their density and uniformity were evaluated by two independent examiners and documented without knowledge of the clinical findings. The clinical activity (active vs. remission) was assessed according to clinical criteria (diarrhea, steatorrhea, weight loss). 38 healthy subjects and 50 patients with Crohn's disease served as controls. RESULTS: In all 138 patients and controls adequate visualization of the bowel was achieved. In 16/50 (32%) patients with active celiac sprue changes of motility and reduction of Kerckring's plicae circulares with a loss of their density and uniformity were detected, whereas all 34/50 (68%) of patients with celiac sprue in remission did not have this pattern. In none of the controls with Crohn's disease or in the healthy subjects comparative sonographic signs of active celiac sprue were observed. In four patients with active celiac sprue a circumscript echopoor tumor of the small bowel wall could be sonographically detected, which turned out to be T-cell lymphoma in three and a carcinoma of the small intestine in one patient. An increased number of and/or enlarged mesenteric lymph nodes were found in patients with active celiac sprue. CONCLUSION: Changes of motility and reduction of Kerckring's plicae circulares with loss of density and uniformity at ultrasonography are a reliable indicator of active celiac sprue.

Adult↗

[Contamination of the small intestine as a cause for ileus attacks in children after intestinal surgery].

During the last two years eight children aged 3 months to 7 years were treated successfully for contaminated small bowel syndrome (CSBS). All patients had a history of a laparotomy in the neonatal period and showed bile stained vomiting and diarrhoea. On examination, a painful distended abdomen with hyperactive bowel sounds was found. Plain abdominal x-rays showed signs of mechanical intestinal obstruction. The diagnosis of CSBS was made by positive gram stain and cultures of samples taken via a nasogastric tube. After antibiotic treatment the symptoms disappeared within a few days. We therefore believe that CSBS should always be considered in the differential diagnosis of abdominal emergencies. Our views agree with those of other authors in so far as we feel that antibiotic therapy may help to avoid unnecessary laparotomies in such cases.

Child↗

[Nitrogen metabolism in the large intestine of ruminants. 1. Metabolism of i.v. infused 15N-urea without additional carbohydrate supply to the large intestine].

The experiments were carried out on 3 bulls (body weight: 172, 229 and 193 kg), equipped with ileo-caecal cannulas and with catheters in the jugular veins on both sides. The offered pelleted ration consisted of straw 73%, molasses 12%, cereals 10%, ammonium hydrogen carbonate 3% and urea 2%. Feed intake amounted to about 3 kg per animal and day. During a preliminary period of 5 days 50% of ileal digesta were collected for 12 hours daily, deep-freezed and stored. In the main experiment 15N-urea was infused intravenously for 24 hours. During this period and during the following 6 hours ileal digesta were collected and replaced by precollected, unlabelled digesta. The urea metabolism was estimated by the 15N-labelling of blood urea, by the 15N-excretion via faeces and urea, by the appearance of 15N in ileal digesta and by the 15N-labelling of faecal NAN, NH3 and bacterial fraction. The time course of the 15N-labelling of plasma urea during infusion can be described by an exponential function. The urea flux rate was estimated from the calculated plateau value. The flux rate for the 3 animals was 28.8, 30.7 and 34.8 mumol per minute per kg0.75, respectively. During the infusion of 15N-urea 1.0-2.4% of the infused amount of 15N' appeared in ileal digesta, half of it in the TCA precipitable fraction. At the same time the 15N-labelling of faecal NH3 increased sharply, however, the 15N-labelling of the faecal bacterial fraction was smaller by one order of magnitude. Deficiency of fermentable substrates and problems of inhomogenity of the NH3 pool are supposed as reasons for this result. 30 to 50% of the urea flux entered the digestive tract, the direct entry of urea into the large intestine seems to be only very low.

Animal Feed↗

[Nitrogen and amino acid absorption in the small intestine of growing pigs. 2. Apparent and true crude protein digestibility and amino acid absorption to the end of the small intestine].

Studies of the apparent and true ileal digestibility of crude protein and the apparent and true amino acid (AA) absorption were carried out with 7 female pigs in the live weight range between 22 and 34 kg after ileorectostomy. Protein sources of varied quality with regard to the method of treatment (toasted) and untoasted soybean oilmeal) and to the AA composition (vital gluten or vital gluten + L-lysine X HCl) were used. Distinct differences regarding the apparent ileal digestibility of crude protein and the apparent absorption of amino acids were discovered between the protein sources, the cause of which is to be seen in the different amounts of endogenous N. The true ileal digestibility of crude protein and amino acid absorption were partly distinctly higher than the values of the apparent ileal digestibility of crude protein and AA absorption. On the whole one can say that the results of the investigation gained by means of ileorectostomy can be compared to the values gained from cannulated animals. Thus the experimental method used is suitable for studying the absorption process to the end of the small intestine of pigs.

Amino Acids↗

Intestinal absorption of cholecalciferol and 25-hydroxycholecalciferol in patients with both Crohn's disease and intestinal resection.

We compared the intestinal absorption of cholecalciferol and 25-hydroxycholecalciferol in patients with Crohn's disease and resections of the small bowel. Patients were subgrouped into those with small (less than 100 cm), intermediate (100-300 cm), and large (greater than 300 cm) resections. [3H]cholecalciferol or [3H]25-hydroxycholecalciferol were given orally and serial blood samples were taken for measurement of plasma radiolabeled vitamin. Absorption of both forms of the vitamin decreased with extent of resection but 25-hydroxycholecalciferol absorption was always greater than that of cholecalciferol. When compared with normal control subjects, 25-hydroxycholecalciferol absorption in these patients was better maintained than that of cholecalciferol. These data indicate that vitamin D malabsorption reflects the extent of distal small-bowel resection in Crohn's disease. Treatment with oral cholecalciferol is sufficient in those with small or moderate resections but oral 25-hydroxycholecalciferol supplementation may be preferred in those with a severe short-bowel syndrome.

Absorption↗

Effect of intestinal resection on phospholipid class distribution and fatty acid composition of mucosal cells in the rat large intestine.

In this study we examined the effect of 50% distal small bowel resection (DSBR) on the lipid composition of rat colonic mucosa, including the fatty acid composition of major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), and sphingomyelin (SM). Phospholipid, free cholesterol, triacylglycerols, diacylglycerols, and cholesterol ester levels were not affected by the surgical operation. PE together with PC accounted for more than 60% of total phospholipid, in both groups of animals. The relative percentages of PE, PC, PI, SM, and cardiolipin (CL) remained unaffected by DSBR. However, a decrease in the content of LPC was observed in 50%-resected animals. Different fatty acid composition changes in the colonic phospholipid classes were observed after resection. PC fraction contained the highest amounts of saturated fatty acids, but monounsaturated fatty acids were present in high levels in PI fraction, in both groups of animals. DSBR produces different changes in the levels of linoleic and arachidonic acids. These results suggest that the lipid composition and the fatty acid profile of the different phospholipid fractions change in the adaptive response of colon to intestinal resection.

Animals↗

Laparoscopic mid sagittal hemicystectomy and bladder reconstruction with small intestinal submucosa and reimplantation of ureter into small intestinal submucosa: 1-year followup.

PURPOSE: We evaluated the long-term results of laparoscopic hemicystectomy and bladder replacement with small intestinal submucosa (SIS) with ureteral reimplantation into the SIS material. MATERIALS AND METHODS: A total of 12 minipigs underwent laparoscopic hemicystectomy. Six pigs underwent bladder reconstruction with SIS and ipsilateral ureteral reimplantation. The remaining 6 control pigs underwent hemicystectomy and primary bladder closure with ipsilateral nephroureterectomy. Preoperative and followup evaluations included blood chemistry, radiography and urodynamic evaluations. The 6, 3, 6 and 9-week, and 12-month followup evaluations included biopsies. At 1 year the animals were sacrificed. Histopathological and contractility studies, and reverse transcriptase-polymerase chain reaction for growth factors and basement membrane components were performed. RESULTS: Bladder capacity and bladder compliance were similar in the 2 groups at all time points. One pig per group died, that is a control at the 9-month evaluation due to an anesthetic complication and an SIS pig 7 months after bladder reconstruction due to spontaneous bladder rupture at the anastomotic site. In the SIS group 4 of 5 surviving pigs had unobstructed reimplanted ureters without evidence of hydroureteronephrosis, while 1 had high grade obstruction at the reimplantation site. Histopathology study after 1 year revealed muscle at the graft periphery and center but it consisted of small fused bundles with significant fibrosis. Nerves were present at the graft periphery and center but they were decreased in number. CONCLUSIONS: Laparoscopic SIS bladder reconstruction and ureteral reimplantation into the SIS after hemicystectomy are technically feasible. However, compared to primary bladder closure no advantage in bladder capacity or compliance was documented.

Animals↗

Endogenous ethanol production in a patient with chronic intestinal pseudo-obstruction and small intestinal bacterial overgrowth.

The case of the gastrointestinal production of ethanol from Candida albicans and Saccharomyces cerevisiae in a Caucasian man with chronic intestinal pseudo-obstruction is reported. The patient, who declared to have always abstained from alcohol, was hospitalized for abdominal pain, belching and mental confusion. The laboratory findings showed the presence of ethanol in the blood. Gastric juice and faecal microbiological cultures were positive for C. albicans and S. cerevisiae. At home, he was on oral antibiotic therapy with amoxicillin plus clavulanic acid for a small bowel bacterial overgrowth, associated with a simple sugar-rich diet. Twenty-four hours after stopping both the antibiotic therapy and the simple sugar-rich diet, the blood ethanol disappeared. A provocative test, performed by giving amoxicillin plus clavulanic acid associated with the simple sugar-rich diet was followed by the reappearance of ethanol in the blood. A review of the literature is reported.

Adult↗

Purification and characterization of two aminopeptidases from guinea-pig small-intestinal mucosa. Cavian intestinal tripeptide hydrolases.

Two electrophoretically distinct cytosolic peptide hydrolases from guinea-pig small-intestinal mucosa have been highly purified by a six-step procedure comprising extraction from mucosal homogenate, ammonium sulphate fractionation, DEAE-cellulose chromatography, chromatofocusing, calcium phosphate chromatography and Sephadex G-100 gel filtration. They have similar apparent molecular masses as determined by gel filtration (Mr = 68 000) or by sodium dodecyl sulphate gel electrophoresis (Mr = 72 000). Both are aminopeptidases with optimum activity at pH 7.6. They are strongly inhibited by p-hydroxymercuribenzoate, o-phenanthroline and bestatin. Although both hydrolyse some dipeptides they have a distinctive kinetic preference for tripeptides composed of aromatic or non-polar residues. Their affinities for some tripeptides are particularly high and also the hydrolysis of some substrates exhibits biphasic kinetics. These two aminotripeptidases are similar but they can be differentiated from each other and from a number of other aminopeptidases.

Aminopeptidases↗

Intestinal absorption of drugs. The influence of mixed micelles on on the disappearance kinetics of drugs from the small intestine of the rat.

The solubilization of the hydrophilic drugs paracetamol and theophylline, and the lipophilic drugs dantrolene, griseofulvin and ketoconazole has been determined in mixed micellar aqueous dispersions composed of 10 mM taurocholate + 5 mM oleic acid. The solubilization of dantrolene and paracetamol has also been determined in aqueous (mixed) micellar dispersions of 1 g L-1 lysophosphatidyl-choline (LPC), or taurocholate/LPC. The influence of these (mixed) micelles on the absorption of the model drugs from solution was studied in the rat chronically isolated internal loop. Absorption kinetics of the drugs were evaluated on the basis of the disappearance rate of the drug dissolved in the perfusion medium in this loop. Absorption experiments with taurocholate/oleic acid in the perfusate resulted in a reduction of the disappearance rate for the lipophilic drugs and the hydrophilic drug theophylline. This could partly be ascribed to the decreased fraction of drug free in solution as a result of its micellar solubilization for dantrolene, griseofulvin and ketoconazole, but the decrease in the disappearance rate of theophylline was unexpected. Taurocholate/oleic acid, LPC and taurocholate/LPC micelles had no effect on the disappearance of paracetamol. The disappearance rate of dantrolene in the presence of LPC alone was not altered, in spite of the decreased fraction of the drug free in solution owing to its micellar solubilization. In contrast, taurocholate/LPC micelles caused a reduction in the rate of disappearance of dantrolene, as expected according to the phase-separation model. In-vitro, taurocholate and taurocholate/LPC reduced the molecular cohesion of porcine intestinal mucus, whereas LPC alone did not exhibit an effect on the gel structure of mucus.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaminophen↗

Vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide receptor chimeras reveal domains that determine specificity of vasoactive intestinal polypeptide binding and activation.

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are closely related G protein-coupled receptors with seven-transmembrane domains. The VIP receptor can bind both VIP and PACAP with high affinity, whereas the PACAP receptor binds only PACAP with high affinity. To elucidate the structural domains involved in a selectivity for VIP binding and the subsequent receptor activation, a series of chimeric receptors between the VIP and PACAP receptors was constructed, expressed in COS-7 cells, and analyzed for ligand binding and cAMP generation. All chimeric constructs bound PACAP with high affinity and subsequently activated cAMP generation similarly to the wild-type receptors. In contrast, profound differences were observed in the potencies of VIP for competition of 125I-labeled PACAP binding to both wild-type receptors and the chimeric receptors. The cAMP responses of these receptors generally correlated with the ability of VIP to compete for PACAP radioligand binding with the exceptions for some particular chimeras. In this report we demonstrate that several domains, including the amino-terminal extracellular domain, the transmembrane domains I and II, and the first extracellular loop of the VIP receptor, are important for the selectivity for VIP binding and responsiveness to VIP. We further show that the third extracellular loop and its proximal domains of the VIP receptor appear to be involved in the VIP recognition, especially the receptor activation process. On the other hand, the direct binding experiments of the VIP radioligand demonstrated that both wild-type receptors and all chimeric receptors have a high affinity binding site for VIP, although this high affinity VIP binding resulted in a biological response only in the VIP receptor or VIP receptor-like chimeras. This suggests that there is a nonbiologically relevant high affinity VIP-binding site within the rat PACAP receptor.

Amino Acid Sequence↗

Identification of novel genes in intestinal tissue that are regulated after infection with an intestinal nematode parasite.

Infection of resistant or susceptible mice with Trichuris muris provokes mesenteric lymph node responses which are polarized towards Th2 or Th1, respectively. These responses are well documented in the literature. In contrast, little is known about the local responses occurring within the infected intestine. Through microarray analyses, we demonstrate that the gene expression profile of infected gut tissue differs according to whether the parasite is expelled or not. Genes differentially regulated postinfection in resistant BALB/c mice include several antimicrobial genes, in particular, intelectin (Itln). In contrast, analyses in AKR mice which ultimately progress to chronic infection provide evidence for a Th1-dominated mucosa with up-regulated expression of genes regulated by gamma interferon. Increases in the expression of genes associated with tryptophan metabolism were also apparent with the coinduction of tryptophanyl tRNA synthetase (Wars) and indoleamine-2,3-dioxygenase (Indo). With the emerging literature on the role of these gene products in the suppression of T-cell responses in vitro and in vivo, their up-regulated expression here may suggest a role for tryptophan metabolism in the parasite survival strategy.

Animals↗

Studies of intestinal lymphoid tissue. I. Electron microscopic evidence of 'blast transformation' in epithelial lymphocytes of mouse small intestinal mucosa.

The morphology of epithelial lymphocytes in osmium-fixed, Epon-embedded jejunum of adult mice was studied by light and electron microscopy. Toluidine blue-stained 1 mum 'thick' plastic sections were compared with adjacent thin sections, thereby permitting precise ultrastructural identification and description of selected epithelial lymphocytes. Their size and appearances varied considerably, ranging from typical small lymphocytes through medium-sized lymphocytes to large immunoblasts. A high proportion of medium-sized epithelial lymphocytes (mean diameter 6-9 +/- 1-1 mum) contained several lysosomes, extensive Golgi complexes, prominent centrioles and abundant ribosomes. Their appearances, therefore, corresponded directly to mitogen-stimulated lymphocytes. In contrast, immunoblasts were big cells (mean diameter 11-0 +/- 0-8 mum) with large, euchromatic nuclei and prominent nucleoli. The majority had pale-staining, ribosome-studded cytoplasm and thus resembled type I, or T blasts. Very rarely, densely staining blasts containing ribosomes and well developed rough endoplasmic reticulum were observed; these corresponded to type II or B blasts. These observations indicate that transformation of lymphocytes occurs within the interepithelial cell spaces of the small intestinal mucosa, suggesting that epithelial lymphocytes are immunocompetent cells which may be responsive to local antigenic stimulation.

Animals↗

Mucosal surface pH of the large intestine of the rat and of normal and inflamed large intestine in man.

The surface pH of rat distal colonic mucosa and human rectal mucosa was measured in vitro using first a small pH electrode with a flattened tip. In buffer with pH 7.56 the mean rat colonic surface pH was 6.72. Lowering the buffer pH in steps resulted in a small fall in surface pH, the values being buffer pH 7.06 surface pH 6.64, buffer pH 6.58 surface pH 6.61 and finally buffer pH 6.09 surface pH 6.39. Similar results were obtained with a buffer where butyrate, 30 mmol/l replaced chloride and when a CO2/bicarbonate buffer was used. During the time taken for the study transmural potential difference only changed by 1-2 mV. Serosal surface pH changed with buffer pH, suggesting that the maintained surface pH is a property of the mucosal surface only. The surface pH of human rectal mucosa was similar to that of rat distal colonic mucosa. As buffer pH fell from pH 7.51 to 5.96 mucosal surface pH only fell from pH 6.80 to 6.26. The values obtained in ulcerative proctitis did not differ from normal mucosa. Secondly pH microelectrodes were used to measure the juxta mucosal pH and the pH-microclimate thickness when luminal pH was controlled. The microclimate had a pH 6.63 adjacent to the mucosa with a thickness of 840 micron. The importance of mucus in maintaining the microclimate was shown by n-acetyl cysteine thinning and prostaglandin E2 thickening the layer. These results describe a surface microclimate in the large intestine of appreciable thickness and a constant juxta mucosal pH. Luminal pH changes produce only a small change in microclimate pH.

Adult↗

Relative contribution of Peyer's patches to intestinal DNA content and tritiated thymidine content of the mouse small intestine.

The quantification of mucosal DNA as a measure of epithelial cellularity has been criticized because it would include the non-epithelial cell populations present in the mucosa. The weight, DNA content and tritiated thymidine uptake of Peyer's patches and the remaining mucosa were measured and compared. Although the Peyer's patches only contribute 2.5% to the weight of the small intestine, they contained 12% of the DNA and 7% of the tritiated thymidine which would be found in a mucosal scrape.

Animals↗

Studies on intestinal absorption of sulpiride (2): transepithelial transport of sulpiride across the human intestinal cell line Caco-2.

To determine the transport mechanism of sulpiride in an in vitro model of the human intestine, we investigated the transepithelial transport of this agent in Caco-2 cells. The transepithelial transport and intracellular accumulation of sulpiride were measured using Caco-2 cell monolayers cultured on a permeable membrane. The transepithelial transport of sulpiride in Caco-2 cells showed temperature dependence, and the transport was enhanced at weakly acidic pH on the apical side. These results demonstrate that the transepithelial transport of sulpiride is carrier mediated. To identify the drug transporter species that take part in the transepithelial transport of sulpiride, we examined the effects with the addition and preloading with specific substrates and inhibitors of various drug transporters. The results obtained from these examinations indicated that the apical-to-basolateral transport of sulpiride is mediated by the peptide transporter PEPT1, organic cation transporters OCTN1 and OCTN2 on the apical membrane, and the basolateral peptide transporter on the basolateral membrane. The basolateral-to-apical transport is mediated by the basolateral peptide transporter and organic cation transporter OCT1 on the basolateral membrane and by P-glycoprotein on the apical membrane. A decrease in the absorption of sulpiride may occur in coadministration protocols involving PEPT1-, OCTN1-, and OCTN2-transported drugs. Coadministration using the P-glycoprotein-transported drugs, in contrast, may enhance the absorption of sulpiride.

Biological Transport↗