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We investigated light and electron microscopic localization of ornithine transcarbamylase (OTC) in rat intestinal mucosa. In the immunoblotting assay of OTC-related protein, a single protein band with a molecular weight of about 36,500 is observed in extracts of liver and small intestinal mucosa but is not observed in those of stomach and large intestine. For light microscopy, tissue slices of the digestive system were embedded in Epon and stained by using anti-bovine OTC rabbit IgG and the immunoenzyme technique. For electron microscopy, slices of these and the liver tissues were embedded in Lowicryl K4M and stained by the protein A-gold technique. By light microscopy, the absorptive epithelial cells of duodenum, jejunum, and ileum stained positively for OTC, but stomach, large intestine, rectum, and propria mucosa of small intestine were not stained. Electron microscopy showed that gold particles representing the antigenic sites for OTC were confined to the mitochondrial matrix of hepatocytes and small intestinal epithelial cells. However, the enzyme was detected in mitochondria of neither liver endothelial cells, submucosal cells of small intestine, nor large intestinal epithelial cells. Labeling density of mitochondria in the absorptive epithelial cells of duodenum, jejunum, and ileum was about half of that in liver cells.
After ligation of the vascular arcades of the upper jejunum in rats, the ischemic damage to the intestinal mucosa and its regenerative behavior after ischemia lasting 120 minutes were investigated with histological and enzyme-histochemical methods. During the ischemic injury of the jejunal mucosa, there is rejection of hydropically swollen epithelial cells into the intestinal lumen advancing from the tip to the base of the villi without a previously detectable loss of activity of the enzymes investigated. At the end of ischemia lasting 120 minutes, there is complete destruction of the villi as well as the upper portions of the crypts. After rapid re-epithelialization of the mucosal surface by a flat epithelium, reformation of villi begins already after a 24 h period of regeneration. On the third day of regeneration, the intestinal mucosa has plump villi again. Their epithelia already show the morphological characteristics of mature enterocytes. On the other hand, enzymatic maturation of the enterocytes is only concluded between the first and second week of regeneration. An altered proliferation kinetics of the crypt epithelia due to the repair process is discussed as cause of this delayed enzymatic maturation of the enterocytes relative to the morphological differentiation. Although the intestinal mucosa has a regular villous structure again after regeneration lasting 8 days, enzyme histochemical finding in the lamina propria indicate that the process of repair is only complete here in the sixth week of regeneration.
OBJECTIVE: To observe the effects of Stragalus membranaceus injection on nitric oxide and endothelin levels of intestinal mucosa in reperfusion injury after hemorrhage shock. METHOD: 32 SD rats were randomly divided into four groups: normal group, model group, low dosage group, (treated with Astragalus membranaceus 10 g x kg(-1)); high dosage group (treated with Astragalus membranaceus 20 g x kg(-1)). Models of hemorrhagic shock for 60 minutes and reperfusion for 90 minutes were created. The animals were administrated 3 mL therapeutic solution before reperfusion. At the end of study, intestinal pathology was observed, and the concentration of lactic acid (LD), nitric oxide (NO), endothelin (ET) of intestinal mucosa were detected. RESULT: The intestinal pathology showed that intestinal mucosa epithelial cells damage in model group was severe, in low dosage group was medium, in high dosage group was slight, and no obvious damage was found in normal group. The concentration of LD and NO of small intestine mucous membrane in model group and low dosage group were significantly higher than those in high dosage group and normal group (P < 0.05), but there were no significant differences between high dosage group and normal group (P > 0.05). The concentration of ET of small intestine mucous membrane in model group was the highest of the four groups (P < 0.05). The concentration of ET in low dosage group was significantly higher than that in high dosage group and normal group (P < 0.05), but there were no significant differences between high dosage group and normal group (P > 0.05). CONCLUSION: Stragalus membranaceus injection can reduce small intestine mucous damage by protecting endothelium function in injury after hemorrhage shock-reperfusion.
RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.
Studies into the activity of adenosine triphosphatase (ATPase) in homogenates of liver, cerebral cortex, renal cortex, and mucosa of small intestine of swine have shown differentiated activity patterns, with peak activity developing in the liver. This has been related to a particularly high metabolism performance of the liver in fattening pigs. No difference was found to exist between magnesium activation of ATPase of swine tissue homogenates and that in tissue obtained from ruminants. ATPase which could be activated by sodium and potassium ions and inhibited by ouabain was detectable from cerebral and renal cortex. Sodium and potassium ATPases accounts from some 25 per cent of the total activity. ATPase that could be stimulated by calcium ions was recorded only from liver homogenate. The optimum pH values of ATPase were between 7.5 and 8 in the liver, 9 in mucosa of small intestine, and 9.5 in cerebral and renal cortex.
A dipeptide hydrolase from the brush border of guinea-pig intestinal mucosa was purified. The enzyme resembles another dipeptide hydrolase isolated from the cytosol fraction of intestinal mucosa. Studies on the binding of cytosol peptide hydrolase to brush-border membranes indicate that the enzyme found in the brush border may be a cytoplasmic contaminant.
The authors examined the quantitative changes in the glycogen content in rat's small intestinal mucosa under the effect of insulin. They found that glycogen content increases significantly during the first 60 minutes, then it decreases gradually, and regains the control value only after 6 hours. It appears that as regards its metabolic activity, the small intestinal mucosa is not an insulin resistant organ.
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Benzo(a)pyene hydroxylation activity was solubilized from rabbit intestinal mucosa microsomes and reconstituted with a cytochrome P-450 preparation obtained by fractionation with 6-amino-n-hexyl Sepharose 4B, hydroxylapatite and CM-Sephadex C-50, and partially purified NADPH-cytochrome c reductase. Phosphatidylserine was required for the maximal activity, while phosphatidylcholine had no stimulatory effect. The carbon monoxide difference spectrum of the cytochrome P-450 fraction showed a maximum peak at 450 nm. Although another cytochrome P-450 fraction was active for hexadecane hydroxylation, this fraction had little activity. The results indicate that more than one cytochrome P-450 exists in the intestinal mucosa microsomes.
1. The presence of an active acyl-CoA-lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-(14)C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-(14)C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the beta-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-(14)C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order.
Migration of intraepithelial lymphocytes (IELs) into intestinal epithelium is not yet well understood. We established an IEL-cell line from ovalbumin (OVA) 23-3 transgenic (Tg) mice and investigated the effect of antigen stimulation on the dynamic process of IEL migration into small intestinal mucosa. The cell line was a T cell receptor (TCR) alphabeta(+) CD4(+) CD8(-) phenotype, expressing alphaEbeta7 integrin in 90% of cells. Under intravital microscopy, the lined IELs adhered selectively to the microvessels of the intestinal villus tip of the Tg mice. The accumulation of IELs was significantly inhibited by an antibody against beta7-integrin and MAdCAM-1. When IELs were stimulated with OVA, the accumulation was attenuated compared to that of resting cells, with decreased expression of alphaEbeta7 integrin. In Tg mice fed with OVA, the number of IELs which migrated in the villus mucosa was significantly smaller than in the non-fed controls. The preferential migratory capacity of IELs to villus mucosa may be altered by specific antigen stimulations.
Intestinal mucosa of rats was prepared by squeezing the frozen and thawed intestine. The method was much easier compared to the conventional method in which intestine was cut longitudinally or everted and mucosa was scraped. Jejunal mucosal weight prepared by the two methods was not different, but ileal mucosal weight prepared by squeezing was significantly heavier than that by scraped. No significant difference was observed between activity of some brush border enzymes (arylamidase, aminopeptidase, alkaline phosphatase, and sucrase) in mucosa prepared by squeezing and that by conventional scraping method in jejunum. Activity of brush border aminopeptidase and sucrase in ileal mucosa prepared by squeezing was significantly higher than that prepared by scraping.
A total of 67 specimens of proximal intestinal mucosa, one from each subject, were examined under dissecting microscopy (DM) and light microscopy (LM). Majority of the subjects were adults and males. The overall per-oral biopsy failure was 13. DM was found to be useful to demonstrate gross villous atrophy. It also helped to orientate the specimens properly before fixation for embedding and sectioning. DM and LM findings did not concord in 17% of the specimens. In all of these specimens the mucosal surface which appeared normal under DM revealed grade-2 change under LM. The present study showed that dissecting microscopy (DM) of the intestinal mucosa is not an alternative for histologic examination under LM. However, scanner of light microscope (SLM) can be used as an alternative for DM.
Peptides and peptidomimetics often exhibit poor oral bioavailability due to their metabolic instability and low permeation across the intestinal mucosa. N-Methylation has been used successfully in peptide-based drug design in an attempt to improve the metabolic stability of a peptide-based lead compound. However, the effect of N-methylation on the absorption of peptides through the intestinal mucosa is not well understood, particularly when transporters, i.e. the oligopeptide transporter (OPT) and P-glycoprotein (P-gp), modulate the passive diffusion of these types of molecules. To examine this, terminally free and terminally modified (N-acetylated and C-amidated) analogs of H-Ala-Phe-Ala-OH with N-methyl groups on either the Ala-Phe or Phe-Ala peptide bond were synthesized. Transport studies using Caco-2 cell monolayers, an in vitro model of the intestinal mucosa, showed that N-methylation of the Ala-Phe peptide bond of H-Ala-Phe-Ala-OH stabilized the molecule to protease degradation, and the resulting analog exhibited significant substrate activity for OPT. However, N-methylation of the Phe-Ala peptide bond of H-Ala-Phe-Ala-OH did not stabilize the molecule to protease degradation, and the substrate activity of the resulting molecule for OPT could not be determined. Interestingly, N-methylation of the Phe-Ala peptide bond of the terminally modified tripeptide Ac-Ala-Phe-Ala-NH2 decreased the substrate activity of the molecule for the efflux transporter P-gp. In contrast, N-methylation of the Ala-Phe peptide bond of the terminally modified tripeptide Ac-Ala-Phe-Ala-NH2 increased the substrate activity of the molecule for P-gp.
As early as the beginning of the twentieth century some data indicated that macromolecules are able to cross the intestinal mucosa to reach the blood. Further evidence was added over the years; however, pathways for this transport still remain to be established. We report here the transfer of two pancreatic enzymes, amylase and lipase, from the intestinal lumen to the blood. Both are present in higher concentrations in the intestinal mucosa and in blood of fed rats. Upon cholinergic stimulation of pancreatic secretion, there was not only an increase in blood enzyme concentrations, but evidence for internalization by duodenal enterocytes was obtained. Following insertion of fluorochrome-tagged amylase and lipase into the duodenal lumen of fasting rats, blood and intestinal tissues were sampled at different time points. Serum activities for both enzymes clearly increased with time. Light microscopy established internalization of both proteins by duodenal enterocytes, and immunogold outlined the pathway taken by both proteins across the enterocytes. From the intestinal lumen, enzymes are channeled through the endosomal compartment to the Golgi apparatus and to the basolateral membrane reaching the interstitial space and blood circulation. Transcytosis through the intestinal mucosa thereby represents an access route for pancreatic enzymes to reach blood circulation.
PURPOSE: The aim of this study is to investigate species differences in the stereoselective hydrolysis for propranolol ester prodrugs in mammalian intestinal mucosa and Caco-2 cells. METHODS: Hydrolase activities for propranolol prodrugs and p-nitrophenylacetate in man (age: 51-71 years), the beagle dog (age: 4 years) and Wistar rat (age: 8 weeks) intestinal mucosa, and also in Caco-2 cells (passage between 60-70) were estimated by determining the rate of production of proparanolol and p-nitrophenol, respectively. RESULTS: The hydrolase activities for both propranolol prodrugs and p-nitrophenylacetate were in the order of man > rat >> Caco-2 cells > dog for intestinal microsomes, and rat > Caco-2 cells = man > dog for intestinal cytosol. Dog microsomes showed stereoselective hydrolysis for propranolol prodrugs, but not those from human or rat. Interestingly, both subcellular fractions of Caco-2 cells showed remarkable R-enantioselectivity except acetyl propranolol. Enzyme kinetic experiments for each enantiomer of butyryl propranolol in microsomes revealed that dog possesses both low and high affinity hydrolases. Both Km and Vmax values in rat were largest among examined microsomes, while Vmax/Km was largest in man. Finally, it was shown that the carboxylesterases might contribute to the hydrolysis of propranolol prodrug in all species by inhibition experiments. CONCLUSIONS: The hydrolase activities for propranolol prodrugs and p-nitrophenylacetate in intestinal mucosa showed great species differences and those in human intestine were closer to those of rat intestine than dog intestine or Caco-2 cells.
We have investigated the longitudinal distribution of glutathione S-transferase (GST) isozymes in the trisected small intestine mucosa of rats. Only GST subunits 1 and 7 were detected by Western blot analysis of the intestinal cytosol using antiserum for GST1-1, 1-2, 3-4 and 7-7. Cytosolic GST1-1, 3-4 and 7-7 were assayed by the quantitative ELISA. There was a marked decline of the concentration of GST1-1 from proximal (35.17 nmol/g tissue) to distal intestine (1.67 nmol/g tissue). GST3-4 was hardly detected in the intestinal mucosa. Among the GSTs, GST7-7 existed in the highest concentration in any segment of intestine, i.e. 58.76 nmol/g tissue (61% of GSTs) in the proximal intestine and 32.38 nmol/g tissue (93% of GSTs) in the distal intestine.