Intestinal Slices Research Articles

Purification and properties of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

A phosphatidylinositol-specific phospholipase C was purified from the culture broth of Bacillus thuringiensis to a homogeneous state as indicated by polyacrylamide gel electrophoresis. Specific activity of purified enzyme was 312 units/mg, and the recovery of the enzyme activity was 27.2%. The purified enzyme (molecular weight: 23 000 ± 1000) was maximally active at pH 7.5 and not influenced by EDTA. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and sphingomyelin. The products from phosphatidylinositol of enzyme reaction were diacylglycerol and myo-inositol 1,2-cyclic phosphate. The enzyme activity was stimulated by sodium deoxycholate or Triton X-100. Divalent cations such as Ca 2+, Mg 2+ and Zn 2+ were inhibitory at concentrations above 10 −3 M. KCl and NaCl were inhibitory at the concentration higher than 10 −2 M. Alkaline phosphatase, an ecto-enzyme located on the surface of plasma membrane, was released from the slices of rat liver, kidney, pancreas and intestine by the treatment with this phospholipase.

Effect of sucrose feeding upon intestinal and hepatic lipid synthesis

Sucrose induces hypertriglyceridemia and increases intestinal triacylglycerol (TG), cholesterol (C), and very low density lipoprotein (VLDL) secretion in the rat. The effect of feeding a sucrose-enriched diet for 2 to 3 weeks upon rat intestinal and hepatic C, fatty acid (FA), and TG synthesis was measured and compared to animals fed chow and glucose-enriched diets. Sucrose did not alter intestinal lipid composition but increased hepatic weight and TG content. Rates of FA and C synthesis were estimated from the incorporation of 14C-2-acetate and of 3H2O into slices of proximal and distal intestine and of liver. Jejunal, but not ileal, C synthesis was stimulated by sucrose feeding, which resulted in elimination of the jejunoileal gradient for C synthesis found in rats fed control diets. There was no change in intestinal FA synthesis and 14C palmitate incorporation into intestinal TG was reduced. In the liver, no change in hepatic C synthesis was observed, but incorporation of 14C-2-acetate into total fatty acid was increased 3-fold and 3H2O incorporation over 2-fold by sucrose feeding. The incorporation of palmitate into hepatic TG was relatively enhanced and into hepatic phosphoglycerides depressed by sucrose. It is concluded that sucrose feeding stimulates jejunal C synthesis and transport in intestinal lipoproteins but that hepatic C synthesis may be modulated to prevent hypercholesterolemia. Since sucrose feeding docs not increase intestinal lipogenesis, circulating FA may be precursors for VLDL TG secreted in lymph.

Effect of chemotaxis on the interaction of cholera vibrios with intestinal mucosa

Earlier reports from this laboratory have shown that chemotaxis is an important mechanism that expedites the in vitro association of cholera vibrios with intestinal slices and that affects the in vivo colonization and virulence of these bacteria to a significant degree. The data reported in the present communication indicate that there appears to be a chemotatic gradient attracting cholera vibrios not only to the surface of the mucus gel, but that this gradient continues for at least a considerable distance toward the base of the villi. It is shown further that a strain of Vibro cholerae was attracted by all 20 amino acids tested, in contrast to Escherichia coli AW405 which is repelled by several of these. Finally, experiments are described that show that superior in vivo colonization of chemotatic vibrios (compared to nonchemotactic mutants) was correlated with a significantly higher degree of mucosal association. Such increased mucosal association of chemotatic vibrios has previously been shown only with mucosal slices in vitro.

A Study of D-Glucose Uptake by Small Intestine of Rats following <i>in vivo</i> Vinblastine Treatment

The pharmacokinetics of tritiated vinblastine in rats has indicated a considerable excretion of the alkaloid through bile into the intestinal lumen. The present study was undertaken to find out whether the presence of the alkaloid in the lumen of small intestine alters the normal transport of nutrients across the small intestine. This was done by measuring the in vitro D-glucose uptake by slices of small intestine of rats pretreated with vinblastine. Vinblastine pretreatment was done by filling the lumen of in vivo segments of small intestine of rats with different concentrations of the alkaloid for 30 and 60 min before uptake was measured. A significant dose-dependent reduction in D-glucose uptake was observed.

Experimental clindamycin-associated colitis in rabbits: Evidence for toxin-mediated mucosal damage

Rabbits given clindamycin by nasogastric tube at a dosage of 15 mg per kg per day developed diarrhea within 48 to 72 hr which was uniformly fatal by 72 to 96 hr. Control rabbits given comparable doses of ampicillin, tetracycline, neomycin, vancomycin, or saline remained well. At autopsy the major gross pathological finding was limited to the cecum which was massively dilated with watery stool. The light microscopic findings in the cecum included necrosis of the surface epithelium and a prominent inflammatory infiltrate in the lamina propria. Fatal clindamycin colitis was prevented in 10 of 10 rabbits by co-administration of vancomycin, 30 mg per kg per day; coadministration of penicillin or neomycin prevented the ileocolitis in 3 of 5 and 1 of 5 animals, respectively. Cell proliferation, measured by radioautography after [3H]thymidine injection, was significantly increased in cecal mucosa but not more distal colonic or small intestinal mucosa during clindamycin administration. Protein synthesis, as determined by incorporation of [3H]leucine into intestinal slices, was significantly increased in both the ileum and cecum after 48 hr of clindamycin administration. Cell-free, sterile extracts of cecal contents of clindamycin-treated animals were injected into ileal loops of untreated rabbits. These fecal extracts produced a severe, hemorrhagic ileitis, although control filtrates from clindamycin-vancomycin-treated animals produced no histological changes. Using selective anaerobic culture media there was an increase in clostridial species from 105 organisms of cecal content per g in controls to 108 organisms per g after 48 hr of clindamycin treatment and a reduction to 104 organisms per g in clindamycin-vancomycin-treated animals. The pathogenesis of clindamycin-associated colitis in rabbits appears to be mediated by a bacterial toxin present in the cecal contents.

Properties and function of indoleamine 2,3-dioxygenase.

Available evidence indicates that indoleamine 2,3-dioxygenase, which is particularly abundant in the lung, colon, and intestine, catalyzes the oxidative ring cleavage of the pyrrole moiety of not only tryptophan and 5-hydroxytryptophan but also serotonin, melatonin, and other indoleamine derivatives. Our recent experiments with slices of intestine, together with those with organ culture of pineal glands, perfusion of heart and lung preparations and intact animals, which I did not have time to mention today, indicate that the oxygenative ring cleavages of various indoleamines do occur in vivo to a significant extent. Secondly, this enzyme appears to utilize superoxide anion rather than molecular oxygen. If superoxide anion is demonstrated, unequivocally, to be the true substrate of this enzyme, not only in vitro but also in vivo, then indoleamine 2,3-dioxygenase may be the first example of a new class of enzymes which utilize superoxide anion as an oxidizing agent just as peroxidases utilize hydrogen peroxide. Lastly, this novel enzyme produced a new class of biogenic amines, which may be referred to, generically, as "anthraniloylamines." Although their natural occurrence has not yet been established, they may have hitherto unknown biological or pharmacological activities and are currently under investigation in our laboratory.

A phylogenetic study of the role of cyclic AMP in lipid synthesis in vertebrates

The effect of cyclic AMP on the incorporation of acetate-2-14C into sterols and fatty acid in vitro in slices of liver and intestine was examined in various representatives of the vertebrate group. In no instance was an effect on lipid synthesis noted in intestine. Cyclic AMP exerted no significant effects on hepatic lipogenesis in lower vertebrates, including the nurse shark, catfish, toad, or iguana. However, the nucleotide strongly inhibited the incorporation of acetate-2-14C into fatty acid by the chicken liver. Similar inhibition of fatty acid synthesis was also noted in rat liver, but in this mammalian species hepatic sterol synthesis was also strikingly suppressed by cyclic AMP. Interruption of the enterohepatic circuit in the rat, while enhancing rates of sterol synthesis in both liver and intestine, neither enhanced nor diminished hepatic susceptibility to suppressed sterologenesis by cyclic AMP, nor did it confer on the intestine any newfound capacity for cyclic AMP-regulated lipid synthesis.

Acute and chronic effects of ethanol on intestinal lipid metabolism

To assess the effects of ethanol on intestinal lipid metabolism, fatty acid oxidation and triacylglycerol synthesis were measured in intestinal slices incubated with ethanol. Ethanol, when used in concentrations likely to be achieved in the upper jejunum after moderate drinking, inhibited both palmitate and acetate oxidation, CO 2 production and triacylglycerol synthesis, whereas it enhanced the esterification of fatty acid with ethanol. The concentrations required for the inhibitory effect were much higher than those needed to saturate enzyme systems known to participate in ethanol oxidation. In vivo administration of ethanol-containing diets produced persistent changes of the intestinal slices with respect to fatty acid oxidation and triacyl-glycerol synthesis. Acute intragastric administration of ethanol (3 g/kg) one hour prior to sacrifice, inhibited both processes in slices obtained from the jejunum, but not in those derived from the ileum. By contrast, chronic ethanol feeding increased the ability for fatty acid oxidation and triacylglycerol synthesis both in the jejunum and in the ileum. This stimulatory effect was associated with significant enhancement of palmitoyl-CoA synthetase activity, suggesting increased fatty acid activation. The inhibition by ethanol in high concentrations of intestinal fatty acid oxidation and triacylglycerol synthesis probably reflects epithelial cell damage; by contrast, prolonged administration of ethanol results in a persistent enhancement of lipid metabolism which may reflect the presence of a different cell population in the intestine.

A study of the cytoplasmic receptors for glucocorticoids in intestine of pre- and postweanling rats.

Glucocorticoids cause both enzymic and morphologic changes in the rat intestine during the time of weaning. To obtain information regarding the mechanism of these actions, we examined the cytoplasmic fraction of intestines from 18-day-old rats for the presence of specific glucocorticoid-binding proteins which are characteristics of target tissues. Incubation of slices of intestine with [3H]dexamethasone in a physiological medium at 2 degrees showed the presence of a cytoplasmic binding macromolecule with high specificity for steroids having glucocorticoid activity. The binding reaction was saturable (concentration of binding sites equals 0.24 pmol per mg of protein) and of high affinity (dissociation constant equals 9.3 nM). Binding was reversible on addition of nonlabeled dexamethasone (t 1/2 equals 5.2 hours), indicating that the usual assay procedure measured both corticosterone-filled and unoccupied binding sites. Sucrose density gradient centrifugation showed that the receptor-dexamethasone complex from intestinal cytosol sedimented at the same rate as that from liver (8.2 S). The receptor-dexamethasone complex was stable at 2 degrees for at least 24 hours in intestinal slices, but in isolated cytosol fractions there was considerable loss of binding even in the presence of high concentrations of [3H]dexamethasone. Furthermore, mixing experiments showed that the presence of cytosol from intestinal mucosa (but not from the muscle layers) caused a dissociation of dexamethasone from receptors of liver cytosol. This suggested the presence of some interfering factor in isolated mucosal cytosol and meant that quantitative studies had to be confined to intact slices. Although the reasons for the instability of steroid-receptor complexes in the presence of isolated intestinal cytosol are not understood, the instability is believed to be associated with homogenization and, therefore, is believed to have no physiological significance. Finally, the ontogenesis of cytoplasmic glucocorticoid receptors in intestinal slices was examined and the pattern compared with that in liver and lung. Receptor activity was present in intestine from late fetal life through adulthood, but concentrations were significantly higher during the first two postnatal weeks than at all other times. By contrast, receptor activity detected in cytosol prepared from rat lung was high around the time of birth, while that in liver rose steadily during the first postnatal week and remained at high levels. Thus specific receptors for glucocorticoids are present in the rat intestine during periods of both responsiveness and unresponsiveness. This suggests that although corticosteroids exert their effects through the cytoplasmic receptors, this early event in glucocorticoid action may not be a controlling step for changes in responsiveness during development.

The biosynthesis of a fucose-containing glycoprotein from intestinal mucosa of normal and vitamin a-deficient rats

Abstract The intestinal mucosa of rats which are vitamin A-deficient yield a fucose-containing glycopeptide with a 3-fold lower specific activity in glucosamine when labeled in vivo or in vitro and compared to pair-fed controls (De Luca, L., Schumacher, M. and Wolf, G. (1970) J. Biol. Chem. 245, 4551–4558). The glycoprotein from which the glycopeptide has been derived, has also been isolated and characterized (Kleinman, H. and Wolf, G. (1974) Biochim. Biophys. Acta, in the press). Glucosamine, fucose, galactose and threonine all showed markedly reduced incorporation in vivo into the fucose-containing glycoprotein in vitamin A deficiency at various time points up to 135 min. Galactose was verified as the principal source of the label in the glycoprotein after labeled galactose administration. Uptake was rapid, followed by a decline for glucosamine and fucose; uptake rose slowly to the end of the experimental period for threonine and galactose. An in vitro secretion study revealed that the fucose-containing glycoprotein is present in the incubation medium of an intestinal slice system, with reduced incorporation of glucosamine from vitamin A-deficient compared to normal rats. It seems likely that the vitamin A-sensitive glycoprotein is not secreted into the medium in soluble form, but resides in desquamated cells.

Synthesis of intestinal glycoproteins inhibitions of [1-14C]glucosamine incorporation by phenylbutazone in vitro

Abstract 1. 1. Phenylbutazone (5 mM) inhibits incorporation of [1- 14 C]glucosamine and [ 14 c]leucine into acid precipitable protein by rat intestinal slices. 2. 2. Inhibition of [1- 14 C]glucosamine incorporation was independent of medium glucosamine concentration. 3. 3. Phenylbutazone inhibited [1- 14 C]glucosamine incorporation by cycloheximide-treated slices. Reduced [1- 14 C]glucosamine incorporation did not result solely from inhibition of protein synthesis. 4. 4. Analysis of mucosal acid soluble intermediate labelling revealed an increase in glucosamine and a reduction in all hexosamine intermediates following phenylbutazone. 5. 5. These effects closely parallel previous experience with sodium salicylate and may represent a common basis for their adverse gastrointestinal effects.

Effect of actinomycin D on vitamin D-mediated uptake of 45Ca by intestinal slices.

Vitamin D significantly increases the uptake and transport of calcium in the duodenum of D-deficient rats fed either high or low calcium diets. In previous work by others showing that actinomycin D blocked the effect of vitamin D, low calcium: adequate phosphorus animals were used, leaving unanswered the question as to whether actinomycin D would have a similar effect in animals fed a high calcium: low phosphorus diet. With treatment combining ±vitamin D and ±actinomycin D, slices of duodenum were tested for passivevs. active uptake of calciumin vitro. The transport of calcium across the intestine into blood was measuredin vivo. With regard to intestinal uptake, there was no interaction between actinomycin D and vitamin D in animals fed 1.2% Ca: 0.06% P diet for 20 days before treatment. In those animals fed the 0.02% Ca: 0.3% P diet for the same period of time, actinomycin D reduced the uptake of intestinal slicesin vitro but, when combined with vitamin D, the vitamin significantly enhanced the uptake of calcium, though to a lesser extent, in the presence of the antibiotic. Vitamin D tended to increase the transport of calcium into blood of animals on both diets in the presence or absence of actinomycin D.

Gluconeogenesis and Amino Acid Metabolism<subtitle>III. Uptake of Glutamine and Output of Alanine and Ammonia by Non-hepatic Splanchnic Organs of Fasted Rats and Their Metabolic Significance</subtitle>

Amino acid metabolism in non-hepatic splanchnic organs of fasted rats was studied both in vitro and in vivo to determine the roles of glutamine, glutamate, alanine, and ammonia in the amino acid metabolism of these organs and of the whole body. First, arterial-portal vein differences in the plasma concentrations of alanine and ammonia, but not of glutamate, were shown to increase 1 to 5min after loading-glutamine through the femoral vein, suggesting conversion of the nitrogen of glutamine to that of alanine and ammonia. Second, glutamine was incubated with slices of small intestine and the production of large amounts of glutamate and ammonia, but only a small production of alanine, were observed with a concomitant decrease in glutamine. This result is in sharp contrast to in vivo findings. Third, non-hepatic splanchnic organs were perfused to investigate the problem further by reproducing the in vivo metabolism in vitro. When the organas were perfused without addition of glutamine, the amounts of glutamine in the perfusate and in the-, intestine did not change appreciably, but alanine and ammonia increased both in the-perfusate and in the perfused intestine. The increases in alanine and ammonia in the perfusate were stimulated by the addition of glutamine to the perfusate. Production of ammonia was also demonstrated on perfusing the same organs from germ-free rats. These results are largely consistent with in vivo findings and indicate, that the nitrogen of glutamine and other amino acids is converted to that of alanine and ammonia in non-hepatic splanchnic organs. In these organs (presumably of the intestine), alanine and ammonia are considered to be “ end products” of amino acid metabolism that are conveyed to the liver. This is in contrast to the fact that the “ end products” of amino acid metabolism in the kidney are serine and ammonia, while those in peripheral tissues, including the skeletal muscle, skin, and adipose tissue, are alanine and glutamine. Finally, evidence was obtained which suggested that non-hepatic splanchnic organs may play a major role in the trunover of plasma, glutamine and in the supply of alanine for hepatic gluconeogenesis in fasted rats.

Cyclic AMP and intestinal glycoprotein synthesis: the effect of -adrenergic agents, theophylline, and dibutyryl cyclic AMP.

Incorporation of [1-14C]glucosamine by acid-precipitable protein was enhanced in intestinal slices by isoproterenol, epinephrine, theophylline, and dibutyryl cyclic AMP. Moreover, the effect of either isoproterenol or epinephrine on [1-14C]glucosamine incorporation was markedly potentiated by the addition of theophylline. Uptake of [1-14C]glucosamine into the acid-soluble compartment of intestinal slices was not significantly affected by isoproterenol, epinephrine, or theophylline.The stimulatory effect of isoproterenol and theophylline was completely inhibited by cycloheximide. Subfractionation of intestinal slice homogenates indicated that increased synthesis was not confined to one glycoprotein class but appeared to effect mucins and glycocalyx glycoproteins equally. These results indicate that β-adrenergic hormones and cyclic AMP stimulate intestinal glycoprotein synthesis and may control the production of glycoproteins located at the intestinal surface.

Effect of soy sterols on cholesterol synthesis in the rat.

AbstractSoy sterols increased the incorporation of acetate into cholesterol by rat liver slices and prevented the depression of cholesterogenesis caused by dietary cholesterol. Acetate incorporation into cholesterol by intestinal slices was not affected by soy sterols but was decreased by cholesterol. The results with liver slices agree with the view that soy sterols, by interfering with the intestinal absorption of cholesterol, prevent the feedback inhibition of hepatic cholesterogenesis.

Uptake of vitamin E by rat small intestinal slices

1. 1. The uptake of vitamin E (α-tocopherol) by rat small intestinal slices from micellar incubation media of varying composition has been examined. 2. 2. About 82% of the radioactivity recovered from tissue lipids was associated with the mucosal layer, and was shown by thin-layer chromatography to be mostly unchanged α-tocopherol. 3. 3. Uptake of α-tocopherol (30 min, 37°C) was not substantially affected by: adding 5 mM monoolein, with or without 5 mM oleic acid, to 10 mM sodium taurodeoxycholate; increasing sodium taurodeoxycholate from 10 to 30 mM, or changing pH from 5.3 to 7.2. Uptake was greatly reduced by addition of an oil phase (triolein) to the micellar solution or by absence of a micellar phase. 4. 4. When the concentration of α-tocopherol in the incubation medium was increased beyond the turbidity point a substantial change in uptake occurred only in one-quarter of the experiments.

Influence of glucose on intestinal transport of phenylalanine in the presence and absence of dinitrophenol

Glucose inhibits the accumulation ofl-phenylalanine by rat intestinal slices during 45-minute incubations. In the presence of DNP (which alone abolishes all active transport), glucose, but not galactose, significantly stimulates amino-acid uptake. However, during short incubations, glucose inhibits the entry of phenylalamine into the tissue, both in the presence and absence of DNP.

Synthesis of intestinal clycoproteins inhibition of [1- 14C]glucosamine incorporation by sodium salicylate in vitro

1. 1. Sodium salicylate at concentrations of 7.5 mM and 15.0 mM inhibited incorporation of [I- 14C]glucosamine into glycoprotein by slices of rat small intestine. This inhibition was dose dependant and not due to an inhibition of glucosamine uptake by the intestinal mucosa. It was also unaffected by the concentration of glucosamine in the incubation medium. 2. 2. A comparison of the effect of sodium salicylate on both [1- 14C]glucosamine and L-[U- 14C]leucine incorporation revealed that leucine incorporation was more markedly inhibited at early time periods. These results indicate that the inhibition of glucosamine incorporation may in part result from a reduced synthesis of peptide acceptor. 3. 3. The addition of salicylate to slices in which protein synthesis had been reduced to 2.0% by cycloheximide resulted in an additional 38% inhibition of glucosamine incorporation. 4. 4. Radioactive free glucosamine was increased in acid-soluble intermediates of slices exposed to salicylate and cycloheximide as compared to those exposed to cycloheximide alone, while radioactivity in acetylated acid soluble intermediates was decreased. 5. 5. Salicylate therefore inhibits the incorporation of glucosamine which occurs independently of protein synthesis, and thus appears to have a specific inhibitory effect on oligosaccharide assembly. Inhibition primarily effects acetylation of glucosamine.

Intestinal Lipogenesis by Alcohol

Investigators keep coming up with new bits of evidence that add to the already numerous evils of alcohol and so nibble away at the joys of drinking. Carter and his colleagues¹at Harvard now report that the feeding of a high-alcohol liquid diet to rats over a four-week period resulted in a significant increase in the synthesis of triglyceride from palmitate as measured in intestinal slices and microsomes. The controls were treated with isocaloric glucose in place of alcohol. The alcohol diet caused a 25- to 54-fold increase in microsomal incorporation of palmitate to triglyceride; this was minimized when pyrazole, an inhibitor of alcohol metabolism, was added to the alcohol feedings. The authors introduce their study by recalling previous work on the effects of alcohol on intestinal physiology. Some functions are inhibited, such as the uptake and transport of amino acids and glucose and the absorption of vitamin B

Role of the free amino acid pool of the intestine in protein synthesis

1. The accumulation of glycine and its incorporation into protein have been studied in rat intestinal slices in vitro. 2. Pyridoxal HCl, which raises intracellular pool concentration of glycine, similarly increases the rate of glycine incorporation into protein. 3. Kinetic data demonstrates a lag in glycine incorporation into protein, suggesting that the glycine passes through an intracellular pool (or pools) before its incorporation into protein. 4. In vivo experiments using leucine demonstrate that specific activity in the protein is a reflection of the specific activity of the soluble “pool” of leucine. 5. These data are consistent with a model in the intestinal mucosa in which glycine in the extracellular pool is not the direct precursor for protein synthesis, but suggest that glycine and perhaps other amino acids first pass through some intracellular pool(s).