Rat Intestinal Slices Research Articles

Rat intestinal precision-cut slices as an in vitro model to study xenobiotic interaction with transporters

ATP-binding cassette (ABC) proteins play key role in tissue defence by transporting metabolic waste and toxic chemicals out of the cells. Consequently, intact cell systems are required to study xenobiotic interactions with ATP-dependent transporters. The aim of the present study was to set up an intestinal precision-cut slice technique to study the interactions of ABC transporters with xenobiotics. Rat intestinal slices were incubated with verapamil, indomethacin and glibenclamide, and the ability of the above-mentioned drugs to inhibit the multidrug resistance glycoprotein (MDR) and/or multidrug-resistance-associated protein (MRP) was assessed by measuring the intracellular conversion of calcein-AM to fluorescent calcein. The ABC transporters’ inhibitors caused a time-dependent florescence increase which reached the maximum value at 30 min. Verapamil and glibenclamide promoted a concentration-dependent intracellular accumulation of calcein (IC 50 8.1 × 10 −6 M, 1.9 × 10 −4 M, respectively). The effect of glibenclamide was fully reversed by washing the slices, suggesting the reversible nature of calcein accumulation. These data suggest that the precision-cut intestinal slices are a reliable, simple and fast system to evaluate xenobiotic interactions with ABC transporters in rat and, hopefully, in human intestine.

A new technique for preparing precision-cut slices from small intestine and colon for drug biotransformation studies

Introduction: A new technique was developed to prepare precision-cut slices from small intestine and colon with the object of studying the biotransformation of drugs in these organs. Methods: Rat intestinal slices were prepared in two different ways. In the first method, slices were punched out of the small intestine. In the second method, precision-cut slices were made from agarose-filled and -embedded intestines, using the Krumdieck tissue slicer. This method was also applied to colon tissue. Viability of the slices was determined by analysis of intracellular ATP and RNA levels and morphology. Drug metabolizing activity was studied using lidocaine, testosterone, and 7-ethoxycoumarin (7-EC) as phase I substrates, and 7-hydroxycoumarin (7-HC) as a phase II substrate. Results: Precision-cut slices made from agarose-filled and -embedded intestine better preserved ATP levels than tissue that was punched out of the intestinal wall. After 24 h of incubation, morphology in precision cut-slices showed was quite well preserved while punched out tissue was almost completely autolytic after incubation. In addition, total RNA amount and quality was much better maintained in precision-cut slices, when compared to punched out tissue. Both intestinal slices and punched-out tissue showed high, and comparable, phase I and phase II biotransformation activities. Discussion: It is concluded that preparing precision-cut 0.25 mm slices out of agarose-filled and -embedded intestine provides an improvement, compared with punched-out tissue, and that both intestinal and colon slices are useful preparations for in vitro biotransformation studies.

Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine

Mucin secretion in situ from rat intestinal loops was promoted more effectively by dialysed crude cholera filtrate than by an equivalent amount of purified enterotoxin. The filtrate could be rendered inactive by incubation with mixed gangliosides or passage through a G M1-affinity column, which indicated that the secretory action of the filtrate depended upon its enterotoxin component. In an effort to explain the greater potency of the filtrate, we established the presence of a metalloproteinase in the filtrate and demonstrated that this enzyme was capable of degrading purified rat intestinal mucin. Sufficient degradation occurred to cause a substantial decrease in viscosity (57% in 120 min). Biochemical analysis of the mucin before and after exposure to filtrate revealed a rise in the combined percentage of serine, threonine and proline (53–58%), suggesting that poorly glycosylated areas (which are less abundant in these amino acids) were being partly removed from the mucin. The carbohydrate composition was essentially unaltered. Inhibition of the filtrate metalloproteinase by Zincov and α 2-macroglobulin significantly ( P < 0.005) reduced the ability of cholera filtrate to degrade mucin or to stimulate mucin secretion from rat intestinal slices in vitro. Purified cholera enterotoxin added to enterotoxin-depleted filtrate was a more potent secretagogue (secretory stimulant) in intestinal loops than an equivalent amount of enterotoxin alone. We therefore propose that mucin secretion induced by cholera filtrate is caused by cholera enterotoxin, but that degradation of the protective epithelial mucus layer by a constituent metalloproteinase may assist the toxin by allowing increased access to mucosal G M1 receptor sites.

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.

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.

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).

Intestinal Drug Absorption and Metabolism III: Glycine Conjugation and Accumulation of Benzoic Acid in Rat Intestinal Tissue

Glycine conjugation of 14C-benzoic acid to 14C-hippuric acid was demonstrated in in vitro rat intestinal slices and everted intestinal preparations. Hippuric acid was detected and quantified by two independent analytical techniques, a quantitative TLC method, and a reverse isotope dilution method. Comparison of hippuric acid concentration after 2 hr. in mucosal and serosal fluids when hippuric acid was formed endogenously from benzoic acid, and with mucosal and serosal concentrations when hippuric acid was added exogenously to the mucosal fluid, indicated that hippuric acid is formed in the tissue compartment rather than by bacterial or intestinal enzymes in the mucosal fluid. Hippuric acid is apparently formed enzymatically in the mucosal cell according to a previously proposed cell compartment model and subsequently diffuses to both mucosal and serosal fluids. The net amount of hippuric acid formed under the conditions of the study was small (about 1%) and was found to be due to limited synthesis rather than to extensive hydrolysis. Both exogenously added benzoic acid and hippuric acid accumulated in the intestinal tissue compartment (11.2 and 6.4%, respectively) during transfer from the mucosal fluid.

Dietary fat effect on incorporation and release of lipids and cholesterol by rat intestinal slices

The effect of saturated and unsaturated fats on in vitro formation and release of lipids and cholesterol from(14)C acetate by rat intestinal tissue was investigated. The rats were fed a basal diet enriched with either 25% corn oil or lard and then sacrificed after a 10- or 25-day feeding period. It was observed that a similar(14)C lipid content but a greater(14)C cholesterol content was found in the intestinal tissue of rats fed corn oil than in rats fed lard for 10 days. After a longer period of feeding of 25 days, the intestinal tissue(14)C cholesterol level was decreased in the corn oil fed rats without any significant effect on other lipids. These data suggest that corn oil in some way influences cholesterol biosynthesis depending upon its degree of unsaturation and the period of time for which it is fed. The decrease at the later time might involve some mechanism which aids in getting rid of accumulated tissue cholesterol. Less(14)C lipid and(14)C cholesterol were released by the intestinal tissue of rats fed the unsaturated fat as compared with those fed the saturated fat, suggesting a possible role in vivo in reducing blood lipids and blood cholesterol levels.

Inhibition of protein synthesis in rat intestinal slices by tetracycline.

THE tetracycline group of drugs have been found to inhibit protein synthesis in in vitro systems composed of ribosome and supernatant fractions derived from microbial as well as mammalian cells1–3. It has also been reported that chlortetracycline reduces the in vivo incorporation of 35S-methionine into liver, spleen, kidney, and gastric mucosa proteins in the rabbit4. In this report tetracycline was administered to rats and intestinal slices were later tested in vitro for incorporation of 14C-L-leucine into protein. Tetracycline clearly inhibited protein synthesis by rat jejunal slices.

Absorption and metabolism of lecithin and lysolecithin by intestinal slices

1. 1. Intestinal absorption and metabolism of lysolecithin and lecithin has been studied using hamster and rat intestinal slices and inverted sacs, and [ choline- Me- 3H]-[ acyl- 3H]lysolecithin and lecithin as substrates. 2. 2. The physicochemical form of the substrates has been, for lecithin, either a liquid crystalline dispersion in buffer or mixed lecithin-sodium taurodeoxycholate micellar solutions, and for lysolecithin, either a micellar solution in buffer or mixed lysolecithin-sodium taurodeoxycholate micellar solution. The classification of the physicochemical form of the substrates has been derived from their behaviour on gel filtration. 3. 3. Lysolecithin is well absorbed and metabolized mainly to lecithin. 4. 4. The uptake and metabolism of lysolecithin is not influenced by the presence of sodium taurodeoxycholate in the medium. 5. 5. The absorption and metabolism of lysolecithin is the same in slices from all levels of the small intestine except the most distal part. 6. 6. No definite evidence could be obtained that lecithin is absorbed intact.

Studies on the permeability barrier to amino-acid penetration of the intestinal mucosa during incubation in vitroin a Na+-free buffer.

After an incubation of rat intestinal slices in vitro in a solution of L-phenylalanine in sodium-free, potassium-substituted buffer, even of long duration, the mean tissue concentration of the substrate never attains the level of the surrounding medium. The amino-acid uptake under such conditions kinetically resembles free diffusion, and is directly proportional to the concentration of the substrate in the incubation medium.

Metabolism of 1c14 octanoic and 1c14 palmitic acid by rat intestinal slices.

SummaryMeasurements were carried out on the in vitro metabolism of C14-octanoic and C14-palmitic acid by rat intestinal slices. Significantly more octanoic acid was catabolized to CO2 and water soluble products and less recovered as lipid soluble products compared to when palmitic acid was the substrate. Analysis of these lipid soluble products by thin layer chromatography revealed that more of the palmitic acid was esterified to triglyceride compared to octanoic acid. Further analysis of the lipid soluble products by gas liquid chromatography demonstrated that a small but significant amount (9%) of the label from C14-octanoic acid was recovered in fatty acids with chain lengths of 10-20 carbon atoms. This suggests that some of the C14-octanoic acid may have been catabolized to C14-acetate which was then incorporated into long chain fatty acids. In contrast, virtually all of the label from C14-palmitic acid was recovered as such, indicating that very little mucosal catabolism of palmitic acid had occurred.