Everted Intestinal Sacs Research Articles

Mechanism for the enhancement of paracellular and transcellular permeability by absorption promoters.

The effects of the absorption promoters on two possible pathways, paracellular and transcellular routes, were investigated physiologically or biochemically. Assuming the paracellular route to be a water channel since the permeation through this route related to water absorption, the pore size was obtained from the equivalent pore concept using intestinal everted sac. Sodium caprate, sodium laurate, mixed micelles composed of sodium oleate and sodium taurocholate, EDTA disodium salts, which had larger promoting effects on the intestinal absorption of cefmetazole, fosfomycin and phenol red than other promoters, enlarged the pore size to the extent that inulin could permeate. For the transcellular route, the membrane perturbing actions of the promoters were examined using brush border membrane vesicles, where its protein and lipid portions were labelled with fluorescent probes, and a fluorescence polarization technique. The interaction of the potent promoter such as sodium caprate and membrane protein or lipids was found to cause membrane perturbation and increase the membrane permeability. In this minireview, one methodology for the mechanistic study on drug absorption promotion was introduced as described above. Consequently, the changes in the paracellular and transcellular routes could be found specifically, to explain the in situ absorption promotion of poorly absorbed drugs quantitatively.

Intestinal cholesterol and oleic acid uptake from solutions supersaturated with lipids.

To test the role of nonmicellar phases in lipid absorption, intestinal uptake of fatty acids and cholesterol has been studied in vitro from supersaturated and micellar solutions. The micellar solubility limit at equilibrium was established for cholesterol and oleate/monoolein (2:1) at pH 6.7 with 10 mM taurocholate. Uptake by rat intestinal everted sacs was measured during incubation of 5 min. Cholesterol uptake increased linearly with the cholesterol content of micellar or supersaturated solutions up to a supersaturation of 150%. Oleate uptake, by contrast, remained essentially the same from either saturated or supersaturated (130-280%) mixtures. The difference between cholesterol and oleate uptake rates is explained by their distinct effects on micellar size, which is unchanged by cholesterol supersaturation but is increased by oleate. Solutions largely supersaturated (280%) with oleate-monoolein are polydisperse and contain viscous isotropic and paracrystalline phases similar to those observed during lipid absorption. These results suggest that, in the presence of such solutions, uptake occurs from both the micellar saturated and nonmicellar supersaturated phases.

Effect of human milk folate binding protein on folate intestinal transport

The present study examined the effect of human milk folate binding protein (FBP) on the intestinal transport of 5-methyltetrahydrofolate (5-CH 3H 4PteGlu). This was performed by examining the transport of radiolabeled 5-CH 3H 4PteGlu bound to FBP using everted sacs of rat intestine. In the jejunum at pH 6, transport of 27 n m bound 5-CH 3H 4PteGlu was linear with time for 30 min of incubation. Transport of 13 n m bound 5-CH 3H 4PteGlu was higher in the jejunum than in the ileum at both pH 6 (2.1 ± 0.3 and 0.36 ± 0.03 pmol/g wet wt/25 min, respectively) and pH 8 (1.9 ± 0.3 and 0.32 ± 0.02 pmol/g wet wt/25 min, respectively). In the jejunum, transport of 13 n m bound 5-CH 3H 4PteGlu at pH 6 was less than transport of an equimolar concentration of free 5-CH 3H 4PteGlu (2.1 ± 0.3 and 5.1 ± 0.5 pmol/g wet wt/25 min, respectively) but was similar at pH 8 (1.9 ± 0.3 and 2.47 ± 0.3 pmol/g wet wt/25 min, respectively). In the ileum transport of bound and free 5-CH 3H 4PteGlu was similar at pH 6 (0.36 ± 0.03 and 0.41 ± 0.06 pmol/g wet wt/25 min, respectively) and pH 8 (0.32 ± 0.02 and 0.43 ± 0.1 pmol/g wet wt/25 min, respectively). The transport process of bound 5-CH 3H 4PteGlu in the jejunum was energy, temperature, and Na + dependent, but not pH dependent, and was competitively inhibited by sulfasalazine. Ninety-two percent of the transport substrate that appeared in the serosal compartment following incubation with bound 5-CH 3H 4PteGlu was found to be free (unbound) 5-CH 3H 4PteGlu. These results show that human milk FBP decreases the rate of transport of 5-CH 3H 4PteGlu in the jejunum and suggest that FBP-bound 5-CH 3H 4PteGlu may utilize the same transport system as free 5-CH 3H 4PteGlu. The results also suggest a role for human milk FBP in regulating the nutritional bioavailability of folate.

Age-related changes in calcium and phosphorus uptake by rat small intestine

The purpose of these experiments was to determine whether there are changes in intestinal Ca and P uptake with age and whether the regulation of Ca and P uptake changes with age. Experiments were performed in male Fischer 344 rats aged 2–3 months (young), 12–14 months (adult) and 22–24 months (old). Ca and P uptake were measured simultaneously by incubating everted intestinal sacs in a buffered salt solution containing radiolabeled 0.25 mM Ca and 1.0 mM P for 15 min. Ca uptake declined by over 50% with age in the duodenum, and P uptake showed a similar decline in both the duodenum and jejunum. The biggest decrease was seen between the young and adult age groups. These decreases in uptake were paralleled by decreases in serum 1,25-dihydroxyvitamin D with age. Administration of 1,25-dihydroxyvitamin D-3 increased Ca uptake by 50–65% in the duodenum and increased P uptake by 85–120% in the duodenum and jejunum of both young and adult rats. Although 1,25-dihydroxyvitamin D-3 increased uptake by about the same percentage in each age group, the maximal uptake was much greater in the young than in the adult. Feeding a low-Ca diet increased duodenal Ca uptake by 68% and increased serum 1,25-dihydroxyvitamin D over 2-fold in young rats. There was no significant increase in either parameter in adult rats fed a low-Ca diet. However, duodenal P uptake was stimulated by a low-Ca diet by 87% in young rats and by 51% in adult rats. These results demonstrate that there is an age-related decline in Ca and P uptake by the intestinal mucosa. In addition, there is decreased capacity of 1,25-dihydroxyvitamin D-3 and a low-Ca diet to stimulate intestinal uptake in the adult.

Heavy metal-induced inhibition of active transport in the rat small intestine in vitro. Interaction with other ions

1. 1. The effect of Hg 2+, Cd 2+ and Pb 2+ on the intestinal transport of d-glucose and l-tyrosine was studied in everted sacs of small intestine. 2. 2. Hg 2+, Cd 2+ and Pb 2+ (10 −5 and 10 −4) singly or in combination inhibit the active transport of d-glucose (10mM) and l-tyrosine (2mM) in rat and pigeon small intestine. 3. 3. The presence of other ions (Mg 2+, Zn 2+ and SeO 3 2) diminishes the toxic effect of heavy metal ions, rendering them less effective.

Uptake of 450191-S, a 1H-1,2,4-triazolyl benzophenone derivative, in the everted sac of rat small intestine: role of intestinal aminopeptidases.

5-[(2-Aminoacetamido)methyl]-1-[p-chloro-2-(o-chlorobenzoyl)phenyl ]-N,N-dimethyl-1 H-1,2,4-triazole-3-carboxamide hydrochloride dihydrate (450191-S), a sleep inducer, is a ring-opened derivative of 1,4-benzodiazepine and has been reported to be activated by intestinal aminopeptidases in a step of absorption. In this study, we investigated the role of the intestinal aminopeptidases in the uptake of 450191-S by the intestine by using various aminopeptidase inhibitors in everted sacs of rat small intestine in vitro. Glycylglycine did not affect the desglycylation of 450191-S but glycyl-L-leucine or L-leucyl-L-leucine inhibited the reaction. This inhibition was accompanied by reduced concentration of 450191-S metabolites in the intestinal tissue. When the incubation was carried out at 0 degrees C or with puromycin, the desglycylation was also inhibited and the concentration was similarly reduced. Since the extent of inhibition of the desglycylation of 450191-S was negatively correlated with the total concentration of 450191-S metabolites in the intestinal tissue (r = 0.9660), the aminopeptidases must play an important role in the uptake of 450191-S by the intestine. To confirm this, we examined the uptake of the desglycylated product of 450191-S, 8-chloro-6-(2-chlorophenyl)-N,N-dimethyl-4H-1,2,4-triazolo- [1,5-alpha] [1,4]-benzodiazepine-2-carboxamide (M-1) in the same manner. No effect of dipeptides, puromycin or reduced temperature was found on M-1 uptake by the intestine, leading to the conclusion that the aminopeptidases are important for enhancing the uptake of 450191-S in the intestine.

Digestion and absorption of sugars and sugar substitutes in rat small intestine.

The bioavailability of newly developed sugar substitutes was observed by measuring the transmural potential difference (delta PD) evoked by Na+-dependent active transport of glucose, which is supposed to be produced by the hydrolysis of sugar substitutes. delta PD was measured using everted intestinal sac prepared from jejunum of adult rats and compared with the digestibility of sugar substitutes in the mucosal homogenate of everted sac. delta PDs evoked by glucose, maltose or maltosylfructose had almost the same levels, however, the delta PD evoked by sucrose was a little lower. delta PDs evoked by maltitol or palatinose were low, and delta PDs evoked by fructo-oligosaccharides were negligible. The hydrolyzing activities of these sugars and sugar substitutes by the mucosal homogenate were correlated with the delta PDs. A significant positive correlation was observed between delta PDmax of various sugars and sugar substitutes and the Vmax of their corresponding hydrolyzing activities. Also, a significant positive correlation was observed between Kt and Km values of these sugars. These results suggest that the absorption of sugar substitutes is dependent on digestibility by membrane digestive enzymes.

Development maturation of riboflavin intestinal transport in the rat.

The intestinal transport of riboflavin in the immature intestine of the suckling rat (14 day old) and its subsequent maturation in weanling (22 day old) and adult (90 day old) rats were investigated using the intestinal everted sac technique. The mucosal-to-serosal transport of 0.5 microM riboflavin was linear with time for 30-min incubation and occurred at a rate of 4.6, 3.6, and 1.6 pmol/g initial tissue wet wt/min in suckling, weanling, and adult rats, respectively. The transport of 0.5 microM riboflavin was higher in the jejunum than the ileum in all age groups. The transport system of riboflavin in all age groups was saturable, energy-, temperature-, and Na+-dependent. Kinetic parameters of the transport process were different. Apparent Kt of the transport process was the same in suckling and weanling rats (0.12 and 0.11 microM, respectively) but tripled in adult rats (0.35 microM). On the other hand, a progressive decrease in Vmax from 166 to 122 to 54 pmol/g initial tissue wet weight/30 min was observed in the suckling, weanling, and adult rats, respectively. The present study demonstrates that the characteristics of the transport process of riboflavin is similar in suckling, weanling, and adult rats and occurs by an energy-, temperature-, and Na+-dependent carrier-mediated process. However, the affinity and the activity (or the number) of the transport carriers of riboflavin decrease with maturation.

Tryptophan transport through transport system T in the human erythrocyte, the Ehrlich cell and the rat intestine

We studied the transport of tryptophan through transport system T in the human red cell, the Ehrlich ascites-tumour cell and in everted sacs of rat intestine. In red cells we confirmed earlier results on Na +-independence and aromatic amino acid specificity (Rosenberg, R., Young, J.D. and Ellory, J.C. (1980) Biochim. Biophys. Acta 598, 375–384). In addition we observed that N-methylation or N-acetylation did not reduce the affinity of the substrates for system T, hydroxylation could increase or decrease substrate affinity, and system T was insensitive to pH changes in the medium. These results characterize reactive differences between system T and other known amino acid transport systems. We also found that d-isomers were about 1 3 as effective as l-isomers to inhibit l-tryptophan uptake. d-Tryptophan competitively inhibited l-tryptophan uptake, but was not taken up by system T. l-Tryptophan produced trans-stimulation of the uptake (influx) and trans-inhibition of the release (efflux) of l-[ 3H]tryptophan; d-tryptophan produced trans-inhibition of the efflux but did not affect significantly the uptake. These results show that in red cells the transport properties of transport system T are asymmetric. Transport system T seems to be absent in the other two preparations studied, the Ehrlich ascites-tumour cell and the rat intestine.

Intestinal absorption mechanisms of gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide citrate (DN-1417) and thyrotropin-releasing hormone (TRH).

The absorption mechanisms of gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide citrate (DN-1417) and thyrotropin-releasing hormone (TRH) were studied in the rat. In situ absorption experiments were carried out by the radioimmunoassay, and experiments using everted sacs of small intestine were by radioactivity measurements with 14C-labeled DN-1417 or 3H-labeled TRH in the low concentration range of drug and by a high pressure liquid chromatography in the rather high concentration range of drug. The site specificity of absorption in the small intestine of rats could not be found with DN-1417, whereas TRH-T was absorbed from only the upper part of small intestine. Dose-proportional absorption of DN-1417 was observed in experiments of in situ as well as in vitro. Dose-proportional transfer of DN-1417 through the everted small intestine was also found within the concentration range from 120 ng/ml to 27 mg/ml, whereas the transfer ratio of TRH decreased with increase in the concentration of TRH. DN-1417 transfer from mucosal to serosal fluid was not inhibited by the replacement of medium Na ions by K ions, pretreatment of intestinal mucosa with HgCl2, the existence of an oligopeptide, or the existence of beta-lactam antibiotics which had been reported to be absorbed by active transport or carrier-mediated transport systems. While, TRH transfer was inhibited by the replacement of medium Na ions by K ions, pretreatment of intestinal mucosa with HgCl2, the existence of an oligopeptide, and the existence of beta-lactam antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the metabolic fate of sucrose esters in rats

The metabolism in rats of sucrose esters of stearic acid and palmitic acid was studied in vivo and in vitro using esters labelled with 14C at the sucrose or fatty-acid moiety. In excretion studies, the ratio of expired radioactivity to absorbed radioactivity after oral administration of the sucrose esters labelled at the sucrose moiety was similar to that after the administration of [ 14C]sucrose. A similar correlation between the ester labelled at the fatty-acid moiety and the free [ 14C]fatty acid was also observed. No intact sucrose ester was detected in the urine. Studies in vitro using everted intestinal sacs showed that there was virtually no transport of 14C-labelled sucrose esters from the mucosal to the serosal solution through the intestinal tissues, and that the enzymes in the intestinal mucosa played a more important role in the hydrolysis of sucrose esters than did those in the digestive fluid. In studies of intestinal absorption through the mesenteric lymphatic system, during the 24 hr after ingestion 1.8% of the administered radioactivity was recovered in the lymph after dosing with [U- 14C]sucrose monostearate whereas 20% was recovered in the lymph after dosing with sucrose [1- 14C]monostearate. This difference in levels of recovery of administered radioactivity indicated that sucrose monostearate was absorbed only after hydrolysis. No intact ester was detected in the lymph or in the portal or femoral blood. The results of all of these experiments show that the sucrose esters are hydrolysed to sucrose and fatty acids prior to intestinal absorption.

Drug absorption from the gastrointestinal tract and immunity: the mechanism of the decreased absorption of salicylic acid during systemic anaphylaxis. I.

Previous studies have demonstrated the decrease of intestinal salicylic acid absorption in ovalbumin-immunized rats during systemic anaphylaxis. In the present study, the mechanism whereby systemic anaphylaxis interferes with the intestinal absorption of salicylic acid was studied. The pH of the luminal solution was not affected by the intravenous challenge with ovalbumin. A significant increase of the intraluminal protein was observed in rats under systemic anaphylaxis. However, there was no significant difference between ovalbumin-treated rats and saline-treated ones on the binding of salicylic acid with intraluminal macromolecular substances. Enhanced mucus release in the perfusate was also observed in sensitized rats but the extent of decrease in absorption of salicylic acid did not correlate with the increase in amount of the intraluminal mucus in the same animals. In addition, no significant effect was observed on the uptake by the intestinal everted sac of rats with systemic anaphylaxis. These findings suggested that mucus as well as protein is not responsible for the decrease of absorption of salicylic acid induced by systemic anaphylaxis. From these observations, it would appear that the circulatory changes in the gastrointestinal tract may play an important role in the decreased absorption of salicylic acid during systemic anaphylaxis.

Sites of dipeptide hydrolysis in relation to sites of histidine and glucose active transport in hamster intestine.

The effects of dipeptides and amino acids on the active transport of L-histidine and D-glucose by sacs of everted small intestine of the hamster have been used to determine the sites of final hydrolysis of the dipeptides in relation to the sites of active transport of L-histidine and D-glucose. The results, plus earlier observations (Wiseman, 1977), show that (a) dipeptide active transport occurs at a superficial site, followed by progressively deeper sites for (b) final hydrolysis of glycyl-phenylalanine and phenylalanyl-glycine, then deeper (c) L-histidine active transport, then (d) final hydrolysis of alanyl-alanine, alanyl-leucine, glycyl-alanine, glycyl-proline, leucyl-alanine and leucyl-leucine, then (e) D-glucose active transport, then (f) final hydrolysis of alanyl-glycine, alanyl-valine, glycyl-glycine, prolyl-glycine, valyl-alanine and valyl-valine. The site of D-glucose active transport (2e) and all the sites superficial to it (2a-d) lie in the intestinal epithelial cell's brush-border. The location within the cell of site(s) 2f is not known; it may lie in the cytosol. All the dipeptides appeared to inhibit L-histidine active transport by the release of free amino acid and not by action of intact dipeptide, supporting the view that dipeptides and free amino acids do not share a common transport pathway in the epithelium of the small intestine.

Biochemical and Ultrastructural Characterization of the Molecular Topography of the Rat Intestinal Microvillous Membrane

The topography of the intestinal microvillous membrane and its surface components was examined using biochemical and ultrastructural techniques. Microvillous membrane surface glycoproteins were labeled using everted intact intestinal sacs, prepared from rat proximal small intestine. Galactosyl residues were identified by labeling with galactose oxidase/sodium boro[3H]hydride and free amino groups with pyridoxal phosphate/sodium boro[3H]hydride. Membranes were purified, solubilized in sodium dodecyl sulfate, and protein labeling analyzed by acrylamide electrophoresis. Using the intact intestine, only microvillous membrane surface amino groups were labeled. However, when microvillous membrane vesicles were purified first and then labeled, radioligand binding to galactoproteins and free amino groups substantially increased. Ultrastructural and cytochemical studies with ruthenium red revealed the intact intestinal surface to have a strong electronegative charge due to the presence of anionic sites in both the thick mucopolysaccharide surface coat and its underlying glycocalyx. During purification of microvillous membrane, the mucous coat was detached from the membrane surface, leaving the glycocalyx directly exposed to the external environment. Enzymatic probing of microvillous membrane vesicles with papain left the vesicles intact and revealed the membrane to be asymmetric with the majority of its integral proteins located at the outer membrane surface. This orientation of galactosyl and amino groups towards the intestinal lumen plus the overlying thick mucopolysaccharide coat should theoretically afford a greater degree of protection against destruction by luminal proteases and bile acids. Moreover, the shedding of membrane-bound hydrolases into the mucous layer may allow the "membrane surface" phase of digestion to commence before nutrients have diffused completely through the surface coat to reach the enterocyte surface.

Inhibition of the active transport of d-glucose and l-tyrosine by DDT and DDE in the rat small intestine

1. The effect of DDT and DDE on the active transport of d-glucose and l-tyrosine was studied in everted sacs of small intestine. 2. DDT and DDE (10 −5 and 10 −4 M) inhibit the active transport of d-glucose and l-tyrosine. 3. These organochlorine compounds also inhibit the (Na + + K +)-ATPase from the same tissue, to about the same extent. 4. These results suggest that the inhibition of the active transport is due to an action of DDT and DDE on the sodium pump.

Permeation and hydrolysis of trichloroethyl phosphate in the rat intestine.

The hydrolysis and permeation of trichloroethyl phosphate were examined in vitro by using everted sacs of rat intestine. The concentration of trichloroethanol was higher in serosal solutions than in mucosal solutions at a high concentration of the phosphate ester whereas the opposite was the case at a low concentration of the ester at pH 7.4. The concentration of trichloroethanol in serosal solutions differed in different regions of the small intestine. The phosphate ester permeated into serosal solutions as such at pH 3.5. Therefore the ester is expected to permeate into serosal solutions as such and then to be hydrolyzed at a high concentration of the phosphate ester at pH 7.4. Percentage losses of the phosphate ester from the intestinal loop in situ differed slightly in different regions of intestinal tracts. The ester is expected to be hydrolyzed in the intestinal lumen before absorption under normal conditions, or be absorbed as such when present at a high concentration.

Uptake and esterification of plant sterols by rat small intestine.

The commonly found plant sterols, beta-sitosterol, campesterol, and stigmasterol, differ structurally from cholesterol only in side chains but are absorbed in much smaller amounts than cholesterol. Because intestinal mucosal cell uptake and esterification are important steps in absorption, these were studied in vivo after feeding the sterols and in vitro using everted sacs of rat small intestine. The studies showed that campesterol uptake was significantly higher than that of beta-sitosterol, whereas stigmasterol uptake was extremely low throughout the intestine. The total intestinal content of campesterol was 2.223 mg/g or about 14% of the dose fed as compared with 1.496 mg/g or 7.4% for beta-sitosterol and only 0.392 mg/g or 2.3% for stigmasterol. Intestinal tissue concentration of esterified campesterol was higher than that of beta-sitosterol, whereas that of esterified stigmasterol was extremely low. The results suggest that campesterol absorption would be higher than that of beta-sitosterol; stigmasterol probably would not be absorbed in any significant amount because of its negligible uptake due to its inability to partition out of the mixed micelles. It appears that the structure of the side chain of a sterol is an important determinant for uptake and esterification, and probably absorption, in the small intestine.

Stereospecific inversion of (R)-(-)-benoxaprofen in rat and man.

(R)-(-)-benoxaprofen is stereospecifically inverted to the (S)-(+)-enantiomer by rats and humans. The rate of inversion is much faster in rats (t 1/2 ca. 2.5 h) than in humans (t 1/2 108 h). Inversion in rats apparently does not occur in the liver, but can be brought about in vitro by an everted intestinal sac preparation, suggesting that the transformation takes place while passing through the gut wall.

Absorption of nicotinic acid and nicotinamide from rat small intestine in vitro

Intestinal absorption of nicotinic acid and nicotinamide was studied using everted sacs of rat small intestine. Transport down the concentration gradient showed saturation kinetics at low concentrations and linear kinetics at higher concentrations. Addition of ouabain or omission of sodium ions decreased absorption. Neither compound was absorbed against a concentration gradient. The mode of transport was thought to be carrier-mediated facilitated diffusion at lower concentrations masked by passive diffusion at higher concentrations.

Oxidation and subsequent glucuronidation of 3,4-benzopyrene in everted intestinal sacs in control and 3-methylcholanthrene-pretreated rats.

The metabolism of 3,4-benzopyrene in the wall of isolated everted intestinal sacs was studied in control and 3-methylcholanthrene induced rats. The luminal (outer) side of the intestine was perfused with a 100-mumol/l concentration of 3,4-benzopyrene in Krebs-Ringer's bicarbonate buffer supplemented with 5% glucose and 2% albumin. A thin-layer chromatography assay method was used to analyze benzo(a)pyrene metabolites. After the pretreatment of rats with 3-methylcholanthrene, the amount of hydroxylated metabolites was about 1.5-fold and the amount of consequent glucuronides was about 3-fold in comparison with the controls. 3-Methylcholanthrene induction mainly increased the production of 3- and 9-hydroxybenzopyrene and dihydrodiols. The glucuronidation of 3- and 9-hydroxybenzopyrene was enhanced by 3-methylcholanthrene but the conjugation of dihydrodiols was nonresponsive to induction. Only small amounts of glucuronides were present in the inner (serosal) side of the intestinal sac. The data suggest different responses of various benzo(a)pyrene metabolites to induction in the intestinal mucosa. In addition to metabolic functions the intestinal mucosa may function as an excretory organ for polycyclic aromatic hydrocarbons entering the body.