Everted Sacs Research Articles

Mechanism of pH-Modulated Transport of 5-Methyltetrahydrofolate in Rat Jejunal Everted Sacs.

Absorption and transport of 5-methyltetrahydrofolate, the dietary form of the vitamin after intestinal hydrolysis, was studied in rat everted small intestinal segments. The folate transport from the mucosal to the serosal compartment was pH dependent and optimal at experimental pH4.0, and the transport was only 40-50% of that level at the physiological pH of the gut, i.e., 6.5-7.0. The methyltetrahydrofolate transport to the serosal site was not linked with any cotransport with H+, but the optimum transport was dependent on the mucosal acidic pH of 4 irrespective of the serosal pH. Methyltetrahydrofolates were, however, bound to their mucosal cell receptor optimally at pH6.5. Addition of ATP at the mucosal site enhanced mucosal to serosal folate transport at pH6.5 of the gut. A role for an intestinal mucosal Mg+-ATPase in the hydrolysis of ATP and formation of an acidic microclimate were observed. Sodium azide, an inhibitor of Mg+-ATPase, hindered methyltetrahydrofolate transport at pH6.5 but not at pH4.0. The overall results suggest that 5-methyltetrahydrofolate is absorbed in the intestine by a passive process in an acid microclimate at pH4.0 generated by intestinal mucosal brush-border Mg+-ATPase.

Inhibition of neuronally mediated secretion in rat colonic mucosa by prostaglandin D2

The effect of prostaglandin D2 (PGD2) on ion transport across the mucosa of the descending colon was studied in rats. PGD2 dose-dependently decreased baseline short-circuit current of mucosa-submucosal preparations mounted either in the Ussing chamber or mounted as an everted sac. However, with the everted sac technique, the tissue was about 1000 times more sensitive to PGD2. Concomitant with the decrease in short-circuit current, PGD2 increased the mucosal-to-serosal fluxes of sodium and chloride and decreased the serosal-tomucosal flux of chloride. PGD2 inhibited the secretory action of the PGI2 analogue iloprost, PGD2α, and neurotensin. The action of these secretagogues was dependent on the presence of the submucosal plexus. In contrast, PGD2 had no effect on the increase in short-circuit current caused by PGD2, substance P, or serotonin, the actions of which were not dependent on the presence of the submucosal plexus. The results indicate that the action site of the antisecretory mechanism of PGD2 is localized in the secretomotor neurons.

The study of leucine and methionine transport in the gut of rats intoxicated with thiram.

The effects of thiram on amino acids transport in rats jejunum were investigated. Using the gut everted sac method it was shown that 4 weeks intoxication with the pesticide (5% DL50 per os daily) caused time dependent inhibition in transport of C14-leucine and C14-methionine in the rat jejunum. In order to obtain further information, the influx of both amino acids into the jejunum slices was investigated in rats treated with thiram for 2 weeks. Over a range of amino acid concentrations (0-20 mM) the influx of these compounds was defined as the sum of a saturable term (active transport) and a linear term (passive transport). It was shown that thiram affected only the active transport of amino acids. The analysis of this transport for leucine using Lineweaver-Burk plots showed a decrease of the half-saturation constant (Kt), while the maximal value (Jm) remained unchanged. In the methionine transport study both kinetic constants were changed.

Intraluminal and intracellular phases of fat absorption are impaired in essential fatty acid deficiency.

The structure and function of enterocyte membranes are particularly sensitive to the degree of fatty acid saturation. The objective of the present study was to assess intestinal fat transport in essential fatty acid (EFA)-deficient animal models. Both the digestive and absorptive phases leading to the formation and the secretion of triglyceride (TG)-rich lipoproteins were investigated. After an intraduodenal fat infusion, the percentage increase of plasma TG over fasting values was examined over a period of 4 h in two groups of control and EFA-deficient rats. Lower values at 1 and 2 h (P less than 0.05) were observed in EFA-deficient rats, suggesting fat malabsorption. Likewise, postprandial chylomicronemia was diminished. In a separate group of rats, EFA deficiency was associated with reduced TG and chylomicron-TG transport into lymph. Although pancreatic lipase activity did not change (47.1 vs. 46.2 mumol free fatty acids.mg protein-1.h-1), bile flow was decreased over the 8-h period of collection. Concomitantly, a significant decline (nmol.min-1.g liver-1, P less than 0.05) was discernible in the biliary secretory rate of bile salts (14.09 +/- 2.13 vs. 35.09 +/- 3.73), phospholipids (7.01 +/- 0.61 vs. 11.79 +/- 1.65) and cholesterol (0.19 +/- 0.01 vs. 0.83 +/- 0.06). In vitro studies, utilizing everted sacs incubated with mixed micelles, revealed that EFA-deficient jejunal segments of rats incorporated and esterified less [14C]oleic acid (21 and 32%, respectively). Moreover, the synthesis and secretion of TG-rich lipoproteins were found markedly reduced in mouse jejunal explant cultures. We conclude that EFA deficiency modifies both the intraluminal and intracellular phases of fat absorption.

Influence of milk factors on riboflavin uptake by rat intestinal everted sac.

The uptake of riboflavin was measured in Krebs' medium and a system consisting of riboflavin-free dialyzed milk (providing milk factors) equalized to Krebs' medium with respect to different salt concentration and pH. The final riboflavin concentration in the medium ranged between 0.1 and 0.5 microM. Total riboflavin incorporation was greater in the milk system and the increase ranged between 120 and 134% in comparison with Krebs' medium (100%). The Michaelis-Menten constant (Km) and Vmax for labelled riboflavin uptake were calculated by Lineweaver-Burk plotting of data. Km value decreased from 1.23 microM for Krebs' medium to 0.75 microM in the presence of milk factor(s). Vmax values were 62 and 60 pmol/100 mg tissue/min for Krebs' and milk systems, respectively. It was concluded that milk factor(s) enhanced intestinal uptake of riboflavin.

Chronic ethanol feeding and acute ethanol exposure in vitro: effect on intestinal transport of biotin

This study examined the effects of chronic ethanol feeding and acute ethanol exposure in vitro on biotin transport in rat intestine. Transport studies were performed with intestinal everted sacs. Ethanol was fed to rats for 6-7 wk. Compared with pair-fed controls, ethanol feeding significantly decreased plasma biotin concentrations and transport at physiological concentrations (0.01, 0.1, and 0.3 mumol/L) but not at pharmacological concentration (100 mumol/L). When added to the incubation medium of everted jejunal sacs from dry-food-fed rats, ethanol (2% vol:vol) significantly inhibited the transport of biotin at a physiological concentration (0.1 mumol/L) but not at a pharmacological concentration (100 mumol/L). The inhibitory effect of ethanol on the transport of 0.1 mumol biotin/L increased with increasing concentration of ethanol in the incubation medium [0.5% to 5% (vol:vol)]. Acetaldehyde, the major ethanol metabolite, also significantly inhibited biotin transport at 0.2% (vol:vol). These data demonstrate that chronic ethanol feeding and acute ethanol exposure in vitro inhibit the intestinal transport of biotin by the carrier-mediated process. Chronic ethanol feeding is also associated with a significant decrease in plasma biotin concentrations. The ethanol-induced inhibition in intestinal transport of biotin may be a contributing factor in reducing plasma biotin concentrations.

Processing and transfer of epidermal growth factor in developing rat jejunum and ileum

Using everted sac technique we demonstrated the transfer of 125I-mEGF across the jejunal and ileal walls of suckling, weanling and adult rats. The transfer by the suckling rat jejunum and ileum was significantly inhibited by the presence of dinitrophenol and sodium azide or by the replacement of sodium with potassium or choline. RP-HPLC analysis detected carboxy-terminal processing of 125I-mEGF in suckling and adult rat jejunum and ileum. Suckling rat jejunum produced 125I-des(53)mEGF and 125I-des(49–53)mEGF, whereas 125I-des(48–53)mEGF was detected in suckling rat ileum or adult rat jejunum and ileum. All three forms of 125I-mEGF bound to anti-EGF antibody and EGF receptors. The receptor binding of 125I-des(53)mEGF was higher than that of 125I-mEGF, but those of 125-des(49–53)mEGF and 125I-des(48–53)mEGF were greatly diminished. Results indicate a carboxy-terminal processing of mouse EGF during uptake and transfer in the small intestine of developing and adult rats, and the resulting products showed altered receptor binding. An identical amino acid sequence of the C-terminal pentapeptide of EGF from mouse, human and possibly rat may suggest a biological significance of C-terminal processing of EGF in the small intestine.

Factors affecting zinc-histidine kinetics in rat jejunal everted sacs

Factors affecting zinc-histidine kinetics in rat jejunal everted sacs

Use of Exogenous Glutathione for Metabolism of Peroxidized Methyl Linoleate in Rat Small Intestine

The potential effects of dietary glutathione on the metabolism of peroxidized lipid ingested in the diet were studied using everted sacs of rat small intestine and peroxidized methyl linoleate. Peroxidized methyl linoleate was added to the luminal side, and the appearance of thiobarbituric acid-reactive (TBA-reactive) material on the contraluminal side was measured. Incubation with N,N,bis(2-chloroethyl)-N-nitrosourea (BCNU) under conditions in which it inhibits the glutathione disulfide reductase/glutathione peroxidase system increased the appearance of TBA-reactive material, indicating that at least a portion of the TBA-reactive material passing through the epithelium is peroxide in nature. Adding glutathione (GSH) to the luminal side substantially decreased the appearance of TBA-reactive material on the contraluminal side, either without or with BCNU treatment. Inhibition of GSH transport and control experiments with GSH, peroxidized methyl linoleate and purified brush border membranes showed that this decrease was due primarily to uptake of luminal GSH and its use to support intracellular GSH-dependent reactions. Thus, the results indicate that exogenous GSH, which can exist in certain diets, can be taken up by the small intestine and used to protect against absorption of lipid peroxidation products.

Uptake of Riboflavin by Rat Intestinal Mucosa In Vitro

The uptake of riboflavin was studied in everted sacs of rat intestine using [14C]riboflavin and [3H]polyethylene glycol to define the mechanism of mucosal membrane transport. Three-minute incubations were used throughout. Initial studies indicated the presence of saturable uptake in duodenum, jejunum and ileum. Studies in jejunum at low riboflavin concentrations demonstrated saturable uptake [Km = 0.154-0.177 mumol/L, Vmax = 19.6-25.8 pmol/(100 mg dry tissue.min)]. In contrast, uptake was linear with respect to higher concentrations of vitamin (10-50 mumol/L). Uptake at low (0.1 mumol/L) but not high (20 mumol/L) riboflavin concentrations was inhibited by 50 mumol/L lumiflavin, anoxia, 5 mmol/L indoacetamide, Na(+)-free buffer and low temperature (Q10 = 1.64). Conclusions are as follows: 1) Saturable uptake of riboflavin occurs throughout the rat small intestine; 2) uptake by the jejunal mucosa is competitively inhibited and is consistent with a transport carrier located in the brush border membrane; 3) saturable uptake is energy-dependent and may be directly or indirectly driven by a Na+ gradient; and 4) riboflavin is also taken up by rat intestinal mucosa by a nonsaturable, energy-independent mechanism consistent with simple, passive diffusion.

Studies on selenate and selenite absorption by sheep ileum using an everted SAC method and an isolated, vascularly perfused system

1. 1. Using everted sacs of sheep ileum, selenate uptake was shown to be more rapid than selenite uptake. 2. 2. Sulphate, thiosulphate and ouabain all inhibited the uptake of selenate. A Na + K +-ATPase is probably responsible for energizing the ileal brush-border transport of selenate. 3. 3. The isolated, vascularly perfused ileum was shown to absorb selenate more rapidly than selenite. 4. 4. The perfusion system described would appear to be a useful tool for studying the kinetics of intestinal absorption and the measurement of metabolic transformations within the mucosa.

Simultaneous isolation and characterization of brush border and basolateral membrane vesicles from bovine small intestine.

Purified brush border and basolateral membranes were isolated from homogenized intestinal enterocytes of Holstein steers by divalent cation precipitation followed by differential and sucrose density gradient centrifugation. Alkaline phosphatase and Na/K adenosine triphosphatase served as marker enzymes for the brush border and basolateral membranes, respectively. The brush border and basolateral membrane fractions were enriched 5.1- and 10.1-fold, respectively, over the cellular homogenate. Electron micrographs, obtained with transmission electron microscopy, confirmed the vesicular nature of the membranes and revealed that basolateral membrane vesicles generally were smaller and more irregular in shape than brush border membrane vesicles. The vesicular nature of isolated membrane preparations was confirmed with osmotic activity experiments. Enrichment of brush border and basolateral membrane fractions compared to the initial homogenate and the vesicular configuration of both preparations indicate that the isolated brush border and basolateral membrane preparations were suitable models for evaluating nutrient transport properties of bovine small intestine. The number of transport experiments possible per animal using the membrane vesicle technique is many times more efficient than some other in vitro techniques (i.e., intestinal rings or everted sacs).

Effect of Dietary Proteins and/or Their Digestive Products on Intestinal Taurocholate Absorption

[14C]Taurocholate was orally administered to rats together with a definite amount of either casein- or soy protein-based diet and its postprandial movement along the digestive tract was investigated. A difference was observed between both dietary groups in intraluminal transit as well as mucosal accumulation of [14C]taurocholate in the ileum; namely the soy protein intake led to a decrease in the bile acid incorporation into the ileal mucosa relative to the casein intake, although raising its intraluminal stay. In addition, the digestive products from these and other food proteins by pepsin-pancreatin digestion (peptides with molecular weights of more than 1,000) were examined for their inhibitory effects on in vitro absorption of taurocholate with ileal everted sacs. As the digestive product affinity for taurocholate increased, the rate of taurocholate absorption decreased. It thus seems likely that a food protein more abundant in hydrophobic peptides following intraluminal digestion adsorbs much more bile acids in the gut, thereby disturbing their intestinal absorption.

Uptake of cadmium and metallothionein by rat everted intestinal sacs

The gastrointestinal uptake and transport of cadmium (Cd) and the role of metallothionein (MT) were studied in everted sacs of rat intestine (ESRI). When ESRI were incubated for 30 min in a medium containing various Cd concentrations (1–5 × 10 −4 m) as CdCl 2, Cd-cysteine (Cd-Cyst) or rat liver Cd-MT-II (Cd-MT), a dose-dependent tissue uptake of Cd was observed. In ESRI incubated with Cd-MT, total Cd uptake was lower than that of CdCl 2 (25% of CdCl 2). Fractionation of the tissue showed that about 80% of Cd in the tissue was recovered in the particulate fraction after CdCl 2 and Cd-Cyst incubation, while that after Cd-MT incubation was present mainly in the cytosol fraction (about 80%). Most of the Cd in the cytosol fraction of Cd-MT-incubated ESRI was associated with a 10,000 molecular weight protein on Sephadex G-75 column fractionation. Similar results were obtained after incubation of ESRI from Zn-pretreated rats with 109CdCl 2 solution. In addition to the Cd-MT peak, there was a small peak of Cd associated with a high molecular weight fraction. Only a small percentage of Cd was leaked to serosal fluid in the everted sacs incubated at a low concentration of CdCl 2 (0.8%) but this leakage of cadmium was increased at higher concentration and was higher after incubation with Cd-MT. The results suggest that the uptake of Cd from CdCl 2 and Cd-MT is different. Although Cd-MT was taken up intact by everted sacs, the uptake was slow as compared to Cd salts. The intracellular presence of MT had little effect on the uptake of CdCl 2 but the Cd was sequestered by MT in the intestine.

Influence of unsaturated oils on intestinal absorption of cholesterol

The influence of dietary fatty acid composition on intestinal cholesterol uptake, absorption and synthesis was examined in rats fed diets enriched with lard, sunflower or corn oil (28% w/w). When cholesterol uptake was measured from intestinal everted sacs and brush border vesicles, no significant difference was observed between saturated, polyunsaturated and control diets. The cholesterol absorption measured in vivo was significantly lower in the group fed on corn oil diet as compared to groups fed on control or lard diets. This result was assessed by the fact that hepatic cholesterol synthesis which is inversely related to cholesterol absorption was three-fold higher with corn oil diet than with control or lard diets. When lard and vegetable oil diets were adjusted for β-sitosterol content, cholesterol absorption was similar with the three fat enriched diets. These results show that cholesterol absorption is not influenced by the fatty acid composition of the diet because the low concentration of plant sterols found in vegetable oils is sufficient to decrease significantly in vivo cholesterol absorption.

Drug-delivery by ion-exchange. Stability of ester prodrugs of propranolol in surfactant and enzymatic systems

The degradation of O-acetyl and O- n-propanoyl propranolol with surfactants follows the pathways afforded by chemical hydrolysis and yields both propranolol, by hydrolysis, and the corresponding N-acyl propranolol by O-to- N rearrangement. The extent of rearrangement is controlled by the surfactant charge with cationic systems favouring rearrangement while anionic surfactants suppress this reaction. In contrast, in the presence of isolated esterases, the rearrangement becomes unimportant and is detected only with O-acetylpropranolol. Longer chain members of the series ( O- n-propanoyl to O- n-decanoyl propranolol) act solely as precursors of propranolol in enzymatic systems with the rates of reaction showing a maximum at the O- n-hexanoyl derivative. Hydrolysis to propranolol is also observed with isolated intestinal segments and in everted sacs the serosal chamber showed greater activity than the mucosal compartment.

In vitro intestinal processing of prostaglandin F2 alpha in rats: developmental aspects.

Cytoprotective prostaglandins (PGs) such as PGF2 alpha are present in milk and can be absorbed intact from the gastrointestinal tract. Developmental differences in gastrointestinal PG metabolism in vivo have been previously reported. To evaluate the role of small intestinal tissue, detailed analysis of PG processing in vitro of small intestinal segments of suckling and weanling rats using everted sacs was performed. Sacs were incubated in Krebs-Ringer bicarbonate buffer, pH 7.4, at 37 degrees C. Bathing mucosal fluid contained [3H]PGF2 alpha. Proximal and middle intestinal segment everted sacs of suckling rats exhibited greater transfer of radioactivity in comparison with those of weanling rats. Whereas in suckling rats transfer of radioactivity decreased from proximal to distal regions of the intestine, no segmental differences were seen in weanlings. Maturational differences were also present in the capacity for PG catabolism. Suckling rat everted sacs had a greater proportion of radioactivity present as intact, unmetabolized PGF2 alpha present in sac tissue and serosal fluid in all intestinal segments in comparison with those of weanling rats. Weanling everted sacs contained greater amounts of less polar metabolites. These results indicate that there are quantitative and qualitative age-related differences in transfer and processing of exogenous PGF2 alpha in small intestinal tissue of developing rats.

In Vitro Intestinal Processing of Prostaglandin F2α in Rats

SummaryCytoprotective prostaglandins (PGs) such as PGF2α are present in milk and can be absorbed intact from the gastrointestinal tract. Developmental differences in gastrointestinal PG metabolism in vivo have been previously reported. To evaluate the role of small intestinal tissue. detailed analysis of PG processing in vitro of small intestinal segments of suckling and weanling rats using everted sacs was performed. Sacs were incubated in Krebs‐Ringer bicarbonate buffer, pH 7.4, at 37°C. Bathing mucosal fluid contained [H]PGF2α. Proximal and middle intestinal segment everted sacs of suckling rats exhibited greater transfer of radioactivity in comparison with those of weanling rats. Whereas in suckling rats transfer of radioactivity decreased from proximal to distal regions of the intestine, no segmental differences were seen in weanlings. Maturational differences were also present in the capacity for PG catabolism. Suckling rat everted sacs had a greater proportion of radioactivity present as intact, unmetabolized PGF2α present in sac tissue and serosal fluid in all intestinal segments in comparison with those of weanling rats. Weanling everted sacs contained greater amounts of less polar metabolites. These results indicate that there are quantitative and qualitiative age‐related differences in transfer and processing of exogenous PGF2α in small intestinal tissue of developing rats.

Influence of lipid-rich diets on intestinal cholesterol uptake.

The purpose of this work was to determine whether or not cholesterol transport by intestinal brush border is influenced by fat-enriched diets and therefore plays a role in the regulation of cholesterol absorption. This study was carried out on rats divided into three groups according to diet: (1) control with a diet containing 1.2% cholesterol (diet T), (2) diet T plus 28% saturated fat (lard) and (3) diet T plus 28% polyunsaturated fat (corn oil). Uptake of cholesterol and oleic acid from defined mixed micellar solutions was studied on two experimental models: everted sacs and brush border vesicles of the intestinal membrane. In vivo cholesterol absorption was measured by the dual isotope plasma ratio method of Zilversmit. Fat-enriched diets decreased both in vivo cholesterol absorption and in vitro cholesterol uptake without any specific effect of unsaturated fats. This suggests that the mechanisms involved in the transport of cholesterol across the brush border membrane may be rate-limiting for cholesterol absorption. Oleate and butyrate uptakes, in contrast, were unaffected by the fat content of the diet.

Enhancement of colonic drug absorption by the paracellular permeation route.

Colonic absorption of poorly absorbable cefmetazole was shown to increase considerably by the addition of 1% sodium caprate, sodium laurate, and mixed micelles composed of sodium oleate and sodium taurocholate. At 0.25%, their effects were weaker but still significant. Colonic absorption of inulin was also increased by the promoters at a concentration of 0.25%. These results suggest that there is a common route between inulin and cefmetazole absorption, i.e., the paracellular route. Sodium taurocholate, sodium caprylate, and EDTA disodium salts (EDTA-2Na) at 1% enhanced cefmetazole absorption less than caprate, laurate, or mixed micelles, but no such effect was found at 0.25%. The colonic pore radius was determined from the equivalent pore theory using an everted sac procedure. Caprate, laurate, and mixed micelles at 0.25% caused this radius to increase significantly, thus making it possible for inulin to permeate the everted sac from the mucosal to the serosal side. The effects of taurocholate, caprylate, and EDTA-2Na for increasing colonic pore sizes and the degree of inulin permeation were less than those of caprate, laurate, or mixed micelles. The change in the paracellular route is thus considered to result from the increase in pore size.