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.     

Differential effect of clofibrate on inflammation-induced alterations in plasma proteins in the rat

Daily intramuscular injections of clofibrate begun 6h before the initiation of inflammation induced by the subcutaneous injection of turpentine exerted a differential, dose-dependent inhibition of the anticipated acute-phase globulin response. Specifically, clofibrate at 140mg/kg muted the increase in alpha(2)-macrofoetoprotein, but did not affect that of seromucoid or haptoglobin and only transiently inhibited the rise in copper and the rebound in transferrin. A higher dose, 280mg/kg, markedly suppressed alpha(2)-macrofoetoprotein appearance and the rebound in transferrin, somewhat inhibited the increase in seromucoid and haptoglobin and only transiently affected the rise in plasma copper; 420mg of clofibrate/kg very nearly abolished the appearance of alpha(2)-macrofoetoprotein, markedly suppressed the transferrin rebound and the increases in seromucoid and haptoglobin and again only transiently affected the increase in copper. Clofibrate did not diminish the localized inflammatory response, did not cause microscopically detectable liver damage and did not prevent the hypozincaemia, hypoalbuminaemia and enhanced amino acid uptake by liver usually associated with inflammation. Thus it is unlikely that clofibrate exerted its dose-dependent selective inhibition by muting the initial stimulus or by impairing hepatic metabolism. This seemingly selective action of clofibrate on plasma-protein alterations during inflammation may provide a means of elucidating the function of individual acute-phase globulin during disease. Clofibrate of itself, apart from inflammation, produced decreases in plasma zinc, copper, transferrin and seromucoid and an increase in hepatic amino acid uptake that were to some extent dependent on the dose of the drug.     

Inhibition of pyruvate transport by fatty acids in isolated cells from rat small intestine.

1. A technique is described for the rapid separation of intestinal epithelial cells from the incubation medium by passage through a silicon-oil layer and collection in acid, in which their soluble constituents are released. 2. The inhibition by fatty acids of pyruvate oxidation is further studied. Measurement of pyruvate transport in epithelial cells at 0 degree C showed that short- and medium-chain fatty acids as well as ricinoleate inhibit this transport. Propionate inhibits pyruvate transport by another mechanism than octanoate. 3. Differences between pyruvate propionate and octanoate transport across the epithelial cell membrane were obtained in efflux studies. These studies revealed that acetate, propionate, butyrate and high concentrations of bicarbonate readily stimulate the efflux of pyruvate, probably by anionic counter-transport. No effects were seen with octanoate and hexanoate. The data obtained in these efflux studies suggest that lipophilicity and the pKa values of the monocarboxylic acids determine the contribution of non-ionic diffusion to overall transport. 4. Saturation kinetics, competitive inhibition by short-chain fatty acids and counter-transport suggest a carrier-mediated transport of pyruvate.     

Extracts from peppermint leaves,lemon balm leaves and in particular angelica roots mimic the pro-secretory action of the herbal preparation STW 5 in the human intestine

AimThe herbal preparation STW 5 contains fresh plant extracts from bitter candytuft whole plant, extracts from greater celandine herb, angelica root, lemon balm leaves, peppermint leaves, caraway fruit, liquorice root, chamomile flower and milk thistle fruit. We recently reported that STW 5 increased intestinal chloride secretion and proposed that this action may be involved in its clinical efficacy in the treatment of irritable bowel syndrome. The aim of this study was to identify the extracts responsible for the secretory action in order to provide the basis to develop novel target oriented herbal combinations.MethodsWe used the Ussing chamber voltage clamp technique to study the effects of individual extracts of STW 5 on short circuit current (Isc, reflecting electrogenic ion transport across epithelial cells) in mucosal/submucosal preparations of human small or large intestinal specimens and the human epithelial cell line T84.ResultsSTW 5 at concentrations of 512 µg/ml and 5120 µg/ml evoked an increase in Isc. The increase at the lower concentration was due to pro-secretory effects of angelica which were nerve mediated. The increase at the higher concentration was additionally mimicked by peppermint and lemon balm. The remaining extracts did not influence I_SC in the large intestine. The results were similar in T84 cells except that angelica had no effect while chamomile induced secretion. These pro-secretory effects were reduced by adenylate cyclase inhibitor MDL-12330A, cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR_inh-172 and calcium activated chloride channels blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid (SITS). Liquorice decreased I_SC only in small intestine which was reversed by the epithelial sodium channel blocker amiloride.ConclusionsResults suggested that the pro-secretory action of STW 5 is mainly due to angelica with lesser contribution of peppermint and lemon balm. Their effects involve activation of cAMP- and Ca⁺⁺-activated Cl⁻ channels. We suggest that peppermint, lemon balm and in particular angelica may be the basis to develop novel herbal preparations to specifically treat secretory disorder based on impaired epithelial secretion, such as constipation.     

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine

Background  

Several aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel.     

Glucose metabolism in the mucosa of the small intestine. Glycolysis in subcellular preparations from the cat and rat

1. Lactic acid formation in supernatant fractions of homogenates of cat or rat small-intestinal mucosa was measured under optimum conditions with glucose, fructose, glucose 6-phosphate, fructose 1,6-diphosphate or 3-phosphoglycerate as substrate. 2. Between 80 and 107% of the glycolytic activity of the homogenate was recovered in these particle-free preparations when glucose, fructose, glucose 6-phosphate or fructose 1,6-diphosphate was used as substrate. 3. Evidence was obtained that hexokinase and phosphofructokinase were the rate-limiting enzymes in the initial sequence of glycolytic reactions. The limitation of rate by hexokinase was much more pronounced in preparations from the cat than in those from the rat. 4. With subcellular preparations from cat or rat small intestine lactic acid was also formed from ribose 5-phosphate and at rates similar to those observed with glucose. 5. A higher rate of glycolysis was observed with glucose 6-phosphate as substrate with preparations from the proximal half of the small intestine of the rat as compared with the distal half. 6. Mucosal preparations from rats starved for 24-48hr. exhibited only about one-quarter of the glycolytic activity of those of fed control groups. The decreased rate of formation of lactic acid from either glucose or fructose was mainly due to a decrease in the activity of hexokinase(s). The activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and a number of other enzymes were not significantly decreased by starvation. 7. The results are discussed in relation to metabolic control of glycolysis in other mammalian tissues.     

Lactate formation by rat small intestine in vitro

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na⁺ in the incubating medium were replaced by K⁺, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

Slow-waves in rat small intestine

Rat small intestine generates rhythmic slow-wave activity. The slow-waves are not eliminated by ouabain application or incubation in potassium free solution. Exposure to low sodium or calcium free solution decreases slow-wave activity. Incubation in sodium and calcium free solution eliminates activity. It is concluded that rat small intestinal slow-waves may not result from the same mechanism as in the cat.     

Novel sequential stress model for functional dyspepsia: Efficacy of the herbal preparation STW5

BackgroundMany screening procedures for agents with potential usefulness in functional dyspepsia (FD) rely on animals exposed to stress early in life (neonatal maternal separation, NMS) or in adulthood (restraint stress, RS).PurposeSince many clinical cases of FD have been associated with stress in early life followed by stress in adulthood, a sequential model simulating the clinical situation is described. To explore the validity of the model, the efficacy of STW5, a multicomponent herbal preparation of proven usefulness in FD, was tested.Study design/methodsA sequential stress model established where rats are exposed to NMS after birth followed later by RS in adulthood. Stress hormones and ghrelin were measured in plasma, while responsiveness of stomach fundus strips to smooth muscle stimulants and relaxants was assessed ex-vivo. The effectiveness of treatment with STW5 a few days before and during exposure to RS in preventing changes induced by the stress model is reported and compared to its efficacy when used in animals subjected to RS alone.ResultsResponses to both stimulants and relaxants were reduced to various extents in the studied models, but treatment with STW5 tended to normalize gastric responsiveness. Plasma levels of ghrelin, corticosterone releasing factor, and corticosterone were raised by RS as well as the sequential model. Treatment with STW5 tended to prevent the deranged parameters.ConclusionThe sequential stress model has a place in drug screening for potential usefulness in FD as it simulates more the clinical setting. Furthermore, the findings shed more light on the mechanisms of action of STW5 in FD.     

Inhibition of carnitine palmitoyltransferase in the rat small intestine reduces export of triacylglycerol into the lymph

Following digestion of dietary triacylglycerol (TAG), intestinal epithelial cells absorb fatty acids and monoacylglycerols that are resynthesized into TAG by enzymes located on the endoplasmic reticulum (ER). A study in rat liver (Abo-Hashema, K. A., M. H. Cake, G. W. Power, and D. J. Clarke. 1999. Evidence for TAG synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases. J. Biol. Chem. 274: 35577-35582) showed that there is a carnitine-dependent ER lumenal synthesis of TAG. We wanted to test the hypothesis that a similar pathway was present in rat intestine by utilizing etomoxir, a specific inhibitor of carnitine palmitoyltransferase (CPT). Intraduodenal infusion of etomoxir inhibited CPT activity in the ER by 69%. Etomoxir did not affect either the uptake of intraduodenally infused [3H]glyceryltrioleate by the intestinal mucosa or the production of mucosal [3H]TAG, excluding the possibility that etomoxir interfered with TAG absorption or synthesis. Etomoxir did not inhibit protein synthesis, glucose, cholesterol or palmitate absorption or metabolism, or ATP concentrations. Etomoxir substantially (74%) diminished lymph TAG output from intralumenally infused glyceryltrioleate.In conclusion, these data strongly support the hypothesis that an ER CPT system exists and is necessary for processing dietary TAG into chylomicrons. The significant reduction in lymphatic output of chylomicron TAG on etomoxir treatment suggests that the major source of chylomicron TAG is a diacylglyceroltransferase on the lumenal surface of the ER.     

Ammonia production by the small intestine of the rat.

1. Slices of duodenum and jejunum produce ammonia from glutamine in vitro. 2. Ammoniagenesis does not increase in response to acidosis or potassium deficiency, two conditions known to cause enhanced ammoniagenesis in the kidney. 3. Gut contains glutaminase 1 as well as gamma-glutamyl transpeptidase. 4. These enzymes do not show any increase during starvation.     

Lactate formation by rat small intestine in vitro.

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na+ in the incubating medium were replaced by K+, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

Anti-inflammatory and tight junction protective activity of the herbal preparation STW 5-II on mouse intestinal organoids

BackgroundIrritable bowel syndrome (IBS) is a functional bowel disorder, in which recurrent abdominal pain is associated with defecation or a change in bowel habits. STW 5-II is a combination of six medicinal herbs with a clinically proven efficacy in managing IBS.AimThis study aims to establish an in vitro IBS model using mouse intestinal organoids and to explore the anti-inflammatory and tight junction protective activities of the multi-herbal preparation STW 5-II.MethodsIntestinal organoids were cultured in 1:1 Matrigel™ and medium domes. Inflammation and tight junction disruption were induced by a cocktail of cytokines (TNFα, IFNγ, IL-1β, IL-6) and bacterial proteins (LPS, flagellin). Organoids were treated with different concentrations of STW 5-II, and its multi-target activity was assessed using microarray analyses, RT-qPCR, immunofluorescence, western blot, immunohistochemistry, and a FITC permeability assay. In addition, we analyzed the expression of pNF-κB, pSTAT1, iNOS and ZO-1. In silico analyses were conducted to predict and identify the active components that may be responsible in mediating the multi-target anti-inflammatory activity of STW 5-II.ResultsAn organoid based IBS model was successfully established. STW 5-II effectively reduced the cytokines-induced overexpression of the pro-inflammatory mediators pNF-κB, pSTAT1 and iNOS. Moreover, STW 5-II attenuated cytokine-mediated downregulation of the tight junction protein, ZO-1. This finding was confirmed by a FITC permeability assay. In silico analyses revealed a promising inhibitory activity of some isolated compounds from STW 5-II against NF-κB, STAT1 and iNOS.ConclusionSTW 5-II possesses multiple anti-inflammatory as well as tight junction protective activities that could explain its clinically proven efficacy in managing IBS symptoms.     

Enhanced glucose absorption in the rat small intestine following repeated doses of 5-fluorouracil

Many studies demonstrated that 5-fluorouracil (5-FU) treatment of rodents caused the damage of small intestine, resulting in the malabsorption, while we recently found that repeated administration of 5-FU to rats increased Na(+)-dependent glucose absorption in the small intestine. This study investigated the cause of enhanced glucose absorption. 3-O-methyl-d-glucose (3-OMG) absorption was examined using the everted intestine technique. d-Glucose uptake, phlorizin binding, Western blot analysis and membrane fluidity were examined using small intestinal brush-border membrane vesicles (BBMV). Repeated oral administration of 5-FU to rats increased Na(+)-dependent 3-OMG absorption in the small intestine, while alkaline phosphatase activity in the small intestine decreased. Na(+)/K(+)-ATPase activity of 5-FU-treated rats was about three-fold higher than that of control rats. Although the amount of Na(+)-dependent glucose co-transporter (SGLT1) in 5-FU-treated rats decreased, the overshoot magnitude of d-glucose uptake in BBMV was not altered. Maximum binding of phlorizin in 5-FU-treated rats was 1.5-fold larger than that of control rats, but not altered the maximal rate of d-glucose absorption, Michaelis constant of d-glucose and dissociation constant of phlorizin. The membrane fluidity of 5-FU-treated rats increased. The enhanced d-glucose absorption in 5-FU-treated rats seems to occur secondarily due to the activation of Na(+)/K(+)-ATPase activity in basolateral membranes (BLM). Because the amounts of SGLT1 in 5-FU-treated rats decreased, the increase of turnover rate of SGLT1 and/or an expression of unknown Na(+)-dependent glucose co-transporter with high affinity for d-glucose and phlorizin sensitivity would contribute to the enhancement of d-glucose transport in 5-FU-treated rats.     

Inhibition by cadmium of D-galactose and L-phenylalanine transport by rat intestine in vitro

The effect of cadmium (CdCl2) on galactose and phenylalanine uptake by rat everted intestinal rings has been studied. The rings were preincubated (15 min) and incubated (5 min) in the presence of Cd. Galactose uptake (from 0.5 mM to 10 mM) was inhibited by 0.5 mM Cd about 25%. Only the phlorizin-dependent galactose transport was affected by cadmium, being a non-competitive type inhibition. A 15 min washing with saline solution significantly reduced the cadmium induced inhibition, which was practically reversed by washing with 5 mM EDTA. The uptake of 0.5 mM phenylalanine was not affected by 0.5 mM Cd but it was depressed by 1 mM Cd. Such inhibition was exerted on the sodium-dependent phenylaline transport. Washing with 5 mM EDTA diminished only slightly the inhibition of the transport by cadmium. It is suggested that the inhibition of intestinal transport of galactose and phenylalanine by cadmium may be due to its reversible interaction with metal-binding ligands, possibly sulfhydryl groups, related to the luminal transport systems.