Rat Intestinal Tract Research Articles

Intestinal tract is an important organ for lowering serum uric acid in rats.

The kidney was recognized as a dominant organ for uric acid excretion. The main aim of the study demonstrated intestinal tract was an even more important organ for serum uric acid (SUA) lowering. Sprague-Dawley rats were treated normally or with antibiotics, uric acid, adenine, or inosine of the same molar dose orally or intraperitoneally for 5 days. Rat’s intestinal tract was equally divided into 20 segments except the cecum. Uric acid in serum and intestinal segment juice was assayed. Total RNA in the initial intestinal tract and at the end ileum was extracted and sequenced. Protein expression of xanthine dehydrogenase (XDH) and urate oxidase (UOX) was tested by Western blot analysis. The effect of oral UOX in lowering SUA was investigated in model rats treated with adenine and an inhibitor of uric oxidase for 5 days. SUA in the normal rats was 20.93±6.98 μg/ml, and total uric acid in the intestinal juice was 308.27±16.37 μg, which is two times more than the total SUA. The uric acid was very low in stomach juice, and attained maximum in the juice of the first segment (duodenum) and then declined all the way till the intestinal end. The level of uric acid in the initial intestinal tissue was very high, where XDH and most of the proteins associated with bicarbonate secretion were up-regulated. In addition, SUA was decreased by oral UOX in model rats. The results suggested that intestinal juice was an important pool for uric acid, and intestinal tract was an important organ for SUA lowering. The uric acid distribution was associated with uric acid synthesis and secretion in the upper intestinal tract, and reclamation in the lower.

Upregulation of VEGFR1 in a rat model of esophagogastric anastomotic healing

Introduction: Anastomotic leakage after gastrointestinal surgery is a significant cause of morbidity and mortality. Esophagogastric and colorectal anastomoses are vulnerable to leakage. Extended knowledge of growth factors and their receptors is needed to understand anatomic healing.Methods: The expression pattern of vascular growth factor receptor (VEGFR1-3), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFRα/β) and keratinocyte growth factor receptor (KGFR) were analyzed by semiquantitative-PCR in the rat intestinal tract and in esophagogastric anastomosis 5d after surgery.Results: VEGFR1, VEGFR2, EGFR, KGFR and PDGFRα expression was observed throughout the intestinal tract including esophagus, stomach, small bowl and colon. VEGFR3 was not found in gastric samples and PDGFRβ expression was not detected in the small bowl.Semiquantitative analyses of the VEGFR1, PDGFRα and EGFR expression in esophagogastric anastomotic tissues revealed a 2-fold upregulation of the VEGFR1 in gastric samples, while no change was observed in the esophageal anastomotic side.Conclusion: Our results revealed a distinct expression pattern of the investigated growth factor receptors in rat intestinal tract. Showing higher expression levels of growth factor receptors at the gastric anastomotic tissue at the fifth postoperative day suggests a different contribution of the gastric and the esophageal side to the anastomotic healing.

Effect of moxibustion on the expression levels of proteins of neuron and neuropeptide in the intestinal tract of rats with Crohn's disease

ObjectiveTo explore the regulating effect of moxibustion on the enteric nervous system of rats with Crohn's disease. MethodsTen SD rats were selected randomly from 40 rats as normal control (group A), and the other 30 rats were established into Crohn's disease rat models by adopting clysis method with TNBS. On the basis of modeling successfully, the model rats were randomly divided into model group (group B), herbs-partitioned moxibustion group (group C) and mild moxibustion group (group D) with 8 rats in each group (4 rats were dead during modeling. After modeling, 2 rats were selected from group A, and 2 rats were selected from models for determination, at last, 8 rats were included in each group). In group C and group D, herbs-partitioned moxibustion or mild moxibustion was applied on “Tiānshū (▪ ST 25)” bilaterally, and the rats in group A and group B were fixed as in treatment groups. HE stain was conducted, and morphological observation was performed on the colonic tissue of rats; the expression levels of proteins of S-100, SP, NPY and their receptors were observed by adopting immunohistochemical method. ResultsCompared with group A, the expression levels of proteins of S-100, SP and its receptor NK1R, NPY and its receptors NPY1R and NPY2R in the intestinal tract of rats in model groups increased obviously, and the differences were statistically significant. (Ps-100<0.01, PSP<0.05, PNK1R<0.01, PNPY<0.05, PNPY1Y<0.05, PNPY2R<0.01), after treatment with drug-paste interposed moxibustion and mild moxibustion, the levels reduced significantly (PS-100<0.05, PSP<0.05, PNK1R<0.01, PNPY<0.05, PNPY1R<0.05, PNPY2R<0.01). ConclusionMoxibustion treatment may regulate the expression levels of proteins of S-100, SP, NK1R, NPY, NPYIR and NPY2R through warm stimulation, alleviate inflammatory response of colonic tissue, and repair impaired colonic mucosa, thus achieving the goal of treating Crohn's disease.

Vinblastine, an anticancer drug, causes constipation and oxidative stress as well as others disruptions in intestinal tract in rat.

The purpose of this study is to examine the gastrointestinal disorders after injection of vinblastine (2mgkg-1b.w. i.v.) in rats. Animals were divided into two equal groups: Group 1 was considered as a control group (NaCl, 0.9%). Group 2 was treated with intravenous injection of vinblastine for 7days. Loperamide (2mgkg-1) was injected in a saline solution subcutaneously to induce constipation in another group of rats during the same period. Fecal parameters of the different groups have been determined. At the end of the experiment, animals were anaesthetized and sacrificed by decapitation. The intestinal mucosa specimens were examined for lipid peroxidation, sulfhydryl groups (-SH) and protein carbonylation as well as antioxidant enzyme activities and intracellular mediators. Gastrointestinal motility was realized by the test meal (10% charcoal in 5% gum arabic). In result, statistically significant decreases in the fecal number and water content collected during 24h were detected in the vinblastine group, but less important than loperamide control group. The animals treated with vinblastine, showed also a significant decrease (13%) of GIT, lower than that of loperamide (34%). The intestinal tissues from vinblastine-treated rats were showed a significant increase in lipoperoxydation and H2O2 production as well as a significant depletion of enzymatic and non-enzymatic antioxidants. Added to that, a disruption of intracellular iron and calcium levels was observed. Therefore, the present study provide the first strong evidence that vinblastine induced numerous disruptions in gastrointestinal which are related to oxidative stress and intracellular mediators disorders.

Enzyme-linked immunosorbent assay for S100A9 in the stool of rats with dextran sulfate sodium-induced colitis

Calprotectin, a heterodimer of S100A8 and S100A9, has been reported to be a useful biomarker in inflammatory bowel disease (IBD); however, the relationship between the fecal level of S100A9 and the extent of inflammation in IBD remains unclear. Our aim was to develop a new enzyme-linked immunosorbent assay (ELISA) for rat S100A9, and to investigate whether changes in fecal S100A9 levels reflect the inflammatory conditions in the intestinal tracts of rats with dextran sulfate sodium (DSS)-induced colitis. Anti-rat S100A9 monoclonal antibodies were raised in mice and used for the development of a novel ELISA for rat S100A9. The performance of our ELISA was assessed by dilution and recovery tests, and the detection range was 3.75–240ng/mL. The dilution test showed good linearity. The recovery of fecal S100A9 was 95.1% (mean), with a range of 86.1%–108.8%. Colitis was induced in rats by oral administration of 3% DSS/drinking water (DW) for 11days (D group), while DW alone was provided to rats of the control group (C group) during the same period. The extent of inflammation was evaluated with the disease activity index (DAI), and the concentration of fecal S100A9 was determined by ELISA. Both the DAI scores and the fecal S100A9 levels were significantly higher in the D group than in the C group. Microscopic observation revealed that S100A9 was dominantly produced in many immune cells of myeloid origin in rat rectal tissues. These results indicate that the novel ELISA may be applied to clinically evaluate IBD in rats with high sensitivity. In conclusion, our ELISA is useful in toxicological and pharmacological evaluations.

Inclusion complexes of HP-β-cyclodextrin with agomelatine: Preparation, characterization, mechanism study and in vivo evaluation

Agomelatine (AGM), is efficacious in both the acute phase and the continuation phase of depression. However, its poor water-solubility, low bioavailability and polymorphism limit its pharmacological effects. To address these problems, agomelatine-hydroxypropyl-β-cyclodextrin inclusion complex (AGM/HPβ-CD) was prepared successfully by freeze-drying. The products was evaluated by structural characterization, solubilization test, in-situ absorption of rat intestinal tract and pharmacokinetic study. In addition, thermodynamic studies were performed, the results indicated that the inclusion process was enthalpy-determined and exothermic nature of complexation, signifying the role of steric interactions in complex formation. Molecular docking of AGM with HPβ-CD has been conducted as well to verify the experimental findings and predict the stable molecular structure of the inclusion complex. The in vivo data showed that, AGM was mainly absorbed in duodenum and jejunum by passive diffusion. AGM/HPβ-CD inclusion complex displayed earlier Tmax and higher Cmax, and the AUC0-12h was approximately twice larger than its physical mixture. These results suggested that AGM/HPβ-CD inclusion complex was established with 1:1 stoichiometry through the naphthalene group of AGM and it was deeply inserted into the cavity of HPβ-CD, and the inclusion complex could significantly enhance the oral bioavailability of AGM.

Divergent Effect of Dezocine, Morphine and Sufentanil on Intestinal Motor Function in Rats

Background: Opioid induced bowel dysfunction is the most common side effect of preoperatively administrated morphine, fentanyl and its derivative. However, the influence of dezocine on intestinal mobility is rarely reported. This study was designed to investigate the effects of dezocine, morphine and sufentanil on both intestinal smooth muscle contraction and propulsion in rats.Methods: Contractile tension and frequency of isolated rat small intestine smooth muscle were measured using tension transducer after incubation with different concentrations of dezocine, morphine and sufentanil. The propulsive rate of methylene blue in rat intestinal tract was measured 30 minutes after intraperitoneal injection of morphine, sufentanil and dezocine. Percent of change in contractile tension and contraction frequency compared to baseline level were calculated to evaluate muscle contraction. Propulsive rate of methylene blue was calculated as the percentage of methylene blue moving distance in intestinal tract compared to the length of the small intestine.Results: Morphine and sufentanil significantly increased the contractile tension of isolated small intestine smooth muscle at high doses. The contraction frequency did not change significantly among the 3 tested doses. Increasing the dose of dezocine from 1.7 mg.L-1 to 10.2 mg.L-1 did not change either the contractile tension or the contraction frequency. The propulsive rate of methylene blue in intestinal tract was significantly decreased after the treatment with morphine, sufentanil and dezocine (45.6%, 43.7%, and 42.1% respectively) compared to control group(57.1%), while the difference among the 3 drug groups were not significant.Conclusion: Morphine and sufentanil may dose dependently increase the contractile tension and contraction ability of isolated rat small intestine smooth muscle, while dezocine has no significant effect on intestine smooth muscle contraction. However, all these opioids might impair small intestinal propulsion.

Bioavailability of andrographolide and protection against carbon tetrachloride-induced oxidative damage in rats

Andrographolide, a bioactive diterpenoid, is identified in Andrographis paniculata. In this study, we investigated the pharmacokinetics and bioavailability of andrographolide in rats and studied whether andrographolide enhances antioxidant defense in a variety of tissues and protects against carbon tetrachloride-induced oxidative damage. After a single 50-mg/kg administration, the maximum plasma concentration of andrographolide was 1μM which peaked at 30min. The bioavailability of andrographolide was 1.19%. In a hepatoprotection study, rats were intragastrically dosed with 30 or 50mg/kg andrographolide for 5 consecutive days. The results showed that andrographolide up-regulated glutamate cysteine ligase (GCL) catalytic and modifier subunits, superoxide dismutase (SOD)-1, heme oxygenase (HO)-1, and glutathione (GSH) S-transferase (GST) Ya/Yb protein and mRNA expression in the liver, heart, and kidneys. The activity of SOD, GST, and GSH reductase was also increased in rats dosed with andrographolide (p<0.05). Immunoblot analysis and EMSA revealed that andrographolide increased nuclear Nrf2 contents and Nrf2 binding to DNA, respectively. After the 5-day andrographolide treatment, one group of animals was intraperitoneally injected with carbon tetrachloride (CCl4) at day 6. Andrographolide pretreatment suppressed CCl4-induced plasma aminotransferase activity and hepatic lipid peroxidation (p<0.05). These results suggest that andrographolide is quickly absorbed in the intestinal tract in rats with a bioavailability of 1.19%. Andrographolide protects against chemical-induced oxidative damage by up-regulating the gene transcription and activity of antioxidant enzymes in various tissues.

Comparison of the effects of Mg–6Zn and Ti–3Al–2.5V alloys on TGF-β/TNF-α/VEGF/b-FGF in the healing of the intestinal tract in vivo

To evaluate the different effects of Mg–6Zn alloy and Ti–3Al–2.5V alloy implants in intestinal tract healing, we compared these two different alloys with respect to their effect on a rat's intestinal tract, using serum magnesium, radiology, pathology and immunohistochemistry in vivo. Itwas found using the scanning electron microscope that the Mg–6Zn alloy began to degrade during the first week and that the Ti–3Al–2.5V alloy was non-degradable throughout the process. The Mg–6Zn alloy did not have an impact on serum magnesium. Superior to the Ti–3Al–2.5V alloy, the Mg–6Zn alloy enhanced the expression of transforming growth factor-β1 in healing tissue, and promoted the expression of both the vascular endothelial growth factor and the basic fibroblast growth factor, which helped angiogenesis and healing. The Mg–6Zn alloy reduced the expression of the tumor necrosis factor (TNF-α) at different stages and decreased inflammatory response, which may have been related to the zinc inhibiting TNF-α. In general, the Mg–6Zn alloy performed better than Ti–3Al–2.5V at promoting healing and reducing inflammation. The Mg–6Zn alloy may be a promising candidate for use in the pins of circular staplers for gastrointestinal reconstruction in medicine.

Oral absorption enhancement of salmon calcitonin by using both N-trimethyl chitosan chloride and oligoarginines-modified liposomes as the carriers

In this study both N-trimethyl chitosan chloride (TMC) and oligoarginine (Arg8) were utilized to modify liposomes as the multifunctional carriers (TMC-Arg8-Lips) for enhancing the oral absorption of salmon calcitonin. Two permeation enhancers with positive charges were sequentially adsorbed on the liposomal surface with negative charges by electrostatic interaction. Instead of salmon calcitonin, fluorescein isothiocyanate dextran (FD4) was loaded in TMC-Arg8-Lips for Caco-2 cell permeation test in vitro and penetration examination in rat intestinal tract in vivo. The results showed that the apparent permeability coefficient (Papp) of TMC-Arg8-Lips containing FD4 were 7.0-, 4.4-, 1.8- and 1.4-folds higher than FD4 solution, FD4-TMC solution, non-modified liposomes (Non-Lips) and TMC modified liposomes (TMC-Lips), respectively. A strong fluorescence was observed by confocal laser scanning microscope (CLSM) at rat intestinal wall isolated in different times after the FD4 loaded carriers were intragastrically administrated. Furthermore, the images revealed that TMC-Arg8-Lips could penetrate deeply inside the mucosal membrane. The pharmacodynamic study indicated that TMC-Arg8-Lips containing calcitonin were more efficient in enhancing the absorption and prolonging the reduction of blood calcemia in rats. The area above the plasma calcium concentration–time curve (AAC) of TMC-Arg8-Lips containing calcitonin was increased by more than 16.6- and 1.6-fold when compared to Non-Lips and TMC-Lips, respectively.

Antidiarrheal Activity of 19-Deoxyicetexone Isolated from Salvia ballotiflora Benth in Mice and Rats

The antidiarrheal properties of 19-deoxyicetexone, a diterpenoid isolated from Salvia ballotiflora were evaluated on castor oil-, arachidonic acid (AA)- and prostaglandin (PGE2)-induced diarrhea in rodent models. The structure of 19-deoxyicetexone was determined by X-ray crystallography, mass spectrometry (EI-MS), as well as ultraviolet (UV-Vis), infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopies. This compound significantly and dose-dependently reduced frequency of stooling in castor oil-induced diarrhea, and at dose of 25 mg/kg it also inhibited diarrhea induced with AA, while it had no effect on PGE2-induced diarrhea. This compound at doses of 25 mg/kg also diminished castor oil-induced enteropooling and intestinal motility, and inhibited the contraction of the rats’ ileum induced by carbachol chloride at a concentration of 100 µg/mL. 19-Deoxyicetexone did not present acute toxicity at doses of 625 mg/kg. Its antidiarrheal activity may be due to increased reabsorption of NaCl and water and inhibition of the release of prostaglandins, gastrointestinal motility and fluid accumulation in the intestinal tracts of rats. These findings suggest that 19-deoxyicetexone may be used in the treatment of diarrhea, although more studies must be carried out to confirm this.

Comparison of the effects of Mg–6Zn and titanium on intestinal tract in vivo

To evaluate the ability of Mg-6Zn to replace titanium nails in the reconstruction of the intestinal tract in general surgery, we compared the Mg-6Zn and titanium implants with respect to their effects on rat's intestinal tract by biochemical, radiological, pathological and immunohistochemical methods. The results indicated that Mg-6Zn implants started to degrade at the third week and disintegrate at the fourth week. No bubbles appeared, which may be associated with intestinal absorption of the Mg-6Zn implants. Pathological analyses (containing liver, kidney and cecum tissues) and biochemical measurements, including serum magnesium, creatinine, blood urea nitrogen, glutamic-pyruvic-transaminase and glutamic-oxaloacetic-transaminase proved that degradation of Mg-6Zn did not harm the important organs, which is an improvement over titanium implants. Immunohistochemical results showed that Mg-6Zn could enhance the expression of transforming growth factor-β1. Mg-6Zn reduced the expression of tumor necrosis factor at different stages. In general, our study demonstrates that the Mg-6Zn alloy had good biocompatibility in vivo and performed better than titanium at promoting healing and reducing inflammation. It may be a promising candidate for stapler pins in intestinal reconstruction.

Metaproteome Analysis and Molecular Genetics of Rat Intestinal Microbiota Reveals Section and Localization Resolved Species Distribution and Enzymatic Functionalities

The digestion of food ingredients depends on the action of the gut microbiota and has a significant influence on the health, especially in the case of metabolic diseases, of the host organism. Despite the relevance of the structure and functionalities in the microbiota for the metabolism of the host, the spatial resolution of microbial consortia and the functionalities in the different gut sections of the rat are mostly unknown. Since there are suitable rat models for human metabolic diseases, the microbiota of the rat is of special interest. Samples along the intestinal tract of rats were investigated using metaproteomics and 16S rRNA gene pyrosequencing. The procedures for harvesting bacteria from the mucus and the content of the gut sections and feces were optimized leading to 2802 nonredundant bacterial protein groups in total that were assigned to spectra measured by liquid chromatography-tandem mass spectrometry. The majority of 16S rRNA genes and protein groups belonged to members of Firmicutes, Bacteroidetes and Proteobacteria. The functionalities in the enzyme repertoire were compared between the mucus and the content of the large intestine sections and the feces samples. This spatial resolution allowed pinpointing changes in the community to specific metabolic capacities like carbohydrate transport and energy conservation. The results showed that the mere analysis of feces samples reflects the functions of the gut microbiota only to a minor extent and sheds light on the metabolic interchange between the microbiota and the host organism.

Effect of cryopreservation and microencapsulation of lactic acid bacterium Enterococcus faecium MC13 for long-term storage

The aim of this work was to investigate the effect of cryoprotectants on the survival of probiotic bacterium Enterococcus faecium MC13 during freeze drying and storage. The maximum relative cell viabilities were observed when cells were freeze dried and stored at −20 °C, which is optimum temperature for the preservation of E. faecium. At all storage temperatures, trehalose was found to be retaining the highest relative cell viability than other cryoprotectants. In addition, alginate–chitosan capsules were produced to encapsulate E. faecium with the aim of enhancing survival of probiotic cells and keeping the probiotic during exposure to the harsh gastro-intestinal conditions. Encapsulation of probiotic into alginate–chitosan capsules found to be retaining higher survival of probiotic cells (4.342 ± 0.26 Log CFU mL −1) at −20 °C for six months. Microencapsulated cells were resistant to simulated gastric (pH 2.0) and intestinal fluids (pH 7.5), resulting in significantly enhanced survival when compared with free cells. During in vivo treatment, capsules were broken and probiotic cells were directly released into the intestinal tract of rat. This result showed that microencapsulation of E. faecium MC13 with alginate and a chitosan coating offers an effective means of delivery of viable cells to the colon and maintains their survival during the adverse gastro-intestinal conditions.

Pharmacological Modulation by Shakuyakukanzoto (Shao-Yao-Gan-Cao-Tang) and the Ingredients in Rat Intestinal Smooth Muscle

Shakuyakukanzoto (Shao-Yao-Gan-Cao-Tang), a formulation of Japanese herbal (Kampo) medicines, is composed of Paeoniae Radix and Glycyrrhizae Radix. Effects of Shakuyakukanzoto and the ingredients on rat intestinal tract were examined. Shakuyakukanzoto (0.01 - 0.3 mg/ml) relaxed a carbachol (CCh, 0.3 μM) - induced contraction in a concentration-dependent manner. Both components (Paeoniae radix and Glycyrrhizae radix) also relaxed the CCh-induced contraction. At 0.1 to 1 mM, their constituents (paeoniflorin and glycyrrhetic acid) and the metabolic products (18-α- and 18-β-glycyrrhetinic acids) exerted almost the same actions. The relaxations induced by Shakuyakukanzoto were not modified by 1 μM nicardipine, 10 μM suramin (ATP receptor inhibitor) and several K+ channel inhibitors, but was attenuated by 20 μM IBMX (a phosphodiesterase inhibitor). Also, IBMX inhibited the relaxations induced by paeoniflorin and glycyrrhetic acid, but not by other ingredients. Nicardipine decreased the relaxation of just 18-α-glycyrrhetinic acid. Even in non-treatment with CCh, Shakuyakukanzoto relaxed the intestinal tract. CCh (0.3 μM) elicited spontaneous contractions in 23% specimens, depressed by application of Shakuyakukanzoto. These results indicate that Shakuyakukanzoto causes a remarkable relaxation by the anti-cholinergic and the PDE inhibitory actions, but by minor contribution of Ca2+ channel inhibition. Thus, Shakuyakukanzoto exerts an anti-spasmodic action due to the interaction with pharmacological effects of its ingredients.

Metabolism of Gambogic Acid in Rats: A Rare Intestinal Metabolic Pathway Responsible for Its Final Disposition

Gambogic acid (GA) is a promising natural anticancer candidate. Although the anticancer activity of GA has been well demonstrated, information regarding the metabolic fate of GA is limited. Previous studies suggested that GA is mainly excreted into intestinal tract in rats through bile after intravenous administration, whereas only traces appeared in the feces, suggesting that GA is metabolized extensively in the intestine. However, there has been no report about the intestinal metabolism of GA either in animals or humans. In this study, large amounts of two sulfonic acid metabolites of GA were found in the feces samples of rats after intravenous administration, and their structures were identified as 10-α sulfonic acid GA and 10-β sulfonic acid GA by comparison of the retention times and spectral data with those of synthesized reference substances using liquid chromatography-diode array detector-tandem mass spectrometry. This rare intestinal metabolic pathway mainly involves Michael addition of the sulfite ion to the 9,10 carbon-carbon double bond of α,β-unsaturated ketone. In addition, a more detailed metabolic profile in rats is proposed, according to the results of in vitro and in vivo studies. It was found that GA can be metabolized by a variety of routes, including monooxidation, hydration, glutathionylation, glucuronidation, and glucosidation in the liver of rats. These findings provide information on the major metabolic soft spot of GA in the intestine and liver of rats, which is not only useful in the future human metabolic study of this compound but also of value in the metabolic studies of GA analogs.

Hydroxypropyl-Substituted β-Cyclodextrins: Influence of Degree of Substitution on the Thermodynamics of Complexation with Tauroconjugated and Glycoconjugated Bile Salts

The effect of the degree of substitution (DS) on the ability of hydroxypropylated β-cyclodextrin (HPβCD) to form inclusion complexes with six different bile salts, found within the intestinal tracts of rats, dogs, and humans, was studied by isothermal titration calorimetry. The composition and molecular structure of the cyclodextrin samples were characterized by MALDI-TOF mass spectrometry together with 1D and 2D-NMR, and some of the complexes were studied by 2D ROESY NMR. The stability and structure of the complexes were mainly determined by the position of hydroxyl groups on the bile salts and depended relatively little on the number of hydroxypropyl side chains on the CDs. The enthalpy and entropy of complexation exhibited a strong linear increase as the DS increased from 0 to 1, and a pronounced enthalpy-entropy compensation was observed. These observations are interpreted as an increased release of ordered water from the hydration shells of the bile salts, caused by the hydroxypropyl substituents on the rim of the CD. It is estimated that each CD hydroxypropyl substituent dehydrates a hydrophobic surface area of approximately 10 Å(2).

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na(+) channel, we examined the effect of Nedd4-2 on the apical Ca(2+) channel TRPV6, which is involved in transcellular Ca(2+) transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When co-expressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca(2+) influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.

Identification of key residues in WW domains of ubiquitin‐protein ligase Nedd4‐2 that bind to calcium channel TRPV6

We previously observed that ubiquitin‐protein ligase Nedd4‐2 mediated degradation of calcium channel TRPV6 in Xenopus oocytes. The goal of this study was to identify the domains and key residues in Nedd4‐2 that interact with TRPV6 as an association between TRPV6 and Nedd4‐2 was demonstrated by Co‐IP upon co‐expression in oocytes. As revealed by far‐western approach, TRPV6 interacted with WW1 and WW2 domains, but not with WW3, WW4, C2 or HECT domain of Nedd4‐2. To identify the residues in WW1 and WW2 domains responsible for the interactions, 5 residues in WW1 that are conserved in WW1‐2 but not in WW3‐4 domains were mutated to their corresponding residues in WW3‐4. Only D204H mutation abolished the binding of WW1 to TRPV6. Similarly, the corresponding D376H mutation in WW2 also impaired its binding to TRPV6. On the other hand, the H539D mutation in the corresponding position of WW4 enabled its interaction to TRPV6. However, the 2 sites appeared not necessary for the association between TRPV6 and Nedd4‐2 as assessed by Co‐IP. Interestingly, both TRPV6 ubiquitination and degradation were increased by Nedd4‐2 with D204H and D376H mutations. This suggests that the binding between TRPV6 and WW1‐2 domains may prevent the ubiquitination of TRPV6 by Nedd4‐2. Nedd4‐2 co‐localized with TRPV6 in rat intestinal tract as observed by immunostaining with antibodies against TRPV6 and Nedd4‐2. Our results provide evidence that Nedd4‐2 plays a role in regulating TRPV6 stability under physiological conditions.

Montmorillonite adsorbs creatinine and accelerates creatinine excretion from the intestine

Abstract Objectives This study aims to evaluate the sorption by montmorillonite of creatinine and the accelerating effect of montmorillonite on creatinine excretion from the intestine. Methods The sorption of montmorillonite was observed in vitro. Also, rat intestinal tract and blood vessels were perfused circularly with perfusate with or without creatinine, respectively, to study the promotion of creatinine diffusion from the blood vessel to the intestine and the inhibition of creatinine absorption in the intestinal tract. The effect of decreasing the serum concentration of creatinine was studied in an acute hypercreatininaemia mouse model. The concentration of creatinine was determined by the basic picric acid method. Key findings Montmorillonite adsorbed creatinine markedly in the simulated intestinal solution in a concentration-dependent manner. The sorption–time curve of montmorillonite with creatinine showed that the sorption was fast. The adsorption rate reached a maximum in 10 min. The pH of the solution influenced the sorption, the rate of which was higher at a low pH than at a high pH. Creatinine could diffuse from the blood vessel to the intestine and was reabsorbed in the intestine. Montmorillonite promoted the diffusion and inhibited the absorption. Montmorillonite decreased the serum creatinine level of hypercreatininaemia mice prepared by injecting creatinine intraperitoneally. Conclusions Montmorillonite adsorbs creatinine and accelerates its excretion from the intestine.