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Malabsorption of NaCl is the primary cause of diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption occurs via the dual operation of Na:H and Cl:HCO3 exchange in the brush border membrane (BBM) of villus cells. Cl:HCO3 exchange is mediated by BBM transporters DRA (downregulated in adenoma) and PAT1 (putative anion transporter 1) in the mammalian small intestine. DRA/PAT1-mediated Cl:HCO3 exchange was significantly downregulated in the BBM of villus cells in a rabbit model of chronic ileitis, while Na:H exchange was unaffected. The inhibition of Cl:HCO3 exchange was restored in the rabbits when treated with a broad-spectrum immunomodulator, i.e. a glucocorticoid, indicating that the downregulation of DRA/PAT1 is likely to be immune-mediated during chronic enteritis. Mucosal mast cells are one type of key immune cells that are known to proliferate and release immune inflammatory mediators, thus playing a significant role in the pathogenesis of IBD. However, how mast cells may regulate DRA- and PAT1-mediated Cl:HCO3 exchange in a rabbit model of chronic ileitis is unknown. In this study, treatment of rabbits with chronic intestinal inflammation with the mast cell stabilizer ketotifen did not affect the mucosal architecture of the inflamed intestine. However, ketotifen treatment reversed the inhibition of Cl:HCO3 activity in the BBM of villus cells. This restoration of Cl:HCO3 activity to normal levels by ketotifen was found to be secondary to restoring the affinity of the exchangers for its substrate chloride. This observation was consistent with molecular studies, where the mRNA and BBM protein expressions of DRA and PAT1 remained unaffected in the villus cells under all experimental conditions. Thus, this study indicates that mast cells mediated the inhibition of coupled NaCl absorption by inhibiting Cl:HCO3 exchange in a rabbit model of chronic enteritis. [ABSTRACT FROM AUTHOR]

In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models. To investigate this, we generated 2D enterocyte monolayers from human jejunal 3D organoids and used small interfering RNAs (siRNAs) to knock down NHE3 or SGLT1. Molecular and uptake studies were performed to determine the effects on NHE3 and SGLT1 expression and activity. Knockdown of NHE3 by siRNA in enterocyte monolayers was verified by qPCR and Western blot analysis and resulted in reduced NHE3 activity. However, in NHE3 siRNA-transfected cells, SGLT1 activity was significantly increased. siRNA knockdown of SGLT1 was confirmed by qPCR and Western blot analysis and resulted in reduced SGLT1 activity. However, in SGLT1 siRNA-transfected cells, NHE3 activity was significantly increased. These results demonstrate for the first time the functionality of siRNA in patient-derived organoid monolayers. Furthermore, they show that the two primary Na absorptive pathways in human enterocytes reciprocally regulate one another. [ABSTRACT FROM AUTHOR]

BACKGROUND: The general assumption that prebiotics reach the colon without any alterations has been challenged. Some in vitro and in vivo studies have demonstrated that 'non‐digestible' oligosaccharides are digested to different degrees depending on their structural composition. In the present study, we compared different methods aiming to assess the digestibility of oligosaccharides synthesized by β‐galactosidase (β‐gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450‐β‐gal) from lactose, lactulose and lactitol. RESULTS: In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450‐β‐gal were partially hydrolyzed in vitro, depending on their structure and composition, with rat small intestinal extract (RSIE) and small intestinal brush‐border membrane vesicles (BBMV) from pig. Digestion of some oligosaccharides increased when mixtures were fed to C57BL/6 mice used as in vivo model; however, lactulose‐oligosaccharides were the most resistant to the physiological conditions of mice. In general β (1→6) linked products showed higher resistance compared to β (1→3) oligosaccharides. CONCLUSION: In vitro digestion methods, without disaccharidases, may underestimate the importance of carbohydrates hydrolysis in the small intestine. Although BVMM and RSIE digestion assays are appropriate in vitro methods for these studies, in vivo studies remain the most reliable for understanding what actually happens in the digestion of oligosaccharides. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Aim: Trafficking, membrane retention, and signal‐specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ‐adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP‐dependent kinase II (cGKII) within detergent‐resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. Methods: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3‐deficient, cGMP‐kinase II‐deficient, and NHERF2‐deficient mice, after oral application of the heat‐stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non‐raft fractions were separated by Optiprep density gradient centrifugation of Triton X‐solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. Results: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide‐induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII‐deficient mice, and strongly reduced in NHERF2‐deficient mice. Conclusion: NHE3, cGKII, and NHERF2 form a lipid raft‐associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft‐associated activated cGKII. [ABSTRACT FROM AUTHOR]

Fasting is known to alter the function of various organs and the mechanisms of glucose metabolism, which affect health outcomes and slow aging. However, it remains unclear how fasting and feeding affects glucose absorption function in the small intestine. We studied the effects of the fasting and feeding on glucose-induced short-circuit current (Isc) in vitro using an Ussing chamber technique. Glucose-induced Isc by SGLT1 was observed in the ileum, but little or no Isc was observed in the jejunum in ad libitum-fed mice. However, in mice fasted for 24–48 h, in addition to the ileum, robust glucose-induced Isc was observed over time in the jejunum. The expression of SGLT1 in the brush border membranes was significantly decreased in the jejunum under fed conditions compared to 48 h fasting, as analyzed by western blotting. Additionally, when mice were fed a 60% high glucose diet for 3 days, the increase in glucose-induced Isc was observed only in the ileum, and totally suppressed in the jejunum. An increase in Na+ permeability between epithelial cells was concomitantly observed in the jejunum of fasted mice. Transepithelial glucose flux was assessed using a non-metabolizable glucose analog, 14C-methyl α-d-glucopyranoside glucose (MGP). Regardless of whether fed or fasted, no glucose diffusion mechanism was observed. Fasting increased the SGLT1-mediated MGP flux in the jejunum. In conclusion, segment-dependent up- and down-regulation mechanisms during fasting and feeding are important for efficient glucose absorption once the fast is broken. Additionally, these mechanisms may play a crucial role in the small intestine's ability to autoregulate glucose absorption, preventing acute hyperglycemia when large amounts of glucose are ingested. [ABSTRACT FROM AUTHOR]

Background/Aims The liver and small bowel are known to be closely interrelated. In addition, the expression of tight junction proteins (TJPs) is suggested to be associated with the development of non-alcoholic fatty liver disease (NAFLD). This study aimed to investigate the effects of age and sex on the hepatic steatosis and the small bowel inflammation induced by the high fructose, high fat diet (HFHFD) in rats. Methods 6-week-old male and female rats, along with 2-year-old, were used. The HFHFD groups were provided with the high fat diet (60% kcal from fat) and high fructose water (20%). After 8 weeks, both liver and small intestinal tissues were evaluated by histological analysis. TJP expression in the jejunum mucosa was analyzed by qRT-PCR. Results The histological inflammation scores of old control groups in small bowel were higher than those of young control groups regardless of sex, particularly in the jejunum (Table). The jejunum villus/crypt ratio of 6-week-old rats significantly decreased only among male HFHFD group (Table). In contrast, in the 2-year-old rats, the villus/crypt ratio resulted in a reduction only among female HFHFD group. The hepatic steatosis were significantly higher in the both age of male HFHFD groups and only old female HFHFD (Table). The mRNA expression of claudin-2 significantly elevated in female HFHFD (Table). Conclusion HFHFD induced inflammation of small bowel and hepatic steatosis, especially in old rats, suggesting that NAFLD might be related with damage of small bowel brush border membrane. The upregulated gene expression of claudin-2 in the aged female rats suggests a potential role of TJPs in provoking NAFLD in postmenopausal females. [ABSTRACT FROM AUTHOR]

Although the functions of small intestine are largely regulated by enteric nervous system (ENS), an independent intrinsic innervation, as well as central nervous system (CNS), the neural regulation of drug absorption from the small intestine still remains to be clarified. To obtain some information on it, the effect of adrenergic agonists on P-glycoprotein (P-gp) function was investigated by utilizing a vascular-luminal perfused rat small intestine. Adrenaline significantly decreased the secretion of rhodamine-123 (R-123) into the intestinal lumen, but dibutyryl cAMP (DBcAMP) significantly enhanced R-123 secretion. The inhibition study with quinidine clearly indicated that the decrease in secretory clearance of R-123 by adrenaline or the increase by DBcAMP would be attributed to the decrease or increase in P-gp activity, respectively. Expression levels of P-gp in whole mucosal homogenates were not changed at all by any chemicals examined, but those on brush border membrane (BBM) of intestinal epithelial cells were significantly decreased or increased by adrenaline or DBcAMP, respectively. Furthermore, changes in P-gp activity caused by adrenergic agonists and DBcAMP were significantly correlated with changes in expression level of P-gp in BBM, suggesting that the trafficking of P-gp from cytosolic pool to BBM would be regulated by adrenergic agonists and DBcAMP. [ABSTRACT FROM AUTHOR]

[Display omitted] • Membrane filtered IMF increases goblet cell number and acidic mucus. • Membrane filtered IMF increases trypsin activity in the duodenum. • Membrane filtered IMF increases lactase activity in brush border membrane vesicles. Human breast milk promotes maturation of the infant gastrointestinal barrier, including the promotion of mucus production. In the quest to produce next generation infant milk formula (IMF), we have produced IMF by membrane filtration (MEM-IMF). With a higher quantity of native whey protein, MEM-IMF more closely mimics human breast milk than IMF produced using conventional heat treatment (HT-IMF). After a 4-week dietary intervention in young pigs, animals fed a MEM-IMF diet had a higher number of goblet cells, acidic mucus and mucin-2 in the jejunum compared to pigs fed HT-IMF (P < 0.05). In the duodenum, MEM-IMF fed pigs had increased trypsin activity in the gut lumen, increased mRNA transcript levels of claudin 1 in the mucosal scrapings and increased lactase activity in brush border membrane vesicles than those pigs fed HT-IMF (P < 0.05). In conclusion, MEM-IMF is superior to HT-IMF in the promotion of mucus production in the young gut. [ABSTRACT FROM AUTHOR]

Cyclic nigerosylnigerose (CNN) is a cyclic tetrasaccharide with properties distinct from those of other conventional cyclodextrins. We investigated the relative available energy of CNN in healthy humans. CNN digestibility was determined using brush border membrane vesicles from the small intestines of rats. CNN was not hydrolyzed by rat intestinal enzymes. To investigate breath hydrogen excretion, 13 human subjects were included in a double-blind cross-over, randomized, placebo-controlled study. The effects of CNN on hydrogen excretion were compared with those of a typical nondigestible, fermentable fructooligosaccharide (FOS). In the study participants, hydrogen excretion hardly increased upon CNN and was remarkably lower than for FOS. The available energy value was determined using the fermentability based on breath hydrogen excretion and was evaluated as 0 kcal/g for CNN. CNN was hardly metabolized and hence may be used as a low-energy dietary fiber. [ABSTRACT FROM AUTHOR]

Absorption of monosaccharides is mainly mediated by Na+-d-glucose cotransporter SGLT1 and the facititative transporters GLUT2 and GLUT5. SGLT1 and GLUT2 are relevant for absorption of d-glucose and d-galactose while GLUT5 is relevant for d-fructose absorption. SGLT1 and GLUT5 are constantly localized in the brush border membrane (BBM) of enterocytes, whereas GLUT2 is localized in the basolateral membrane (BLM) or the BBM plus BLM at low and high luminal d-glucose concentrations, respectively. At high luminal d-glucose, the abundance SGLT1 in the BBM is increased. Hence, d-glucose absorption at low luminal glucose is mediated via SGLT1 in the BBM and GLUT2 in the BLM whereas high-capacity d-glucose absorption at high luminal glucose is mediated by SGLT1 plus GLUT2 in the BBM and GLUT2 in the BLM. The review describes functions and regulations of SGLT1, GLUT2, and GLUT5 in the small intestine including diurnal variations and carbohydrate-dependent regulations. Also, the roles of SGLT1 and GLUT2 for secretion of enterohormones are discussed. Furthermore, diseases are described that are caused by malfunctions of small intestinal monosaccharide transporters, such as glucose-galactose malabsorption, Fanconi syndrome, and fructose intolerance. Moreover, it is reported how diabetes, small intestinal inflammation, parental nutrition, bariatric surgery, and metformin treatment affect expression of monosaccharide transporters in the small intestine. Finally, food components that decrease d-glucose absorption and drugs in development that inhibit or downregulate SGLT1 in the small intestine are compiled. Models for regulations and combined functions of glucose transporters, and for interplay between d-fructose transport and metabolism, are discussed. [ABSTRACT FROM AUTHOR]

Maltobionic acid (MA), formed by a gluconic acid and glucose linked by an α‐1,4 bond, may have the properties of a nondigestible oligosaccharide. The objective of this study was to elucidate the bioavailability of MA in rats and humans by observing digestion of MA by small intestinal enzymes, the fermentation of MA by gut microbiota, and the effect of adaptation following prolonged ingestion of MA. MA digestion was assessed using brush border membrane vesicles (BBMV) from rat small intestine. A within‐subject repeated measures design was used for ingestion experiments in 10 healthy female participants. After MA ingestion, postprandial plasma glucose and insulin levels, breath hydrogen excretion, and urinary MA were measured. The effect of adaptation following prolonged MA ingestion was investigated in rats. MA was minimally hydrolyzed by BBMV. Ingestion of 10 g of MA by healthy females did not elevate postprandial plasma glucose and insulin levels. Breath hydrogen and urinary MA were negligibly excreted over 8 hr following ingestion. Adaptation to prolonged MA ingestion produced no significant difference in exhaled hydrogen levels over 8 hr following administration compared with controls. MA is a new food material that is highly resistant to digestion and fermentation. It expresses the characteristics of a nondigestible oligosaccharide, including being low energy, improving the flavor of food and juice, and mineral solubilization. [ABSTRACT FROM AUTHOR]

Transmembrane mucin MUC17 is an integral part of the glycocalyx as it covers the brush border membrane of small intestinal enterocytes and presents an extended O-glycosylated mucin domain to the intestinal lumen. Here, we identified two unknown phosphorylated serine residues, S4428 and S4492, in the cytoplasmic tail of human MUC17. We have previously demonstrated that MUC17 is anchored to the apical membrane domain via an interaction with the scaffolding protein PDZK1. S4492, localized in the C-terminal PDZ binding motif of MUC17, was mutated to generate phosphomimetic and phosphodeficient variants of MUC17. Using Caco-2 cells as a model system, we found that induction of an inflammatory state by long-term stimulation with the proinflammatory cytokine TNFa resulted in an increase of MUC17 protein levels and enhanced insertion of MUC17 and its two phospho-variants into apical membranes. Up-regulation and apical insertion of MUC17 was followed by shedding of MUC17-containing vesicles. Transmembrane mucins have previously been shown to play a role in the prevention of bacterial colonization by acting as sheddable decoys for encroaching bacteria. Overexpression and increased presentation at the plasma membrane of wild-type MUC17 and its phosphodeficient variant MUC17 S-4492A protected Caco-2 cells against adhesion of enteropathogenic Escherichia coli, indicating that C-terminal phosphorylation of MUC17 may play a functional role in epithelial cell protection. We propose a new function for MUC17 in inflammation, where MUC17 acts as a second line of defense by preventing attachment of bacteria to the epithelial cell glycocalyx in the small intestine. [ABSTRACT FROM AUTHOR]

“Cell penetrating peptides” (CPPs) are natural or synthetic peptides with the ability to interact with cell membranes in order to enter cells and/or deliver cargo. They attract considerable interest as permeation enhancers for oral delivery of therapeutic drugs with poor bioavailability, such as proteins or DNA. A main barrier is the intestinal epithelium where passage needs to proceed through a paracellular -and/or a transcellular pathway. Using an organ cultured mucosal explant model system and a selection of fluorescent polar -and lipophilic tracers, the aim of the present study was to investigate the interaction of two CPPs, melittin and Hiv-1 Tat, with the enterocyte brush border. Melittin belongs to the amphipathic class of CPPs, and within 0.5–1 h it bound to, and penetrated, the enterocyte brush border, causing leakage into the cytosol and increased paracellular passage into the lamina propria. Surprisingly, melittin also abolished endocytosis of tracers from the brush border into early endosomes in the terminal web region (TWEEs), excluding any permeation enhancing effect via such an uptake mechanism. Electron microscopy revealed that melittin caused an elongation of the brush border microvilli and a reduction in their diameter. HIV-1 Tat is a cationic CPP that is internalized by cells due to a sequence, mainly of arginines, from residue 49 to 57, and a peptide containing this sequence permeabilized enterocytes to a polar tracer by a leakage into the cytosol. In conclusion, the CPPs studied acted by causing leakage of tracers into the enterocyte cytosol, not by inducing endocytosis. [ABSTRACT FROM AUTHOR]

Immunoglobulin G (IgG) transfer in opposite directions across the small intestinal brush border serves different purposes in early life and in adulthood. In the neonate, maternal IgG is taken up from the gut lumen into the blood, conferring passive immunity to the offspring, whereas in the adult immunoglobulins, including IgG made by plasma cells in the lamina propria, are secreted via the brush border to the lumen as part of the mucosal defense. Here, IgG has been proposed to perform a luminal immune surveillance which eventually includes a reuptake through the brush border as pathogen-containing immune complexes. In the present work, we studied luminal uptake of FITC-conjugated and gold-conjugated IgG in cultured pig jejunal mucosal explants. After 1 h, binding to the brush border was seen in upper crypts and lower parts of the villi. However, no endocytotic uptake into EEA-1-positive compartments was detected, neither at neutral nor acidic pH, despite an ongoing constitutive endocytosis from the brush border, visualized by the polar tracer CF594. The 40-kDa neonatal Fc receptor, FcRn, was present in the microvillus fraction, but noteworthy, a 37 kDa band, most likely a proteolytic cleavage product, bound IgG in a pH-dependent manner more efficiently than did the full-length FcRn. In conclusion, our work does not support the theory that bidirectional transfer of IgG across the intestinal brush border is part of the luminal immune surveillance in the adult. [ABSTRACT FROM AUTHOR]

Background: Endurance athletes search for diet regimens that will improve performance and decrease gastrointestinal disturbances during training and events. Although the intestine can adapt to changes in the amount and composition of dietary inputs, the responses to the combination of endurance exercise and diet are poorly understood. Methods: We evaluated small intestinal dimensions and mucosal architecture and calculated the capacities of the entire small intestine to digest maltose and maltodextrin and absorb glucose in response to two different diet types; a western human diet and the Daniel Fast, a vegan style diet, and with moderate intensity endurance training or a no-exercise sedentary lifestyle for a 13 week period (n = 7 per group). The influences of diet and exercise, alone and in combination, were analyzed by analysis of variation. Results: Rats fed the western diet gained more weight (P < 0.05) due to more fat mass (P < 0.05), with a similar response for the sedentary compared with the exercised rats in each diet group (P < 0.05). The Daniel Fast rats had longer and heavier intestines with deeper crypts with villi that were wider (P < 0.05), but not taller. Despite increased energetic demands, the exercised rats had shorter and lighter intestines with shorter villi (P < 0.05). Yet, the percentage of mucosa did not differ among groups. Total small intestinal activities for maltase and α-glucoamylase, and capacities for glucose absorption were similar regardless of diet or exercise. Conclusions: These findings indicate the structural responses of the small intestine to a vegan style diet are modified by exercise, but without altering the capacities of the brush border membrane to digest and absorb carbohydrates. [ABSTRACT FROM AUTHOR]

The gluten exorphins (GEs) A5 and C5 are peptides with a potential opioid-like activity derived from enzymatic degradation of wheat gluten. In this study, a 70% Caco2/30% HT-29 co-culture layer was used as an in vitro model of human small intestine to investigate the transport of the two GEs and their resistance to brush border peptidases. Thirty, 150 or 600 μg of the peptides, GE A5 and GE C5, were added to the apical chamber of the transwells. We previously demonstrated these amounts to be released during in vitro digestion of wheat-derived bread and pasta samples. The solutions in apical and basolateral chambers were analyzed for intact GEs or related fragments by UPLC/HRMS. The two peptides behaved differently since after 120 min higher amount of intact GE A5 (0.55–24.60%) compared to GE C5 (0.20–1.00%) was recovered in the basolateral chamber, depending on the initial dose that was added to the transwell apical compartment. The GE C5 was strongly hydrolyzed by cellular peptidases. The fluxes of two GEs through the in vitro intestinal epithelium were concentration- and time-dependent. The transport inhibitors Wortmannin and Glycyl-sarcosine did not significantly affect the apical-to-basolateral fluxes of the two GEs. Differently, Cytochalasin D slightly increased the transepithelial transport of both GEs, and decreased the TEER values of the co-culture cells. These results suggest the paracellular route as a possible transport mechanism of both GEs. Overall, this study indicates the possibility that food-derived GEs can cross the human intestinal epithelium as intact peptides, and improves the knowledge for exploiting the opioid-like activity potentially associated with the ingestion of wheat-derived foods. [ABSTRACT FROM AUTHOR]

Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen ³H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given ³H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given ³H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5- fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux to dispose of endogenous cholesterol efficiently for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Prebiotics synthesized by probiotic genera are of interest due to their specific selectivity. However, modifications of their composition and concentrations by digestion may affect their properties and effects in the host. In the present study we assessed small intestinal digestibility of oligosaccharides synthesized by Acidipropionibacterium acidipropionici LET120-β-gal (β-galactosidase) from lactose (LET120-GOS), lactulose (LET120-OsLu) and lactitol (LET120-LOS) by incubating them with brush border membrane vesicles (BBMV) from pig and a rat small intestinal extract (RSIE) as in vitro models of mammalian mucosa. Both BBMVs and RSIE partially degraded the potential prebiotic oligosaccharides synthesized by dairy propionibacteria and hydrolysis was influenced by the structure and composition of the oligosaccharides. The highest degradation was observed for LET120–GOS, with 46.8% and 58.1% of hydrolysis after 5 h of digestion with BBMV and RSIE, respectively. On the contrary, LET120-OsLu and LET120-LOS showed higher resistance to intestinal degradation. β(1 → 6) linked products showed higher resistance to mammalian digestive enzymes compared to β(1 → 4) and β(1 → 3) linked oligosaccharides. Results emphasize the need of quantifying prebiotics that would reach the colon to anticipate their impact on health and the dose needed to exert that effect. [Display omitted] • Oligosaccharides formed by propionibacteria β-galactosidase were in vitro digested. • Pig and rat small intestine extracts partially hydrolyzed oligosaccharides (OS). • OS from lactitol and lactulose were less degraded than those obtained from lactose. • Hydrolysis degree was influenced by β-linkage type and monomers composition. [ABSTRACT FROM AUTHOR]

Pinocytosis at the small intestinal brush border was studied in postweaned porcine cultured mucosal explants, using the fluorescent polar probes Alexa hydrazide (AH, MW 570), Texas red dextran (TRD, MW ~ 3000), and Cascade blue dextran (CBD, MW ~ 10,000). Within 1 h, AH appeared in a string of subapical punctae in enterocytes, indicative of an ongoing constitutive pinocytosis. By comparison, TRD was taken up less efficiently into the same compartment, and no intracellular labeling of CBD was detectable, indicating that only small molecules are pinocytosed from the postweaned gut lumen. AH remained in the terminal web region in EEA-1-positive endosomes (“TWEEs”) for at least 2 h, implying that the pinocytic uptake does not proceed towards a transcytic pathway. Like AH, cholera toxin B subunit (CTB) was readily internalized, but the two probes appeared in completely non-overlapping subapical compartments, indicating the existence of two different uptake mechanisms operating simultaneously at the brush border. CTB is internalized by clathrin-dependent receptor mediated endocytosis, but surprisingly the toxin also caused a rapid disappearance from the apical cell surface of two major brush border enzymes, alkaline phosphatase and aminopeptidase N, demonstrating the disruptive effect of this pathway. By immunofluorescence, caveolin-1 was hardly detectable in enterocytes, arguing against a caveolae-mediated uptake of AH, whereas the pinocytosis/phagocytosis inhibitors dimethyl amiloride and cytochalasin D both arrested AH uptake. We propose that the constitutive pinocytic mechanism visualized by AH contributes to maintenance of membrane homeostasis and to enrich the contents of lipid raft constituents at the brush border. [ABSTRACT FROM AUTHOR]

Na-K-ATPase located on the basolateral membrane (BLM) of intestinal epithelial cells provides a favorable intracellular Na + gradient to promote all Na dependent co-transport processes across the brush border membrane (BBM). Down-regulation of Na-K-ATPase activity has been postulated to alter the absorption via Na-solute co-transporters in human inflammatory bowel disease (IBD). Further, the altered activity of a variety of Na-solute co-transporters in intact villus cells has been reported in animal models of chronic enteritis. But the molecular mechanism of down-regulation of Na-K-ATPase is not known. In the present study, using a rabbit model of chronic intestinal inflammation, which resembles human IBD, Na-K-ATPase in villus cells was shown to decrease. The relative mRNA abundance of α -1 and β -1 subunits was not altered in villus cells during chronic intestinal inflammation. Similarly, the protein levels of these subunits were also not altered in villus cells during chronic enteritis. However, the BLM concentration of α-1 and β -1 subunits was diminished in the chronically inflamed intestinal villus cells. An ankyrin–spectrin skeleton is necessary for the proper trafficking of Na-K-ATPase to the BLM of the cell. In the present study, ankyrin expression was markedly diminished in villus cells from the chronically inflamed intestine resulting in depolarization of ankyrin-G protein. The decrease of Na-K-ATPase activity was comparable to that seen in ankyrin knockdown IEC-18 cells. Therefore, altered localization of Na-K-ATPase as a result of transcriptional down-regulation of ankyrin-G mediates the down-regulation of Na-K-ATPase activity during chronic intestinal inflammation. [ABSTRACT FROM AUTHOR]

Highlights: [•] Monocrotophos elevated the activities of brush border enzymes in small intestine of diabetic rats. [•] Monocrotophos enhanced Na+/K+-ATPase activity in small intestine of diabetic rats. [•] Monocrotophos altered redox state markers in small intestine of diabetic rats. [•] Monocrotophos augmented goblet cell hyperplasia and inflammation in small intestine of diabetic rats. [ABSTRACT FROM AUTHOR]

Enterotoxins of Staphylococcus aureus are among the most common causes of food poisoning. Acting as superantigens they intoxicate the organism by causing a massive uncontrolled T cell activation that ultimately may lead to toxic shock and death. In contrast to our detailed knowledge regarding their interaction with the immune system, little is known about how they penetrate the epithelial barrier to gain access to their targets. We therefore studied the uptake of two staphylococcal enterotoxins (SEs), SEA and SEB, using organ cultured porcine jejunal explants as model system. Attachment of both toxins to the villus surface was scarce and patchy compared with that of cholera toxin B (CTB). SEA and SEB also bound to microvillus membrane vesicles in vitro, but less efficiently than CTB, and the binding was sensitive to treatment with endoglycoceramidase II, indicating that a glycolipid, possibly digalactosylceramide, acts as cell surface receptor at the brush border. Both SEs partitioned poorly with detergent resistant membranes (DRMs) of the microvillus, suggesting a weak association with lipid raft microdomains. Where attachment occurred, cellular uptake of SEA and SEB was also observed. In enterocytes, constitutive apical endocytosis normally proceeds only to subapical early endosomes present in the actomyosin-rich 'terminal web' region. But, like CTB, both SEA and SEB penetrated deep into the cytoplasm. In conclusion, the data show that after binding to the enterocyte brush border SEA and SEB perturb the apical membrane trafficking, enabling them to engage in transcytosis to reach their target cells in the subepithelial lamina propria. [ABSTRACT FROM AUTHOR]

In enterocytes of the small intestine, endocytic trafficking of CFTR channels from the brush border membrane ( BBM) to the subapical endosomes requires the minus-end motor, myosin VI (Myo6). The subapical localization of Myo6 is dependent on myosin Ia (Myo1a) the major plus-end motor associated with the BBM, suggestive of functional synergy between these two motors. In villus enterocytes of the Myo1a KO mouse small intestine, CFTR accumulated in syntaxin-3 positive subapical endosomes, redistributed to the basolateral domain and was absent from the BBM. In colon, where villi are absent and Myo1a expression is low, CFTR exhibited normal localization to the BBM in the Myo1a KO similar to WT. cAMP-stimulated CFTR anion transport in the small intestine was reduced by 58% in the KO, while anion transport in the colon was comparable to WT. Co-immunoprecipitation confirmed the association of CFTR with Myo1a. These data indicate that Myo1a is an important regulator of CFTR traffic and anion transport in the BBM of villus enterocytes and suggest that Myo1a may power apical CFTR movement into the BBM from subapical endosomes. Alternatively, it may anchor CFTR channels in the BBM of villus enterocytes as was proposed for Myo1a's role in BBM localization of sucrase-isomaltase. [ABSTRACT FROM AUTHOR]

Abstract: This study aimed at studying the effect of age and Enterococcus faecium NCIMB 10415 on jejunal physiological measures in suckling and weaned piglets. A total of 112 piglets from 16 landrace sows were used. Sows received either no probiotic (n =8, control) or were fed a diet with 4.2 to 4.3×106 cfu/g E. faecium NCIMB 10415 (n =8, EF) from 28days ante partum onwards. Piglets were offered creep feed beginning at 12 d of age and piglets were weaned to a pre-starter diet at 26 d of age. For EF piglets, the creep feed (pre-starter) and starter diets contained 5.1×106 cfu/g and 3.6×106 cfu EF/g, respectively. Twenty-seven randomly selected piglets from each group were euthanized at 12 (n =6), 26 (n =6), 34 (n =7), and 54d (n =8) of age and a 45cm mid-jejunal segment was taken. Histomorphometry was performed and relative transcript abundance of caspase-3, proliferating cell nuclear antigen, intestinal alkaline phosphatase (IAP), lactase-phlorizin hydrolase (LPH), sucrase (SUC), aminopeptidase-N (APN) and sodium/glucose cotransporter 1 (SGLT1) was determined. Lactase (LAC), maltase (MAL), SUC, APN and IAP activities were measured in brush border membranes. The EF treatment did not affect (P>0.05) performance variables like the pre- or post-weaning daily weight gain, feed intake, incidence of diarrhoea and ileal and total tract nutrient digestibility, except the feed conversion ratio which was reduced (P=0.033). Jejunal villus length and crypt depth did not differ between groups (P>0.05) but increased age-dependently (P=0.003 and P<0.001, respectively). Feeding EF did not markedly influence brush border enzyme expression or activity. Enzyme activity increased (SUC, MAL) or decreased (LAC, IAP) with age reflecting maturation and adaptation to creep feed (P<0.001). The gene expression of SUC, SGLT1 and IAP changed over time (P=0.012, P=0.003 and P=0.013). Furthermore, gene expression was not affected by treatment. The current study suggests that EF did not markedly affect physiological variables associated with digestive function. Age of piglets seems to be the main factor determining function and morphology of the jejunal tissue, reflecting both maturation and adaptation to creep feed. [Copyright &y& Elsevier]

Sodium absorption in the mammalian small intestine occurs predominantly by two primary pathways that include Na/H exchange (NHE3) and Na-glucose cotransport (SGLT1) on the brush border membrane (BBM) of villus cells. However, whether NHE3 and SGLT1 function together to regulate intestinal sodium absorption is unknown. Nontransformed small intestinal epithelial cells (IEC-18) were transfected with either NHE3 or SGLT1 small interfering RNAs (siRNAs) and were grown in confluent monolayers on transwell plates to measure the effects on Na absorption. Uptake studies were performed as well as molecular studies to determine the effects on NHE3 and SGLT1 activity. When IEC-18 monolayers were transfected with silencing NHE3 RNA, the cells demonstrated decreased NHE3 activity as well as decreased NHE3 mRNA and protein. However, in NHE3 siRNA-transected cells, SGLT1 activity, mRNA, and protein in the BBM were significantly increased. Thus, inhibition of NHE3 expression regulates the expression and function of SGLT1 in the BBM of intestinal epithelial cells. In addition, IEC-18 cells transected with silencing SGLT1 RNA demonstrated an inhibition of Na-dependent glucose uptake and a decrease in SGLT1 activity, mRNA, and protein levels. However, in these cells, Na/H exchange activity was significantly increased. Furthermore, NHE3 mRNA and protein levels were also increased. Therefore, the inhibition of SGLT1 expression stimulates the transcription and function of NHE3 and vice versa in the BBM of intestinal epithelial cells. Thus this study demonstrates that the major sodium absorptive pathways together function to regulate sodium absorption in epithelial cells. [ABSTRACT FROM AUTHOR]

Background: Lactoferrin (Lf) belongs to the transferrin family of non-heme iron-binding proteins and is found in milk and mucosal secretions. Consequently, it is now considered a multifunctional protein mainly involved in both the innate and adaptive immune defenses of the organism against various pathogens, and Lf receptors have been identified at the surfaces of a number of different cells. In the small intestine Lf binds to the luminal surface, but its further interaction with the epithelial cells is controversial. Methods: In the present work, we studied the uptake of Lf in cultured mucosal explants of pig small intestine by immunofluorescence and immunogold microscopy. Results: Lf rapidly bound to the brush border and subsequently appeared in punctae in the apical cytoplasm, indicating internalization into an endosomal compartment. Essentially, no labeling was detected elsewhere in the enterocytes by 2 h incubation. However, in addition to enterocytes, a distinct subpopulation of cells in the lamina propria also took up Lf, most likely from the serosal side of the explants. None of these cells were apoptotic, nor did they belong to the predominant group of immunoglobulin-synthesizing plasma cells in the lamina propria. However, they were CD3, identifying them as T lymphocytes. Lf labeling of these cells was mainly seen in the cytosol, but occasionally nuclear staining was seen as well, suggesting a direct regulatory role of Lf. Conclusion: We propose that Lf functions in the immune defense of the small intestinal mucosa by targeting the population of T cells in the lamina propria. [ABSTRACT FROM AUTHOR]

The small intestinal brush border functions as the body's main portal for uptake of dietary nutrients and simultaneously acts as the largest permeability barrier against pathogens. To enable this, the digestive enzymes of the brush border are organized in lipid raft microdomains stabilized by cross-linking galectins and intelectin, but little is known about the dynamic properties of this highly specialized membrane. Here, we probed the endocytic membrane trafficking from the brush border of organ-cultured pig intestinal mucosal explants by use of a fixable, lipophilic FM dye. The fluorescent dye readily incorporated into the brush border, and by 15 mm faint but distinct punctae were detectable 1 μm beneath the brush border, indicative of a constitutive endocytosis. The punctae represented a subpopulation of early endosomes confined to the actomyosin-rich terminal web region, and their number and intensity increased by 1 h, but trafficking further into the enterocyte was not observed except in immature epithelial cells of the crypts. A powerful ligand for receptor-mediated endocytosis, cholera toxin B subunit, increased apical endocytosis and caused membrane trafficking to proceed to compartments localized deeper into the cytoplasm of the enterocytes. Two major raft-associated brush border enzymes, alkaline phosphatase and aminopeptidase N, were excluded from endocytosis. We propose that the terminal web cytoskeleton, by inhibiting traffic from apical early endosomes further into the cell, contributes to the overall permeability barrier of the gut. [ABSTRACT FROM AUTHOR]

Oxidative stress and mitochondrial dysfunction have been implicated in the pathogenesis of indomethacin-induced enteropathy. We evaluated the potential of curcumin, a known cytoprotectant, as an agent to protect against such effects. Rats were pretreated with curcumin (40 mg/kg by intra-peritoneal injection) before administration of indomethacin (20 mg/kg by gavage). One hour later, the small intestine was isolated and used for assessment of parameters of oxidative stress. Mitochondria, brush border membranes (BBM) and surfactant-like particles (SLP) were also isolated from the tissue. Mitochondria were used for assessment of functional integrity, estimation of products of lipid peroxidation and lipid content. BBM were used for estimation of products of lipid peroxidation and lipid content, while the SLP were used for measurement of lipid content. The results showed that oxidative stress and mitochondrial dysfunction occurred in the small intestine of indomethacin-treated rats. Pre-treatment with curcumin was found to ameliorate these drug-induced changes. Significant changes were seen in some of the lipids in the mitochondria, BBM and SLP in response to indomethacin. However, curcumin did not have any significant effect on these drug-induced changes. We conclude that curcumin, by attenuating oxidative stress and mitochondrial dysfunction, holds promise as an agent that can potentially reduce NSAID-induced adverse effects in the small intestine. [ABSTRACT FROM AUTHOR]

We have characterized the Na-glutamine cotransporter in the rabbit intestinal crypt cell brush border membrane vesicles (BBMV). Substrate specificity experiments showed that crypt cell glutamine uptake is mediated by system N. Real-time PCR experiments showed that SN2 (SLC38A5) mRNA is more abundant in crypt cells compared with SN1 (SLC38A3), indicating that SN2 is the major glutamine transporter present in the apical membrane of the crypt cells. SN2 cDNA was obtained by screening a rabbit intestinal cDNA library with human SN1 used as probe. Rabbit SN2 cDNA encompassed a 473- amino-acid-long open reading frame. SN2 protein displayed 87% identity and 91% similarity to human SN2. Functional characterization studies of rabbit SN2 were performed by using vaccinia virus- mediated transient expression system. Substrate specificity of the cloned transporter was identical to that of SN2 described in the literature and matched well with substrate specificity experiments performed using crypt cell BBMV. Cloned rabbit SN2, analogous to its human counterpart, is Li+ tolerant. Hill coefficient for Li+ activation of rabbit SN2-mediated uptake was 1. Taken together, functional data from the crypt cell BBMV and the cloned SN2 cDNA indicate that the crypt cell glutamine transport is most likely mediated by SN2. [ABSTRACT FROM AUTHOR]

Inşammatory fibroid polyps are uncommon gastrointestinal tract lesions. The stomach and ileum are the most commonly affected sites. Clinical symptoms depend on the location and size of the lesion. Obstruction or intussusception is the most frequent initial symptom when an inşammatory fibroid polyp is located in the small intestine. We describe two cases of inşammatory fibroid polyps of the small intestine causing ileocecal and ileoileal intussusception. [ABSTRACT FROM AUTHOR]

In the mammalian small intestine, coupled NaCI absorption occurs via the dual operation of Na/H and Cl/HCO3 exchange on the villus cell brush border membrane (BBM). Although constitutive nitric oxide (cNO) has been demonstrated to alter gastrointestinal tract functions, how cNO may specifically alter these two transporters to regulate coupled NaCI absorption is unknown. In villus cells, inhibition of cNO synthase (cNOS) with L-NG-nitroarginine methylester (L-NAME) stimulated Na/H exchange whereas Cl/HCO3 exchange was unaffected. In villus cell BBM vesicles (BBMV) prepared from rabbits treated with L-NAME, Na/H exchange was also stimulated. D-NAME, an inactive analog of L-NAME, and N6-(1-imonoethyl)-L-lysine dihydrochloride, a more selective inhibitor of inducible NO synthase, did not affect Na/H exchange. Kinetic studies demonstrated that the mechanism of stimulation is secondary to an increase in the maximal rate of uptake of Na, without an alteration in the affinity of the transporter for Na. Northern blot studies demonstrated an increase in the message for the BBM Na/H exchanger NHE3, and Western blot studies showed that the immunoreactive protein levels of NHE3 was increased when cNOS was inhibited. Thus these results indicate that cNO under nominal physiological states most likely maintains an inhibitory tone on small intestinal coupled NaCI absorption by specifically inhibiting BBM Na/H expression. [ABSTRACT FROM AUTHOR]

Synthesis and deposition of immunoglobulins in the brush border was studied in organ-cultured pig small intestinal mucosal explants. Surprisingly, comparable amounts of IgM and IgA were synthesized during a 6-h pulse, and also newly made IgG was detected in media and explants, including the microvillar fraction. For IgA and IgM, this subcellular distribution is consistent with basolateral-to-apical transcytosis, mediated by the polymeric immunoglobulin receptor. IgG is a ligand for the Fc receptor FcRn, and β2-microglobulin, the light chain of FcRn, coclustered in immunogold double labeling with IgG in subapical endosomes and in the basolateral membrane of enterocytes. In addition, β2-microglobulin was copurified with IgG on protein G-Sepharose. Apical endocytosis of IgG, as judged by internalization of fluorescent protein G, was not detectable except in a few isolated cells. This suggests that IgG in the adult small intestine is transported across the enterocyte mainly in the basolateral to apical direction. Significant fractions of all immunoglobulins bound to lactoseagarose, indicating that ‘anti-glycosyl’ antibodies, raised against commensal gut bacteria, are synthesized locally in the small intestine. By partial deposition in the brush border, these antibodies therefore may have a protective function by preventing lectin-like pathogens from gaining access to the brush border surface. [ABSTRACT FROM AUTHOR]

Abstract: The divalent metal ion transporter DMT1 is localized in the brush border membrane (BBM) of the upper small intestine and has been shown to be able to transport Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. Belgrade rats have a glycine-to-arginine (G185R) mutation in DMT1, which affects its function. We investigated copper transport with BBM vesicles of Belgrade rats loaded with calcein, which exhibits fluorescence quenching by various metal ions. Transport of copper was disrupted in unenergized BBM vesicle of b/b Belgrade rats, as had been described for iron transport, while +/b vesicles exhibited normal transport by DMT1. When either b/b or +/b vesicles were loaded with ATP and magnesium, similar high-affinity accumulation of copper was observed in both types of vesicles. Thus, brush border membranes possess an ATP-driven, high-affinity copper transport system which could serve as the primary route for copper uptake by the intestine. [Copyright &y& Elsevier]

The effect of 1-, 3-, and 5-day fasting on enzymes of brush border membrane and carbohydrate metabolism in the rat small intestine has been studied. Fasting led to significant decrease in the specific activities of brush border marker enzymes alkaline phosphatase and sucrase, as compared to levels in unfasted controls, both in the homogenates and in brush border membrane preparations. Kinetic studies revealed that these changes were due to changes in the maximal velocity (Vmax) only of alkaline phosphatase, but both the Michaelis constant (Km) and Vmax were altered for sucrase. Fasting decreased the activities of lactate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase but increased the activities of glucose-6-phosphatase and fructose 1,6-bisphosphatase in mucosal homogenates. The activities of glucose-6-phosphate dehydrogenase and malic enzyme were also decreased by fasting. Thus fasting caused changes in the activities of various enzymes involved in the metabolism of glucose. The results of these enzyme studies suggest that the degradation of glucose is decreased but its production by gluconeogenesis is enhanced upon fasting. [Copyright &y& Elsevier]

In the rabbit small intestine, there are three functionally different brush-border membrane (BBM) anion/HCO[sup -, sub 3] exchangers: 1) Cl/HCO[sup -, sub 3] exchange on the BBM of villus cells responsible for coupled NaCl absorption; 2) Cl/HCO[sup -, sub 3] exchange on the BBM of crypt cells possibly involved in HCO[sup -, sub 3] secretion; and 3) short-chain fatty acid (SCFA)/HCO[sup -, sub 3] exchange on the BBM of villus cells, which facilitates SCFA absorption. Although constitutive nitric oxide (cNO) has been postulated to alter many gastrointestinal tract functions, how cNO may specifically alter these three transporters is unknown. Inhibition of cNO synthase with N[sup G]-nitro-L-arginine methyl ester (L-NAME) 1) did not affect villus cell BBM Cl/HCO[sup -, sub 3] exchange, 2) stimulated crypt cell BBM Cl/HCO[sup -, sub 3] exchange, and 3) inhibited villus cell BBM SCFA/HCO[sup -, sub 3] exchange. D-NAME, an inactive analog of L-NAME, and L-N[sup 6]-(1-iminoethyl)lysine, a more selective inhibitor of inducible NO, did not affect these transport processes. Kinetic studies demonstrated that 1) the mechanism of inhibition of crypt cell BBM Cl/HCO[sup -, sub 3] exchange is secondary to a decrease in the maximal rate of uptake of Cl, without an alteration in the affinity of the transporter for Cl, and 2) the mechanism of stimulation of villus cell BBM SCFA/HCO[sup -, sub 3] exchange is secondary to an increase in the affinity of the transporter for SCFA without an alteration in the maximal rate of uptake of SCFA. These results indicate that cNO uniquely regulates the three BBM anion/HCO[sup -, sub 3] transporters in the rabbit small intestine. [ABSTRACT FROM AUTHOR]

This study presents a method of brush border membrane (BBM) preparation from the human small intestine using polyethylene glycol (PEG) precipitation and also looks at the effect of in vitro oxidant exposure on structural and functional alterations in the membrane. Isolated BBM were relatively pure as judged by 10- to 14-fold enrichment of marker enzymes with less than 1% contamination by other subcellular organelles. These membranes showed uphill transport of glucose and lipid analysis showed a cholesterol-phospholipid (C/P) ratio of 1.19. Isolated BBM were found to be susceptible to superoxide generated by xanthine oxidase (XO), resulting in lipid and protein oxidation along with altered glucose uptake. Superoxide exposure also resulted in phospholipid alterations, especially generation of lyso phospholipids. These changes were prevented by inhibiting XO by allopurinol or scavenging superoxide by superoxide dismutase (SOD). Other oxidants studied did not have significant affect on these membranes. These studies suggest that PEG can be used for preparation of BBM from the human small intestine and these membranes undergo structural and functional alterations on exposure to superoxide. [ABSTRACT FROM AUTHOR]

Dipeptidyl peptidase IV (EC 3.4.14.-) of pig small intestinal brush border membrane was solubilized by Triton X-100 and purified by immunoadsorption on Sepharose conjugated to antibodies against kidney dipeptidyl peptidase IV. The recovery was around 20% and the purification factor around 500. The enzyme appeared homogeneous in crossed immunoelectrophoresis and migrated in polyacrylamide gel electrophoresis in dodecyl sulphate as a single polypeptide chain corresponding to a molecular weight of 137000. Gel filtration under non-denaturing conditions indicated a molecular weight around 230000, which suggested a dimeric structure for the isolated form of dipeptidyl peptidase IV. The enzyme showed a strong tendency to aggregate and its hydrophobic properties were further characterized in studies on detergent binding. Interaction with sodium deoxycholate or cetyltrimethylammonium bromide in charge-shift crossed immunoelectrophoresis thus resulted in an anodic or cathodic shift in mobility, respectively. Furthermore, by crossed immunoelectrophoresis in the presence of 14C-labelled Triton X-100 the detergent binding could be directly visualized by autoradiography. After trypsin treatment the enzyme no longer formed complexes with the detergents. The intestinal dipeptidyl peptidase IV released the N-terminal dipeptides from glycyl-L-proline-4-nitroanilide, glycyl-L-proline-2-naphthylamide, glycyl-L-prolyl-L-alanine, L-alanyl-L-alanyl-Lalanyl-L-alanine, and L-leucyl-L-leucyl-L-valyl-L-tyrosyl-L-serine. It had a very low activity on L-alanine-4-n-itroanilide and showed no endopeptidase activity. The activity on all hydrolyzed substrates was sensitive to the serine protease inhibitor diisopropylfluorophosphate. The Km for the hydrolysis of glycyl-L-proline-4-nitroanilide was 0.24 mM. The dipeptides glycyl-L-proline and glycyl-L-leucine competitively inhibited the hydrolysis of glycyl-L-proline-4-nitroanilide with Ki values of 3.7 mM and 1.7 mM, respectively. [ABSTRACT FROM AUTHOR]

Presents a study on the regional brush-border uptakes of alpha-methyl-D-glucose and L-proline from birth until adulthood in guinea pig small intestine. Materials and methods; Results and discussion.

Provides evidence of a series of major species differences in the transport of imino acids and non-alpha-amino acids. Influx across the brush-border membrane of beta-alanine and gamma-aminobutyric acid; Influx of proline across the brush-border membrane; Influx of taurine across the brush-border membrane; Interaction of alpha-amino-monocarboxylic acids with the imino acid carrier.

In order to determine whether the neonatal mouse small intestine has the ability to be selective in the binding of homologous immunoglobulin isotypes. mouse immunoglobulins were tested for their ability to bind to brush borders isolated from jejunal enterocytes of neonatal and adult mice. The binding of immunoglobulins to brush borders was detected by fluorescence-activated cell sorter (FACS) analysis. The results showed that brush borders from 12-dayold mice bound mouse immunoglobulins selectively, binding IgG2a, IgG2b, IgG3 and IgE but not IgG1, IgM or IgA. Similarly, at 16 days, only IgG2a, IgG2b, IgG3 and IgE were bound significantly. The ability of jejunal brush borders to bind gamma-globulins declined with age and their selectivity was lost such that in mice of 18 days and over not one of the immunoglobulins tested was bound. It was concluded that neonatal mouse jejunal Fcγ receptors bind IgG2a and IgG2b almost exclusively. The sudden drop in binding between 16 and 18 days coincides with the previously reported age at which immunoglobulin transmission across the gut in the neonatal mouse ceases (closure). [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the presence of a K+-H+ exchanger in brush-border membrane vesicles (BBMV) isolated from chick small intestine. 86Rb+, as a tracer for K+ transport, was used to probe for the exchange mechanism. An outwardly directed proton gradient (pH 5.5 inside, pH 7.5 outside) stimulated 86Rb+ uptake into voltage-clamped BBMV. H+-driven 86Rb+ uptake was only weakly inhibited by 5-(N-ethyl-N-isopropyl)amiloride, whereas this agent strongly inhibited H+-driven Na+ uptake. At initial rates, proton-driven 86Rb+ uptake was significantly reduced by external K+ but it was not significantly affected by external Na+. Conversely, extravesicular Na+ inhibited proton-driven Na+ uptake, whilst K+ had little effect. H+-driven K+ uptake tended to saturate with increasing external K+ concentrations and Lineweaver-Burk analysis of the data revealed a Km, lot external K+ of 2 mM. These findings are consistent with the presence of K+-H+ exchange activity in the chicken jejunal brush-border membrane. [ABSTRACT FROM AUTHOR]

Resection of distal small intestine causes calcium malabsorption in humans and in a rat model of 50% distal resection. We tested the hypothesis that this calcium malabsorption is caused in the rat model by a brush border defect. We compared brush border membrane vesicles from the proximal small intestine ofcontrol (transection and anastomosis at midsmall intestine) with distally resected rats. Mucosal protein was 25% greater in the resected group and the vesicles were enriched 37-fold in sucrase activity when compared with homogenate. Kinetic constants vmax (maximal initial rate ofsaturable calcium uptake at infinite concentration), kΤ (calcium concentration for saturable calcium uptake rate at half vmax, and kD (rate constant for nonsaturable calcium uptake per unit concentration) were slightly but not significantly greater in the resected as compared with the transected group, ruling out the brush border as the cause for decreased transmucosal calcium transport. [ABSTRACT FROM AUTHOR]

To investigate maturational changes of membrane food protein binding capacity, we studied binding characteristics of brush border membranes isolated from small intestines of newborn and adult rats. Binding of biotinylated gliadin peptides, cow's milk proteins (alpha-casein, beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin) and lectins was assessed by a sensitive chemiluminescence blot assay. We found specific food protein binding with regard to saturation and inhibition. Maximal binding of most food proteins and several lectins to brush border membranes of newborn and adult rats was comparable, whereas binding of beta-lactoglobulin was substantially less. Common or adjoining binding sites for the different food proteins tested were indicated by corresponding membrane protein binding patterns and by inhibition properties of unrelated proteins. Compared to newborns, adult membrane vesicles as well as isolated membrane proteins showed higher binding capacities. Thus postnatal maturation of small intestinal brush border membranes correlated with increased food protein binding capacity. [ABSTRACT FROM AUTHOR]

The osmotic water permeability P of brush border (BBM) and basolateral (BLM) membrane vesicles from rat small intestine and renal cortex was studied by means of stopped-flow spectrophotometry. Scattered light intensity was used to follow vesicular volume changes upon osmotic perturbation with hypertonic mannitol solutions. A theoretical analysis of the relationship of scattered light intensity and vesicular volume justified a simple exponential approximation of the change in scattered light intensity. The rate constants extracted from fits to an exponential function were proportional to the final medium osmolarity as predicted by theory. For intestinal membranes, computer analysis of optical responses fitted well with a single-exponential treatment. For renal membranes a double-exponential treatment was needed, implying two distinct vesicle populations. P values for BBM and BLM preparations of small intestine were equal and amount to 60 μm/sec. For renal preparations, P values amount to 600 μm/sec for the fast component, BBM as well as BLM, and to 50 (BBM) and 99 (BLM) μm/sec for the slow component. The apparent activation energy for water permeation in intestinal membranes was 13.3±0.6 and in renal membranes, 1.0±0.3 kCal/mole, between 25 and 35°C. The mercurial sulfhydryl reagent pCMBS inhibited completely and reversibly the high P value in renal brush border preparations. These observations suggest that in intestinal membranes water moves through the lipid matrix but that in renal plasma membranes water channels may be involved. From the high P values of renal membrane vesicles a transcellular water permeability for proximal tubules can be calculated which amounts to ∼1 cm/sec. This value allows for an entirely transcellular route for water flow during volume reabsorption. [ABSTRACT FROM AUTHOR]

We examined the effects of seven n-alkyl alcohols (from n-butyl to n-undecyl alcohol), isoamyl alcohol and benzyl alcohol on the activity of membrane enzyme Mg-ATPase of the rabbit small intestinal brush border membrane. Their relationships with the changes in the fluidity of the membrane lipid bilayer were examined through studies on the fluorescence anisotropies of diphenylhexatriene (DPH) and its ionic derivatives. Good linear correlations were found both between the partition coefficients of the alcohols and their concentrations causing similar decreases in the activity of Mg-ATPase and between their partition coefficients and the alcohol-induced changes in fluorescence anisotropies. Within the concentration range of the alcohols tested, including isoamyl alcohol and benzyl alcohol, the decreases in activity of the membrane enzyme Mg-ATPase clearly corresponded with the decreases in fluorescence anisotropy of DPH, which is thought to be located within the hydrophobic core of the membrane. From these findings, one possible explanation is that inhibition of this enzyme by the alcohols is due to perturbation of the lipid bilayer of the brush border membrane. [ABSTRACT FROM AUTHOR]