Colonic Secretion Research Articles

Effects of Hemp seed soft capsule on colonic ion transport in rats.

AIMTo investigate the effect of Hemp seed soft capsule (HSCC) on colonic ion transport and its related mechanisms in constipation rats.METHODSSprague-Dawley male rats were randomly divided into three groups: normal group, constipation group and HSSC group. Rats in the constipation and HSSC groups were administrated loperamide 3 mg/kg per day orally for 12 d to induce the constipation model. Then, the HSSC group was given HSSC 0.126 g/kg per day by gavage for 7 d. The normal and constipation groups were treated with distilled water. After the treatment, the fecal wet weight and water content were measured. The basal short-circuit current (Isc) and resistance were measured by an Ussing Chamber. Besides the in vivo drug delivery experiment above, an in vitro drug application experiment was also conducted. The accumulative concentrations of HSSC (0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 2.5 mg/mL, 5.0 mg/mL, 10.0 mg/mL and 25.0 mg/mL) were added to the normal isolated colonic mucosa and the Isc was recorded. Further, after the application of either ion (Cl- or HCO3-) substitution, ion channel-related inhibitor (N-phenylanthranilic acid, glybenclamide, 4,4-diisothiocyano-2,2-stilbenedisulfonic acid or bumetanide) or neural pathway inhibitor [tetrodotoxin (TTX), atropine, or hexamethonium], the Isc induced by HSSC was also measured.RESULTSIn the constipation group, the fecal wet weight and the water content were decreased in comparison with the normal group (P < 0.01). After the treatment with HSSC, the fecal wet weight and the water content in the HSSC group were increased, compared with the constipation group (P < 0.01). In the constipation group, the basal Isc was decreased and resistance was increased, in comparison with the normal group (P < 0.01). After the treatment with HSSC, the basal Isc was increased (P < 0.05) and resistance was decreased (P < 0.01) in the HSSC group compared with the constipation group. In the in vitro experiment, beginning with the concentration of 1.0 mg/mL, differences in Isc were found between the experimental mucosa (with HSSC added) and control mucosa. The Isc of experimental mucosa was higher than that of control mucosa under the same concentration (1.0 mg/mL, P < 0.05; 2.5-25 mg/mL, P < 0.01). After the Cl- or HCO3- removal and pretreated with different inhibitors (cAMP-dependent and Ca2+-dependent Cl- channels, Na+-K+-2Cl- cotransporter (NKCC), Na+-HCO3- cotransporter or Cl-/HCO3- exchanger inhibitor), there were differences between experimental mucosa and control mucosa; the Isc of experimental mucosa was lower than that of control mucosa under the same concentration (P < 0.05). Meanwhile, after pretreatment with neural pathway inhibitor (TTX, atropine, or hexamethonium), there were no differences between experimental mucosa and control mucosa under the same concentration (P > 0.05).CONCLUSIONHSSC ameliorates constipation by increasing colonic secretion, which is mediated via the coaction of cAMP-dependent and Ca2+-dependent Cl- channels, NKCC, Na+-HCO3- cotransporter or Cl-/HCO3- exchanger.

Isothiocyanate-enriched moringa seed extract alleviates ulcerative colitis symptoms in mice.

Moringa (Moringa oleifera Lam.) seed extract (MSE) has anti-inflammatory and antioxidant activities. We investigated the effects of MSE enriched in moringa isothiocyanate-1 (MIC-1), its putative bioactive, on ulcerative colitis (UC) and its anti-inflammatory/antioxidant mechanism likely mediated through Nrf2-signaling pathway. Dextran sulfate sodium (DSS)-induced acute (n = 8/group; 3% DSS for 5 d) and chronic (n = 6/group; cyclic rotations of 2.5% DSS/water for 30 d) UC was induced in mice that were assigned to 4 experimental groups: healthy control (water/vehicle), disease control (DSS/vehicle), MSE treatment (DSS/MSE), or 5-aminosalicyic acid (5-ASA) treatment (positive control; DSS/5-ASA). Following UC induction, water (vehicle), 150 mg/kg MSE, or 50 mg/kg 5-ASA were orally administered for 1 or 2 wks. Disease activity index (DAI), spleen/colon sizes, and colonic histopathology were measured. From colon and/or fecal samples, pro-inflammatory biomarkers, tight-junction proteins, and Nrf2-mediated enzymes were analyzed at protein and/or gene expression levels. Compared to disease control, MSE decreased DAI scores, and showed an increase in colon lengths and decrease in colon weight/length ratios in both UC models. MSE also reduced colonic inflammation/damage and histopathological scores (modestly) in acute UC. MSE decreased colonic secretions of pro-inflammatory keratinocyte-derived cytokine (KC), tumor necrosis factor (TNF)-α, nitric oxide (NO), and myeloperoxidase (MPO) in acute and chronic UC; reduced fecal lipocalin-2 in acute UC; downregulated gene expression of pro-inflammatory interleukin (IL)-1, IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) in acute UC; upregulated expression of claudin-1 and ZO-1 in acute and chronic UC; and upregulated GSTP1, an Nrf2-mediated phase II detoxifying enzyme, in chronic UC. MSE was effective in mitigating UC symptoms and reducing UC-induced colonic pathologies, likely by suppressing pro-inflammatory biomarkers and increasing tight-junction proteins. This effect is consistent with Nrf2-mediated anti-inflammatory/antioxidant signaling pathway documented for other isothiocyanates similar to MIC-1. Therefore, MSE, enriched with MIC-1, may be useful in prevention and treatment of UC.

Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells.

Bile acids and epithelial-derived human β-defensins (HβDs) are known to be important factors in the regulation of colonic mucosal barrier function and inflammation. We hypothesized that bile acids regulate colonic HβD expression and aimed to test this by investigating the effects of deoxycholic acid (DCA) and ursodeoxycholic acid on the expression and release of HβD1 and HβD2 from colonic epithelial cells and mucosal tissues. DCA (10-150 µM) stimulated the release of both HβD1 and HβD2 from epithelial cell monolayers and human colonic mucosal tissue in vitro In contrast, ursodeoxycholic acid (50-200 µM) inhibited both basal and DCA-induced defensin release. Effects of DCA were mimicked by the Takeda GPCR 5 agonist, INT-777 (50 μM), but not by the farnesoid X receptor agonist, GW4064 (10 μM). INT-777 also stimulated colonic HβD1 and HβD2 release from wild-type, but not Takeda GPCR 5-/-, mice. DCA stimulated phosphorylation of the p65 subunit of NF-κB, an effect that was attenuated by ursodeoxycholic acid, whereas an NF-κB inhibitor, BMS-345541 (25 μM), inhibited DCA-induced HβD2, but not HβD1, release. We conclude that bile acids can differentially regulate colonic epithelial HβD expression and secretion and discuss the implications of our findings for intestinal health and disease.-Lajczak, N. K., Saint-Criq, V., O'Dwyer, A. M., Perino, A., Adorini, L., Schoonjans, K., Keely, S. J. Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells.

α-melanocyte stimulating hormone modulates the central acyl ghrelin-induced stimulation of feeding, gastrointestinal motility, and colonic secretion.

BackgroundAcyl ghrelin-induced intake depends on hypothalamic neuropeptide Y and agouti-related protein (AgRP) neurotransmitters. Intracerebroventricular (ICV) injection of AgRP increases feeding through competitive antagonism at melanocortin receptors. ICV administration of α-melanocyte stimulating hormone (α-MSH), a natural antagonist of AgRP, may modulate the acyl ghrelin-induced orexigenic effect.ObjectiveThis study aimed to investigate the modulating effect of α-MSH on the central acyl ghrelin-induced food intake, gastrointestinal motility, and colonic secretion in rats.Methods and proceduresWe examined the effects of α-MSH and acyl ghrelin on food intake, gastric emptying, small intestinal transit, colonic motility, and secretion in conscious rats with a chronic implant of ICV catheters.ResultsICV injection of O-n-octanoylated ghrelin (0.1 nmol/rat) significantly increased the cumulative food intake up to 8 h (P<0.01), enhanced non-nutrient semi-liquid gastric emptying (P<0.001), increased the geometric center and running percentage of small intestinal transit (P<0.001), accelerated colonic transit time (P<0.05), and increased fecal pellet output (P<0.01) and total fecal weight (P<0.01). Pretreatment with ICV injection of α-MSH (1.0 and 2.0 nmol/rat) attenuated the acyl ghrelin-induced hyperphagic effect, fecal pellet output, and total fecal weight, while higher dose of α-MSH (2.0 nmol/rat) attenuated the increase in the geometric center of small intestinal transit (P<0.01). However, neither dose of α-MSH altered acyl ghrelin-stimulated gastroprokinetic effect, increase in the running percentage of small intestinal transit, nor accelerated colonic transit time.Conclusionα-MSH is involved in central acyl ghrelin-elicited feeding, small intestinal transit, fecal pellet output, and fecal weight. α-MSH does not affect central acyl ghrelin-induced acceleration of gastric emptying and colonic transit time in rats.

Human-derived gut microbiota modulates colonic secretion in mice by regulating 5-HT3 receptor expression via acetate production.

Serotonin [5-hydroxytryptamine (5-HT)], an important neurotransmitter and a paracrine messenger in the gastrointestinal tract, regulates intestinal secretion by its action primarily on 5-HT3 and 5-HT4 receptors. Recent studies highlight the role of gut microbiota in 5-HT biosynthesis. In this study, we determine whether human-derived gut microbiota affects host secretory response to 5-HT and 5-HT receptor expression. We used proximal colonic mucosa-submucosa preparation from age-matched Swiss Webster germ-free (GF) and humanized (HM; ex-GF colonized with human gut microbiota) mice. 5-HT evoked a significantly greater increase in short-circuit current (ΔIsc) in GF compared with HM mice. Additionally, 5-HT3 receptor mRNA and protein expression was significantly higher in GF compared with HM mice. Ondansetron, a 5-HT3 receptor antagonist, inhibited 5-HT-evoked ΔIsc in GF mice but not in HM mice. Furthermore, a 5-HT3 receptor-selective agonist, 2-methyl-5-hydroxytryptamine hydrochloride, evoked a significantly higher ΔIsc in GF compared with HM mice. Immunohistochemistry in 5-HT3A-green fluorescent protein mice localized 5-HT3 receptor expression to enterochromaffin cells in addition to nerve fibers. The significant difference in 5-HT-evoked ΔIsc between GF and HM mice persisted in the presence of tetrodotoxin (TTX) but was lost after ondansetron application in the presence of TTX. Application of acetate (10 mM) significantly lowered 5-HT3 receptor mRNA in GF mouse colonoids. We conclude that host secretory response to 5-HT may be modulated by gut microbiota regulation of 5-HT3 receptor expression via acetate production. Epithelial 5-HT3 receptor may function as a mediator of gut microbiota-driven change in intestinal secretion.NEW & NOTEWORTHY We found that gut microbiota alters serotonin (5-HT)-evoked intestinal secretion in a 5-HT3 receptor-dependent mechanism and gut microbiota metabolite acetate alters 5-HT3 receptor expression in colonoids.View this article's corresponding video summary at https://www.youtube.com/watch?v=aOMYJMuLTcw&feature=youtu.be.

Forskolin (cAMP) Promotes Insertion of Large Conductance Potassium (KCa1.1) Channels into Apical Membranes of Rat Distal Colon

Mammalian colon contributes significantly to overall K+ homeostasis. Under normal conditions, the proximal colon secretes and distal colon absorbs K+. Distal colon also has a secretory capacity, which becomes important to K+ homeostasis under pathological conditions, such as end‐stage renal failure. K+ secretion in the distal colon is primarily mediated through apical large conductance (BK) channels. We hypothesized that the BK opener BMS‐204352 (BMS) and the adenylate cyclase activator forskolin (FSK) induce K+ secretion through separate mechanisms. K+ flux studies were performed in distal colonic mucosal tissues obtained from male Sprague‐Dawley rats (200–225g). Unidirectional 86Rb fluxes (K+ surrogate), and short‐circuit current (ISC) were measured under voltage‐clamped conditions in an Ussing chamber setup. Fluxes were performed in the presence of amiloride (10 mM) to inhibit ENaC‐mediated ISC, as well as VO4 (1mM) to inhibit apical H+/K+ ATPase and unmask BK‐mediated K+ secretion. Results indicate that BMS (10mM) and FSK (10mM) induced a net K+ secretion of 0.083 ± 0.04 and 0.274 ± 0.05 mEq/cm2·hr, respectively (n=4, p&lt;0.05). FSK, but not BMS, increased ISC compared to baseline (220.5 ± 30.5 vs 132.2 ± 18.7 uA/cm2). Immunofluorescence studies performed on the same tissues used in flux experiments showed markedly increased apical BK staining in tissues exposed to FSK, but not in those exposed to only BMS. Taken together, these data suggest that while BMS may only open BK channels already present on apical membranes, FSK may cause further K+ secretion by triggering the insertion of more BK channels into the apical membrane.Support or Funding Information• R01 DK104791/DK/NIDDK NIH HHS/United States

Altered Colonic Permeability and Epithelial Secretion in Subgroups of Irritable Bowel Syndrome Patients

Altered Colonic Permeability and Epithelial Secretion in Subgroups of Irritable Bowel Syndrome Patients

Human derived Gut Microbiota Modulates Colonic Secretion in Mice by Regulating 5‐HT3 Receptor Expression

The human gastrointestinal (GI) tract is home to trillions of microorganisms. There is dynamic interaction between the gut microbiota and the human host, but how gut microbiota modulates GI physiology is largely unknown. A recent study from our laboratory showed that human gut microbiota increases host serotonin (5‐hydroxytryptamine, 5‐HT) biosynthesis. 5‐HT is an important neurotransmitter and a paracrine messenger in the GI tract that increases intestinal secretion primarily through 5‐HT3 and 5‐HT4 receptors. In this study we investigated whether gut microbiota modulate 5‐HT3R and 5‐HT4R expression and host secretory response to 5‐HT.MethodsWe used proximal colon mucosa‐submucosa preparations from germ free (GF) and humanized (ex‐GF colonized with human bacteria for 4–5 weeks; HM) male mice. The change in short circuit current (ΔIsc) was determined in response to increasing concentrations of 5‐HT (0.003–300μM) on the submucosal side either alone or in the presence of 5‐HT3R antagonist ondansetron (100nM) in an Ussing chamber. Receptor expression was quantified using rt‐PCR (normalized to L32) and western blot (normalized to GAPDH). Immunohistochemistry (IHC) was used to determine localization of 5‐HT3R in the paraformaldehyde‐fixed 10μm sections from proximal colonic epithelium. Differences between groups were tested using Mann‐Whitney U (Mdn; IQ range) with P‐value lower than 0.05 considered significant.Results5‐HT induced ΔIsc was significantly higher in GF than HM mice (218; 184–274 vs 163; 132–182 μA/cm2, n=6, P = 0.04). To determine if this change was due to an alteration in 5‐HTR, we quantified receptor expression. We found significantly higher 5‐HT3R mRNA expression (1.82; 1.08–2.80 vs 1.00; 0.33–1.38, n=8–9, P = 0.046) in GF compared to HM mice, but no change in 5‐HT4R expression. 5‐HT3R protein expression was also significantly higher in GF mice compared to HM (4.62; 1.06–6.14 vs 1.00; 1–1.3, n= 11–12, P = 0.01) mice. There was a significantly higher ΔIsc in GF compared to HM (87.1; 51.7–149 vs 37.0; 19.5–44 μA/cm2, n=5, P = 0.02) mice, following application of 2‐methyl 5‐hydroxytryptamine hydrochloride (selective 5‐HT3R agonist) suggesting increased receptor expression augments secretory response. Furthermore, ondansetron significantly blocked 5‐HT induced maximum ΔIsc in GF mice (201; 189–260 vs 117.6; 92–149 μA/cm2, n=6–8, P = 0.001) but not in HM mice (163; 132–182 vs 120; 107–150 μA/cm2, n=6, P = 0.24). To determine the expression of 5‐HT3R in mouse colon mucosa‐submucosa we used 5‐HT3–tagged GFP mice. IHC identified 5‐HT3R‐GFP positive cells in the mouse colonic epithelium. To ascertain if the significant difference in 5‐HT‐induced ΔIsc between GF and HM mice (188; 152–200 vs 125; 99–139 μA/cm2, n=5, P = 0.008) is driven by epithelial 5‐HT3R, we measured the response in the presence of TTX (block neuronal transmission). TTX (500 nM) had no effect of ondansetron inhibition of 5‐HT‐induced ΔIsc, suggesting a role for epithelial 5HT3R.ConclusionGut microbial colonization decreases host secretory response to 5‐HT by downregulating 5‐HT3R expression. Epithelial 5‐HT3R may be important in gut microbiota modulation of intestinal secretionSupport or Funding InformationNIH K08 DK

Brachyspira hyodysenteriae Infection Regulates Mucin Glycosylation Synthesis Inducing an Increased Expression of Core-2 O-Glycans in Porcine Colon.

Brachyspira hyodysenteriae causes swine dysentery (SD), leading to global financial losses to the pig industry. Infection with this pathogen results in an increase in B. hyodysenteriae binding sites on mucins, along with increased colonic mucin secretion. We predict that B. hyodysenteriae modifies the glycosylation pattern of the porcine intestinal mucus layer to optimize its host niche. We characterized the swine colonic mucin O-glycome and identified the differences in glycosylation between B. hyodysenteriae-infected and noninfected pigs. O-Glycans were chemically released from soluble and insoluble mucins isolated from five infected and five healthy colon tissues and analyzed using porous graphitized carbon liquid chromatography tandem mass spectrometry. In total, 94 O-glycans were identified, with healthy pigs having higher interindividual variation, although a larger array of glycan structures was present in infected pigs. This implied that infection induced loss of individual variation and that specific infection-related glycans were induced. The dominating structures shifted from core-4-type O-glycans in noninfected pigs toward core-2-type O-glycans in infected animals, which correlated with increased levels of the C2GnT glycosyl transferase. Overall, glycan chains from infected pigs were shorter and had a higher abundance of structures that were neutral or predominantly contained NeuGc instead of NeuAc, whereas they had a lower abundance of structures that were fucosylated, acidic, or sulfated than those from noninfected pigs. Therefore, we conclude that B. hyodysenteriae plays a major role in regulating colonic mucin glycosylation in pigs during SD. The changes in mucin O-glycosylation thus resulted in a glycan fingerprint in porcine colonic mucus that may provide increased exposure of epitopes important for host-pathogen interactions. The results from this study provide potential therapeutic targets and a platform for investigations of B. hyodysenteriae interactions with the host via mucin glycans.

Oxalobacter formigenes colonization normalizes oxalate excretion in a gastric bypass model of hyperoxaluria

BackgroundHyperoxaluria and oxalate kidney stones frequently develop after Roux-en-Y gastric bypass (RYGB). Oxalobacter formigenes can degrade ingested oxalate. ObjectivesExamine the effect of O. formigenes wild rat strain (OXWR) colonization on urinary oxalate excretion and intestinal oxalate transport in a hyperoxaluric RYGB model. SettingBasic Science Laboratory, United States. MethodsAt 21 weeks of age, 28 obese male Sprague-Dawley rats survived Sham (n = 10) or RYGB (n = 18) surgery and were maintained on a 1.5% potassium oxalate, 40% fat diet. At 12 weeks postoperatively, half the animals in each group were gavaged with OXWR. At 16 weeks, percent dietary fat content was lowered to 10%. Urine and stool were collected weekly to determine oxalate and colonization status, respectively. At week 20, [14 C]-oxalate fluxes and electrical parameters were measured in vitro across isolated distal colon and jejunal (Roux limb) tissue mounted in Ussing Chambers. ResultsRYGB animals lost 22% total weight while Shams gained 5%. On a moderate oxalate diet, urinary oxalate excretion was 4-fold higher in RYGB than Sham controls. OXWR colonization, obtained in all gavaged animals, reduced urinary oxalate excretion 74% in RYGB and 39% in Sham and was further augmented by lowering the percentage of dietary fat. Finally, OXWR colonization significantly enhanced basal net colonic oxalate secretion in both groups. ConclusionsIn our model, OXWR lowered urinary oxalate by luminal oxalate degradation in concert with promotion of enteric oxalate elimination. Trials of O. formigenes colonization and low-fat diet are warranted in calcium oxalate stone formers with gastric bypass and resistant hyperoxaluria.

Establishment and effect observation on three kinds of Ti-Xu with constipation rat models

To establish three types constipation models with the syndrome of Xue-Xu, Yin-Xu and Yang-Xu in rats, and to compare the difference of colonic motility, colonic water metabolism, colonic mucus secretion and the level of aquaporin-2 (AQP2). Forty Sprague Dawley rats, male and female in half, were randomly divided into four groups:normal control group(N), Xue-Xu with constipation group(XC), Yin-Xu with constipation group(YC) and Yang-Xu with constipation group(YAC). Bloodletting and loperamide were used to induce the Xue-Xu with constipation in rats, thyroxin and loperamide were used to induce the Yin-Xu with constipation in rats, ice-water stimulation and loperamide were used to induce the Yang-Xu with constipation in rats. Bloodletting was coducted once a week,drug was ad-ministrated orally to rats once a day for continuous 42 days. Rats'state, body weights, stool character, transfer time between mouth and anal, small intestinal propulsion rate were measured. The colon tissues of rats were stained with alcian blue-periodic acid schiff (AB-PAS) to analyze the changes of mucus secretion. The aquaporin-2 expressions in proximal and distal colon of rats were measured by immunohistochemical method. Compared to normal control group, weight-increasing speed of the rats were reduced in three models. The order of indepen-dent movement change was YC, XC and YAC. Solid stool appeared at 30 d, fecal scores were increased. Transfer time between mouth and anal was significantly extended, small intestinal propulsion rate were significantly decreased(P < 0.05, P < 0.01). Water content in stool were significantly decreased in three type models(P < 0.05, P < 0.01), the content of water in colon were decreased in XC and YAC group (P < 0.05, P < 0.01). The colonic gland and goblet cells were narrowed in some extent, the mucus excretion were decreased. The levels of aquaporin-2 expressions in proximal and distal colon of rats were increased(P < 0.05, P < 0.01), increasing order of AQP2 expression in proximal colon was YAC, YC and XC, increasing order of AQP2 expression in distal colon was YAC, XC and YC. Long term stimulation of complex factors could induce Ti-Xu with constipation animal model, and there were some differences in colonic motility, water metabolism, colonic mucus secretion and AQP2 in rats.

Campylobacter jejuni infection of conventionally colonized mice lacking nucleotide-oligomerization-domain-2

BackgroundThe nucleotide-binding oligomerisaton protein 2 (NOD2) constitutes a pivotal sensor of bacterial muramyl dipeptide and assures expression of distinct antimicrobial peptides and mediators produced by enterocytes and immune cells directed against pathogens including Campylobacter jejuni. We here elucidated the role of NOD2 during murine C. jejuni infection in more detail.ResultsConventionally colonized NOD2 deficient (NOD2−/−) mice and corresponding wildtype (WT) counterparts were perorally infected with C. jejuni strain 81–176 on three consecutive days. The pathogen colonized both WT and NOD2−/− mice only sporadically until day 14 post infection (p.i.). However, the slightly higher prevalence of C. jejuni in NOD2−/− mice was accompanied by higher intestinal Escherichia coli loads known to facilitate C. jejuni colonization. Neither overt macroscopic (clinical) nor microscopic sequelae (such as colonic epithelial apoptosis) could be observed upon murine C. jejuni infection of either genotype. Innate immune responses were less distinctly induced in C. jejuni infected NOD2−/− versus WT mice as indicated by lower colonic numbers of neutrophils in the former. Conversely, adaptive immune cell counts including T lymphocytes were higher in large intestines of NOD2−/− as compared to WT mice that were paralleled by increased colonic IL-6 secretion and higher TNF and IL-18 mRNA expression levels in large intestines of the former. Only in NOD2−/− mice, however, colonic IL-22 mRNA expression was down-regulated at day 14 p.i. Whereas viable commensal intestinal bacteria could exclusively be detected in mesenteric lymph nodes and livers of NOD2−/− mice, bacterial translocation rates to kidneys and spleen were NOD2 independent. Notably, large intestinal mRNA expression levels of mucin-2, constituting a pivotal factor involved in epithelial barrier integrity, were comparable in naive and C. jejuni infected mice of either genotype.ConclusionNOD2 is involved in the well-balanced regulation of innate and adaptive pro-inflammatory immune responses of conventional mice upon C. jejuni infection.

Oxalobacter formigenes-Derived Bioactive Factors Stimulate Oxalate Transport by Intestinal Epithelial Cells.

Hyperoxaluria is a major risk factor for kidney stones and has no specific therapy, although Oxalobacter formigenes colonization is associated with reduced stone risk. O. formigenes interacts with colonic epithelium and induces colonic oxalate secretion, thereby reducing urinary oxalate excretion, via an unknown secretagogue. The difficulties in sustaining O. formigenes colonization underscore the need to identify the derived factors inducing colonic oxalate secretion. We therefore evaluated the effects of O. formigenes culture conditioned medium (CM) on apical 14C-oxalate uptake by human intestinal Caco-2-BBE cells. Compared with control medium, O. formigenes CM significantly stimulated oxalate uptake (>2.4-fold), whereas CM from Lactobacillus acidophilus did not. Treating the O. formigenes CM with heat or pepsin completely abolished this bioactivity, and selective ultrafiltration of the CM revealed that the O. formigenes-derived factors have molecular masses of 10-30 kDa. Treatment with the protein kinase A inhibitor H89 or the anion exchange inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid completely blocked the CM-induced oxalate transport. Knockdown of the oxalate transporter SLC26A6 also significantly restricted the induction of oxalate transport by CM. In a mouse model of primary hyperoxaluria type 1, rectal administration of O. formigenes CM significantly reduced (>32.5%) urinary oxalate excretion and stimulated (>42%) distal colonic oxalate secretion. We conclude that O. formigenes-derived bioactive factors stimulate oxalate transport in intestinal cells through mechanisms including PKA activation. The reduction in urinary oxalate excretion in hyperoxaluric mice treated with O. formigenes CM reflects the in vivo retention of biologic activity and the therapeutic potential of these factors.

Changes in the intestinal bacterial community, short-chain fatty acid profile, and intestinal development of preweaned Holstein calves. 1. Effects of prebiotic supplementation depend on site and age.

Digestive disorders are common during the first few weeks of life of newborn calves. Prebiotics are nondigestible but fermentable oligosaccharides that modulate growth and activity of beneficial microbial populations, which can result in enhanced gut health and function. Galactooligosaccharides (GOS) have demonstrated such prebiotic potential. In this study, the effect of GOS supplementation on intestinal bacterial community composition and fermentation profiles; intestinal health, development, and function; and growth was evaluated in dairy calves fed for high rates of growth. Eighty male Holstein calves were assigned either to a control treatment consisting of commercial milk replacer or to a GOS-rich (i.e., 3.4% of dry matter) milk replacer treatment. After 2 and 4wk, 8 calves per treatment were slaughtered at each age. Samples of intestinal digesta and tissue were collected for assessment of bacterial communities, short-chain fatty acid concentrations, in vitro measurement of nutrient transport and permeability, histomorphology, and gastrointestinal organ size. The remaining 48 calves continued to wk 8 to measure body growth, nutrient intake, and fecal and respiratory scores. Calves fed GOS displayed greater Lactobacillus and Bifidobacterium relative abundance and more developed intestinal epithelial structures, but also had greater fecal scores presumably related to greater colonic water secretion. Control calves showed slightly better growth and milk dry matter intake. Size of intestinal organs, intestinal nutrient transport, and epithelium paracellular resistance were not affected by treatment. Excessive GOS supplementation had both prebiotic and laxative effects, which led to slightly lower growth performance while promoting commensal bacteria population and greater intestinal epithelium growth.

Segmental differences in upregulated apical potassium channels in mammalian colon during potassium adaptation.

Rat proximal and distal colon are net K+ secretory and net K+ absorptive epithelia, respectively. Chronic dietary K+ loading increases net K+ secretion in the proximal colon and transforms net K+ absorption to net K+ secretion in the distal colon, but changes in apical K+ channel expression are unclear. We evaluated expression/activity of apical K+ (BK) channels in surface colonocytes in proximal and distal colon of control and K+-loaded animals using patch-clamp recording, immunohistochemistry, and Western blot analyses. In controls, BK channels were more abundant in surface colonocytes from K+ secretory proximal colon (39% of patches) than in those from K+-absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K+ loading had no clear-cut effects on the abundance, immunolocalization, or expression of BK channels in proximal colon. By contrast, in distal colon, K+ loading 1) increased BK channel abundance in patches from 12 to 41%; 2) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and 3) increased expression of BK channel α-subunit protein when assessed by Western blotting (P < 0.001). Thus apical BK channels are normally more abundant in K+ secretory proximal colon than in K+ absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K+ secretory response to dietary K+ loading.

The Farnesoid X Receptor: Good for BAD

Diarrhea is a feature of several chronic intestinal disorders that are associated with increased delivery of bile acids into the colon. Although the prevalence of bile acid diarrhea is high, affecting approximately 1% of the adult population, current therapies often are unsatisfactory. By virtue of its capacity to inhibit colonic epithelial fluid secretion and to down-regulate hepatic bile acid synthesis through induction of the ileal fibroblast growth factor 19 release, the nuclear bile acid receptor, farnesoid X receptor, represents a promising target for the development of new therapeutic approaches. Here, we review our current understanding of the pathophysiology of bile acid diarrhea and the current evidence supporting a role for farnesoid X receptor agonists in treatment of the disease.

Chenodeoxycholic acid requires activation of EGFR, EPAC, and Ca2+ to stimulate CFTR-dependent Cl- secretion in human colonic T84 cells.

Bile acids are known to initiate intricate signaling events in a variety of tissues, primarily in the liver and gastrointestinal tract. Of the known bile acids, only the 7α-dihydroxy species, deoxycholic acid and chenodeoxycholic acid (CDCA), and their conjugates, activate processes that stimulate epithelial Cl- secretion. We have previously published that CDCA acts in a rapid manner to stimulate colonic ion secretion via protein kinase A (PKA)-mediated activation of the dominant Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (Ao M, Sarathy J, Domingue J, Alrefai WA, and Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013); however, PKA signaling did not account for the entire CDCA response. Here we show that in human colonic T84 cells, CDCA's induction of CFTR activity, measured as changes in short-circuit current (Isc), is dependent on epidermal growth factor receptor (EGFR) activation and does not involve the bile acid receptors TGR5 or farnesoid X receptor. CDCA activation of Cl- secretion does not require Src, mitogen-activated protein kinases, or phosphoinositide 3-kinase downstream of EGFR but does require an increase in cytosolic Ca2+ In addition to PKA signaling, we found that the CDCA response requires the novel involvement of the exchange protein directly activated by cAMP (EPAC). EPAC is a known hub for cAMP and Ca2+ cross talk. Downstream of EPAC, CDCA activates Rap2, and changes in free cytosolic Ca2+ were dependent on both EPAC and EGFR activation. This study establishes the complexity of CDCA signaling in the colonic epithelium and shows the contribution of EGFR, EPAC, and Ca2+ in CDCA-induced activation of CFTR-dependent Cl- secretion.

Mechanism of Action and Pharmacology of Patiromer, a Nonabsorbed Cross-Linked Polymer That Lowers Serum Potassium Concentration in Patients With Hyperkalemia.

Hyperkalemia is a potentially life-threatening condition, and patients who have chronic kidney disease, who are diabetic, or who are taking renin–angiotensin–aldosterone system inhibitors are at increased risk. Therapeutic options for hyperkalemia tend to have limited effectiveness and can be associated with serious side effects. Colonic potassium secretion can increase to compensate when urinary potassium excretion decreases in patients with renal impairment, but this adaptation is insufficient and hyperkalemia still results. Patiromer is a novel, spherical, nonabsorbed polymer designed to bind and remove potassium, primarily in the colon, thereby decreasing serum potassium in patients with hyperkalemia. Patiromer has been found to decrease serum potassium in patients with hyperkalemia having chronic kidney disease who were on renin–angiotensin–aldosterone system inhibitors. Results of nonclinical studies and an early phase clinical study are reported here. Two studies with radiolabeled drug, one in rats and the other in dogs, confirmed that patiromer was not absorbed into the systemic circulation. Results of an in vitro study showed that patiromer was able to bind 8.5 to 8.8 mEq of potassium per gram of polymer at a pH similar to that found in the colon and had a much higher potassium-binding capacity compared with other resins, including polystyrene sulfonate. In a study in hyperkalemic rats, a decrease in serum potassium was observed via an increase in fecal potassium excretion. In a clinical study in healthy adult volunteers, a significant increase in fecal potassium excretion and a significant decrease in urinary potassium excretion were observed. Overall, patiromer is a high-capacity potassium binder, and the chemical and physical characteristics of patiromer may lead to good clinical efficacy, tolerability, and patient acceptance.

The Extracellular Calcium-Sensing Receptor in the Intestine: Evidence for Regulation of Colonic Absorption, Secretion, Motility, and Immunity.

Different from other epithelia, the intestinal epithelium has the complex task of providing a barrier impeding the entry of toxins, food antigens, and microbes, while at the same time allowing for the transfer of nutrients, electrolytes, water, and microbial metabolites. These molecules/organisms are transported either transcellularly, crossing the apical and basolateral membranes of enterocytes, or paracellularly, passing through the space between enterocytes. Accordingly, the intestinal epithelium can affect energy metabolism, fluid balance, as well as immune response and tolerance. To help accomplish these complex tasks, the intestinal epithelium has evolved many sensing receptor mechanisms. Yet, their roles and functions are only now beginning to be elucidated. This article explores one such sensing receptor mechanism, carried out by the extracellular calcium-sensing receptor (CaSR). In addition to its established function as a nutrient sensor, coordinating food digestion, nutrient absorption, and regulating energy metabolism, we present evidence for the emerging role of CaSR in the control of intestinal fluid homeostasis and immune balance. An additional role in the modulation of the enteric nerve activity and motility is also discussed. Clearly, CaSR has profound effects on many aspects of intestinal function. Nevertheless, more work is needed to fully understand all functions of CaSR in the intestine, including detailed mechanisms of action and specific pathways involved. Considering the essential roles CaSR plays in gastrointestinal physiology and immunology, research may lead to a translational opportunity for the development of novel therapies that are based on CaSR's unique property of using simple nutrients such as calcium, polyamines, and certain amino acids/oligopeptides as activators. It is possible that, through targeting of intestinal CaSR with a combination of specific nutrients, oral solutions that are both inexpensive and practical may be developed to help in conditioning the gut microenvironment and in maintaining digestive health.

Reduction of epithelial secretion in male rat distal colonic mucosa by bile acid receptor TGR5 agonist, INT-777: role of submucosal neurons.

Recent evidence from rat neuron-free mucosa study suggests that the membrane bile acid receptor TGR5 decreases colonic secretion under basal and stimulated conditions. As submucosal neurons are key players in secretory processes and highly express TGR5, we investigated their role in TGR5 agonist-induced inhibition of secretion and the pathways recruited. TGR5 expression and localization were assessed in rat proximal (pC) and distal (dC) colon by qPCR and immunohistochemistry with double labeling for cholinergic neurons in whole-mount preparations. The influence of a selective (INT-777) or weak (ursodeoxycholic acid, UDCA) TGR5 agonist on colonic secretion was assessed in Ussing chambers, in dC preparation removing seromuscular ± submucosal tissues, in the presence of different inhibitors of secretion pathways. TGR5 mRNA is expressed in full thickness dC and pC and immunoreactivity is located in colonocytes and pChAT-positive neurons. Addition of INT-777, and less potently UDCA, decreased colonic secretion in seromuscular stripped dC by -58.17± 2.6%. INT-777 effect on basal secretion was reduced in neuron-free and TTX-treated mucosal-submucosal preparations. Atropine, hexamethonium, indomethacin, and L-NAME all reduced significantly INT-777's inhibitory effect while the 5-HT4 antagonist, RS-39604, and lidocaine abolished it. INT-777 inhibited stimulated colonic secretion induced by nicotine, but not cisapride, carbachol or PGE2. TGR5 activation inhibits basal and stimulated distal colonic secretion in rats by acting directly on epithelial cells and also inhibiting submucosal neurons. This could represent a counter-regulatory mechanism, at the submucosal level, of the known prosecretory effect of bile acids in the colon.