Involvement of butyrate in electrogenic K+ secretion in rat rectal colon.

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are synthesized from dietary carbohydrates by colonic bacterial fermentation. These SCFAs supply energy, suppress cancer, and affect ion transport. However, their roles in ion transport and regulation in the intracellular environment remain unknown. In order to elucidate the roles of SCFAs, we measured short-circuit currents (ISC) and performed RT-PCR and immunohistochemical analyses of ion transporters in rat rectal colon. The application of 30 mM butyrate shifted ISC in a negative direction, but did not attenuate the activity of epithelial Na+ channels (ENaC). The application of bumetanide, a Na+-K+-2Cl- cotransporter inhibitor, to the basolateral side reduced the negative ISC shift induced by butyrate. The application of XE991, a KCNQ-type K+ channel inhibitor, to the apical side decreased the ISC shift induced by butyrate in a dose-dependent manner. The ISC shift was independent of HCO3- and insensitive to ibuprofen, an SMCT1 inhibitor. The mucosa from rat rectal colon expressed mRNAs of H+-coupled monocarboxylate transporters (MCT1, MCT4, and MCT5, also referred to as SLC16A1, SLC16A3, and SLC16A4, respectively). RT-PCR and immunofluorescence analyses demonstrated that KCNQ2 and KCNQ4 localized to the apical membrane of surface cells in rat rectal colon. These results indicate that butyrate, which may be transported by H+-coupled monocarboxylate transporters, activates K+ secretion through KCNQ-type K+ channels on the apical membrane in rat rectal colon. KCNQ-type K+ channels may play a role in intestinal secretion and defense mechanisms in the gastrointestinal tract.

Prediction of Bioequivalence and Food Effect Using Flux- and Solubility-Based Methods.

In this work, two different approaches have been developed to predict the food effect and the bioequivalence of marketed itraconazole (ITRA) formulations. Kinetic solubility and simultaneous dissolution-permeation tests of three (ITRA) formulations (Sporanox capsules and solution and SUBA-ITRA capsules) were carried out in simulated fasted and fed states. Fraction of dose absorbed ratios estimating food effect and bioequivalence were calculated based on these results and were compared to the in vivo study results published by Medicines Agencies. The comparison demonstrated that kinetic solubility and flux values could be used as input parameters for biopharmaceutics modeling and simulations to estimate food effect and bioequivalence. Both prediction methods were able to determine a slightly negative food effect in the case of the Sporanox solution and also a pronounced positive food effect for the Sporanox capsule. Superior bioavailability was predicted when the Sporanox solution was compared to the Sporanox capsule (in agreement with in vivo data).

Discovery of a novel chalcone derivative inhibiting CFTR chloride channel via AMPK activation and its anti-diarrheal application.

Secretory diarrhea is one of the most common causes of death world-wide especially in children under 5 years old. Isoliquiritigenin (ISLQ), a plant-derived chalcone, has previously been shown to exert anti-secretory action in vitro and in vivo by inhibiting CFTR Cl- channels. However, its CFTR inhibition potency is considerably low (IC50 > 10 µM) with unknown mechanism of action. This study aimed to identify novel chalcone derivatives with improved potency and explore their mechanism of action. Screening of 27 chalcone derivatives identified CHAL-025 as the most potent chalcone analog that reversibly inhibited CFTR-mediated Cl- secretion in T84 cells with an IC50 of ∼1.5 µM. As analyzed by electrophysiological and biochemical analyses, the mechanism of CFTR inhibition by CHAL-025 is through AMP-activated protein kinase (AMPK), a negative regulator of CFTR activity. Furthermore, Western blot analyses and molecular dynamics (MD) results suggest that CHAL-025 activates AMPK by binding at the allosteric site of an upstream kinase calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß). Interestingly, CHAL-025 inhibited both cholera toxin (CT) and bile acid-induced Cl- secretion in T84 cells and prevented CT-induced intestinal fluid secretion in mice. Therefore, CHAL-025 represents a promising anti-diarrheal agent that inhibits CFTR Cl- channel activity via CaMKKß-AMPK pathways.

Antidiarrheal effect of extract from the bark of Combretum leprosum in mice.

This study investigated the effects of the ethanolic extract from the bark of Combretum leprosum (ECL) on intestinal transit and castor-oil induced diarrhea in mice. The oral administration of ECL (750 and 1000 mg/kg) slowed intestinal transit (ID50 of 455 mg/kg). The ECL (250-1000 mg/kg) reduced castor-oil induced diarrhea, in a time- and dose-dependent manner (p < 0.05). To determine if antidiarrheal effect of ECL involves α2-adrenergic or opioid receptor activation, the mice were pretreated with antagonists of these receptors, yohimbine or naloxone respectively. None of these drugs inhibited the antidiarrheal effect of ECL. To test if antidiarrheal effect of ECL is due to an antisecretory action, we realized the enteropooling assay on rats. The ECL increased bowel content and did not inhibit intestinal fluid secretion increase induced by misoprostol (100 µg/kg, s.c.). To determine if antimotility effect of ECL is due to a reduction on gastric motility, we realized the organ bath assay in the rat fundus stomach. Isotonic recordings show that the carbachol /KCl - induced contraction was not reversed by the addition of ECL. In conclusion, our results suggest that ECL contains antidiarrheal compounds and these compounds could induce a reduction of intestinal tract motility.

Cellular mechanisms underlying the inhibitory effect of flufenamic acid on chloride secretion in human intestinal epithelial cells.

Intestinal Cl- secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA) suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl- secretion in human intestinal epithelial (T84) cells. FFA inhibited cAMP-dependent Cl- secretion in T84 cell monolayers with IC50 of ∼8 µM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl- channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K+ channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca2+-dependent Cl- secretion with IC50 of ∼10 µM. FFA inhibited activities of Ca2+-activated Cl- channels and KCa3.1, a Ca2+-activated basolateral K+ channels, but had no effect on activities of Na+-K+-Cl- cotransporters and Na+-K+ ATPases. These results indicate that FFA inhibits both cAMP and Ca2+-dependent Cl- secretion by suppressing activities of both apical Cl- channels and basolateral K+ channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas.

In vitro physiological and antibacterial characterization of ZnO nanoparticle composites in simulated porcine gastric and enteric fluids.

BACKGROUND: Diarrhea in piglets is one of the main causes of animal death after weaning; zinc oxide (ZnO) has been used in high doses for the control of this sickness. The aim of this study was to determine the physicochemical properties of ZnO nanoparticles synthesized and immobilized on a chitosan/alginate (CH/SA) complex and investigate the antimicrobial activity and in vitro release profile of zinc (Zn2+) from these new compounds. The ZnO nanoparticles composites were prepared and combined with CH/SA or CH/SA and sodium tripolyphosphate (TPP). The structure and morphology of the composites were analyzed by characterization methods such as X-ray diffraction, FTIR spectroscopy, thermogravimetric analysis, atomic absorption spectrophotometry and scanning electron microscopy. RESULTS: The crystallite size of ZnO nano was 17 nm and the novel ZnO composites were effective in protecting ZnO in simulated gastric fluid, where Zn2+ reached a concentration six-fold higher than the levels obtained with the unprotected commercial-zinc oxide. In addition, the novel composites suggest effective antimicrobial activity against Escherichia coli and Staphylococcus aureus. CONCLUSIONS: The results described herein suggest that the novel nano composites may work as an alternative product for pig feeding as verified by the in vitro assays, and may also contribute to lower the zinc released in the environment by fecal excretion in animals waste.

Lithocholic acid attenuates cAMP-dependent Cl- secretion in human colonic epithelial T84 cells.

Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl(-) secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 µM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current (Isc), it did not alter carbachol-stimulated Isc LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca(2+) concentration ([Ca(2+)]i), chelation by BAPTA-AM did not reverse LCA's effect on Isc LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on Isc Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl(-) current and inhibits a basolateral K(+) current. In summary, 50 µM LCA greatly inhibits cAMP-stimulated Cl(-) secretion, making low doses of LCA of potential therapeutic interest for diarrheal diseases.

Guanylin activates Cl(-) secretion into the lumen of seawater eel intestine via apical Cl(-) channel under simulated in vivo conditions.

Guanylin (GN) action on seawater eel intestine was examined under simulated in vivo conditions, where isotonic luminal fluid has low NaCl and high MgSO4 (MgSO4 Ringer). In Ussing chamber, MgSO4 Ringer induced serosa-negative potential difference (PD) even after bumetanide treatment, which is due to the higher paracellular Na(+) permeability over Cl(-), as confirmed by the replacement by MgCl2 (no Cl(-) gradient) or Na2SO4 Ringer (no Na(+) gradient). Luminal GN reversed serosa-negative PD, probably by enhancing Cl(-) secretion into the lumen, as the GN effect was blocked by apical Cl(-) channel blockers [diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid, glibenclamide but not cystic fibrosis transmembrane regulator (CFTR)inh-172] or replacement of luminal fluid by MgCl2 Ringer. The blockers' effect was undetectable when normal Ringer was on both sides. In the sac preparation, NaCl secretion occurred into the lumen (Na(+) > Cl(-)), and GN further enhanced Cl(-) secretion (Cl(-) > Na(+)), resulting in water secretion. These GN effects were also blocked by DPC. Quantitative analyses showed that isotonic NaCl is absorbed when luminal fluid is normal Ringer, but, when luminal fluid is MgSO4 Ringer, hypertonic NaCl, almost equivalent to seawater, is secreted into the lumen after GN. These results indicate that GN stimulates the secretion of hypertonic NaCl into the lumen of seawater eel intestine, like rectal gland of marine elasmobranchs, to get rid of excess NaCl although marine teleost intestine is thought to have only absorptive-type cells with a unique Na-K-Cl cotransport system. The secreted NaCl may activate the cotransport system and further help absorb water in the final segment of seawater eel intestine.

Muscarinic cholinoceptor-mediated activation of JNK negatively regulates intestinal secretion in mice.

Regulation of intestinal secretion is important for body fluid homeostasis. We investigated the role of three MAP kinases (MAPKs) as negative regulators in muscarinic cholinoceptor (mAChR)-mediated intestinal secretion in mice. Electrophysiological analyses revealed that mAChR stimulation enhanced intestinal chloride secretion, which was further augmented by the inhibition of JNK but not by that of ERK or p38 with specific inhibitors SP600125, U0126 or SB203580, respectively. Immunoblot analyses in colonic mucosa showed that mAChR stimulation increased MAPKs phosphorylation that was suppressed by the specific inhibitor for each MAPK. This suggests that JNK is a major negative regulator in mAChR-induced intestinal secretion.

Genistein stimulates jejunal chloride secretion via sex-dependent, estrogen receptor or adenylate cyclase mechanisms.

BACKGROUND/AIMS: Daily subcutaneous injections with the phytoestrogen genistein, 600 mg/ kg genistein/day (600G) significantly increased intestinal chloride (Cl(-)) secretion (I(sc), µA/cm(2)) in C57BL/6J female and male murine jejunum after 1-2-weeks treatment. METHODS AND RESULTS: In 600G females, basolateral application of the adenylate cyclase inhibitor MDL-12330A (10 µM) significantly reduced basal and total I(sc) in the presence of forskolin (27 and 40% respectively, P < 0.05), with no effect in 600G males, suggesting that 600G-mediated increases in I(sc) in females are due to an adenylate cyclase-dependent mechanism. Concomitant injections with the non-selective estrogen receptor (ER) antagonist ICI-182780 (25 mg/kg/day) resulted in a significant inhibition of basal I(sc) in males (38%, P < 0.05), but was without effect in females (further reinforcing an ER-independent mechanism of action). The ERα-selective antagonist (MPP, 25 mg/kg/day) similarly significantly inhibited the basal I(sc) (37%, P < 0.05) in males, whereas the ERß-selective antagonist (PHTPP, 25 mg/kg/day) was without effect, suggesting that 600G-mediated increases in I (sc) in male mice are due to an ERα-dependent mechanism. Jejunum ERα/actin expression was significantly increased by 600G in males. Compared to intact mice, orchiectomy has differing effects on 600G-mediated basal Isc; castration (CAST) abolished the 600G-mediated increases in I(sc), and ovariectomy (OVX) had no effect on the 600G-stimulated increases in I(sc). Daily estradiol injections (10-20 mg/kg body weight estradiol (10E2 or 20E2) had no effect in intact females, whereas 10E2 significantly increased basal I(sc) in OVX females. CONCLUSION: These data suggest that daily estradiol and genistein injections have differential sex-dependent mechanisms of action on murine intestinal Cl(-) secretion.

A tannic acid-based medical food, Cesinex(®), exhibits broad-spectrum antidiarrheal properties: a mechanistic and clinical study.

BACKGROUND: The purpose of this investigation was to evaluate the efficacy and tolerability of a tannic acid-based medical food, Cesinex(®), in the treatment of diarrhea and to investigate the mechanisms underlying its antidiarrheal effect. METHODS: Cesinex(®) was prescribed to six children and four adults with diarrhea. Patient records were retrospectively reviewed for the primary outcome. Cesinex(®) and its major component, tannic acid, were tested for their effects on cholera toxin-induced intestinal fluid secretion in mice. Polarized human gut epithelial cells (HT29-CL19A cells) were used to investigate the effects of tannic acid on epithelial barrier properties, transepithelial chloride secretion, and cell viability. RESULTS: Successful resolution of diarrheal symptoms was reported in nine of ten patients receiving Cesinex(®). The treatment of HT29-CL19A cells with clinically relevant concentrations of tannic acid (0.01-1 mg/ml) significantly increased transepithelial resistance (TER) and inhibited the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent or the calcium-activated Cl(-) secretion. Tannic acid could also improve the impaired epithelial barrier function induced by tumor necrosis factor alpha (TNFα) and inhibited the disrupting effect of TNFα on the epithelial barrier function in these cells. Cholera toxin (CTX)-induced mouse intestinal fluid secretion was significantly reduced by the administration of Cesinex(®) or tannic acid. Cesinex(®) has high antioxidant capacity. CONCLUSIONS: Cesinex(®) demonstrates efficacy and a good safety profile in the treatment of diarrhea. The broad-spectrum antidiarrheal effect of Cesinex(®) can be attributed to a combination of factors: its ability to improve the epithelial barrier properties, to inhibit intestinal fluid secretion, and the high antioxidant capacity.

CFTR chloride channel as a molecular target of anthraquinone compounds in herbal laxatives.

AIM: To clarify whether CFTR is a molecular target of intestinal fluid secretion caused by the anthraquinone compounds from laxative herbal plants. METHODS: A cell-based fluorescent assay to measure I(-) influx through CFTR chloride channel. A short-circuit current assay to measure transcellular Cl(-) current across single layer FRT cells and freshly isolated colon mucosa. A closed loop experiment to measure colon fluid secretion in vivo. RESULTS: Anthraquinone compounds rhein, aloe-emodin and 1,8-dihydroxyanthraquinone (DHAN) stimulated I(-) influx through CFTR chloride channel in a dose-dependent manner in the presence of physiological concentration of cAMP. In the short-circuit current assay, the three compound enhanced Cl(-) currents in epithelia formed by CFTR-expressing FRT cells with EC(50) values of 73 ± 1.4, 56 ± 1.7, and 50 ± 0.5 µmol/L, respectively, and Rhein also enhanced Cl(-) current in freshly isolated rat colonic mucosa with a similar potency. These effects were completely reversed by the CFTR selective blocker CFTR(inh)-172. In in vivo closed loop experiments, rhein 2 mmol/L stimulated colonic fluid accumulation that was largely blocked by CFTR(inh)-172. The anthraquinone compounds did not elevate cAMP level in cultured FRT cells and rat colonic mucosa, suggesting a direct effect on CFTR activity. CONCLUSION: Natural anthraquinone compounds in vegetable laxative drugs are CFTR potentiators that stimulated colonic chloride and fluid secretion. Thus CFTR chloride channel is a molecular target of vegetable laxative drugs.

Berberine Slows the Progression of Prediabetes to Diabetes in Zucker Diabetic Fatty Rats by Enhancing Intestinal Secretion of Glucagon-Like Peptide-2 and Improving the Gut Microbiota.

Background: Berberine is a plant alkaloid that has multiple beneficial effects against intestine inflammation. In our previous study, we have found that berberine also possesses an antidiabetic effect. However, whether berberine is useful in the prevention of type 2 diabetes mellitus (T2DM) through its effect on intestine endocrine function and gut microbiota is unclear. Aim: To investigate the effects of berberine in the prevention of T2DM, as well as its effects on intestine GLP-2 secretion and gut microbiota in ZDF rats. Methods: Twenty Zucker Diabetic Fatty (ZDF) rats were fed a high-energy diet until they exhibited impaired glucose tolerance (IGT). The rats were then divided into two groups to receive berberine (100 mg/kg/d; berberine group) or vehicle (IGT group) by gavage for 3 weeks. Five Zucker Lean (ZL) rats were used as controls. Fasting blood glucose (FBG) was measured, an oral glucose tolerance test was performed, and the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was calculated. Intestinal expression of TLR-4, NF-κB, TNF-α, mucin, zona occludens-1 (ZO-1) and occludin were assessed (immunohistochemistry). Plasma levels and glutamine-induced intestinal secretion of glucagon-like peptide-1 (GLP-1) and GLP-2 were measured (enzyme-linked immunosorbent assay). The plasma lipopolysaccharide (LPS) level was measured. Fecal DNA extraction, pyrosequencing, and bioinformatics analysis were performed. Results: After 3 weeks of intervention, diabetes developed in all rats in the IGT group, but only 30% of rats in the berberine group. Treatment with berberine was associated with reductions in food intake, FBG level, insulin resistance, and plasma LPS level, as well as increases in fasting plasma GLP-2 level and glutamine-induced intestinal GLP-2 secretion. Berberine could increase the goblet cell number and villi length, and also reverse the suppressed expressions of mucin, occludin, ZO-1 and the upregulated expressions of TLR-4, NF-κB and TNF-α induced in IGT rats (P<0.05). Berberine also improved the structure of the gut microbiota and restored species diversity. Conclusion: Berberine may slow the progression of prediabetes to T2DM in ZDF rats by improving GLP-2 secretion, intestinal permeability, and the structure of the gut microbiota.

Emerging pharmacologic therapies for irritable bowel syndrome.

New therapies are being developed for irritable bowel syndrome (IBS). These advances are based on understanding pathophysiology or the development of medications with greater selectivity in classes of agents with known efficacy. Prucalopride, the newest European Medicines Agency-approved 5-hydroxytryptamine receptor 4 (5-HT(4)) agonist, is effective in the treatment of chronic constipation with improved cardiovascular safety relative to older 5-HT(4) drugs; similarly, ramosetron, the 5-hydroxytryptamine receptor 3 (5-HT(3)) antagonist, appears efficacious in diarrhea-predominant IBS. Secretagogues with different mechanisms of action target apical domains in enterocytes that are involved in chloride secretion, such as chloride channels, the cystic fibrosis transmembrane regulator, and guanylate cyclase C. As a class, such secretagogues have high efficacy and safety for constipation. With more data obtained from phase 2 and 3 trials, we expect other classes of medications, including bile acid modulators, anti-inflammatory agents, visceral analgesics, and newer centrally acting agents to be efficacious and enter the armamentarium for the treatment of IBS in the future.

Antidiarrheal activity of aqueous extract of Hermannia incana Cav. leaves in Wistar rats.

Hermannia incana Cav. is a prostrate herb used to treat diarrhea, stomach ache, nausea and vomiting, by the people of Eastern Cape Province, South Africa. The phytochemical screening as well as the antidiarrheal activity of H. incana leaf extract at 200, 400 and 600 mg/kg body weight was evaluated in rats. Phytochemical screening revealed the presence of bioactive agents such as alkaloids, tannins, saponins, phenolics, triterpenes, cardiac glycosides, flavonoids, cardenolides and dienolides. The extract significantly prolonged the time of induction of diarrhea, reduced the frequency of diarrheal episodes and water content of the feces, and inhibited castor oil-induced enteropooling. The extract also suppressed intestinal propulsive movement of a charcoal meal through the gastrointestinal tract. These results demonstrate the antidiarrheal properties of the extract, thereby supporting the folkloric use of the plant as an antidiarrheal agent in the Eastern Cape of South Africa.

Activation of PPARgamma by rosiglitazone attenuates intestinal Cl- secretion.

The thiazolidinedione (TZD) drugs rosiglitazone (Ro) and pioglitazone (Po) are PPARgamma agonists in widespread clinical use as insulin-sensitizing agents in Type 2 diabetes. On the basis of recent evidence implicating PPARgamma as a positive modulator of intestinal epithelial differentiation, we hypothesized that TZD drugs might attenuate intestinal secretory function. To evaluate this possibility, we examined the effects of Ro and Po on electrogenic Cl- secretion [short-circuit current (I(sc))] in mouse intestinal segments and in cultured human intestinal epithelial cells (HT29-Cl.19A). As hypothesized, oral administration of Ro (20 mg.kg(-1).day(-1)) to mice for 8 days markedly reduced intestinal I(sc) responses to cAMP (forskolin)- and Ca2+ (carbachol)-dependent stimuli. In these Ro-treated mice, cholera toxin-induced intestinal fluid accumulation was reduced 65%. With continued Ro treatment, the I(sc) response to carbachol recovered significantly, whereas that to forskolin remained attenuated. Treatment of HT29 cells for 5 days with 10 muM Ro or Po in vitro brought about a similar hyposecretory state. In HT29 cells, the loss of cAMP-dependent Cl- secretion was attributable to a reduced expression of CFTR Cl- channel, KCNQ1 K+ channel, and Na-K-2Cl cotransporter-1 proteins. The transient loss of Ca2+-dependent Cl- secretion involved an impairment of basolateral Ca2+-stimulated K+ channel activity without a detectable loss of K(Ca)3.1 channel protein. Our results establish TZD drugs as important modulators of intestinal Cl- secretory function.

Secretagogue stimulation enhances NBCe1 (electrogenic Na(+)/HCO(3)(-) cotransporter) surface expression in murine colonic crypts.

A Na(+)/HCO(3)(-) cotransporter (NBC) is located in the basolateral membrane of the gastrointestinal epithelium, where it imports HCO(3)(-) during stimulated anion secretion. Having previously demonstrated secretagogue activation of NBC in murine colonic crypts, we now asked whether vesicle traffic and exocytosis are involved in this process. Electrogenic NBCe1-B was expressed at significantly higher levels than electroneutral NBCn1 in colonic crypts as determined by QRT-PCR. In cell surface biotinylation experiments, a time-dependent increase in biotinylated NBCe1 was observed, which occurred with a peak of +54.8% after 20 min with forskolin (P < 0.05) and more rapidly with a peak of +59.8% after 10 min with carbachol (P < 0.05) and which corresponded well with the time course of secretagogue-stimulated colonic bicarbonate secretion in Ussing chamber experiments. Accordingly, in isolated colonic crypts pretreated with forskolin and carbachol for 10 min, respectively, and subjected to immunohistochemistry, the NBCe1 signal showed a markedly stronger colocalization with the E-cadherin signal, which was used as a membrane marker, compared with the untreated control. Cytochalasin D did not change the observed increase in membrane abundance, whereas colchicine alone enhanced NBCe1 membrane expression without an additional increase after carbachol or forskolin, and LY294002 had a marked inhibitory effect. Taken together, our results demonstrate a secretagogue-induced increase of NBCe1 membrane expression. Vesicle traffic and exocytosis might thus represent a novel mechanism of intestinal NBC activation by secretagogues.

Treatment of chronic constipation: current pharmacologic approaches and future directions.

Chronic constipation is a common condition that affects up to 27% of the population. Dietary and lifestyle measures are usually the first-line therapy, but if these fail to have an effect then a variety of prescription and consumer laxatives are available. Traditional laxatives include bulking agents, osmotic agents, stool softeners, and stimulants of the gastrointestinal tract. All have been found to be more effective than placebo at relieving symptoms of constipation, but these results have been obtained primarily in short (4-week) trials and no class of laxative has been shown to be superior to another. Traditional laxatives work in many, but not all, patients and some patients cannot cope with the side effects, unpleasant taste, the requirements of the dosing regimen, or the notion of dose increase. New enterokinetic agents that affect peristalsis through selective interaction with 5-hydroxytryptamine-4 receptors and novel agents acting on intestinal secretion could offer an alternative option for patients with chronic constipation who cannot get adequate relief from current laxatives.

Intestinal secretion: stimulation by peptides.

Two peptides isolated from intestinal mucosa, vasoactive intestinal peptide, and gastric inhibitory peptide, stimulate small intestinal secretion in conscious dogs. Glucagon and pentagastrin also stimulate, but secretin and the octapetide of cholecystokinin do not. The stimulants may participate in regulation of intestinal secretion in health and in diseases with excessive secretion.

Trimethyltin chloride induced chloride secretion across rat distal colon.

TMT (trimethyltin chloride), an organotin, is ubiquitous in the environment. The consumption of contaminated food may cause exposure of the human diet to this toxic compound. The present study was to investigate the effects of TMT on the regulation of ion transport across the rat distal colon. The rat colonic mucosa was mounted in Ussing chambers. The effects of TMT were assessed using the Isc (short-circuit current). Both apical and basolateral TMT induced, dose-dependently, an increase in Isc, which was due to a stimulation of Cl- secretion as measured using ion substitution experiments and pharmacological manoeuvres. The secretion was also inhibited by several K+ channel blockers administrated at the basolateral side. When the apical side was permeabilized by nystatin, the TMT-induced K+ conductance was effectively blocked by tetrapentylammonium, a Ca2+-sensitive K+ channel blocker. The response of TMT was sensitive to the basolateral Ca2+ and the intracellular Ca2+ store, which could be disclosed by applying the inhibitors of ryanodine receptors and inositol 1,4,5-trisphosphate receptors. In conclusion, TMT led to Cl- secretion, which was essentially regulated by basolateral Ca2+-sensitive K+ channels. These results suggest the importance of K+ channels in the toxicity hazard of TMT.