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Bacterially derived short chain fatty acids (SCFAs), such as butyrate, are vital in maintaining the symbiotic relationship that exists between humans and their gastrointestinal microbial populations. A key step in this process is the transport of SCFAs across colonic epithelial cells via MCT1 transporters. This study investigated MCT1 protein abundance in various human intestinal tissues. Initial RT-PCR analysis confirmed the expected MCT1 RNA expression pattern of colon > small intestine > stomach. Using surgical resection samples, immunoblot analysis detected higher abundance of a 45 kDa MCT1 protein in colonic tissue compared to ileum tissue (P

Background and purpose: Prostaglandin F2α (PGF2α) is implicated in the pathogenesis of inflammatory bowel disease and colorectal cancer. This study investigates the effects of PGF2α on electrophysiological parameters in isolated human colonic mucosa. Experimental approach: Ion transport was measured as changes in short-circuit current across human colonic epithelia mounted in Ussing chambers. Colonic crypts were isolated by calcium chelation and cyclic adenosine monophosphate (cAMP) was measured by ELISA. Key Results: PGF2α stimulated chloride secretion in a concentration-dependent manner with an EC50 of 130 nM. The PGF2α induced increase in chloride secretion was inhibited by AL8810 (10 µM), a specific PGF2α receptor antagonist. In addition, PGF2α (1 µM) significantly increased levels of cAMP in isolated colonic crypts. Conclusions and implications: PGF2α stimulated chloride secretion in samples of human colon in vitro through a previously unrecognizd cAMP-mediated mechanism. These findings have implications for inflammatory states.

Background and purpose: Kinins are acknowledged as important regulators of intestinal function during inflammation; however, their effects on human intestinal ion transport have not been reported. Here, we used muscle-stripped human colonic tissue and cultured T84-cell monolayers to study bradykinin (BK) actions on human intestinal ion transport. Experimental approach: Ion transport was measured as changes in short-circuit current (Isc) across colonic epithelia mounted in Ussing chambers. Key results: In intact tissue, there was a distinct polarity to BK-elicited Isc responses. Whereas basolateral BK stimulated sustained responses (EC50=0.5±0.1 μM), those to apical BK were more rapid and transient (EC50=4.1±1.2 nM). In T84 cells, responses to both apical and basolateral BK were similar to those seen upon apical addition to intact tissues. Cross-desensitization between apical and basolateral domains was not observed. BK-induced responses were largely due to Cl− secretion as shown by their sensitivity to bumetanide and removal of Cl− from the bathing solution. Studies using selective agonists and antagonists indicate responses to BK are mediated by B2 receptors. Finally, responses to basolateral BK in intact tissues were inhibited by tetrodotoxin (1 μM), atropine (1 μM), capsaicin (100 μM) and piroxicam (10 μM). BK-stimulated prostaglandin (PG)E2 release from colonic tissue. Conclusions: BK stimulates human colonic Cl− secretion by activation of apical and basolateral B2 receptors. Responses to apical BK reflect a direct action on epithelial cells, whereas those to basolateral BK are amplified by stimulation of enteric nerves and PG synthesis. British Journal of Pharmacology (2008) 155, 558–566; doi:10.1038/bjp.2008.288; published online 7 July 2008

Urea transporters (UTs) encoded by the Slc14a1 (UT-B) and Slc14a2 (UT-A) genes facilitate the movement of urea across plasma membranes. Urea flux into the gastrointestinal tract enhances the growth...

Urea entry into the gastrointestinal tract is facilitated by the urea transporter UT-B1 (Stewart 2011). This process is crucial to our intestinal wellbeing as it is the first step in the urea nitrogen salvaging mechanism, which is necessary for maintaining healthy gut bacterial populations. In a previous study using a bovine UT-B antibody, a 35kDa human UT-B1 protein was found to be highly expressed in the ascending colon (Collins et al., 2010). The aim of this study was to produce a reliable human UT-B antibody to be used for further investigations of human UT-B expression. Initial PCR analysis using primers designed along the UT-B gene confirmed UT-B1 as the major isoform expressed at the RNA level in all human gastrointestinal tissues. Three polyclonal human UT-B antibodies - one designed against the N-terminal (UT-B#N) and two against the C-terminal (UT-B#C and UT-Bc19) - were made according to the UT-B1 amino acid sequence and extensive characterization was carried out using Western blot analysis. Wh...

1. This study examined the effects of chronic exposure of rats to 3,4-methylenedioxymethamphetamine (MDMA) on [(3)H]5-hydroxytryptamine ([(3)H]5-HT) re-uptake into purified rat brain synaptosomes, 5-HT-induced isometric contraction of aortic rings and [(3)H]5-HT re-uptake into rat aorta. 2. Rats were administered MDMA (20 mg kg(-1) i.p.) twice daily over 4 days. One, 7, 14 or 21 days post treatment, whole brain synaptosomes and descending thoracic aortic rings were prepared for investigation. 3. Chronic MDMA treatment significantly reduced the maximum rate (V(max)) of specific high-affinity [(3)H]5-HT re-uptake 1 day after treatment and for up to 21 days post-final administration of MDMA. Direct application of MDMA (100 microM) abolished synaptosomal re-uptake of [(3)H]5-HT in vitro. 4. Chronic MDMA administration significantly reduced the maximum contraction (E(max)) to 5-HT at 1 and 7 days after treatment, but not at 14 or 21 days. 5. Chronic MDMA administration had no effect on sodium-dependent [(3)H]5-HT re-uptake into aorta 1 day after treatment, nor did 100 microM MDMA have any direct effect on [(3)H]5-HT uptake into aortic rings in vitro. 6. These results show, for the first time, an altered responsiveness of vascular tissue to MDMA after chronic administration. In addition, they demonstrate a difference in the sensitivity of central and peripheral 5-HT uptake systems to chronic MDMA exposure, and suggest that the action of MDMA in the cardiovascular system does not arise from a direct effect of MDMA on peripheral 5-HT transport.

This in vitro study was designed to determine the potential use of the NK1 antagonist, SR140333 as an anti-diarrhoeal treatment for food allergy or inflammatory bowel disease. The effect of various immune and neuronal stimuli on human colonic substance P (SP) release and the effect of SR140333 on subsequently stimulated mucosal ion transport was investigated. Submucosal and sensory nerve fibre stimulation using electrical field stimulation (1 ms/7 Hz/7 V) and capsaicin (50 μM) respectively, mast cell activation by anti-IgE (1/250 dilution) and granulocyte stimulation using fMLP (50 μM) each released SP and evoked a secretory response. SP and the NK1 selective agonist, Sar-SP (0.1–1000 nM) stimulated an increase in colonic secretion which was antagonized by SR140333 (pD′2=6.7 and 7.25 versus SP and Sar-SP respectively). SR140333, at a concentration that blocked NK1-mediated secretion (500 nM), also reduced the secretory response to both αIgE and capsaicin. This suggests a pathophysiologic role for NK1 receptors. Capsaicin evoked SP release was increased in tissue taken from Crohn's disease but not ulcerative colitis patients. The response to SP was however reduced by 70 and 89% respectively. Mast cells and sensory afferents contribute to allergic diarrhoea. Since SR140333 reduced the secretory response to mast cell and afferent stimulation this compound may be particularly useful in reducing the symptoms of food allergy. British Journal of Pharmacology (2001) 133, 1346–1354; doi:10.1038/sj.bjp.0704194

Background: The efficacy of cyclosporin in the management of ulcerative colitis is recognized. Not all patients respond to this treatment. Existing clinical and laboratory parameters are of little use in identifying those most likely to respond. Aims: To determine whether in-vitro sensitivity to cyclosporin as measured by a lymphocyte proliferation assay is predictive of in-vivo response to therapy. Methods: The study comprised seven responders with ulcerative colitis, seven non-responders, and 14 healthy matched controls. A lymphocyte proliferation assay was carried out in the presence of a range of concentrations of cyclosporin and a dose–response curve constructed for each subject. The IC50 value, the concentration of cyclosporin that resulted in 50% inhibition of proliferation, was calculated for each subject. IC50 values for responders, non-responders and controls were compared using a Mann–Whitney test. Results: There was a wide range of values obtained for the study group as a whole. IC50 values for non-responders were significantly higher than those of responders (P

1. The effects of the alkaloid berberine on basal and stimulated ion transport were investigated in voltage-clamped rat colonic epithelia. 2. Berberine (100-500 microM) reduced basal short circuit current (SCC) when applied basolaterally but not when applied apically. 3. SCC responses to mast cell activation by anti-rat IgE were significantly attenuated in the presence of berberine. 4. Berberine, applied to the basolateral bathing solution, also reduced SCC responses to the following agents which stimulate chloride secretion in rat colon: carbachol, forskolin, sodium nitroprusside, dibutyryl cyclic-AMP, heat-stable E. coli enterotoxin, 8-bromo-cyclic GMP and thapsigargin. Calcium mediated ion transport responses appear to be more sensitive to berberine inhibition than those which are cyclic GMP-mediated, which in turn are more sensitive than cyclic AMP-mediated responses. 5. Berberine added apically was without effect upon forskolin-stimulated ion transport. Cytochalasin D treatment of the lumenal surface of rat colon conferred apical-side sensitivity to berberine. 6. Berberine (at concentrations up to 500 microM) was without effect on generation of cyclic AMP by forskolin or on generation of cyclic GMP by sodium nitroprusside in isolated mucosal segments. Protein kinase A activity stimulated by dibutyryl cyclic AMP was unaffected by berberine (at concentrations up to 500 microM). 7. The precise mechanism of action of berberine remains to be elucidated. However, its site of action appears to be distal to second messenger production and may be at a level common to all stimuli of colonic chloride secretion.

Objectives Zinc is a useful addition to oral rehydration therapy for acute diarrhoea. We have assessed the mechanism of its epithelial antisecretory action when intestinal epithelial tight junctions were pharmacologically opened. Methods Rat isolated ileal and colonic mucosae were mounted in Ussing chambers and exposed to ZnSO4 (Zn2+) in the presence of secretagogues and inhibition of short circuit current (Isc) was measured. Key findings Pre-incubation with basolateral but not apical Zn2+ reduced Isc stimulated by forskolin, carbachol and A23187. In the presence of the tight junction-opener, cytochalasin D, antisecretory effects of apically-applied Zn2+ were enabled in colon and ileum. The apparent permeability coefficient (Papp) of Zn2+ was increased 1.4- and 2.4-fold across rat ileum and colon, respectively, by cytochalasin D. Basolateral addition of Zn2+ also reduced the Isc stimulated by nystatin in rat colon, confirming K channel inhibition. In comparison with other inhibitors, Zn2+ was a relatively weak blocker of basolateral KATP and K Ca2+ channels. Exposure of ileum and colon to Zn2+ for 60 min had minimal effects on epithelial histology. Conclusions Antisecretory effects of Zn2+ on intestinal epithelia arose in part through nonselective blockade of basolateral K channels, which was enabled when tight junctions were open.

1. Using a number of 5-hydroxytryptamine (5-HT)-antagonists we have compared their activity against the chloride-secretory response in guinea-pig ileal and colonic epithelia when challenged with 5-HT or antigen. Guinea-pigs sensitized to beta-lactoglobulin (beta LG) were used throughout; these were obtained by providing animals with cows' milk for drinking. 2. Of methysergide, ketanserin, cyproheptadine and ICS 205-930, only the latter inhibited both the response to 5-HT and to antigen challenge. Methysergide caused a minor, significant effect on 5-HT but not on beta LG responses. Ketanserin had no effect on the responses to 5-HT, but both ketanserin and cyproheptadine inhibited the challenge to beta LG. 3. The data are considered in relation to the current views of receptor subtypes for 5-HT. Some of the reported inhibitors may be non-specific, while we consider there is evidence to support the view that 5-HT3-receptors (neuronal receptors) are involved both in the responses to 5-HT and to antigen challenge. 4. Tetrodotoxin mimicked the effect of ICS 205-930 on both the response to 5-HT and to antigen challenge in sensitized tissues, confirming a neuronal involvement for both types of stimuli.