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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

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

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.

1. Rabbit small intestinal segments containing Peyer's patches (PP) were examined in Ussing chambers using short-circuit current (Isc) recording. By comparison with control small intestinal mucosal segments, rabbit PP-containing epithelia exhibited decreased basal Isc, increased transepithelial resistance (TER) and unchanged potential difference (PD). 2. Carbachol caused a decrease in Isc in rabbit PP epithelia. Forskolin, dibutyryl cyclic GMP, histamine and the calcium ionophore, A23187, were without effect. In contrast, control epithelial segments of rabbit intestine responded to carbachol and forskolin with an increased Isc, indicative of electrogenic chloride secretion. The EC50 for carbachol was approximately 2 microM in both types of epithelia. Methacholine also caused an outward current in rabbit PP epithelia which had similar properties to that of carbachol. The effect of the cholinomimetics on rabbit PP was basolateral-sided, reversible, and sensitive to low concentrations of the general muscarinic cholinoceptor blockers, atropine, scopolamine and also to the M1 cholinoceptor blocker, pirenzepine. 3. The Isc response to cholinomimetics in rabbit PP was insensitive to bumetanide, amiloride, TEA, barium, acetazolamide, piroxicam and omeprazole, but was attenuated in the presence of ouabain. Using bilaterally-substituted solutions, the carbachol effect on rabbit PP Isc was abolished in chloride/bicarbonate-free, but not in chloride-free solutions, suggestive of stimulation of electrogenic bicarbonate absorption by the agent. Substitution for sodium abolished both the basal current and the Isc response to carbachol. Part of the effect of carbachol on PP Isc appeared to be mediated by submucosal neurones because addition of tetrodotoxin reduced the effect by 60%. 4 As microfold (M) epithelial cells predominate in the PP of the rabbit, the unusual phenotype of cholinomimetic-induced outward current may be used as an electrophysiological marker for these potential sites of oral vaccine delivery, and in particular it may also be of use as a marker for rabbit M cells.

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.

Colonic epithelia from rats infected with the nematode Nippostrongylus brasiliensis have been studied under short circuit conditions and in response to challenge with worm antigen. Challenge from the serosal but not the mucosal side with antigen caused a transient increase in inwardly directed short circuit current. No effects were observed in comparable tissues from noninfected animals. Simultaneous measurements of short circuit current and of the fluxes of sodium or chloride ions showed there was an increase in electrogenic chloride secretion and an inhibition of electroneutral sodium chloride absorption, associated with antigen challenge. This result, together with the inhibitory effects of piretanide on the response to antigen challenge, indicate that chloride ions are a major carrier of the short circuit current response. However, the equivalence of the biophysical response to ion fluxes was not established, there being an excess of chloride secretion. The mast cell stabilizing agent, FPL 52694, significantly inhibited the current responses to antigen, while cromoglycate and doxantrazole were ineffective. Mepyramine, an H1-receptor antagonist, and indomethacin, an inhibitor of fatty acid cyclo-oxygenase, were without effect on the responses to antigen challenge. Anti-rat IgE produced qualitatively similar responses to antigen in both normal and sensitized colonic epithelia. However, the responses were significantly greater in tissues derived from infected animals. Maximally effective antigen concentrations prevented subsequent responses to anti-rat IgE in sensitized tissues, while anti-rat IgE only attenuated the responses to antigen. The ways in which antigen challenge modifies epithelial function is discussed, particularly in relation to its possible role in promoting rejection of the nematodes during secondary infection.

1. Type 1 hypersensitivity reactions in response to antigen challenge have been measured as short circuit current (SCC) responses in reconstructed tissues consisting of syngeneic cell types. 2. In all instances reconstructed tissues consisted of an epithelial monolayer grown on collagen-coated Millipore filters and a pad of peritoneal cells. Monolayers were formed of either HCA-7 or HCA-7-Col 1 cells derived from a human adenocarcinoma. Peritoneal cells were derived from rats or guinea-pigs sensitized to either ovalbumin or beta-lactoglobulin. 3. The SCC responses of the monolayers were dependent upon the 'concentration' of peritoneal cells in the reconstructed tissue. The threshold concentration was 0.4 X 10(6) cells when rat peritoneal cells are combined with an epithelial monolayer of 0.2 cm2. 4. The SCC responses in response to antigen challenge were selectively inhibited by the H1-receptor antagonist, mepyramine. Similarly the effects of exogenously applied histamine were antagonised by mepyramine. 5. The responses to antigen challenge were not inhibited by tetrodotoxin in reconstructed tissues. This result is in contrast to that with isolated intestinal epithelia from sensitized animals where tetrodotoxin inhibits the SCC responses to external field stimulation and to challenge with antigens. The consequences of these results for understanding the mechanisms of epithelial Type 1 hypersensitivity reactions are discussed. Suggestions are made to illustrate how the methods developed here may be employed to ask questions about the nature of mediators released and the types of cell responsible in human disease conditions.