Proceedings of The Physiological Society
University College London (2011) Proc Physiol Soc 24, C17 and PC17
Effects of flavonoids on agonist-stimulated chloride secretion in Caco-2 cells and mouse colon
M. A. Carew1, T. A. Hague2,4, V. Millar3, L. Bowman3, M. Brunet3, R. Jbar4, C. H. Fry4, L. J. MacVinish3
1. School of Pharmacy and Chemistry, Kingston University, Kingston upon Thames, United Kingdom. 2. School of Life Sciences, Kingston University, Kingston upon Thames, United Kingdom. 3. Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom. 4. Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.
The flavonoids quercetin, kaempferol and genistein are present in the diet and known to stimulate chloride secretion in the intestine through a cAMP-dependent pathway (Cermak et al 2001). However, many such in vitro studies have used concentrations of the aglycone form of the flavonoid well in excess of the bioavailable concentrations observed in plasma after a meal. We therefore tested the activity of these flavonoids on colonic chloride secretion at concentrations potentially achievable in vivo (<30 μM serosal, <100 μM mucosal). The possible augmentation of secretory responses to the agonists ATP and forskolin was also studied. Caco-2 human colonic epithelial monolayers and isolated mouse distal colonic mucosae were mounted in Ussing chambers and changes to the short-circuit current (ΔISC) were measured in response to flavonoids and other agents. Values of ISC are presented as mean ± SD (n). Differences between means were analysed by t-test or one-way ANOVA with Dunnett’s post-test and deemed significant at p<0.05. mAll three flavonoids stimulated a transient increase in chloride secretion in Caco-2 monolayers when added to the mucosal side. The EC50 for the response to quercetin was 30 μM with a ΔISC of 8.82 ±3.98 μA/cm2, n=6. ATP (100 μM) stimulated similar increases of ISC from the mucosal side (8.29 ± 2.24 μA/cm2, n=8) and these responses were significantly increased by pre-treatment with quercetin 100 μM or kaempferol 100 μM to 17.04 ± 7.00 μA/cm2, n=7, and 15.68 ± 4.16 μA/cm2, n=4, respectively. Serosal ATP responses were smaller (1.84 ± 1.11 μA/cm2, n=5) but significantly increased by mucosal addition of quercetin 30 μM (39.15 ± 20.5 μA/cm2, n=6), and genistein 30 μM (76.34 ± 17.23 μA/cm2, n=3). MDL12330A, an adenylyl cyclase inhibitor, abolished the responses to quercetin or kaempferol, but not the Ca2+-dependent secretagogue ATP, indicating stimulation of cAMP signalling by the flavonoids. In mouse colon, genistein was effective on the mucosal side in the range 1-100 μM, but the response to serosal ATP was not augmented. However, the lowest concentration of genistein (1 μM), which alone evoked only a small ΔISC (<3 μA/cm2) significantly augmented secretory responses to low concentrations of forksolin: at 0.1 μM, from 25.10 ± 26.95 to 87.65 ± 54.31 μA/cm2, n=11, and at 0.3 μM from 44.10 ± 39.8 to 106.50 ± 56.64 μA/cm2, n=7. Responses to forskolin at 1-10 μM were not augmented. We suggest that genistein at a likely bioavailable concentration may interact with cAMP-dependent agonists to enhance chloride secretion in the colon. The consequence may include a natural secretory laxative effect in the gut.
Where applicable, experiments conform with Society ethical requirements