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Role of Quercetin in Modulating Chloride Transport in the Intestine

View Article: PubMed Central - PubMed

ABSTRACT

Epithelial chloride channels provide the pathways for fluid secretion in the intestine. Cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCCs) are the main chloride channels in the luminal membrane of enterocytes. These transmembrane proteins play important roles in many physiological processes. In this study, we have identified a flavonoid quercetin as a modulator of CaCC chloride channel activity. Fluorescence quenching assay showed that quercetin activated Cl− transport in a dose-dependent manner, with EC50 ~37 μM. Short-circuit current analysis confirmed that quercetin activated CaCC-mediated Cl− currents in HT-29 cells that can be abolished by CaCCinh-A01. Ex vivo studies indicated that application of quercetin to mouse ileum and colon on serosal side resulted in activation of CFTR and CaCC-mediated Cl− currents. Notably, we found that quercetin exhibited inhibitory effect against ANO1 chloride channel activity in ANO1-expressing FRT cells and decreased mouse intestinal motility. Quercetin-stimulated short-circuit currents in mouse ileum was multi-component, which included elevation of Ca2+ concentration through L-type calcium channel and activation of basolateral NKCC, Na+/K+-ATPase, and K+ channels. In vivo studies further revealed that quercetin promoted fluid secretion in mouse ileum. The modulatory effect of quercetin on CaCC chloirde channels may therefore represent a potential therapeutic strategy for treating CaCC-related diseases like constipation, secretory diarrhea and hypertension. The inverse effects of quercetin on CaCCs provided evidence that ANO1 and intestinal epithelial CaCCs are different calcium-activated chloride channels.

No MeSH data available.


Activation effect of quercetin on chloride channel activities in mouse ileum and colon. (A) Activation of CFTR and CaCC-mediated Cl− current in freshly isolated mouse ileum by different concentrations of quercetin. (B) Activation of CFTR and CaCC-mediated Cl− current in colonic epithelia by different concentrations of quercetin. The corresponding histogram of each trace compares the Isc obtained from different concentrations of quercetin. (C) Inhibition of CFTR-mediated Cl− current by indicated CaCCinh-A01 in mouse ileum. (D) Inhibition of CFTR chloride channel activity by CFTRinh-172 (100 μM).
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Figure 5: Activation effect of quercetin on chloride channel activities in mouse ileum and colon. (A) Activation of CFTR and CaCC-mediated Cl− current in freshly isolated mouse ileum by different concentrations of quercetin. (B) Activation of CFTR and CaCC-mediated Cl− current in colonic epithelia by different concentrations of quercetin. The corresponding histogram of each trace compares the Isc obtained from different concentrations of quercetin. (C) Inhibition of CFTR-mediated Cl− current by indicated CaCCinh-A01 in mouse ileum. (D) Inhibition of CFTR chloride channel activity by CFTRinh-172 (100 μM).

Mentions: CaCCs have been shown to exist in mouse intestinal epithelia, and therefore the efficacy of quercetin was tested ex vivo in isolated mouse intestinal mucosa by Ussing chamber short-circuit assay. The results showed that the short-circuit currents in mouse ileal and colonic epithelia on serosal side were increased by quercetin in a dose-dependent manner, reaching a peak at 200 μM in both cases (Figures 5A,B). Isc stimulated by the same concentration of quercetin was slightly higher in mouse ileal mucosa than in colonic mucosa. As expected, the current was slightly inhibited by 20 μM T16Ainh-A01 (inhibition rate ≤ 20%), but was partially inhibited by 100 μM CaCCinh-A01 (inhibition rate ≥ 40%) while the remaining current was abolished by 100 μM CFTRinh-172. The inhibitory effect of CaCCinh-A01 on CFTR chloride channel activity in mouse ileal mucosa was also measured, and CaCCinh-A01 did not appear to inhibit forskolin-induced CFTR-mediated Cl− current at 10–100 μM (Figures 5C,D). These results further confirmed that quercetin activated a CaCC current different from ANO1 in mouse intestinal epithelia.


Role of Quercetin in Modulating Chloride Transport in the Intestine
Activation effect of quercetin on chloride channel activities in mouse ileum and colon. (A) Activation of CFTR and CaCC-mediated Cl− current in freshly isolated mouse ileum by different concentrations of quercetin. (B) Activation of CFTR and CaCC-mediated Cl− current in colonic epithelia by different concentrations of quercetin. The corresponding histogram of each trace compares the Isc obtained from different concentrations of quercetin. (C) Inhibition of CFTR-mediated Cl− current by indicated CaCCinh-A01 in mouse ileum. (D) Inhibition of CFTR chloride channel activity by CFTRinh-172 (100 μM).
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Figure 5: Activation effect of quercetin on chloride channel activities in mouse ileum and colon. (A) Activation of CFTR and CaCC-mediated Cl− current in freshly isolated mouse ileum by different concentrations of quercetin. (B) Activation of CFTR and CaCC-mediated Cl− current in colonic epithelia by different concentrations of quercetin. The corresponding histogram of each trace compares the Isc obtained from different concentrations of quercetin. (C) Inhibition of CFTR-mediated Cl− current by indicated CaCCinh-A01 in mouse ileum. (D) Inhibition of CFTR chloride channel activity by CFTRinh-172 (100 μM).
Mentions: CaCCs have been shown to exist in mouse intestinal epithelia, and therefore the efficacy of quercetin was tested ex vivo in isolated mouse intestinal mucosa by Ussing chamber short-circuit assay. The results showed that the short-circuit currents in mouse ileal and colonic epithelia on serosal side were increased by quercetin in a dose-dependent manner, reaching a peak at 200 μM in both cases (Figures 5A,B). Isc stimulated by the same concentration of quercetin was slightly higher in mouse ileal mucosa than in colonic mucosa. As expected, the current was slightly inhibited by 20 μM T16Ainh-A01 (inhibition rate ≤ 20%), but was partially inhibited by 100 μM CaCCinh-A01 (inhibition rate ≥ 40%) while the remaining current was abolished by 100 μM CFTRinh-172. The inhibitory effect of CaCCinh-A01 on CFTR chloride channel activity in mouse ileal mucosa was also measured, and CaCCinh-A01 did not appear to inhibit forskolin-induced CFTR-mediated Cl− current at 10–100 μM (Figures 5C,D). These results further confirmed that quercetin activated a CaCC current different from ANO1 in mouse intestinal epithelia.

View Article: PubMed Central - PubMed

ABSTRACT

Epithelial chloride channels provide the pathways for fluid secretion in the intestine. Cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCCs) are the main chloride channels in the luminal membrane of enterocytes. These transmembrane proteins play important roles in many physiological processes. In this study, we have identified a flavonoid quercetin as a modulator of CaCC chloride channel activity. Fluorescence quenching assay showed that quercetin activated Cl− transport in a dose-dependent manner, with EC50 ~37 μM. Short-circuit current analysis confirmed that quercetin activated CaCC-mediated Cl− currents in HT-29 cells that can be abolished by CaCCinh-A01. Ex vivo studies indicated that application of quercetin to mouse ileum and colon on serosal side resulted in activation of CFTR and CaCC-mediated Cl− currents. Notably, we found that quercetin exhibited inhibitory effect against ANO1 chloride channel activity in ANO1-expressing FRT cells and decreased mouse intestinal motility. Quercetin-stimulated short-circuit currents in mouse ileum was multi-component, which included elevation of Ca2+ concentration through L-type calcium channel and activation of basolateral NKCC, Na+/K+-ATPase, and K+ channels. In vivo studies further revealed that quercetin promoted fluid secretion in mouse ileum. The modulatory effect of quercetin on CaCC chloirde channels may therefore represent a potential therapeutic strategy for treating CaCC-related diseases like constipation, secretory diarrhea and hypertension. The inverse effects of quercetin on CaCCs provided evidence that ANO1 and intestinal epithelial CaCCs are different calcium-activated chloride channels.

No MeSH data available.