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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.


Inhibitory effect of quercetin on ANO1-mediated short-circuit current in ANO1-expressing FRT cells. (A) Effect of apical addition of quercetin on Eact-induced short-circuit current without or with subsequent addition of T16Ainh-A01. (B) Inhibition of Eact-induced short-circuit current by basolateral application of quercetin without or with subsequent addition of 10 μM T16Ainh-A01. The histograms compare the magnitudes of inhibition as obtained from the corresponding traces (n = 5).
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Figure 3: Inhibitory effect of quercetin on ANO1-mediated short-circuit current in ANO1-expressing FRT cells. (A) Effect of apical addition of quercetin on Eact-induced short-circuit current without or with subsequent addition of T16Ainh-A01. (B) Inhibition of Eact-induced short-circuit current by basolateral application of quercetin without or with subsequent addition of 10 μM T16Ainh-A01. The histograms compare the magnitudes of inhibition as obtained from the corresponding traces (n = 5).

Mentions: ANO1 is a bona fide calcium-activated chloride channel, thus Cl− current measurement was performed in stably transfected FRT cells expressing ANO1 to investigate the effect of quercetin on ANO1 Cl− channel activity. Surprisingly, quercetin did not activate ANO1-mediated Cl− current in the stably transfected FRT cells. On the contrary, after CaCC current was induced by 10 μM of the ANO1 agonist Eact, quercetin exhibited inhibitory activity. When added to the apical side of FRT monolayers, quercetin slightly inhibited the current at low concentrations (10–20 μM) and almost completely inhibited the Cl− current at 200 μM (Figure 3A). The remaining current could be inhibited by the specific inhibitor T16Ainh-A01. Basolateral administration of quercetin resulted in partial inhibition of Eact-induced short-circuit current (~57%) (Figure 3B). The inverse effects of quercetin on ANO1 in transfected FRT cells and CaCC current in HT29 cells suggested the existence of calcium-activated chloride channels in the intestinal epithelial cells that are different from ANO1.


Role of Quercetin in Modulating Chloride Transport in the Intestine
Inhibitory effect of quercetin on ANO1-mediated short-circuit current in ANO1-expressing FRT cells. (A) Effect of apical addition of quercetin on Eact-induced short-circuit current without or with subsequent addition of T16Ainh-A01. (B) Inhibition of Eact-induced short-circuit current by basolateral application of quercetin without or with subsequent addition of 10 μM T16Ainh-A01. The histograms compare the magnitudes of inhibition as obtained from the corresponding traces (n = 5).
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Related In: Results  -  Collection

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Figure 3: Inhibitory effect of quercetin on ANO1-mediated short-circuit current in ANO1-expressing FRT cells. (A) Effect of apical addition of quercetin on Eact-induced short-circuit current without or with subsequent addition of T16Ainh-A01. (B) Inhibition of Eact-induced short-circuit current by basolateral application of quercetin without or with subsequent addition of 10 μM T16Ainh-A01. The histograms compare the magnitudes of inhibition as obtained from the corresponding traces (n = 5).
Mentions: ANO1 is a bona fide calcium-activated chloride channel, thus Cl− current measurement was performed in stably transfected FRT cells expressing ANO1 to investigate the effect of quercetin on ANO1 Cl− channel activity. Surprisingly, quercetin did not activate ANO1-mediated Cl− current in the stably transfected FRT cells. On the contrary, after CaCC current was induced by 10 μM of the ANO1 agonist Eact, quercetin exhibited inhibitory activity. When added to the apical side of FRT monolayers, quercetin slightly inhibited the current at low concentrations (10–20 μM) and almost completely inhibited the Cl− current at 200 μM (Figure 3A). The remaining current could be inhibited by the specific inhibitor T16Ainh-A01. Basolateral administration of quercetin resulted in partial inhibition of Eact-induced short-circuit current (~57%) (Figure 3B). The inverse effects of quercetin on ANO1 in transfected FRT cells and CaCC current in HT29 cells suggested the existence of calcium-activated chloride channels in the intestinal epithelial cells that are different from ANO1.

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.