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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.

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Characterization of quercetin-induced short-circuit current in mouse ileum and colon. (A–H) Activating effect of quercetin after pre-treatment with DMSO (A), nifedipine (B), nifedipine plus CaCCinh-A01 (C), nifedipine plus CFTRinh-172 (D), nifedipine plus CaCCinh-A01 plus CFTRinh-172 (E), CaCCinh-A01 (F), CFTRinh-172 (G), and CFTRinh-172 plus CaCCinh-A01 (H). (I) Comparison of the inhibitory effects of different agents on the short-circuit currents. Data are the means ± SEs from 6 to 7 determinations. “*” and “**” indicate significantly difference from control at the P < 0.05 and P < 0.01 levels.
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Figure 6: Characterization of quercetin-induced short-circuit current in mouse ileum and colon. (A–H) Activating effect of quercetin after pre-treatment with DMSO (A), nifedipine (B), nifedipine plus CaCCinh-A01 (C), nifedipine plus CFTRinh-172 (D), nifedipine plus CaCCinh-A01 plus CFTRinh-172 (E), CaCCinh-A01 (F), CFTRinh-172 (G), and CFTRinh-172 plus CaCCinh-A01 (H). (I) Comparison of the inhibitory effects of different agents on the short-circuit currents. Data are the means ± SEs from 6 to 7 determinations. “*” and “**” indicate significantly difference from control at the P < 0.05 and P < 0.01 levels.

Mentions: Next, we investigated the components of quercetin-induced short-circuit current in mouse ileum. CaCC can be activated by intracellular Ca2+, and Ca2+ influx by calcium channels is a main route to enhance intracellular calcium concentration. In order to prove whether L-type calcium channels were involved in quercetin-induced CaCC activation, 10 μM of an L-type calcium channel inhibitor (nifedipine) was added to the serosal incubation solution. Compared with DMSO control (Figure 6A), the short-circuit current induced by 200 μM quercetin was reduced by 41% after pre-treatment with nifedipine (Figure 6B). The result suggested that L-type calcium channel was involved in the Isc generated by quercetin. We also analyzed the effect of CaCCinh-A01 and CFTRinh-172 on quercetin-induced Isc after inhibition of the L-type calcium channel. Quercetin-stimulated short-circuit current was further decreased by ~6.3 and 43% after the addition of nifedipine plus CaCCinh-A01 (100 μM) and nifedipine plus CFTRinh-172 (100 μM), respectively (Figures 6C,D). No significant difference was observed between the nifedipine group and nifedipine plus CaCCinh-A01 group (Figures 6B,C). These results indicated that Ca2+ influx through L-type calcium channel was an important way to activate intestinal epithelial CaCC. As CFTRinh-172 could further inhibit the short-circuit current by 25% (Figure 6D), it suggested the existence of CFTR-mediated Cl− current. However, CaCCinh-A01 or CFTRinh-172 could only inhibit part of the Isc induced by quercetin. CaCCinh-A01 produced a slightly higher inhibition rate (~52%) (Figure 6F) than CFTRinh-172 (~44%) (Figure 6G). These results were consistent with previous findings. In addition, pre-treatment with nifedipine, CaCCinh-A01 and CFTRinh-172 almost completely abolished quercetin-induced short-circuit current in mouse ileal tissues (Figure 6E), but there remained a small amount of short-circuit current after the addition of CaCCinh-A01 and CFTRinh-172 (Figure 6H). Figure 6I summarizes the result of Isc from the different treatments. The results suggested that quercetin-induced short-circuit currents may involve other Ca2+-activated channels such as Ca2+-activated K+ channel.


Role of Quercetin in Modulating Chloride Transport in the Intestine
Characterization of quercetin-induced short-circuit current in mouse ileum and colon. (A–H) Activating effect of quercetin after pre-treatment with DMSO (A), nifedipine (B), nifedipine plus CaCCinh-A01 (C), nifedipine plus CFTRinh-172 (D), nifedipine plus CaCCinh-A01 plus CFTRinh-172 (E), CaCCinh-A01 (F), CFTRinh-172 (G), and CFTRinh-172 plus CaCCinh-A01 (H). (I) Comparison of the inhibitory effects of different agents on the short-circuit currents. Data are the means ± SEs from 6 to 7 determinations. “*” and “**” indicate significantly difference from control at the P < 0.05 and P < 0.01 levels.
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Figure 6: Characterization of quercetin-induced short-circuit current in mouse ileum and colon. (A–H) Activating effect of quercetin after pre-treatment with DMSO (A), nifedipine (B), nifedipine plus CaCCinh-A01 (C), nifedipine plus CFTRinh-172 (D), nifedipine plus CaCCinh-A01 plus CFTRinh-172 (E), CaCCinh-A01 (F), CFTRinh-172 (G), and CFTRinh-172 plus CaCCinh-A01 (H). (I) Comparison of the inhibitory effects of different agents on the short-circuit currents. Data are the means ± SEs from 6 to 7 determinations. “*” and “**” indicate significantly difference from control at the P < 0.05 and P < 0.01 levels.
Mentions: Next, we investigated the components of quercetin-induced short-circuit current in mouse ileum. CaCC can be activated by intracellular Ca2+, and Ca2+ influx by calcium channels is a main route to enhance intracellular calcium concentration. In order to prove whether L-type calcium channels were involved in quercetin-induced CaCC activation, 10 μM of an L-type calcium channel inhibitor (nifedipine) was added to the serosal incubation solution. Compared with DMSO control (Figure 6A), the short-circuit current induced by 200 μM quercetin was reduced by 41% after pre-treatment with nifedipine (Figure 6B). The result suggested that L-type calcium channel was involved in the Isc generated by quercetin. We also analyzed the effect of CaCCinh-A01 and CFTRinh-172 on quercetin-induced Isc after inhibition of the L-type calcium channel. Quercetin-stimulated short-circuit current was further decreased by ~6.3 and 43% after the addition of nifedipine plus CaCCinh-A01 (100 μM) and nifedipine plus CFTRinh-172 (100 μM), respectively (Figures 6C,D). No significant difference was observed between the nifedipine group and nifedipine plus CaCCinh-A01 group (Figures 6B,C). These results indicated that Ca2+ influx through L-type calcium channel was an important way to activate intestinal epithelial CaCC. As CFTRinh-172 could further inhibit the short-circuit current by 25% (Figure 6D), it suggested the existence of CFTR-mediated Cl− current. However, CaCCinh-A01 or CFTRinh-172 could only inhibit part of the Isc induced by quercetin. CaCCinh-A01 produced a slightly higher inhibition rate (~52%) (Figure 6F) than CFTRinh-172 (~44%) (Figure 6G). These results were consistent with previous findings. In addition, pre-treatment with nifedipine, CaCCinh-A01 and CFTRinh-172 almost completely abolished quercetin-induced short-circuit current in mouse ileal tissues (Figure 6E), but there remained a small amount of short-circuit current after the addition of CaCCinh-A01 and CFTRinh-172 (Figure 6H). Figure 6I summarizes the result of Isc from the different treatments. The results suggested that quercetin-induced short-circuit currents may involve other Ca2+-activated channels such as Ca2+-activated K+ channel.

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&minus; transport in a dose-dependent manner, with EC50 ~37 &mu;M. Short-circuit current analysis confirmed that quercetin activated CaCC-mediated Cl&minus; 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&minus; 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.