Distinct Action of Flavonoids, Myricetin and Quercetin, on Epithelial Cl− Secretion: Useful Tools as Regulators of Cl− Secretion

Epithelial Cl− secretion plays important roles in water secretion preventing bacterial/viral infection and regulation of body fluid. We previously suggested that quercetin would be a useful compound for maintaining epithelial Cl− secretion at a moderate level irrespective of cAMP-induced stimulation. However, we need a compound that stimulates epithelial Cl− secretion even under cAMP-stimulated conditions, since in some cases epithelial Cl− secretion is not large enough even under cAMP-stimulated conditions. We demonstrated that quercetin and myricetin, flavonoids, stimulated epithelial Cl− secretion under basal conditions in epithelial A6 cells. We used forskolin, which activates adenylyl cyclase increasing cytosolic cAMP concentrations, to study the effects of quercetin and myricetin on cAMP-stimulated epithelial Cl− secretion. In the presence of forskolin, quercetin diminished epithelial Cl− secretion to a level similar to that with quercetin alone without forskolin. Conversely, myricetin further stimulated epithelial Cl− secretion even under forskolin-stimulated conditions. This suggests that the action of myricetin is via a cAMP-independent pathway. Therefore, myricetin may be a potentially useful compound to increase epithelial Cl− secretion under cAMP-stimulated conditions. In conclusion, myricetin would be a useful compound for prevention from bacterial/viral infection even under conditions that the amount of water secretion driven by cAMP-stimulated epithelial Cl− secretion is insufficient.


Introduction
Water secretion across epithelial tissues contributes to prevention of our body from bacterial/viral infection and regulation of body fluid content. The water secretion across epithelial tissues is driven by epithelial Cl − secretion [1][2][3]. The epithelial Cl − secretion consists of two steps: (1) the Cl − -entry step across the basolateral membrane via Cl − transporter participating in Cl − uptake into the intracellular space such as Na + -K + -2Cl − cotransporter (NKCC) and (2) the Cl − -releasing step across the apical membrane via Cl − channels such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel at the apical membrane [4,5]. The continuous Cl − secretion requires stimulation of Cl − uptake transporter such as NKCC [6,7]. Flavonoids and flavonoid-like compounds have various actions in cell function [8,9]. Our previous studies have reported that a flavonoid, quercetin, elevates epithelial Cl − secretion under basal conditions, but decreases it under cAMP-stimulated conditions [10] by modifying activity of NKCC [3]. These reports suggest that quercetin regulates the activity of NKCC leading epithelial Cl − secretion to a moderate level irrespective of cAMP-induced stimulation and that quercetin would be a useful compound to achieve a moderate level of epithelial Cl − secretion. However, in some cases, cAMPinduced stimulation is not large enough to maintain an adequate level of epithelial Cl − secretion. Myricetin has been reported to demonstrate promotive and protective effects on intestinal tight junctional barriers of epithelia and it has antiviral function [11][12][13]. Therefore, it would be important to identify a useful compound with stimulatory actions on epithelial Cl − secretion even under cAMP-stimulated conditions. Our study examines the effects of quercetin and myricetin on Cl − secretion in the absence and presence of cAMP-stimulation of renal A6 cells.  [3,14,15]) to the apical solution for detection of NPPB-sensitive conductance. We measured an NPPB-sensitive conductance by calculating the difference between the Gt just before and 30 minutes after application of 100 M NPPB. This difference of Gt represents an NPPBsensitive conductance. In the present study, we use the NPPBsensitive conductance as the apical Cl − conductance. The NPPB-sensitive conductance indicates the apical Cl − channel conductance, since the apical Cl − conductance is much smaller than the basolateral Cl − conductance [16]. Further, apical application of 100 M NPPB diminishes the apical Cl − conductance but not the basolateral Cl − conductance [7,16]. Bumetanide has been shown to have no effects on the NPPBsensitive conductance [7]. These observations [7,16] indicate that the NPPB-sensitive conductance can be used as the apical Cl − conductance.

Measurement of Short-Circuit Current (Isc).
As reported previously [7,16], we measured a short-circuit current (Isc) in A6 cells. The Isc measured directly by clamping the PD to 0 mV was identical to the calculated current as Gt⋅PD (equivalent current); namely, the monolayer had a linear current-voltage relationship. In the present study, we show an equivalent current (Gt⋅PD) as Isc. A positive current represents a net flow of anions from the basolateral solution to the apical one [7].

Temperature.
All experiments shown in the present study were performed at 24-25 ∘ C.

Data Presentation.
Values of Isc and Gt are shown as the mean, and the error bar indicates SEM. ANOVA was used for statistical analysis, and < 0.05 was considered significant.

Effects of Quercetin and Myricetin on Isc under Basal
Conditions. A6 cells can exhibit Cl − secretion and Na + absorption [7,[17][18][19][20][21][22]. Since the purpose of the present study was to examine transport properties of Cl − secretion, we added benzamil (10 M) to the apical solution to block Na + absorption via epithelial Na + channel (ENaC) in A6 cells ( Figure 2). We used 10 M benzamil, because it has been demonstrated to completely block ENaC activity [18,23,24]. DMSO (a solvent control for quercetin and myricetin ( Figure 1)) applied to both apical and basolateral solutions had no effect on Isc (closed triangles as in Figure 2 the benzamil-insensitive Isc (i.e., the residual Isc after application of benzamil was abolished) (Figure 2(a)). Quercetin significantly stimulated Isc (open circles as in Figure 2(a)), and NPPB abolished the quercetin-stimulated Isc (open circles as in Figure 2(a)) suggesting that quercetin stimulated a Cl − -dependent Isc. Myricetin also elevated Isc (closed squares as in Figure 2(a)), and the myricetin-elevated Isc was sensitive to NPPB (closed squares as in Figure 2(a)). Quercetin or myricetin showed no effects on Isc in A6 cells pretreated with NPPB (data not shown). These observations indicate that quercetin and myricetin stimulate the Cl − secretion.

Effects of Quercetin and Myricetin on Isc under Forskolin-Stimulated Conditions.
In order to examine the effects of quercetin and myricetin in the presence of elevated cAMP levels, we used forskolin to activate adenylyl cyclase to increase cellular cAMP. As shown in Figure 2(a) (closed triangles), DMSO, a solvent control for forskolin, had no effect on Isc. Though, forskolin stimulated Isc (open circles, closed squares, and closed triangles in Figure 2(b)), but not in the presence of NPPB (data not shown), suggesting that forskolin stimulated an NPPB-sensitive Isc. Under forskolinstimulated conditions, DMSO (a solvent control for quercetin and myricetin) had no effect on Isc (closed triangles as in Figure 2(b)). However, in the presence of forskolin, quercetin significantly diminished Isc (open circles as in Figure 2(b)) unlike that observed during basal conditions (open circles as in Figure 2(a)). On the other hand, myricetin significantly stimulated Isc (closed squares as in Figure 2(b)) in the presence of forskolin similar to that under basal conditions (closed squares as in Figure 2(a)). Further, we applied bumetanide (a blocker of NKCC) to study the Isc observed in the present study. Application of bumetanide (100 M) almost completely diminished the Isc irrespective of the presence of forskolin, quercetin, or myricetin without any effects on Gt: the Isc in the presence of bumetanide was reduced to ∼0.2 A/cm 2 irrespective of the presence of forskolin, quercetin, or myricetin. Further, the presence of bumetanide did not significantly influence effects of forskolin, quercetin, or myricetin on Gt (data not shown). Thus, these observations indicate that the Isc observed in the present study is mediated by NKCC irrespective of the presence of forskolin, quercetin, or myricetin.

Discussion
Epithelial Cl − secretion plays an important role in prevention from bacterial/viral infection through washout of bacteria and viruses located on the surface of apical membrane using water covering the surface of apical membrane produced by Cl − -secretion-generated osmotic gradient across the epithelial cells and also controls body fluid content by regulating water contents [2,4,7,[25][26][27][28][29][30]. The present study indicates that quercetin is useful for moderate stimulation of epithelial Cl − secretion irrespective of cAMP stimulation and that myricetin can be applied in cases requiring further stimulation of epithelial Cl − secretion with insufficient cAMP stimulation. Various types of flavonoids have been reported to modulate epithelial Cl − secretion [9,[31][32][33][34][35]. For example, genistein stimulates jejunal Cl − secretion via estrogen receptor-mediated pathways [31,32]. Chao and Hamilton [9] have reported that genistein stimulates jejunal Cl − secretion via phosphodiesterase modulation. Further, Fischer and Illek [34] have indicated that trimethoxyflavone, aryl hydrocarbon receptor ligand, activates CFTR Cl − channel stimulating Cl − secretion in lung epithelial cells. Quercetin is also reported to have stimulatory action on Cl − secretion in sinonasal epithelium [35]. Niisato et al. [7] have also reported that flavonoids, genistein, daidzein, and apigenin, stimulate epithelial Cl − secretion.
Although molecular mechanisms of flavonoids' action on epithelial Cl − secretion are still unclear, we have a consensus that flavonoids stimulate epithelial Cl − secretion via activation of the CFTR Cl − channel playing a role in a Cl − -releasing step across the apical membrane and/or Cl − transporter playing a role in a Cl − uptake step across the basolateral membrane [6,7]. Continuous activation of Cl − uptake transporter such as NKCC is required to continuously stimulate epithelial Cl − secretion [6,7]. This continuous activation of Cl − uptake transporter such as NKCC is one of the most important targets from a therapeutic viewpoint for continuous stimulation of epithelial Cl − secretion. The present study and our previous report [3] suggest that quercetin continuously increases activity of NKCC by stimulating translocation of NKCC activating factors to the basolateral membrane from intracellular store sites.
There is little mechanistic information on the effects of flavonoids on the function of NKCC during cAMPdependent Cl − secretion, although many researchers have reported the stimulatory action of flavonoids on epithelial Cl − secretion [9,[31][32][33][34][35][36][37] and the inhibitory action of cAMPactivated Cl − secretion [38]. We were surprised that quercetin reduced the forskolin-stimulated Isc, but myricetin tremendously increased the forskolin-stimulated Isc. As mentioned above, the epithelial Cl − secretion (the NPPB-sensitive Isc) consists of two steps: (1) the Cl − -entry step across the basolateral membrane via Cl − transporter participating in Cl − uptake into the intracellular space such as Na + -K + -2Cl − cotransporter (NKCC) and (2) the Cl − -releasing step across the apical membrane via Cl − channels such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel at the apical membrane [4]. Therefore, the stimulatory action of myricetin on the NPPB-sensitive Isc could be explained by effects of myricetin on the NPPB-sensitive conductance; that is, we suggest that myricetin would increase the epithelial Cl − secretion by activating the CFTR Cl − channel at the apical membrane acting as the Cl − -releasing step. However, the inhibitory action of quercetin on the forskolinstimulated Isc could not be explained by effects of quercetin on the apical NPPB-sensitive conductance, since quercetin increased the NPPB-sensitive conductance (in other words, quercetin activated the Cl − -releasing step via CFTR Cl − channel; see Figure 4). Namely, if the inhibitory action of quercetin on the forskolin-stimulated Isc is explained by effects of quercetin on the NPPB-sensitive conductance (the Cl − -releasing step), quercetin should diminish the NPPBsensitive conductance. Therefore, we should consider other possibilities regarding the inhibitory action of quercetin on the forskolin-stimulated Isc. There are, at least, two possible sites of action that quercetin might have on the cAMPdependent Cl − secretory process and that is either by down regulating the Na + -K + -2Cl − cotransporter and/or the Na + , K + -ATPase. Similar to our data, Schuier et al. [39] have reported that quercetin, morin, and luteolin, all administered at 100 M, reduce forskolin-stimulated Isc of T84 colonic epithelial cells. These authors have offered no explanation for the action of these flavonoids. However, Collins et al. [33] have demonstrated that the flavone, naringenin, added prior to forskolin, reduces the forskolin-activated Isc of human and rat colons. They have surmised that since that action of naringenin is upstream of the activation of CFTR, naringenin inhibits NKCC. Alternatively, the action of quercetin on the forskolin-stimulated Isc might be by downregulating the Na + , K + -ATPase. Indeed, Mezesova et al. [40] have reported that treatment with quercetin (20 mg ⋅ kg −1 ⋅ day −1 ) in both normotensive and hypertensive rats produces impairment in the affinity of Na + binding site of the Na + , K + -ATPase like the inhibitory action on Ca 2+ -ATPase [41,42]. On the other hand, myricetin showed no inhibitory action but stimulatory action on Isc, suggesting that myricetin would not inhibit NKCC or produce impairment in the affinity of Na + binding site of the Na + , K + -ATPase unlike quercetin. Further work is necessary to resolve this complex role of quercetin in cAMP-dependent Cl − secretion.
Although it has been reported that flavonoids including myricetin and quercetin have various action on cell function [3,10,[31][32][33][43][44][45][46][47][48][49][50][51], the present study clearly indicates that myricetin has stimulatory action on cAMPactivated Cl − secretion unlike quercetin. From our knowledge, the present study is the first report showing a flavonoid with stimulatory action on cAMP-activated epithelial Cl − secretion. Thus, using these compounds it could be possible to treat patients with disorders in water secretion across epithelial tissues by regulating epithelial Cl − secretion to ideal levels depending on pathophysiological states of patient.