Quercetin Stimulates Na⁺/K⁺/2Cl⁻ Cotransport via PTK-Dependent Mechanisms in Human Airway Epithelium

Abstract

We investigated regulatory mechanisms of Cl(-) secretion playing an essential role in the maintenance of surface fluid in human airway epithelial Calu-3 cells. The present study reports that quercetin (a flavonoid) stimulated bumetanide-sensitive Cl(-) secretion with reduction of apical Cl(-) conductance, suggesting that quercetin stimulates Cl(-) secretion by activating an entry step of Cl(-) across the basolateral membrane through Na(+)/K(+)/2Cl(-) cotransporter (NKCC1). To clarify the mechanism stimulating NKCC1 by quercetin, we verified involvement of protein kinase (PK)A, PKC, protein tyrosine kinase (PTK), and cytosolic Ca(2+)-dependent pathways. A PKA inhibitor (PKI-14-22 amide), a PKC inhibitor (Gö 6983) or a Ca(2+) chelating agent did not affect the quercetin-stimulated Cl(-) secretion. On the other hand, a PTK inhibitor (AG18) significantly diminished the stimulatory action of quercetin on Cl(-) secretion without inhibitory effects on apical Cl(-) conductance, suggesting that a PTK-mediated pathway is involved in the stimulatory action of quercetin. The quercetin action on Cl(-) secretion was suppressed with brefeldin A (BFA, an inhibitor of vesicular transport from ER to Golgi), and the BFA-sensitive Cl(-) secretion was not observed in the presence of an epidermal growth factor receptor (EGFR) kinase inhibitor (AG1478), suggesting that quercetin stimulates Cl(-) secretion by causing the EGFR kinase-mediated translocation of NKCC1 or an NKC1-activating factor to the basolateral membrane in human airway epithelial Calu-3 cells. However, the surface density of NKCC1 was not increased by quercetin, but quercetin elevated the activity of NKCC1. These observations indicate that quercetin stimulates Cl(-) secretion by activating NKCC1 via translocation of an NKCC1-activating factor through an EGFR kinase-dependent pathway.

Capsaicinoids Regulate Airway Anion Transporters through Rho Kinase– and Cyclic AMP–Dependent Mechanisms

“…Although capsaicin inhibited I SC due to suppression of basolateral channels and transporters, it conversely caused a sustained increase in apical I Cl , reflecting the conductance of CFTR, a cAMP-activated Cl 2 channel (20), indicating that anion uptake across the basolateral membrane is the ratelimiting step that determines the overall secretion capacity (16,22,24,29). In addition, the result that the basolateral Cl 2 conductance increased by forskolin was inhibited by capsaicin excludes the possibility that apical Cl 2 transport would be reduced by intracellular Cl 2 loss as a result of basolateral Cl 2 release.…”

“…In the regulation of basolateral anion transporters, involvement of basolateral membrane Cl 2 and K 1 conductance must be considered (21,24). In the permeabilized monolayer pretreated with apical nystatin (50 mM) under the basolateral-to-apical Cl 2 concentration gradient, forskolin-induced bI Cl (DbI Cl 5 45.7 6 9.4 mA/ cm 2 ; n 5 4) was inhibited by the presence of capsaicin (DbI Cl 5 33.0 6 10.1 mA/cm 2 ; n 5 4; P , 0.01).…”

Hypotonic stress upregulates β- and γ-ENaC expression through suppression of ERK by inducing MKP-1

“…Transepithelial potential (PD) was continuously measured by a high-impedance millivoltmeter that could function as a voltage clamp with automatic fluid resistance compensation (VCC-600, Physiologic Instruments, San Diego, CA) with a pair of calomel electrodes that were immersed in a saturated KCl solution and bridged to the modified Ussing chamber by a pair of polyethylene tubes filled with a solution of 2% (wt/vol) agarose in a 2 M KCl solution (18,19,22,38). I sc was measured by the amplifier (VCC-600) with a pair of silver-silver chloride electrodes that were immersed in a 2 M NaCl solution and bridged to the modified Ussing chamber by a pair of polyethylene tubes filled with a solution of 2% (wt/vol) agarose in a 2 M NaCl solution (1,(42)(43)(44). When the I sc was measured, the PD was clamped to 0 mV for 1 s by the amplifier.…”

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

“…The water secretion across epithelial tissues is driven by epithelial Cl − secretion [1–3]. 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].…”

“…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.…”