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

¹ Department of Molecular Cell Physiology, ² Department of Otolaryngology-Head and Neck Surgery, and ³ Department of Respiratory Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan; and ⁴ Department of Otolaryngology, Kyoto Second Red Cross Hospital, Kyoto, Japan

Correspondence and requests for reprints should be addressed to Yoshinori Marunaka, M.D., Ph.D., Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. E-mail: marunaka{at}koto.kpu-m.ac.jp

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 Ca²⁺-dependent pathways. A PKA inhibitor (PKI-14–22 amide), a PKC inhibitor (Gö 6983) or a Ca²⁺ 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.

Key Words: epidermal growth factor receptor • brefeldin A • ion transport • chloride

CLINICAL RELEVANCE Surface liquid covering airway epithelia protects our bodies from bacterial/viral infection. Quercetin protects our bodies from bacterial/viral infection by producing airway surface liquid via stimulation of Cl– secretion.