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

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 (Go 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.