Asano Junji, Niisato Naomi, Nakajima Ken-ichi, Miyazaki Hiroaki, Yasuda Makoto, Iwasaki Yoshinobu, Hama Takemitsu, Dejima Kenji, Hisa Yasuo, Marunaka Yoshinori
Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
Am J Respir Cell Mol Biol. 2009 Dec;41(6):688-95. doi: 10.1165/rcmb.2008-0338OC. Epub 2009 Feb 27.
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.
我们研究了氯离子（Cl⁻）分泌的调节机制，其在维持人气道上皮Calu-3细胞表面液体方面起着至关重要的作用。本研究报告称，槲皮素（一种类黄酮）刺激了布美他尼敏感的Cl⁻分泌，同时顶端Cl⁻电导降低，这表明槲皮素通过激活Cl⁻经基底外侧膜通过钠/钾/2氯共转运体（NKCC1）的进入步骤来刺激Cl⁻分泌。为了阐明槲皮素刺激NKCC1的机制，我们验证了蛋白激酶（PK）A、PKC、蛋白酪氨酸激酶（PTK）和胞质钙依赖性途径的参与情况。PKA抑制剂（PKI-14-酰胺）、PKC抑制剂（Gö 6983）或钙螯合剂均不影响槲皮素刺激的Cl⁻分泌。另一方面，PTK抑制剂（AG18）显著减弱了槲皮素对Cl⁻分泌的刺激作用，且对顶端Cl⁻电导无抑制作用，这表明PTK介导的途径参与了槲皮素的刺激作用。布美他尼（BFA，一种从内质网到高尔基体的囊泡运输抑制剂）抑制了槲皮素对Cl⁻分泌的作用，如果存在表皮生长因子受体（EGFR）激酶抑制剂（AG1478），则未观察到BFA敏感的Cl⁻分泌，这表明槲皮素通过导致EGFR激酶介导的NKCC1或NKC1激活因子向人气道上皮Calu-3细胞基底外侧膜的转位来刺激Cl⁻分泌。然而，槲皮素并未增加NKCC1的表面密度，但槲皮素提高了NKCC1的活性。这些观察结果表明，槲皮素通过依赖EGFR激酶的途径，通过NKCC1激活因子的转位激活NKCC1来刺激Cl⁻分泌。
