Ueda-Nishimura Tomoko, Niisato Naomi, Miyazaki Hiroaki, Naito Yuji, Yoshida Norimasa, Yoshikawa Toshikazu, Nishino Hoyoku, Marunaka Yoshinori
Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
Biochem Biophys Res Commun. 2005 Jul 15;332(4):1042-52. doi: 10.1016/j.bbrc.2005.05.046.
Transepithelial Cl(-) secretion in polarized renal A6 cells is composed of two steps: (1) Cl(-) entry step across the basolateral membrane mediated by Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and (2) Cl(-) releasing step across the apical membrane via cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We estimated CFTR Cl(-) channel activity and transcellular Cl(-) secretion by measuring 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB, a blocker of CFTR Cl(-) channel)-sensitive transepithelial conductance (Gt) and short-circuit current (Isc), respectively. Pretreatment with 1 microM insulin for 24 h had no effects on NPPB-sensitive Gt or Isc. On the other hand, in A6 cells treated with carbobenzoxy-L-leucyl-leucyl-L-leucinal (MG132; 100 microM for 2 h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space, insulin pretreatment increased the NPPB-sensitive Isc with no effects on NPPB-sensitive Gt. Genistein (100 microM) induced sustained increases in NPPB-sensitive Gt and Isc, which were diminished by brefeldin A (a blocker of protein translocation to Golgi apparatus from endoplasmic reticulum). Co-application of insulin and genistein synergically stimulated the NPPB-sensitive Isc without any effects on NPPB-sensitive Gt. These observations suggest that: (1) insertion and endocytosis of NKCC are stimulated by insulin, (2) the insulin-induced stimulation of NKCC insertion into the basolateral membrane is offset by the stimulatory action on NKCC endocytosis from the basolateral membrane, (3) genistein stimulates insertion of both CFTR Cl(-) channel into the apical membrane and NKCC into the basolateral membrane, and (4) insulin and genistein synergically stimulated NKCC insertion into the basolateral membrane.
极化的肾A6细胞中的跨上皮Cl⁻分泌由两个步骤组成：（1）通过钠/钾/2氯共转运体（NKCC）介导的Cl⁻跨基底外侧膜进入步骤，以及（2）通过囊性纤维化跨膜电导调节因子（CFTR）Cl⁻通道跨顶膜的Cl⁻释放步骤。我们分别通过测量5-硝基2-（3-苯丙基氨基）苯甲酸（NPPB，一种CFTR Cl⁻通道阻滞剂）敏感的跨上皮电导（Gt）和短路电流（Isc）来估计CFTR Cl⁻通道活性和跨细胞Cl⁻分泌。用1 μM胰岛素预处理24小时对NPPB敏感的Gt或Isc没有影响。另一方面，在用苄氧羰基-L-亮氨酰-L-亮氨酰-L-亮氨酸（MG132；100 μM处理2小时）处理的A6细胞中，MG132可抑制质膜上蛋白质向胞质空间的内吞作用，胰岛素预处理增加了NPPB敏感的Isc，而对NPPB敏感的Gt没有影响。染料木黄酮（100 μM）诱导NPPB敏感的Gt和Isc持续增加，这被布雷菲德菌素A（一种从内质网到高尔基体的蛋白质转运阻滞剂）减弱。胰岛素和染料木黄酮共同应用协同刺激NPPB敏感的Isc，而对NPPB敏感的Gt没有任何影响。这些观察结果表明：（1）胰岛素刺激NKCC的插入和内吞作用；（2）胰岛素诱导的NKCC插入基底外侧膜的刺激作用被对基底外侧膜NKCC内吞作用的刺激作用抵消；（3）染料木黄酮刺激CFTR Cl⁻通道插入顶膜以及NKCC插入基底外侧膜；（4）胰岛素和染料木黄酮协同刺激NKCC插入基底外侧膜。
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