Feranchak Andrew P, Kilic Gordan, Wojtaszek Paul A, Qadri Ishtiaq, Fitz J Gregory
Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Biol Chem. 2003 Nov 7;278(45):44632-8. doi: 10.1074/jbc.M301958200. Epub 2003 Aug 25.
In liver cells, the influx of Na+ mediated by nonselective cation (NSC) channels in the plasma membrane contributes importantly to regulation of cell volume. Under basal conditions, channels are closed; but both physiologic (e.g. insulin) and pathologic (e.g. oxidative stress) stimuli that are known to stimulate tyrosine kinases are associated with large increases in membrane Na+ permeability to approximately 80 pA/pF or more. Consequently, the purpose of these studies was to evaluate whether volume-sensitive tyrosine kinases mediate cell volume increases through effects on the activity or distribution of NSC channel proteins. In HTC hepatoma cells, decreases in cell volume evoked by hypertonic exposure increased total cellular tyrosine kinase activity approximately 20-fold. Moreover, hypertonic exposure (320-400 mosM) was followed after a delay by NSC channel activation and partial recovery of cell volume toward basal values (regulatory volume increase (RVI)). The tyrosine kinase inhibitors genistein and erbstatin prevented both NSC channel activation and RVI. Similarly, hypertonic exposure resulted in an increase in p60(c-src) activity, and intracellular dialysis with recombinant p60(c-src) led to activation of NSC currents in the absence of an osmolar gradient. Utilizing FM1-43 fluorescence, exposure to hypertonic media caused a rapid increase in the rate of exocytosis of approximately 40% (p < 0.01), and genistein inhibited both exocytosis and channel activation. These findings indicate that volume-sensitive increases in p60(c-src) and/or related tyrosine kinases play a key role in the regulation of membrane Na+ permeability, suggesting that increases in the NSC conductance may be mediated in part through rapid recruitment of a distinct pool of channel-containing vesicles.
在肝细胞中，质膜上非选择性阳离子（NSC）通道介导的Na⁺内流对细胞体积的调节起着重要作用。在基础条件下，通道是关闭的；但已知刺激酪氨酸激酶的生理刺激（如胰岛素）和病理刺激（如氧化应激）均与膜Na⁺通透性大幅增加至约80 pA/pF或更高有关。因此，这些研究的目的是评估体积敏感性酪氨酸激酶是否通过影响NSC通道蛋白的活性或分布来介导细胞体积增加。在HTC肝癌细胞中，高渗暴露引起的细胞体积减小使总细胞酪氨酸激酶活性增加了约20倍。此外，高渗暴露（320 - 400 mosM）延迟后会出现NSC通道激活以及细胞体积部分恢复至基础值（调节性容积增加（RVI））。酪氨酸激酶抑制剂染料木黄酮和埃伯司他汀可阻止NSC通道激活和RVI。同样，高渗暴露导致p60（c-src）活性增加，用重组p60（c-src）进行细胞内透析可在无渗透压梯度的情况下激活NSC电流。利用FM1 - 43荧光技术，暴露于高渗培养基会使胞吐速率迅速增加约40%（p < 0.01），染料木黄酮可抑制胞吐和通道激活。这些发现表明，p60（c-src）和/或相关酪氨酸激酶的体积敏感性增加在膜Na⁺通透性调节中起关键作用，提示NSC电导增加可能部分是通过快速募集不同的含通道囊泡池介导的。
