Kunzelmann K, Kathöfer S, Greger R
Physiologisches Institut der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Pflugers Arch. 1995 Nov;431(1):1-9. doi: 10.1007/BF00374371.
Na+ and Cl- conductances in the apical membrane of respiratory epithelial cells are essential for electrolyte and water transport in the airways. Apart from the well described defect in adenosine 3' : 5' cyclic monophosphate-(cAMP-) dependent activation of Cl- conductances in cystic fibrosis (CF), an increased Na+ conductance has also been reported from transepithelial measurements. In the present experiments we tried to identify these conductances in nasal epithelial cells using patch-clamp and microelectrode techniques. With these methods we found identical and relatively low membrane voltages of about -36 mV in both freshly isolated and primary cultured normal and CF nasal epithelial cells. A Cl- conductance could be activated by cAMP in normal (deltaG = 0.3 +/- 0.8 nS, n = 10) but not in CF (deltaG = 0.3 +/- 0.1 nS, n = 11) cells, whereas Ca2+-dependent Cl- currents activated by adenosine 5'-triphosphate (ATP) and bradykinin were present in both types of cells. Cell-attached membrane patches from stimulated cells did not reveal discernible single-channel events when activated with any of the agonists. A Na+ conductance was also detected in freshly isolated ciliated respiratory cells in impalement studies, as evidenced by the hyperpolarization induced by 10 micromol/l amiloride (deltaV = -5.2 +/- 0.6 mV, n = 56) and when Na+ was replaced in the bath by N-methyl-D-glucamine (NMDG) (deltaV = -5.7 +/- 0.9 mV, n = 14). In whole-cell patch-clamp experiments, the amiloride-induced hyperpolarization was significantly larger in CF (deltaV = 9.7 +/- 2.4 mV, n = 22) when compared to normal (deltaV = -3.3 +/- 0.9 mV, n = 27) cells in short-term culture. Reverse transcriptase polymerase chain reaction analysis of normal respiratory cells identified messenger RNA of both the cystic fibrosis transmembrane conductance regulator (CFTR) as well as the human epithelial Na+ channel (hNaCh). The present experiments confirm the absence of a cAMP-dependent Cl- conductance in CF respiratory epithelial cells and support previous findings obtained in transepithelial and microelectrode studies which indicate an increased Na+ conductance in respiratory epithelial cells from CF patients.
呼吸道上皮细胞顶端膜中的钠离子（Na⁺）和氯离子（Cl⁻）电导对于气道中的电解质和水转运至关重要。除了在囊性纤维化（CF）中腺苷3':5'环磷酸单酯（cAMP）依赖性激活Cl⁻电导方面描述详尽的缺陷外，经上皮测量还报告了Na⁺电导增加。在本实验中，我们试图使用膜片钳和微电极技术在鼻上皮细胞中鉴定这些电导。通过这些方法，我们发现在新鲜分离的以及原代培养的正常和CF鼻上皮细胞中，膜电压相同且相对较低，约为 -36 mV。cAMP可在正常细胞（ΔG = 0.3 ± 0.8 nS，n = 10）而非CF细胞（ΔG = 0.3 ± 0.1 nS，n = 11）中激活Cl⁻电导，而由三磷酸腺苷（ATP）和缓激肽激活的Ca²⁺依赖性Cl⁻电流在两种类型的细胞中均存在。当用任何一种激动剂激活时，来自受刺激细胞的细胞贴附膜片未显示可辨别的单通道事件。在穿刺研究中，在新鲜分离的纤毛呼吸道细胞中也检测到了Na⁺电导，10 μmol/L氨氯吡咪诱导的超极化（ΔV = -5.2 ± 0.6 mV，n = 56）以及当浴液中的Na⁺被N-甲基-D-葡糖胺（NMDG）替代时（ΔV = -5.7 ± 0.9 mV，n = 14）均证明了这一点。在全细胞膜片钳实验中，与短期培养的正常细胞（ΔV = -3.3 ± 0.9 mV，n = 27）相比，CF细胞（ΔV = 9.7 ± 2.4 mV，n = 22）中氨氯吡咪诱导的超极化明显更大。对正常呼吸道细胞进行逆转录酶聚合酶链反应分析，鉴定出囊性纤维化跨膜电导调节因子（CFTR）以及人上皮Na⁺通道（hNaCh）的信使核糖核酸。本实验证实CF呼吸道上皮细胞中不存在cAMP依赖性Cl⁻电导，并支持先前在经上皮和微电极研究中获得的结果，这些结果表明CF患者呼吸道上皮细胞中的Na⁺电导增加。
