Methods for stable recording of short-circuit current in a Na+-transporting epithelium.
Methods for stable recording of short-circuit current in a Na+-transporting epithelium.
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钠传输上皮中短路电流的稳定记录方法。
DOI：
10.1152/ajpcell.00459.2010
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Awayda,MouhamedS
中科院分区：
文献类型：
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作者：
Gondzik,Veronika;Awayda,MouhamedS
通过包括短路电流技术在内的各种技术测量的上皮Na+转运已被描述为表现出“衰弱”现象。这一现象表现为电流和电阻随时间的减少，并排除了进行旨在检查这种运输调节的长期实验的能力。我们开发了通过改进短路电流技术和商业Ussing类型的小室来延长稳定记录上皮细胞Na+转运的方法。我们利用极化的MDCK细胞系表达上皮Na+通道(ENaC)来描述这些方法。简而言之，对现有的商业室进行了改造，以允许林格溶液的连续流动和对这种流动的精确控制。对腔室歧管和相关管道进行了改进，以实现精确的温度夹具，防止温度波动。对记录电极进行了改进，消除了KCl的使用，防止了KCl膜的去极化。溶液使用标准的碳酸氢盐缓冲液，但所有气体都预先水化以钳制缓冲液渗透压。我们证明了这些改进导致的电流和电阻的测量结果至少在2小时内是稳定的。我们进一步证明，与我们修改之前获得的渗透压漂移类似的渗透压漂移可以导致类似于衰减的电流和电阻的下降。
Epithelial Na+transport as measured by a variety of techniques, including the short-circuit current technique, has been described to exhibit a “rundown” phenomenon. This phenomenon manifests as time-dependent decrease of current and resistance and precludes the ability to carry out prolonged experiments aimed at examining the regulation of this transport. We developed methods for prolonged stable recordings of epithelial Na+transport using modifications of the short-circuit current technique and commercial Ussing-type chambers. We utilize the polarized MDCK cell line expressing the epithelial Na+channel (ENaC) to describe these methods. Briefly, existing commercial chambers were modified to allow continuous flow of Ringer solution and precise control of such flow. Chamber manifolds and associated plumbing were modified to allow precise temperature clamp preventing temperature oscillations. Recording electrodes were modified to eliminate the use of KCl and prevent membrane depolarization from KCl leakage. Solutions utilized standard bicarbonate-based buffers, but all gasses were prehydrated to clamp buffer osmolarity. We demonstrate that these modifications result in measurements of current and resistance that are stable for at least 2 h. We further demonstrate that drifts in osmolarity similar to those obtained before prior to our modifications can lead to a decrease of current and resistance similar to those attributed to rundown.