NA+ & CL FLUXES IN FOETAL DISTAL LUNG EPITHELIAL MONOLAYERS

分离的大鼠胚胎远端肺上皮(FDLE)细胞 镀在Transwell透明支架上并放入塑料中 培养皿，内含1x0.1厘米(直径。X深度)玻璃底板 中锋打得不错。由于文化是融合的，而且可能具有抵抗力， 适用于Transwell Support Act内部的化合物 有效地在根尖表面，而那些应用于 外部(即培养盘侧)作用于基底侧面。 对于氯离子浓度变化超过 浓度范围内，细胞维持在添加100 mM Na+-磷酸盐缓冲液。渗透压维持在297mmol.kg-1。 避免PO4的甘露醇和二价阳离子浓度 降水。因为实验是在倒置的蔡司上进行的 显微镜下电极摆动的角度是垂直的 (Z)-平面；虽然视场略有扭曲，但我们发现 我们可以很容易地将电极定位到距离 单层的顶面，并能清楚地看到是否 有没有和手机有任何接触。请注意，这些结果是 因为它们将被提交以供出版，所以是保密的。钠离子通量 实验：尽管阿米洛利可抑制的钠电流 从Ussing小室维护的单层中检测到我们无法 用Na+离子载体检测有意义的、受调节的Na+流动 我们用过。从时间上讲，我们没有进一步追查这一点； 问题可能源于电极的响应时间太长 (分钟)，令人望而却步的高本底浓度的Na+和， 可能是糟糕的电极设计。氯离子通量实验：这些 实验非常鼓舞人心。显然，部分力量的力量 自引用(SRE)系统是指数据可以 可以获得，尽管是在 使用的电极。从这部作品的主体部分，我们能够确定 使用离子选择性和(特别是氯-)SRE的基础 FDLE单层，通过证明1)我们可以测量 定向氯离子通量，2)可用阻滞剂操纵 CL-传输(未显示NPPB和布美他尼数据)，3)在 未受激态显示了一个净的氯-外流 外部氯离子的生理相关范围(70-140 mm)，类似于 4)可预测的胎肺组织 用？2-肾上腺素受体和P2Y2受体激动剂操纵。未来 这项工作将使用现在安装在我们的 奈威尔斯医院和医学院的部门监督 靶向不同基因的基因敲除寡核苷酸的作用 G蛋白信号通路的一部分。这代表了一部小说 并且特别适合使用其中的系统的数量 阳性转基因细胞的分辨能力低于 其他技术(如ISC测量离子传输特性 单层(参看膜片钳)。

Isolated rat foetal distal lung epithelial (FDLE) cells were plated on Transwell clear supports and placed into plastic petri-dishes containing a 1 x 0.1cm (dia. x depth) glass-bottomed centre well. As cultures were confluent and presumably resistive, compounds applied to the interior of the Transwell support act effectively at the apical surface, whereas those applied to the exterior (ie. the petri-dish side) act at the basolateral surface. For experiments where the concentration of Cl- was altered over a range of concentrations, cells were maintained in supplemented 100mM Na+-phosphate buffer. Osmolality was maintained at 297mmol.kg-1 using mannitol and divalent cation concentrations to avoid PO4 precipitation. As experiments were carried out on an inverted Zeiss microscope the angle of electrode oscillation was in the vertical (Z)-plane; although the field of view was slightly distorted, we found that we could easily position the electrode to within a micron of the apical surface o f the monolayer, and could clearly see whether or not there was any contact with the cell. Notethat these results are confidential as they will be submitted for publication. Na+ flux experiments: Although an amiloride-inhibitable Na+ -current has been detected from monolayers maintained in Ussing chambers we were unable to detect a meaningful, regulatedNa+ fluxwith the Na+ ionophore that we used. In the interestof time, we did not pursue this further; the problem likely stemmed from the long response time of the electrode (minutes), prohibitively high background concentrations of Na+ and, possibly, poor electrode design. Cl- flux experiments: These experiments were highly encouraging. Clearly, part of the strength of the Self-referencing (SrE) system is the rapidity with which data can be obtained, albeit within the operational limitations of the electrode used. From the body of this work we were able to establish a foundation for using ion-selective and (particularly Cl-) SrE with FDLE monolayers by demonstrating that 1) we could measure a directional Cl- flux, 2) which could be manipulated with blockers of Cl- transport (NPPB and bumetanide data not shown), 3) which in the unstimulated state demonstrated a net Cl- efflux over a physiologically relevant range of external Cl- (70-140mM), similar to that of the foetal lung in situ and 4) which could be predictably manipulated with ?2-adrenoreceptors and P2Y2 receptor agonists.Future work will build on this using an SrE system now installed in our Department at Ninewells Hospital and Medical School to monitor the effects of transfected knockout oligonucleotides targeted to various parts of the G-protein signalling pat hway. This represents a novel and particularly appropriate use of the system where the number of positively transfected cells falls below the resolving capability of other techniques (eg Isc measures the ion-transport characteristics of the monolayer (cf patch clamp).