NA+ & CL FLUXES IN FOETAL DISTAL LUNG EPITHELIAL MONOLAYERS

不适用

• 批准号: 6319681
• 负责人: STEPHEN C L
• 金额: $ 1.35万
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6319681
• 关键词: NA+; CL; FLUXES; FOETAL; DISTAL

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).

分离的大鼠胎儿远端肺上皮（FDLE）细胞， 在Transwell透明支持物上铺板并置于塑料中 含有1 x 0.1 cm（直径） x深度）玻璃底 中心好。 由于培养物是融合的并且可能具有抵抗性， 应用于Transwell支持法案内部的化合物 有效地在顶端表面，而那些应用于 外部（即， 培养皿侧）作用于基底外侧表面。 对于Cl-浓度在一定时间内变化的实验， 在浓度范围内，将细胞维持在补充的100mM Na +-磷酸盐缓冲液。 渗透压摩尔浓度维持在297mmol.kg-1，使用 甘露醇和二价阳离子浓度，以避免PO4 降水 当实验在倒置的蔡司上进行时， 在显微镜下，电极摆动角度为垂直方向 （Z）平面;虽然视野略有扭曲，但我们发现 我们可以很容易地将电极定位在 单层的顶面，并可以清楚地看到是否 有任何联系 注意这些结果是 保密，因为它们将被提交出版。 Na+通量 实验：虽然阿米洛利可吸收的Na+电流已经被 从保持在Ussing室中的单层中检测到，我们无法 to detect检测a meaningful有意义，regulated调节Na + flux流with the Na + ionophore离子载体， 我们用的。 为了时间的利益，我们没有进一步追求这一点; 问题可能源于电极的长响应时间 （分钟），过高的Na+背景浓度， 可能是电极设计不良。 Cl-通量实验：这些 实验非常令人鼓舞。 很明显， 自引用（Self-referencing，SrE）系统是指数据可以 虽然在操作限制范围内， 使用电极。 从这部作品的主体中，我们能够建立 使用离子选择性和（特别是Cl-）SrE的基础， FDLE单分子层，证明1）我们可以测量一个 方向性Cl-通量，2）可以用以下阻断剂操纵： Cl-转运（NPPB和布美他尼数据未显示），3） 未受刺激的状态表明，净氯流出超过 生理相关的外部Cl-范围（70 - 140 mM），类似于 胎肺原位的，以及4）可以预测的 被什么人操纵2-肾上腺素受体和P2Y2受体激动剂。 工作将建立在这一点上使用一个SRE系统，现在安装在我们的 Ninewells医院和医学院的部门，以监测 转染的敲除寡核苷酸靶向各种 部分G蛋白信号通路。 这代表了一部小说 特别是适当使用该系统， 阳性转染的细胞福尔斯低于 其他技术（如Isc测量离子传输特性， 单层（CF膜片钳）。