Rat Retina-RPE-Choroid Preparation: Electrophysiological Responses

RPE是色素细胞的单层，与视网膜光感受器相互作用，帮助维持视觉功能。我们开发了一种新的制剂来研究大鼠的RPE生理学，包括神经视网膜。视网膜-视网膜色素上皮-脉络膜(R-RPE-CH)制剂安装在允许视网膜和脉络膜浴液独立灌流的Ussing小室中。完整单层的平均跨上皮电位(+/-SD)为4.3+/-1.0 mV，总跨上皮阻力为152+/-37欧姆*厘米平方(n=10)。与其他哺乳动物RPE制剂一样，我们在顶膜和基侧膜上发现了对钡敏感的钾通道。基底侧膜细胞内微电极显示，递增的基础钾浓度使基底侧膜去极化8 mV(n=2)，并可被Ba2+阻断。降低顶钾浓度可使顶膜超极化，而增加顶钾浓度则使顶膜去极化6 mV(n=3)。Ba2+可阻断钾离子浓度反应。基础浴液中钙浓度增加10倍，从1.8mM增加到18mM，TTP增加0.6mV(p&lt；0.001；n=5)。当DIDS(1 MM)在基底外侧预处理时，这种增加被抑制50%(p&lt；0.01；n=3)。R-RPE-CH制剂是为了研究光诱导的RPE生理变化而开发的，并提供了一种体内功能的体外模型。它可以用来分析发生在Best病中的光诱发的RPE生理学变化，这被认为是基底膜钙激活的氯通道突变所致。

The RPE is a monolayer of pigmented cells that interact with retinal photoreceptors and helps maintain visual function. We have developed a new preparation to study RPE physiology in rat that includes the neural retina. The retina-retinal pigment epithelium-choroid (R-RPE-Ch) preparation was mounted in an Ussing chamber which allowed independent perfusion of retinal and choroidal baths. The intact monolayer had a mean (+/- SD) transepithelial potential of 4.3 +/- 1.0 mV and a total transepithelial resistance of 152 +/- 37 ohm*cm-squared (n =10). As in other mammalian RPE preparations, we found barium-sensitive potassium channels at the apical and basolateral membranes. Intracellular microelectrodes at the basolateral membrane showed that step increases of basal potassium concentration depolarized the basolateral membrane 8 mV (n=2) and were blocked by barium. Decreasing apical potassium concentration mimics light-onset and hyperpolarizes the apical membrane; increasing apical potassium concentration depolarized the apical membrane by 6 mV (n=3). The potassium concentration responses were blocked by barium. Elevation of calcium concentration in the basal bath 10 fold, from 1.8 to 18 mMincreased TTP by 0.6 mV(p<0.001.; n=5). This increase was inhibited by 50% (p<0.01; n=3) when pretreated with DIDS (1mM) on the basolateral side. The R-RPE-Ch preparation was developed to study light-induced changes in RPE physiology, and provides an in-vitro model of in-vivo function. It can be used to analyze light-evoked alterations in RPE physiology that occur in Bests disease, which is thought to result from a mutation in basolateral membrane calcium-activated chloride channels.