Rat Retina-RPE-Choroid Preparation: Electrophysiological Responses

RPE是一种单层色素细胞，与视网膜感光细胞相互作用，帮助维持视觉功能。我们开发了一种新的制备方法来研究大鼠RPE生理学，包括神经视网膜。将视网膜-视网膜色素上皮-脉络膜（R-RPE-Ch）制备物置于Ussing腔中，允许视网膜和脉络膜液独立灌注。完整单层膜的平均（+/- SD）传上皮电位为4.3 +/- 1.0 mV，总传上皮电阻为152 +/- 37 ohm*cm-squared （n =10）。与其他哺乳动物RPE制剂一样，我们在根尖和基底外侧膜上发现了钡敏感的钾通道。基底膜上的细胞内微电极显示，基底钾浓度的阶跃增加使基底膜去极化8 mV (n=2)，并被钡阻断。根尖钾浓度的降低模拟起光现象，使根尖膜发生超极化；增加根尖钾浓度可使根尖膜去极化6 mV （n=3）。钾浓度反应被钡阻断。基础浴钙浓度升高10倍，从1.8到18分钟，使TTP降低0.6 mV（p<0.001; n=5）。在基底外侧使用DIDS （1mM）预处理后，这种增加被抑制了50% （p<0.01; n=3）。R-RPE-Ch的制备是为了研究光诱导的RPE生理变化，并提供了一个体内功能的体外模型。它可以用来分析贝斯特病中发生的RPE生理学的光诱发改变，这被认为是由基底外侧膜钙激活的氯通道突变引起的。

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.