Hu J G, Gallemore R P, Bok D, Frambach D A
Jules Stein Eye Institute, University of California Los Angeles School of Medicine 90033, USA.
Exp Eye Res. 1996 Apr;62(4):443-8. doi: 10.1006/exer.1996.0049.
This study identifies bumetanide-sensitive chloride transport in cultured fetal human retinal pigment epithelium (RPE). Ion flux and electrophysiologic studies were performed on fetal human RPE grown to confluence on microporous culture wells mounted in modified Ussing chambers. Unidirectional transepithelial Cl- fluxes were measured along with the transepithelial potential (TEP), resistance (Rt), and short circuit current (Isc). In separate experiments the apical and basal membrane voltages (Vap and Vba) and membrane resistance ratio (Rap/Rba) were measured using standard electrophysiological techniques. The average electrical parameters under control conditions were TEP = 3.6 +/- 2.1 mV, Rt = 385 +/- 98 ohm cm2, and Isc = 8.8 +/- 3.6 microA cm-2. Apical bumetanide (10(-5) M) reduced the TEP and Isc an average of 0.6 +/- 0.4 mV and 1.6 +/- 0.8 microA cm-2, respectively. Under open-circuit conditions, we measured a net apical to basal 36Cl flux of 0.30 microEq cm-2 hr-1. Bumetanide reduced this net Cl- flux by 83% by reducing the unidirectional apical-to-basal Cl- flux. Apical bumetanide hyperpolarized Vba from -58 +/- 14 to -66 +/- 17 mV and reduced Rap/Rba from 1.17 +/- 0.50 to 0.85 +/- 0.38, consistent with blockade of Cl- uptake and reduction of the Cl- equilibrium potential at a basal membrane Cl- channel. Basal bumetanide had no effect on electrical parameters. We identified a bumetanide sensitive Cl- uptake mechanism at the apical membrane which may represent the Na-K-2Cl cotransporter reported in explant and other cultured preparations of mammalian RPE.
本研究确定了培养的人胎儿视网膜色素上皮（RPE）中布美他尼敏感的氯离子转运。对生长在安装于改良尤斯灌流小室的微孔培养孔上并达到汇合状态的人胎儿RPE进行了离子通量和电生理研究。测量了单向跨上皮氯离子通量以及跨上皮电位（TEP）、电阻（Rt）和短路电流（Isc）。在单独的实验中，使用标准电生理技术测量了顶膜和基底膜电压（Vap和Vba）以及膜电阻比（Rap/Rba）。对照条件下的平均电学参数为TEP = 3.6±2.1 mV，Rt = 385±98 ohm·cm2，Isc = 8.8±3.6 μA·cm-2。顶侧布美他尼（10(-5) M）分别使TEP和Isc平均降低了0.6±0.4 mV和1.6±0.8 μA·cm-2。在开路条件下，我们测量到从顶侧向基底的36Cl净通量为0.30 μEq·cm-2·hr-1。布美他尼通过降低单向顶侧向基底的氯离子通量使该净氯离子通量降低了83%。顶侧布美他尼使Vba从-58±14 mV超极化至-66±17 mV，并使Rap/Rba从1.17±0.50降至0.85±0.38，这与基底膜氯离子通道处氯离子摄取的阻断以及氯离子平衡电位的降低一致。基底侧布美他尼对电学参数无影响。我们在顶膜处确定了一种布美他尼敏感的氯离子摄取机制，其可能代表了在哺乳动物RPE的外植体和其他培养制剂中报道的钠-钾-2氯共转运体。
