pH-dependent ion- transport mechanism in the hfRPE

光感受器是人体中代谢最活跃的神经元细胞;在暗适应时，光感受器内节的耗氧量增加，主要是因为维持暗电流所需的ATP需求增加。 由于光感受器内节的氧消耗在暗适应时增加约1.5-3倍，我们预期CO2产生的成比例增加以及随后视网膜下腔CO2的增加。 CO2在视网膜下腔（SRS）内的积聚导致酸中毒，这对周围细胞的健康有害（即，Muller细胞、光感受器和RPE），因此代谢CO2必须从SRS中快速消散。 我们假设，这种CO2负荷的很大一部分是通过扩散到脉络膜血液供应而消散的，并且这个过程是由RPE介导的。 在这项研究中，我们描述了跨RPE的CO2运输，这涉及多个离子运输机制，从而增加跨RPE的液体吸收。 该项目需要研究hfRPE中的离子转运蛋白，这些蛋白参与眼睛中的光暗转换。 首先，我们表明，跨顶膜的CO2通量高于跨基底外侧膜的CO2通量。 我们研究了跨顶膜的CO2通量由水通道蛋白1介导的可能性，水通道蛋白1在hfRPE培养物中具有高mRNA表达水平，并且在大鼠RPE的顶膜处被发现。 然而，pH成像实验表明，在hfRPE中并非如此。 我们研究了顶端和基底外侧膜CO2通量的差异是否是由RPE中总暴露表面积的差异引起的（顶端膜的表面积比基底外侧膜大大约3-10倍）。 一项涉及削弱紧密连接以允许CO2进一步延伸到顶膜的实验支持了这一假设。 当用经13% CO2平衡的林格氏液灌注心尖浴时，AE 2活性降低，因为已知AE 2在酸性条件下受到抑制。 因为，顶膜处的CO2负荷需要增加HCO 3穿过基底膜的运输。 我们提供了pH成像和电生理学数据来证实RPE基底膜处存在产电Na+/HCO 3-共转运蛋白（NBC）：（1）在基底浴中减少HCO 3 o导致对应于基底膜去极化的TEP升高，（2）TEP升高是DIDS敏感的，（3）TEP升高在零Na条件下不存在。 因此，这种基础Na/HCO 3共转运蛋白可能介导基底外侧膜的HCO 3流出。 与电生理学实验，我们还表明，增加顶端CO2从5%到13%增加基础NBC活动，而减少CO2负荷在顶端膜（从5%到1%）抑制基础NBC活动。 由于CO2转化为HCO 3是由碳酸酐酶II催化的，因此我们进行了一项实验，以表明基底外侧膜Na/HCO 3共转运蛋白活性受到强效碳酸酐酶抑制剂（多佐胺）的抑制。 虽然基础Na/HCO 3共转运蛋白活性依赖于顶端膜的CO2负荷，但有多少HCO 3是由CO2-HCO 3转化提供的？ 与TEP记录，我们表明，基底侧共转运活性被部分抑制时，顶端pNBC 1被DIDS阻断。 这表明顶端Na/HCO 3共转运蛋白（pNBC 1）提供了基础Na/HCO 3共转运蛋白活性所需的HCO 3供应的一部分。 我们还发现，基底外侧Na/HCO 3共转运蛋白的主要底物是HCO 3;抑制顶端Na转运途径，如Na/K ATP酶，Na/K/2Cl共转运蛋白和Na/H交换剂，并不影响基底外侧Na/HCO 3共转运活性。 我们发现，CO2会影响多种离子转运蛋白，最终增加整个RPE的Na、Cl和HCO 3净吸收。 由于液体以渗透梯度流动，溶质转运的增加将增强穿过RPE的稳态液体吸收。 CO2诱导的液体吸收的增加可能具有重要的生理作用，因为视网膜处的代谢水产生速率（如基于视网膜的几何形状和氧消耗速率计算的）是跨越人RPE的稳态液体吸收的大约10%。 因此，未能从视网膜下腔中去除水可能会导致视网膜脱离。

The photoreceptor is the most metabolically active neuronal cell in the human body; oxygen consumption at the inner segment of the photoreceptors increases upon dark adaptation, mainly because of the increased ATP requirements needed to maintain the dark current. Since the oxygen consumption at the inner segment of the photoreceptor increases approximately 1.5-3 times upon dark adaptation, we expect a proportionate increase in CO2 generation and the subsequent increase in CO2 at the subretinal space. The accumulation of CO2 within the subretinal space (SRS) causes acidosis which is detrimental to the health of surrounding cells (i.e., Muller cells, photoreceptors, and RPE), thus metabolic CO2 must be quickly dissipated from the SRS. We hypothesize that a large fraction of this CO2 load is dissipated by diffusion to the choroidal blood supply, and that this process is mediated by the RPE. In this study, we describe the transport of CO2 across the RPE, which involves multiple ion-transport mechanisms that consequently increase fluid-absorption across the RPE. This project entails the study of the ion-transport proteins in the hfRPE that are involved in light-dark transition in the eye. First, we show that CO2 flux across the apical membrane is higher compared to CO2 flux across the basolateral membrane. We investigated the possibility that CO2-flux across the apical membrane is mediated by aquaporin 1, which has high mRNA expression levels in the hfRPE cultures and is found at apical membrane of the rat RPE. However, pH-imaging experiments showed that this was not the case in the hfRPE. We investigated to see if the difference in apical and basolateral membrane CO2 flux is caused by the difference in total exposed surface area in the RPE (the apical membrane has approximately 3-10 times larger surface area than the basolateral membrane). An experiment that involves weakening the tight junction to allow CO2 to extend further to the apical membrane supported this hypothesis. AE2 activity is reduced when the apical bath is perfused with Ringers equilibrated with 13% CO2 because AE2 is known to be inhibited under acidic conditions. Since, a CO2 load at the apical membrane necessitates an increased HCO3 transport across the basal membrane. We present pH-imaging and electrophysiology data to confirm the presence of an electrogenic Na+/HCO3- co-transporter (NBC) at the basal membrane of the RPE: (1) reducing HCO3o at the basal bath caused a TEP-rise that corresponds to the depolarization of the basal membrane, (2) the TEP-rise was DIDS-sensitive, (3) the TEP-rise was absent in zero-Na conditions. Therefore, this basal Na/HCO3 co-transporter may mediate HCO3 efflux at the basolateral membrane. With electrophysiology experiments, we also show that increasing apical CO2 from 5% to 13% increases basal NBC activity, while decreasing CO2 load at the apical membrane (from 5% to 1%) inhibited basal NBC activity. Since the conversion of CO2 to HCO3 is catalyzed by carbonic anhydrase II, we did an experiment to show that basolateral membrane Na/HCO3 co-transporter activity is inhibited by a potent carbonic anhydrase inhibitor (dorzolamide). Although the basal Na/HCO3 co-transporter activity was dependent on CO2 load at the apical membrane, how much of HCO3 is supplied by CO2-HCO3 conversion? With TEP-recordings, we show that basolateral co-transporter activity was partially inhibited when the apical pNBC1 was blocked with DIDS. This suggests that the apical Na/HCO3 co-transporter (pNBC1) provides part of the HCO3-supply necessary for basal Na/HCO3 co-transporter activity. We also showed that the main substrate for the basolateral Na/HCO3 co-transporter is HCO3; inhibiting apical Na-transport pathways such as the Na/K ATPase, Na/K/2Cl co-transporter, and Na/H exchanger did not affect basolateral Na/HCO3 co-transport activity. We showed that CO2 affects multiple ion-transporters that ultimately increases net Na, Cl, and HCO3 absorption across the RPE. Since fluid flows with an osmotic gradient, the increase in solute transport would enhance the steady-state fluid absorption across the RPE. The CO2-induced increase in fluid-absorption may have an important physiological role because the rate of metabolic water production at the retina (as calculated based on the geometry and oxygen consumption rate of the retina) is approximately 10% of the steady state fluid absorption across the human RPE. Therefore failure to remove water from the subretinal space can potentially cause retinal detachment.