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pH-dependent ion- transport mechanism in the hfRPE

hfRPE 中 pH 依赖性离子传输机制

基本信息

批准号：
8149180
负责人：
Sheldon Miller
金额：
$ 26.53万
依托单位：
NATIONAL EYE INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8149180
关键词：
Ion Transport

项目摘要

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.

感光细胞是人体内代谢最活跃的神经细胞；光感受器内段的耗氧量在暗适应时增加，主要是因为维持暗电流所需的 ATP 需求增加。由于暗适应时感光器内段的耗氧量增加约 1.5-3 倍，因此我们预计 CO2 的生成量会成比例增加，随后视网膜下腔的 CO2 也会相应增加。 CO2 在视网膜下腔 (SRS) 内的积累会导致酸中毒，这不利于周围细胞（即 Muller 细胞、光感受器和 RPE）的健康，因此代谢 CO2 必须快速从 SRS 中消散。我们假设大部分二氧化碳负荷通过扩散到脉络膜血液供应而消散，并且该过程是由 RPE 介导的。在这项研究中，我们描述了 CO2 穿过 RPE 的传输，其中涉及多种离子传输机制，从而增加了 RPE 上的液体吸收。该项目需要研究 hfRPE 中参与眼睛明暗转变的离子传输蛋白。首先，我们发现穿过顶膜的 CO2 通量高于穿过基底外侧膜的 CO2 通量。我们研究了跨顶膜的 CO2 通量是由水通道蛋白 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 的顶膜中发现。然而，pH 成像实验表明 hfRPE 的情况并非如此。我们研究了顶膜和基底外侧膜 CO2 通量的差异是否是由 RPE 中总暴露表面积的差异引起的（顶膜的表面积大约是基底外侧膜的 3-10 倍）。一项涉及削弱紧密连接以允许二氧化碳进一步延伸到顶膜的实验支持了这一假设。当根尖浴灌注用 13% CO2 平衡的林格氏液时，AE2 活性会降低，因为已知 AE2 在酸性条件下会受到抑制。因为，顶膜上的 CO2 负荷需要增加穿过基底膜的 HCO3 运输。我们提供 pH 成像和电生理学数据，以确认 RPE 基底膜上存在生电 Na+/HCO3- 协同转运蛋白 (NBC)：(1) 在基底浴中还原 HCO3o 导致 TEP 上升，对应于基底膜去极化，(2) TEP 上升是 DIDS 敏感的，(3) TEP 上升在 RPE 基底膜上不存在。零钠条件。因此，这种基础 Na/HCO3 协同转运蛋白可能介导 HCO3 在基底外侧膜的流出。通过电生理学实验，我们还表明，将顶膜 CO2 浓度从 5% 增加到 13% 会增加基础 NBC 活性，而降低顶膜 CO2 负荷（从 5% 到 1%）会抑制基础 NBC 活性。由于 CO2 转化为 HCO3 是由碳酸酐酶 II 催化的，因此我们进行了一项实验，证明基底外侧膜 Na/HCO3 协同转运蛋白活性受到有效的碳酸酐酶抑制剂（多佐胺）的抑制。尽管基础 Na/HCO3 协同转运蛋白活性取决于顶膜上的 CO2 负载，但有多少 HCO3 是由 CO2-HCO3 转化提供的？通过 TEP 记录，我们发现当顶端 pNBC1 用 DIDS 阻断时，基底外侧协同转运蛋白活性受到部分抑制。这表明顶端 Na/HCO3 协同转运蛋白 (pNBC1) 提供了基础 Na/HCO3 协同转运蛋白活性所需的部分 HCO3 供应。我们还表明基底外侧 Na/HCO3 协同转运蛋白的主要底物是 HCO3；抑制顶端 Na 转运途径（例如 Na/K ATP 酶、Na/K/2Cl 协同转运蛋白和 Na/H 交换器）不会影响基底外侧 Na/HCO3 协同转运活性。我们发现，CO2 会影响多种离子转运蛋白，最终增加 RPE 上的 Na、Cl 和 HCO3 净吸收。由于流体以渗透梯度流动，溶质转运的增加将增强 RPE 上的稳态流体吸收。 CO2 引起的液体吸收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率（根据视网膜的几何形状和耗氧率计算）约为人类 RPE 稳态液体吸收的 10%。因此，未能清除视网膜下腔的水分可能会导致视网膜脱离。