pH-dependent ion- transport mechanism in the hfRPE

hfRPE 中 pH 依赖性离子传输机制

• 批准号: 7734644
• 负责人: Sheldon Miller
• 金额: $ 28.34万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 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/7734644
• 关键词: AQP1 gene; Acidosis; Affect; Apical; Area; Bathing; Bicarbonates; Carbon Dioxide; Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Carrier Proteins; Cells; Complement; Condition; Cultured Cells; Dark Adaptation; Data; Diffusion; Disease; Dorzolamide; Electrophysiology (science); Eye; Failure; Generations; Health; Human; Human body; Image; Ion Transport; Ions; Light; Liquid substance; Mediating; Membrane; Metabolic; Modeling; Muller' s cell; Na(+)-K(+)-Exchanging ATPase; Neurons; Oxygen Consumption; Pathway interactions; Photoreceptors; Physiological; Potassium; Process; Production; Rate; Rattus; Retina; Retinal Detachment; Role; Surface; Tight Junctions; Time; Vascular blood supply; Water; absorption; apical membrane; base; basolateral membrane; design; fluid flow; mRNA Expression; research study; solute; symporter

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.

感光体是人体中最活跃的神经元细胞。在暗适应后，光感受器内部段的氧气消耗增加，这主要是由于维持黑电流所需的ATP要求增加。 由于光感受器的内部段的氧气消耗在黑暗适应后增加了约1.5-3倍，因此我们预计二氧化碳生成比例增加，随后在视网膜下空间中二氧化碳的增加。 CO2在视网膜下空间（SRS）内的积累导致酸中毒，这对周围细胞的健康有害（即Muller细胞，光感受器和RPE），因此必须迅速从SRS中消失代谢CO2。 我们假设，通过扩散到脉络膜血液供应，很大一部分二氧化碳负荷会消散，并且该过程是由RPE介导的。 在这项研究中，我们描述了二氧化碳在RPE跨RPE的运输，该转移涉及多种离子传输机制，从而增加了整个RPE的流体吸收。 该项目需要对HFRPE中的离子传输蛋白进行研究，这些蛋白参与了眼睛的浅黑色过渡。 首先，我们表明，与基底外侧膜的二氧化碳通量相比，跨根部膜的CO2通量更高。 我们研究了跨顶膜的二氧化碳是由Aquaporin 1介导的，Aquaporin 1在HFRPE培养物中具有较高的mRNA表达水平，并且在大鼠RPE的顶端膜上发现。 但是，pH模仿实验表明，在HFRPE中并非如此。 我们研究了顶端和基底外侧膜二氧化碳通量的差异是否是由RPE的总暴露表面积差异引起的（顶膜的表面积比基底外侧膜大约3-10倍）。 一个涉及削弱紧密连接以使CO2进一步扩展到顶端膜的实验支持了这一假设。 当顶端浴被用13％CO2平衡的铃声灌注时，AE2活性会降低，因为已知AE2在酸性条件下被抑制。 由于，顶部膜上的二氧化碳负载需要增加跨基膜的HCO3转运。 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零NA条件。 因此，该基底Na/HCO3共转运蛋白可以介导基底外侧膜上的HCO3外排。 通过电生理实验，我们还表明，将顶端CO2从5％增加到13％会增加基础NBC活性，同时减少顶端膜（从5％到1％）的CO2载荷抑制了基础NBC活性。 由于二氧化碳向HCO3的转化是用碳酸酐酶II催化的，因此我们进行了一个实验，以表明基底外侧膜Na/Hco3共同转运蛋白的活性受到有效的碳酸一次藻酶抑制剂（多唑胺）抑制。 尽管基底Na/HCO3共转运蛋白活性取决于顶部膜上的二氧化碳载荷，但CO2-HCO3转换提供了多少HCO3？ 使用TEP录制，我们表明当顶端PNBC1用DIDS阻断时，基底外侧的共转运蛋白活性被部分抑制。 这表明顶端NA/HCO3共同转运蛋白（PNBC1）提供了基础Na/Hco3共同转运蛋白活性所需的HCO3-供应的一部分。 我们还表明，基底外侧Na/Hco3共转运蛋白的主要底物是HCO3。抑制顶端NA传输途径，例如Na/K ATPase，Na/K/2Cl共转运蛋白和Na/H交换器不会影响基底外侧NA/HCO3 CO-TRANSPORT活动。 我们表明，二氧化碳会影响多个离子转运蛋白，这些离子转运蛋白最终会增加RPE的NA，CL和HCO3吸收。 由于流体与渗透梯度流动，溶质转运的增加将增强整个RPE的稳态吸收。 二氧化碳诱导的吸收流体吸收的增加可能具有重要的生理作用，因为视网膜的代谢水生产速率（基于视网膜的几何形状和氧气消耗率计算出来）约为稳态液体吸收的10％。 因此，未能从视网膜下空间去除水可能会导致视网膜脱离。