Lactate transport and pH-regulation in the human RPE

内层视网膜释放大量的乳酸盐，这与SRS处的高乳酸盐浓度（3.8 - 13 mM）一致，即使在光适应的眼睛中也是如此。 乳酸在暗适应时释放，这是由于（1）外层视网膜的葡萄糖代谢增加，（2）暗适应眼中视网膜氧水平降低和（3）谷氨酸诱导的Mller细胞的乳酸释放。 为了防止SRS处的乳酸蓄积，RPE在顶膜（MCT 1）和基底外侧膜（MCT 3和MCT 4）处表达单羧酸转运蛋白的MCT家族的乳酸转运蛋白。 为了证明乳酸转运在眼睛中的重要性，同一组还显示缺乏MCT1，MCT3和MCT4表达的小鼠迅速失去感光细胞。 乳酸转运也被证明可以增加猪RPE和牛蛙中的液体转运，可能是通过与其他离子转运蛋白相互作用。 Becker及其同事最近的一项研究表明，当产电Na/HCO3共转运蛋白（NBC1）与MCT 1共表达时，MCT 1活性增加。 此外，肌肉细胞中的碳酸酐酶活性可增强MCT 1活性。 因此，在体内RPE中可能存在类似的功能性蛋白质复合物。 我们的研究旨在通过监测细胞内pH值、跨上皮电位（TEP）和总组织电阻（Rt）来确定MCT介导的乳酸转运是否涉及RPE中的CA、Na/HCO3协同转运蛋白或其他离子转运蛋白。 乳酸盐转运穿过RPE的顶膜是一个两阶段的过程;当乳酸盐灌注到顶膜上时，它引起快速的细胞内酸化（阶段1），然后是缓慢的碱化（阶段2）。 这些pH反应是由单羧酸转运蛋白介导的，因为灌注乳酸盐，丙酮酸盐，丙酸盐，或乙酸盐的顶端浴引起类似的pH反应。 我们还能够用MCT 1抑制剂：尼氟灭酸和pCMBS抑制酸化阶段（阶段1）。 阿米洛利和哇巴因阻断恢复期（第2期），表明质子流出通过Na/H交换介导的恢复期。 将乳酸灌注到顶膜上导致由于来自RPE的基底外侧膜的Cl-流出增加而引起的TEP升高。 我们证实了这一点，在培养的hfRPE显示顶端乳酸诱导的TEP反应被削弱的Cl-通道抑制剂（DIDS）在基底外侧膜。 在RPE中，液泡V型H + ATP酶位于顶膜处，并积极地将质子泵出RPE;这通过顶膜暴露于H + ATP酶抑制剂（根皮素、NBD-Cl、DCCD和NEM）后的强细胞内酸化来证明。 因此，H + ATP酶可能介导乳酸盐反应的pH恢复阶段（阶段2），同时引起TEP升高。 在这方面，我们的实验表明，根皮素部分抑制pH恢复阶段和TEP上升。 在培养的hfRPE的顶膜处检测到DIDS可降解的Na/2HCO3共转运蛋白，从而证实了在青蛙和牛RPE中进行的早期实验。 我们的实验表明，阻断pNBC1活性（用DIDS）并不影响H/Lac通过MCT 1的进入，这表明通过pNBC1的HCO 3-内流缓冲H/Lac共转运活性，并且MCT 1转运活性不依赖于pNBC1活性。 由于在肌细胞中已经报道了MCT 1和胞质CA-II之间的相互作用，我们通过用多佐胺抑制CA活性来检测RPE中的这种蛋白质相互作用。 在多佐胺和CO2/HCO3丰富的缓冲液的存在下，我们表明，顶端乳酸酸化放大。 这表明多佐胺不抑制通过MCT 1的H/Lac转运，而是抑制缓冲H/Lac转运的HCO3-进入（通过pNBC1），因此观察到更强的酸化。 在培养的hfRPE细胞中，基底外侧膜处的乳酸转运由MCT3和MCT4介导。 有趣的是，乳酸灌注到基底外侧膜引起缓慢的细胞内碱化，但快速的TEP下降。 灌注任何单羧酸盐（即，乳酸盐、乙酸盐、丙酮酸盐和丙酸盐）添加到基础浴中引起缓慢的细胞内碱化，从而证实碱化是两部分过程：（1）由MCT 3 H/Lac共转运介导的酸化，其被（2）由来自MCT 1、Na/H交换剂或H + ATP酶的质子流出引起的碱化所压倒。 用阿米洛利阻断Na/H交换器在顶膜的质子流出并不能逆转基础乳酸诱导的碱化。 然而，在H + ATP酶抑制剂（pCMBS，根皮素，或NBD-Cl）的存在下，在顶端浴，灌注乳酸盐的基膜引起酸化。 因此，我们的数据表明，质子通过H + ATP酶流出顶端膜和H/Lac通过MCT 1运输出RPE引起20 mM基础乳酸诱导的碱化。

The inner retina releases a lot of lactate, consistent with the high lactate concentration (3.8 - 13 mM) at the SRS even in light-adapted eyes. Lactate is released upon dark-adaptationa result of (1) an increased glucose metabolism at the outer retina, (2) the reduced retinal oxygen level in the dark adapted eye and (3) glutamate-induced lactate release from Mller cells. To prevent lactate accumulation at the SRS, the RPE expresses lactate transporters, of the MCT family of monocarboxylate transporters, at the apical (MCT1) and basolateral membrane (MCT3 and MCT4). To demonstrate the importance of lactate transport in the eye, the same group also showed that mice lacking MCT1, MCT3, and MCT4 expression rapidly lose photoreceptor cells. Lactate transport was also shown to increase fluid transport across the porcine RPE, and in the bullfrog, possibly by interacting with other ion-transporters. A recent study by Becker and colleagues demonstrated that MCT1 activity increases when the electrogenic Na/HCO3 co-transporter (NBC1) is co-expressed with MCT1. In addition, MCT1 activity is enhanced by carbonic anhydrase activity in muscle cells. Therefore, a similar functional protein-complex may exist in vivo in the RPE. Our study aims to determine if MCT mediated lactate-transport involve CAs, Na/HCO3 co-transporter or other ion-transporters in the RPE by monitoring intracellular pH, transepithelial potential (TEP), and total tissue resistance (Rt). Lactate transport across the apical membrane of the RPE is a two phase process; when lactate is perfused onto the apical membrane, it causes a fast intracellular acidification (phase 1), followed by a slow alkalinization (phase 2). These pH-responses are mediated by monocarboxylate transporters because perfusing lactate, pyruvate, propionate, or acetate to the apical bath caused similar pH-responses. We were also able to inhibit the acidification phase (phase 1) with MCT1-inhibitors: niflumic acid and pCMBS. The recovery phase (phase 2) was blocked by amiloride and ouabain, indicating that proton-efflux via the Na/H exchanger mediates the recovery phase. Perfusing lactate onto the apical membrane causes a TEP-rise due to an increased Cl-efflux from the basolateral membrane of the RPE. We confirm this in the cultured hfRPE by showing that the apical lactate-induced TEP-response was weakened by Cl-channel inhibitor (DIDS) at the basolateral membrane. In the RPE, vacuolar V-type H+ATPases are localized at the apical membrane and is actively pumping protons out of the RPE; this was evidenced by the strong intracellular acidification upon apical membrane exposure to H+ATPase inhibitors (phloretin, NBD-Cl, DCCD, and NEM). Thus the H+ATPase may mediate the pH-recovery phase (phase 2) of the lactate-response, and at the same time causes a TEP-rise. In that regard, our experiments show that phloretin partially inhibited the pH-recovery phase and the TEP-rise. A DIDS-inhibitable Na/2HCO3 co-transporter was detected at the apical membrane of the cultured hfRPE, thus corroborating earlier experiments performed in frog and bovine RPE. Our experiments show that blocking pNBC1 activity (with DIDS) did not affect H/Lac entry via MCT1, suggesting that HCO3-influx via pNBC1 was buffering H/Lac co-transport activity and that MCT1 transport activity was independent of pNBC1 activity. Since interactions between MCT1 and the cytosolic CA-II have been reported in muscle cells, we test this protein interaction in the RPE by inhibiting CA activity with dorzolamide. In the presence of dorzolamide and CO2/HCO3-rich buffer, we show that the apical lactate acidification was amplified. This indicates that H/Lac transport via MCT1 was not inhibited by dorzolamide, instead dorzolamide inhibited HCO3-entry (via pNBC1) that was buffering H/Lac transport, thus a stronger acidification was observed. Lactate transport at the basolateral membrane is mediated by MCT3 and MCT4 in cultured hfRPE cells. Interestingly, perfusing lactate to the basolateral membrane caused a slow intracellular alkalinization but a fast TEP-drop. Perfusing any monocarboxylates (i.e., lactate, acetate, pyruvate, and propionate) to the basal bath caused slow intracellular alkalinization, thus confirming that the alkalinization was a two part process: (1) acidification mediated by MCT3 H/Lac co-transport that is overwhelmed by the (2) alkalinization caused by proton efflux from MCT1, Na/H exchanger, or the H+ATPase. Blocking proton efflux from the Na/H exchanger at the apical membrane with amiloride did not reverse basal lactate induced alkalinization. However, in the presence of H+ATPase inhibitors (pCMBS, phloretin, or NBD-Cl) at the apical bath, perfusing lactate to the basal membrane caused acidification. Therefore our data suggests that proton-flux out of the apical membrane via H+ATPase and H/Lac transport out of the RPE via MCT1 caused the 20 mM basal lactate induced alkalinization.