Lactate transport and pH-regulation in the human RPE

视网膜内释放大量乳酸，这与SRS处的高乳酸浓度(3.8-13 mm)一致，即使在光适应的眼睛中也是如此。暗适应时乳酸的释放是以下结果的结果：(1)外视网膜葡萄糖代谢增加，(2)暗适应眼视网膜氧水平降低，(3)谷氨酸诱导的米勒细胞乳酸释放。为了防止乳酸在SRS堆积，RPE在顶端(MCT1)和基侧膜(MCT3和MCT4)表达单羧酸转运体MCT家族的乳酸转运体。为了证明眼内乳酸转运的重要性，同一小组还表明，缺乏MCT1、MCT3和MCT4表达的小鼠迅速失去光感受器细胞。乳酸转运也被证明增加了猪RPE和牛蛙体内的液体转运，这可能是通过与其他离子转运体的相互作用来实现的。Becker及其同事最近的一项研究表明，当发生电的Na/HCO3共转运蛋白(NBC1)与MCT1共表达时，MCT1的活性增加。此外，肌肉细胞中的碳酸氢酶活性增强了MCT1的活性。因此，在RPE体内可能存在类似的功能蛋白复合体。我们的研究旨在通过监测细胞内pH、跨上皮电位(TEP)和总组织阻力(RT)来确定MCT介导的乳酸转运是否涉及RPE中的CaS、Na/HCO3共转运体或其他离子转运体。 乳酸通过RPE顶膜的运输是一个两阶段的过程；当乳酸灌流到顶膜上时，它会导致快速的细胞内酸化(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体介导的，因为将乳酸、丙酮酸、丙酸或醋酸盐灌流到根尖浴会引起类似的pH反应。我们还能用MCT1抑制剂：尼氟米酸和pCMBS抑制酸化阶段(1期)。恢复相(2相)被阿米洛利和哇巴因阻断，表明通过Na/H交换器的质子外流是恢复相的中介。将乳酸盐灌流到根尖膜上，由于RPE基侧膜氯离子外流增加，导致TEP升高。我们在培养的hfRPE中证实了这一点，表明心尖乳酸诱导的TEP-反应被基底膜上的氯通道抑制剂(DIDS)减弱。 在RPE中，液泡V型H+ATPase定位于RPE顶膜，并主动地将质子泵出RPE；这从顶膜暴露于H+ATPase抑制剂(phloretin、NBD-Cl、DCCD和NEM)时的强烈细胞内酸化得到了证明。因此，H+ATPase可能参与了乳酸反应的pH恢复期(2相)，同时也引起了TEP的升高。在这方面，我们的实验表明，根癌素能部分抑制pH-恢复期和TEP-上升。 在培养的hfRPE的顶膜上检测到DIDS抑制的Na/2HCO3共转运体，从而证实了早期在青蛙和牛RPE上进行的实验。我们的实验表明，阻断pNBC1活性(用DIDS)不影响H/Lac通过MCT1的进入，这表明通过pNBC1的HCO3-内流缓冲了H/Lac共转运活性，MCT1的转运活性与pNBC1活性无关。由于MCT1和胞浆CA-II之间的相互作用已经在肌肉细胞中被报道，我们通过用多唑胺抑制CA活性来测试这种蛋白质在RPE中的相互作用。在多唑胺和富含CO2/HCO3的缓冲液存在下，我们发现顶端乳酸酸化被放大。这表明多唑胺不能抑制H/Lac通过MCT1的转运，而是抑制缓冲H/Lac转运的HCO3-进入，因此观察到了更强的酸化作用。 在培养的hfRPE细胞中，乳酸在基底膜上的转运是由MCT3和MCT4介导的。有趣的是，将乳酸灌流到基底膜会导致缓慢的细胞内碱化，但TEP下降很快。向基础浴中灌流任何单羧酸盐(即乳酸、醋酸盐、丙酮酸和丙酸)都会引起缓慢的细胞内碱化，从而证实碱化是一个两部分的过程：(1)MCT3 H/Lac共转运介导的酸化，被MCT1、Na/H交换器或H+ATPase质子外流引起的碱化所压倒。用阿米洛利阻断顶膜Na/H交换器的质子外流不能逆转基础乳酸诱导的碱化。然而，在存在H+-ATPase抑制剂(pCMBS、根皮素或NBD-Cl)的情况下，将乳酸灌流到基底膜会引起酸化。因此，我们的数据表明，通过H+-ATPase的质子流出顶膜和通过MCT1的H/Lac转运出RPE是导致20 mM基础乳酸碱化的原因。

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.