Cotransport of H+, lactate, and H2O in porcine retinal pigment epithelial cells
Cotransport of H+, lactate, and H2O in porcine retinal pigment epithelial cells
复制标题
DOI：
10.1016/s0014-4835(02)00329-9
复制
发表时间：
2003-04-01
影响因子：
3.4
通讯作者：
Zeuthen, T
中科院分区：
文献类型：
--
作者：
Hamann, S;Kiilgaard, JF;Zeuthen, T
眼睛的视网膜色素上皮（RPE）在从视网膜到脉络膜的方向上运输水和乳酸根离子。水的运输在维持视网膜粘连中是重要的，乳酸根离子的运输用于调节视网膜下腔的乳酸根水平和pH。本研究通过非侵入性技术研究了位于哺乳动物RPE顶端（视网膜）膜的单羧酸转运蛋白（MCT 1）中H+、乳酸离子和水的转运之间的耦合机制。将猪RPE细胞的原代培养物生长至汇合，并置于灌注室中，其中可以快速更换面向视网膜膜的溶液。进行了两种类型的实验：通过荧光染料钙黄绿素的自猝灭来测量细胞水体积的变化，并且使用荧光染料BCECF以比率法测量细胞内pH的变化。在不含乳酸盐的溶液中，将甘露醇加入视网膜浴中会引起细胞内酸化和细胞收缩，表现为单一渗透水渗透率为1.2 +/- 0.1 × 10（-4）cm sec（-1）（μ l l（-1））（-1）。然而，在含有50 mmol L（-1）乳酸盐的溶液中，甘露醇诱导的细胞收缩更快，细胞碱化。这些影响不是施加的渗透梯度的大小的线性函数：无论是体积效应和细胞内pH值的变化表现出明显的饱和度与梯度的增加。突然等渗置换的Cl-与乳酸在浓度范围从3至50 mmol l（-1）引起立即细胞肿胀，以及立即细胞内酸化;这两种效果表现出明显的饱和与乳酸浓度的增加。Km值为：水通量为11 +/- 2 mmol l（-1），H+和乳酸通量为13 +/- 4 mmol l（-1）。这些数据表明，H2O是cotransporter沿着与H+和乳酸根离子在MCT 1定位到视网膜膜。这项研究强调了这种协同转运蛋白在维持哺乳动物组织视网膜下腔的水稳态和pH值方面的重要性，并支持我们以前通过侵入性技术在两栖动物组织中进行的研究。(C)2003爱思唯尔科技有限公司版权所有。
The retinal pigment epithelium (RPE) of the eye transports water and lactate ions in the direction from retina to choroid. The water transport is important in maintenance of retinal adhesion and the transport of lactate ions serves to regulate the lactate levels and pH of the subretinal space. This study investigates by means of a non-invasive technique the mechanism of coupling between transport of H+, lactate ion, and water in the monocarboxylate transporter (MCT1) located in the apical (retinal) membrane of a mammalian RPE. Primary cultures of porcine RPE cells were grown to confluence and placed in a perfusion chamber in which the solution facing the retinal membrane could be changed rapidly. Two types of experiments were performed: Changes in cell water volume were measured by self-quenching of the fluorescent dye Calcein, and changes in intracellular pH were measured ratiometrically using the fluorescent dye BCECF. In lactate-free solutions, mannitol addition to the retinal bath caused intracellular acidification and cell shrinkage, given by a single osmotic water permeability of 1.2 +/- 0.1 x 10(-4) cm sec(-1) (osmol l(-1))(-1). In solutions containing 50 mmol l(-1) lactate, however, the mannitol-induced cell shrinkage was faster and the cells alkalinized. These effects were not linear functions of the magnitude of the imposed osmotic gradients: Both volume effects and changes in intracellular pH showed apparent saturation with increasing gradients. Abrupt isosmotic replacement of Cl- with lactate in the concentration range from 3 to 50 mmol l(-1) caused an immediate cell swelling as well as an immediate intracellular acidification; both effects showed apparent saturation with increasing lactate concentration. The K-m values were: 11 +/- 2 mmol l(-1) for the water fluxes and 13 +/- 4 mmol l(-1) for the H+ and lactate fluxes. The data suggest that H2O is cotransported along with H+ and lactate ions in MCT1 localized to the retinal membrane. The study emphasizes the importance of this cotransporter in the maintenance of water homeostasis and pH in the subretinal space of a mammalian tissue and supports our previous study performed by an invasive technique in an amphibian tissue. (C) 2003 Elsevier Science Ltd. All rights reserved.