El-Sherbeny Amira, Naggar Hany, Miyauchi Seiji, Ola M Shamsul, Maddox Dennis M, Martin Pamela Moore, Ganapathy Vadivel, Smith Sylvia B
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000, USA.
Invest Ophthalmol Vis Sci. 2004 Feb;45(2):694-701. doi: 10.1167/iovs.03-0503.
To determine whether taurine transporter (TauT) activity and expression are regulated by hyperosmolarity in RPE, ganglion, and Müller cells.
Uptake of taurine was measured in ARPE-19 cells cultured in DMEM-F12 medium without or with the addition of 50 mM NaCl or 100 mM mannitol. The kinetics of the transport were analyzed. RT-PCR and Northern and Western blot analyses were used to assess TauT mRNA and protein levels. The influence of hyperosmolarity on the uptake of taurine, myo-inositol, and gamma-aminobutyric acid GABA was studied in RPE, RGC-5, and rMC1 cells.
TauT activity was abundant in RPE and was stimulated (3.5-fold) when the cells were exposed to hyperosmolar conditions (DMEM-F12 culture medium plus 50 mM NaCl or 100 mM mannitol). Peak stimulation of taurine uptake occurred after 17 hours of exposure to hyperosmolar medium. Kinetic analysis revealed that the hyperosmolarity-induced stimulation was associated with an increase in V(max) of TauT with no change in K(m). TauT mRNA and protein levels increased in RPE cells exposed to hyperosmolar conditions. Hyperosmolarity also stimulated the uptake of myo-inositol ( approximately 15-fold); GABA uptake was influenced less markedly. Immunofluorescence and functional studies showed that TauT is present in cultured RGC-5 and rMC1 cells. TauT activity was robust in these cells in normal osmolar conditions and increased by approximately twofold in hyperosmolar conditions.
These studies provide the first evidence that hyperosmolarity regulates TauT activity and expression in RPE and that TauT is present in ganglion and Müller cells and is regulated by hypertonicity. The data are relevant to diseases such as diabetes, macular degeneration, and neurodegeneration, in which retinal cell volumes may fluctuate dramatically.
确定牛磺酸转运体（TauT）的活性和表达是否受视网膜色素上皮细胞（RPE）、神经节细胞和米勒细胞中高渗的调节。
在添加或不添加50 mM氯化钠或100 mM甘露醇的DMEM-F12培养基中培养ARPE-19细胞，测量牛磺酸的摄取。分析转运的动力学。采用逆转录聚合酶链反应（RT-PCR）、Northern印迹和Western印迹分析来评估TauT mRNA和蛋白质水平。研究高渗对RPE细胞、视网膜神经节细胞-5（RGC-5）和视网膜米勒细胞系（rMC1）中牛磺酸、肌醇和γ-氨基丁酸（GABA）摄取的影响。
TauT活性在RPE中丰富，当细胞暴露于高渗条件下（DMEM-F12培养基加50 mM氯化钠或100 mM甘露醇）时被刺激（3.5倍）。暴露于高渗培养基17小时后，牛磺酸摄取出现峰值刺激。动力学分析表明，高渗诱导的刺激与TauT的最大转运速率（V(max)）增加有关，而米氏常数（K(m)）不变。暴露于高渗条件下的RPE细胞中TauT mRNA和蛋白质水平增加。高渗也刺激了肌醇的摄取（约15倍）；GABA摄取受到的影响较小。免疫荧光和功能研究表明，TauT存在于培养的RGC-5和rMC1细胞中。在正常渗透压条件下，这些细胞中的TauT活性很强，在高渗条件下增加约两倍。
这些研究提供了首个证据，即高渗调节RPE中TauT的活性和表达，并且TauT存在于神经节细胞和米勒细胞中并受高渗调节。这些数据与糖尿病、黄斑变性和神经退行性疾病等疾病相关，在这些疾病中视网膜细胞体积可能会发生显著波动。
