Talluri Ravi S, Katragadda Suresh, Pal Dhananjay, Mitra Ashim K
School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
Curr Eye Res. 2006 Jun;31(6):481-9. doi: 10.1080/02713680600693629.
To investigate the mechanism of L-ascorbic acid uptake by rabbit corneal epithelial cells and to functionally characterize the specific transporter involved in this translocation process.
Uptake studies were carried out with SIRC (Statens Seruminstitut Rabbit Cornea) and rPCEC (rabbit Primary corneal epithelial cell culture) in 12-well plates using [14C] Ascorbic acid (AA). Uptake was done in the presence of L-ascorbic acid and D-isoascorbic acid to delineate stereospecificity. Inhibition studies were performed in the presence of D-glucose a substrate for GLUT and also para amino hippuric acid (PAHA) a substrate for organic anion transporter. Effects of pH and sodium on the uptake of AA were characterized. Concentration dependency studies were performed. Energy dependence of AA uptake was investigated with ouabain and sodium azide in rPCEC. Reverse Transcription-polymerase chain reaction (RT-PCR) was also performed.
Uptake of AA was inhibited by about 90% and 50% respectively in the presence of L-ascorbic acid and D-isoascorbic acid in both SIRC and rPCEC. Uptake was unaltered by D-glucose and PAHA. The process was sodium dependent and saturable at higher concentrations. Ouabain and sodium azide significantly diminished the uptake process. It also decreased with a reduction in pH. The RT-PCR results showed the presence of SVCT2 but not SVCT1.
Uptake of AA across rabbit corneal epithelial cells appears to be a carrier mediated active process. A saturable, sodium dependent, and pH sensitive transporter with high specificity for L-ascorbic acid was functionally characterized and was identified as SVCT2.
研究兔角膜上皮细胞摄取L-抗坏血酸的机制，并对参与这一转运过程的特定转运体进行功能表征。
在12孔板中使用[14C]抗坏血酸（AA）对SIRC（丹麦国家血清研究所兔角膜）和rPCEC（兔原代角膜上皮细胞培养物）进行摄取研究。在L-抗坏血酸和D-异抗坏血酸存在的情况下进行摄取，以阐明立体特异性。在D-葡萄糖（一种GLUT底物）和对氨基马尿酸（PAHA，一种有机阴离子转运体底物）存在的情况下进行抑制研究。表征了pH和钠对AA摄取的影响。进行了浓度依赖性研究。在rPCEC中用哇巴因和叠氮化钠研究了AA摄取的能量依赖性。还进行了逆转录-聚合酶链反应（RT-PCR）。
在SIRC和rPCEC中，L-抗坏血酸和D-异抗坏血酸存在时，AA的摄取分别被抑制约90%和50%。D-葡萄糖和PAHA对摄取没有影响。该过程依赖于钠，在较高浓度下具有饱和性。哇巴因和叠氮化钠显著降低了摄取过程。随着pH降低，摄取也减少。RT-PCR结果显示存在SVCT2但不存在SVCT1。
兔角膜上皮细胞对AA的摄取似乎是一个载体介导的主动过程。鉴定出一种对L-抗坏血酸具有高特异性、可饱和、依赖钠且对pH敏感的转运体，并将其确定为SVCT2。
