Jain-Vakkalagadda Blisse, Dey Surajit, Pal Dhananjay, Mitra Ashim K
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, USA.
Invest Ophthalmol Vis Sci. 2003 Jul;44(7):2919-27. doi: 10.1167/iovs.02-0907.
The objective of this research was to investigate the presence of an Na(+)-independent, large neutral amino acid transporter, LAT1, on rabbit corneal epithelium and human cornea.
Freshly excised rabbit corneas were used for transport studies and SIRC (a rabbit corneal cell line) cells for uptake studies. Transport and uptake characteristics of [(3)H]-L-phenylalanine were determined at various concentrations and pH. Inhibition studies were conducted in the presence of other L- and D-amino acids and metabolic inhibitors, such as ouabain and sodium azide, and in the absence of sodium to delineate the mechanism of uptake and transport. Reverse transcription-polymerase chain reaction (RT-PCR) for large neutral amino acid transporter-1 (LAT1) was performed on total RNA from rabbit cornea, SIRC cells, and human cornea.
SIRC uptake of L-Phe was found to be saturable, with K(m) of 73 +/- 9 microM, V(max) of 2.0 +/- 0.1 nanomoles/min per milligram protein, and K(d) of 0.44 +/- 0.6 microL/min per milligram protein. Uptake was independent of pH, energy, and Na(+); inhibited by D-Leu, D-Phe, and an L-system-specific inhibitor 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH), but not inhibited by L-Ala and charged amino acids. Transport of L-Phe across rabbit cornea was also saturable (K(m) = 33 +/- 8 microM and V(max) = 0.26 +/- 0.03 nanomoles/min per square centimeter), energy independent, and subject to similar competitive inhibition. LAT1 was identified by RT-PCR in rabbit corneal, SIRC, and human corneal RNA.
A Na(+)-independent, facilitative transport system, LAT1, was identified and functionally characterized on rabbit cornea. LAT1 was also identified on human cornea.
本研究的目的是调查兔角膜上皮和人角膜上是否存在一种不依赖钠离子的大中性氨基酸转运体LAT1。
将新鲜切除的兔角膜用于转运研究，将SIRC（一种兔角膜细胞系）细胞用于摄取研究。在不同浓度和pH值下测定[³H]-L-苯丙氨酸的转运和摄取特性。在存在其他L-和D-氨基酸以及代谢抑制剂（如哇巴因和叠氮化钠）的情况下，以及在无钠条件下进行抑制研究，以阐明摄取和转运机制。对来自兔角膜、SIRC细胞和人角膜的总RNA进行大中性氨基酸转运体-1（LAT1）的逆转录-聚合酶链反应（RT-PCR）。
发现SIRC对L-苯丙氨酸的摄取具有饱和性，米氏常数（K(m)）为73±9微摩尔，最大反应速度（V(max)）为每毫克蛋白质2.0±0.1纳摩尔/分钟，解离常数（K(d)）为每毫克蛋白质0.44±0.6微升/分钟。摄取不依赖于pH值、能量和钠离子；被D-亮氨酸、D-苯丙氨酸和一种L-系统特异性抑制剂2-氨基双环[2,2,1]庚烷-2-羧酸（BCH）抑制，但不被L-丙氨酸和带电荷的氨基酸抑制。L-苯丙氨酸跨兔角膜的转运也具有饱和性（K(m)=33±8微摩尔，V(max)=每平方厘米0.26±0.03纳摩尔/分钟），不依赖能量，并受到类似的竞争性抑制。通过RT-PCR在兔角膜、SIRC和人角膜RNA中鉴定出LAT1。
在兔角膜上鉴定出一种不依赖钠离子的易化转运系统LAT1，并对其进行了功能表征。在人角膜上也鉴定出了LAT1。
