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Involvement of LAT1 and LAT2 in the high- and low-affinity transport of L-leucine in human retinal pigment epithelial cells (ARPE-19 cells)
Authors:Atsushi Yamamoto  Shin-ichi Akanuma  Masanori Tachikawa  Ken-ichi Hosoya
Affiliation:1. Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
Abstract:System L, which is encoded by LAT1 and LAT2, is an amino acid transport system that transports neutral amino acids, including several essential amino acids in an Na+-independent manner. Due to its broad substrate selectivity, system L has been proposed to mediate the transport of amino-acid-related drugs across the blood–tissue barriers. We characterized L-leucine transport and its corresponding transporter in a human retinal pigment epithelial cell line (ARPE-19 cells) as an in vitro model of the outer blood–retinal barrier. [3H]L-leucine uptake by ARPE-19 cells took place in an Na+-, Cl?-independent and saturable manner with Km values of 8.71 and 220 µM. This process was more potently cis-inhibited by substrates of LAT1 than those of LAT2. [3H]L-leucine efflux from ARPE-19 cells was trans-stimulated by substrates of LAT1 and LAT2 through the obligatory exchange mechanism of system L. Although RT-PCR analysis demonstrated that LAT1 and LAT2 mRNA are expressed in ARPE-19 cells, the LAT1 mRNA concentration is 42-fold higher than that of LAT2. Moreover, immunoblot analysis demonstrated that LAT1 is expressed in ARPE-19 cells. In conclusion, although the transport function of LAT1 is greater than that of LAT2, LAT1 and LAT2 are involved in L-leucine transport in ARPE-19 cells. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2475–2482, 2010
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