High- and low-affinity transport ofl-leucine andl-DOPA by the hetero amino acid exchangers LAT1 and LAT2 in LLC-PK1renal cells

Abstract

The present study examined the functional characteristics of the inward and outward l-[14C]DOPA and l-[14C]leucine transporters in LLC-PK1cells. Uptake was initiated by the addition of Hanks' medium with a given concentration of l-[14C]DOPA or l-[14C]leucine. Saturation experiments were performed in cells incubated for 6 min with 0.25 μM concentration of the substrates in the absence and the presence of increasing concentrations of the nonlabeled substrates. Fractional outflow of intracellular l-[14C]DOPA or l-[14C]leucine was evaluated in cells loaded with 2.5 μM l-[14C]DOPA or 1 μM l-[14C]leucine for 6 min and then the corresponding efflux was monitored over 24 min. The high-affinity ( Km= 5.1 μM) uptake of l-[14C]leucine and the low-affinity ( Km= 120.0 μM) uptake of l-[14C]DOPA were largely promoted through a Na+-independent transporter. The uptake of the substrates was insensitive to N-(methylamino)-isobutyric acid but competitively inhibited by 2-aminobicyclo( 2 , 2 , 1 )-heptane-2-carboxylic acid (BCH). l- And d-neutral amino acids, but not acidic and basic amino acids, markedly inhibited l-[14C]DOPA and l-[14C]leucine accumulation. The uptake of l-[14C]leucine was a pH-insensitive process, whereas that of l-[14C]DOPA was sensitive to pH. The efflux of l-[14C]DOPA and l-[14C]leucine was markedly increased ( P < 0.05) by l-cysteine, l-leucine, BCH, and l-DOPA but not by l-arginine. RT-PCR detected LAT1 and LAT2 transcripts in LLC-PK1cells. It is concluded that LLC-PK1cells express both LAT1 and LAT2 transcripts and transport l-[14C]leucine through the Na+-independent pH-insensitive and high-affinity LAT1 transporter, whereas l-[14C]DOPA is mainly transported through the Na+-independent pH-insensitive and low-affinity LAT2 transporter and a minor component through a Na+-dependent transporter.