High- and low-affinity transport of L-leucine and L-DOPA by the hetero amino acid exchangers LAT1 and LAT2 in LLC-PK1 renal cells

The present study examined the functional characteristics of the inward and outward L-[(14)C] DOPA and L-[(14)C] leucine transporters in LLC-PK(1) cells. Uptake was initiated by the addition of Hanks' medium with a given concentration of L-[(14)C] DOPA or L-[(14)C] leucine. Saturation experiments were performed in cells incubated for 6 min with 0.25 muM concentration of the substrates in the absence and the presence of increasing concentrations of the nonlabeled substrates. Fractional outflow of intracellular L-[(14)C] DOPA or L-[(14)C] leucine was evaluated in cells loaded with 2.5 muM L-[(14)C] DOPA or 1 M L-[(14)C] leucine for 6 min and then the corresponding efflux was monitored over 24 min. The high-affinity (K(m) = 5.1 muM) uptake of L-[(14)C] leucine and the low-affinity (K(m) = 120.0 muM) uptake of L-[(14)C] 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-[(14)C] DOPA and L-[(14)C] leucine accumulation. The uptake of L-[(14)C] leucine was a pH-insensitive process, whereas that of L-[(14)C] DOPA was sensitive to pH. The efflux of L-[(14)C] DOPA and L-[(14)C] 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-PK(1) cells. It is concluded that LLC-PK(1) cells express both LAT1 and LAT2 transcripts and transport L-[(14)C] leucine through the Na(+)-independent pH-insensitive and high-affinity LAT1 transporter, whereas L-[(14)C] DOPA is mainly transported through the Na(+)-independent pH-insensitive and low-affinity LAT2 transporter and a minor component through a Na(+)-dependent transporter.