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-[14C]DOPA and L-[14C]leucine transporters in LLC-PK1 cells. 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 microM 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 microM L-[14C]DOPA or 1 microM L-[14C]leucine for 6 min and then the corresponding efflux was monitored over 24 min. The high-affinity (Km = 5.1 microM) uptake of L-[14C]leucine and the low-affinity (Km = 120.0 microM) 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-PK1 cells. It is concluded that LLC-PK1 cells 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.
Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 -319 Porto, Portugal. psoaresdasilva@netcabo.pt
Patrício
P
Soares-da-Silva
Maria Paula
MP
Serrão
D000818:Animals; D001692:Biological Transport; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism*; D018374:LLC-PK1 Cells; D027283:Large Neutral Amino Acid-Transporter 1 / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000493:pharmacokinetics*; D007980:Levodopa / Q000493:pharmacokinetics*; D012964:Sodium / Q000378:metabolism; D013552:Swine; D014158:Transcription, Genetic
D027283:Large Neutral Amino Acid-Transporter 1; D007980:Levodopa; D012964:Sodium; D007930:Leucine