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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

Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200-319 Porto, Portugal

Submitted 2 February 2004 ; accepted in final form 12 April 2004

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 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 (K_m = 5.1 µM) uptake of L-[¹⁴C]leucine and the low-affinity (K_m = 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-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.

renal dopaminergic system; sodium intake