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Cloning and gene silencing of LAT2, the L-3,4-dihydroxyphenylalanine (L-DOPA) transporter, in pig renal LLC-PK₁ epithelial cells

Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal

¹Correspondence: Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal. E-mail: psoaresdasilva{at}netcabo.pt

Organ-specific overexpression of type 2 L-amino acid transporter (LAT2) in the kidney of the spontaneous hypertensive rat (SHR), accompanied by an enhanced ability to take up L-DOPA, may constitute the basis for the enhanced renal production of dopamine in the SHR in an attempt overcome the deficient dopamine-mediated natriuresis. To understand the physiological role of LAT2-mediated L-DOPA handling, we used 21-nucleotide small interfering RNA duplexes (siRNA) to specifically suppress LAT2 expression in LLC-PK₁ cells, a cell line that retains several properties of proximal tubular epithelial cells and takes up L-DOPA largely through Na⁺-independent transporters. After cloning the LLC-PK₁ LAT2 gene, one target region of LAT2 mRNA (nt 97-117) was selected by scanning the length of the LAT2 gene for AA-dinucleotide sequences and downstream 19 nucleotides. Levels of LAT2 cDNA, determined by real-time quantitative RT-PCR, were markedly (P<0.05) reduced by LAT2 siRNA but not by the mismatch LAT2 siRNA. The LAT2 siRNA but not the mismatch LAT2 siRNA, reduced by 85% [¹⁴C]-L-DOPA accumulation, a time- and concentration-dependent effect. The efflux of intracellular [¹⁴C]-L-DOPA was markedly increased (P<0.05) by L-DOPA and L-leucine. The [¹⁴C]-L-DOPA outward transport was decreased 90% by LAT2 siRNA, but not by the mismatch LAT2 siRNA. However, treatment with the siRNA LAT2 did not affect the L-DOPA-induced fractional outflow of [¹⁴C]-L-DOPA. The Na⁺-independent and pH-sensitive L-DOPA transporter may include the hetero amino acid exchanger LAT2, whose activation results in trans-stimulation of L-DOPA outward transfer.—Soares-da-Silva, P., Serrão, M. P., João Pinho, M., Bonifácio, M. J. Cloning and gene silencing of LAT2, the L-3,4-dihydroxyphenylalanine (L-DOPA) transporter, in pig renal LLC-PK₁ epithelial cells.

Key Words: LAT2 • siRNA • LLC-PK₁ cells • amino acid exchanger

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