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Cloning and gene silencing of LAT2, the L‐3,4‐dihydroxyphenylalanine (l‐DOPA) transporter, in pig renal LLC‐PK epithelial cells
Author(s) -
SOARESDASILVA PATRÍCIO,
SERRÃO MARIA PAULA,
PINKO MARIA JOÃO,
BONIFÁCIO MARIA JOÃO
Publication year - 2004
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.04-1787com
Subject(s) - transporter , dopamine , gene , amino acid , biology , complementary dna , amino acid transporter , intracellular , microbiology and biotechnology , chemistry , biochemistry , endocrinology , medicine
Organ‐specific overexpression of type 2 l ‐amino acid transporter (LAT2) in the kidney of the spontaneous hypertensive rat (SKR), 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‐PK 1 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‐PK 1 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% [ 14 C]‐l‐DOPA accumulation, a time‐ and concentration‐dependent effect. The efflux of intracellular [ 14 C]‐l‐DOPA was markedly increased ( P <0.05) by l‐DOPA and l ‐leucine. The [ 14 C]‐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 [ 14 C]‐l‐DOPA. The Na + ‐independent and pH‐sensitive l‐DOPA transporter may include the hetero amino acid exchanger LAT2, whose activation results in irans‐stimulation of l‐DOPA outward transfer.—Soares‐da‐Silva, P., Serrao, M. P., João Pinho, M., Bonifacio, M.J. Cloning and gene silencing of LAT2, the L‐3,4‐dihydroxyphenylalanine (l‐DOPA) transporter, in pig renal LLC‐PK 1 epithelial cells. FASEB J. 18, 1489–1498 (2004)