Cloning and gene silencing of LAT2, the L-3,4-dihydroxyphenylalanine ( L -DOPA) transporter, in pig renal LLC-PK 1 epithelial cells PATRÍCIO SOARES-DA-SILVA 1 , MARIA PAULA SERRÃO, MARIA JOÃO PINHO and MARIA JOÃO BONIFÁCIO Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal 1 Correspondence: Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal. E-mail: psoaresdasilva@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-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 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-PK 1 epithelial cells. Key Words: LAT2 • siRNA • LLC-PK 1 cells • amino acid exchanger
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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
SOARES-DA-SILVA, PATRÍCIO; SERRÃO, MARIA PAULA; PINHO, MARIA JOÃO; BONIFÁCIO, MARIA JOÃO
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, Volume 18 (13): 1489
Fed of American Socs for Experimental Biology – Oct 1, 2004
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