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

• Tipo: Artigo em Revista Científica Internacional
• Data: 2004

Autores

Patrício Soares da Silva (Autor) FMUP; Maria Paula Serrão (Autor) FMUP; Maria João Pinho (Autor) FMUP; Maria João Bonifácio (Autor) Outra

Revista

Título: FASEB Journal

Vol. 18

Páginas: 1489-1498

ISSN: 0892-6638

Editora: Wiley-Blackwell

Indexação

ISI Web of Knowledge - 17 Citações

ISI Web of Science

Scopus - 17 Citações

Pubmed / Medline

Classificação Científica

FOS: Ciências médicas e da saúde > Medicina básica

Outras Informações

ID Authenticus: P-000-88G

DOI: 10.1096/fj.04-1787com

Abstract (EN): 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% [C-14]-L-DOPA accumulation, a time- and concentration-dependent effect. The efflux of intracellular [C-14]-L-DOPA was markedly increased ( P< 0.05) by L-DOPA and L-leucine. The [C-14]-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 [C-14]-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.

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Nº de páginas: 10