Cloning and gene silencing of LAT2, the L-3,4-dihydroxyphenylalanine (L-DOPA) transporter, in pig renal LLC-PK1 epithelial cells.
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.
Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal. psoaresdasilva@netcabo.pt
Patrício
P
Soares-Da-Silva
Maria Paula
MP
Serrão
Maria João
MJ
Pinho
Maria João
MJ
Bonifácio
D027182:Amino Acid Transport System y+ / Q000235:genetics* / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D050483:Dopamine Plasma Membrane Transport Proteins; D004847:Epithelial Cells / Q000378:metabolism*; D020224:Expressed Sequence Tags; D020868:Gene Silencing; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology*; D007980:Levodopa / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000494:pharmacology; D013552:Swine / Q000235:genetics*
D027182:Amino Acid Transport System y+; D000596:Amino Acids; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D007980:Levodopa; D012964:Sodium