Expression and function of LAT1, a neutral amino acid exchanger, in renal porcine epithelial cell line LLC-PK.
OBJECTIVE
The present study examined the expression of LAT1 and the functional characteristics of the inward and outward [14C] l-leucine transporter in the renal porcine epithelial cell line LLC-PK1.


METHODS
LLC-PK1 cells were cultured in polycarbonate filters and accumulation and transepithelial flux of the substrate monitored with [14C] l-leucine. LAT1 transcripts were examined by RT-PCR. LAT1 protein was detected by immunoblotting.


RESULTS
The accumulation of [14C] l-leucine in the cell and the [14C] l-leucine transepithelial flux were four- and twofold, respectively, when the substrate was added from the basal cell side, suggesting that the basolateral membrane is endowed with a high density of transport units, when compared with the apical membrane. Increases in the transepithelial flux of [14C] l-leucine by unlabelled l-leucine were also more pronounced when unlabelled l-leucine was added from the basolateral membrane. In the absence of Na+, unlabelled l-leucine increased the basal and apical fractional outflow of [14C] l-leucine, this being similar at pH 7.4 and pH 6.2. RT-PCR and immunoblotting detected LAT1 transcript and protein, respectively.


CONCLUSIONS
LLC-PK1 cells are endowed with the LAT1 transcript and protein and transport l-leucine through the Na+-independent and pH-insensitive LAT1 transporter. The density of transporter units in LLC-PK1 cells may be higher at the basolateral membranes, although be also present in the apical membranes.
Soares-da-Silva
P
P
Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal. psoaresdasilva@netcabo.pt
Serrão
P
P
Fraga
S
S
Pinho
M J
MJ
D021542:Amino Acids, Neutral / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D027283:Large Neutral Amino Acid-Transporter 1 / Q000378:metabolism* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012964:Sodium / Q000494:pharmacology; D013552:Swine
D021542:Amino Acids, Neutral; D027283:Large Neutral Amino Acid-Transporter 1; D012964:Sodium