Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.
The present study examined the expression of type 1 L-amino acid transporter (LAT1) and its associated glycoprotein 4F2hc in freshly isolated renal proximal tubules and immortalized renal proximal tubular epithelial (PTE) cells from spontaneously hypertensive (SHR) and normotensive (WKY) rats. The study also examined the inward and outward transport of [(14)C]-L-leucine, the preferred substrate of LAT1. The abundance of LAT1 and 4F2hc was greater in SHR than in WKY, both in freshly isolated renal proximal tubules and immortalized renal proximal tubular cells. In the absence of extracellular Na(+) the BCH (2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid)-sensitive [(14)C]-L-leucine uptake in SHR PTE cells was approximately 50% that observed in WKY PTE cells (77+/-4 vs 164+/-7 pmol/mg protein). In the absence of extracellular Na(+) the affinity of the transporter for the substrate in WKY PTE cells was 7.7-fold that in SHR cells, as evidenced by lower K(0.5) values. Gene silencing with a LAT1 siRNA and a 4F2hc siRNA significantly reduced LAT1 and 4F2hc expression, which was accompanied by a marked reduction in Na(+)-independent [(14)C]-L-leucine uptake in both SHR and WKY PTE cells. The spontaneous and L-leucine-stimulated outward transfer of [(14)C]-L-leucine was Na(+)-independent in both SHR and WKY PTE cells. The spontaneous [(14)C]-L-leucine efflux was higher in WKY than in SHR PTE cells and the potency of L-leucine to stimulate [(14)C]-L-leucine efflux in WKY (EC(50) = 9 microM) was greater than in SHR PTE cells (EC(50) = 41 microM). It is concluded that the SHR kidney overexpress LAT1/4F2hc units which display low affinity for L-leucine transport.
Pinho
Maria João
MJ
Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal.
Serrão
Maria Paula
MP
Jose
Pedro A
PA
Soares-da-Silva
Patrício
P
D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism*; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D027283:Large Neutral Amino Acid-Transporter 1 / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011918:Rats, Inbred SHR
D027282:Fusion Regulatory Protein 1, Heavy Chain; D027283:Large Neutral Amino Acid-Transporter 1; D034741:RNA, Small Interfering; D007930:Leucine