Underexpression of the Na⁺-dependent neutral amino acid transporter ASCT2 in the spontaneously hypertensive rat kidney

Abstract

This study examined the inward transport of l-[(14)C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[(14)C]alanine uptake was highly dependent on extracellular Na(+). Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na(+)-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na(+)-dependent l-alanine transport under V(max) conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K(m) values. Differences in magnitude of Na(+)-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na(+)-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.

Cellular distribution of the neutral amino acid transporter subtype ASCT2 in mouse brain

“…The more abundant band appeared at $ 60 kDa, slightly larger than the predicted 58.4 kDa molecular weight for mouse ASCT2 (Utsunomiya-Tate et al 1996). The less abundant band was 35-40 kDa, consistent with previous reports using this antibody in mouse tissues (Dun et al 2007), and was also observed in positive control kidney homogenates, which are rich in ASCT2 protein (Utsunomiya-Tate et al 1996;Pinho et al 2007). Both bands were also evident in P2 synaptosomal membrane fractions ( Fig.…”

“…2007), and was also observed in positive control kidney homogenates, which are rich in ASCT2 protein (Utsunomiya‐Tate et al. 1996; Pinho et al. 2007).…”

Renal amino acid transport systems and essential hypertension

“…Similarly, the abundance of ASCT2 transcript and protein in kidney cortices of SHRs was also lower than that in normotensive WKY rats. The saturable component of sodium‐dependent l ‐alanine transport under V max conditions in SHR PTE cells was half of that in WKY PTE cells, with similar K m values (72). The rat SLC1A5 gene that codes for ASCT2 is located in chromosome 1 and has been previously associated with hypertension by several linkage analysis studies (73).…”

“…Therefore, we hypothesized that H 2 O 2 or O 2 could modulate ASCT2 activity. Lineweaver‐Burk plots revealed the presence of high‐ and low‐affinity states for the sodium‐dependent [ 14 C]‐ l ‐alanine uptake processes in both cell lines (72, 92). At low extracellular sodium concentrations, the sodium‐dependent [ 14 C]‐ l ‐alanine uptake in both WKY and SHR PTE cells is a high‐affinity low‐capacity process and increases in extracellular sodium reduced the affinity for the substrate but increased the capacity to take up [ 14 C]‐ l ‐alanine.…”

Species differences in pharmacokinetic and pharmacodynamic properties of nebicapone

“…SDS-PAGE and Western blot analysis was performed according to a standard procedure of the laboratory [17]. In brief, rat and mouse liver tissues were homogenized (Diax homogenizer, Heidolph GmbH & Co.KG, Schwabach, Germany) in RIPA buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1 % Triton X-100, 0.5 % sodium deoxycholate, 0.1 % SDS, 100 µg/ml PMSF, 2 µg/ml leupeptin and 2 µg/ml aprotinin.…”