Increased Cat3-mediated Cationic Amino Acid Transport Functionally Compensates in Cat1 Knockout Cell Lines

Abstract

Arginine transport is important for a number of biological processes in vertebrates, and its transport may be rate-limiting for the production of nitric oxide. The majority of L-Arg transport is mediated by System y ؉ , although several other carriers have been kinetically defined. System y ؉ cationic amino acid transport is mediated by proteins encoded by a family of genes, Cat1, Cat2, and Cat3. High affinity L-arginine transport was investigated in embryonic fibroblast cells derived from Cat1 knockout mice that lack functional Cat1. Both wild type and knockout cells transport arginine with comparable K m and V max . However, the apparent affinity for lysine transport was 2.4 times lower in Cat1 ؊/؊ cells when compared with wild type cells, a property characteristic of Cat3-mediated transport. Northern analysisdocumented Cat2 mRNA increased 2-fold, whereas Cat3 mRNA levels increased 11-fold in Cat1 ؊/؊ relative to Cat1 ؉/؉ cells. The low affinity Cat2a mRNA was not detectably expressed in these cells. Even though Cat3 expression is normally limited to adult brain, there was a large increase in the amount of Cat3 protein present at the plasma membrane of Cat1 ؊/؊ embryonic fibroblast cells. These results suggest that Cat3 compensates for the loss of functional Cat1 in cells derived from Cat1 knockout mice and mediates the majority of high affinity arginine transport.

Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability

“…Thus, the up-regulation of CAT3 in fibroblasts lacking CD98hc is in accordance with increased AA ϩ intracellular content in such cells (Fig. 4A) as arginine, lysine, and histidine are substrates of murine CAT3 (22,23). Regarding the transport of AA 0 , by analyzing L-Ala uptake, we identified the presence of system A, a co-transporter of Na ϩ and neutral AAs, mainly excluding BCAAs and ARO AAs (24) (Fig.…”

Cationic Amino Acid Transport in the Renal Medulla and Blood Pressure Regulation

“…Previous studies have reported that the expression of CAT-2 mRNA and CAT-3 mRNA and protein were compensatorily upregulated in embryonic fibroblast cells derived from CAT-1 knockout mice. 22 Moreover, other cationic transport systems, ie, system b 0,ϩ and yϩL, have been shown to exist in the mammalian kidney 23,24 and may also contribute to L-arginine uptake in NO-producing cells. A further possibility is that the cationic amino acids have a dual effect on L-arginine: (1) they may competitively inhibit L-arginine uptake by cationic amino acids (cis-inhibition) and (2) the cationic amino acids may also facilitate the exodus of intracellular L-arginine from cells via system y ϩ (transstimulation) and thereby deplete the cells of L-arginine.…”

Sustained Nitric Oxide Production in Macrophages Requires the Arginine Transporter CAT2

“…Prior to RNA preparation, macrophages were incubated in the presence or absence of 20 units ml Ϫ1 IFN␥ (Sigma) for 2 h followed by the addition of 100 ng ml Ϫ1 LPS (Escherichia coli 055:B5, Sigma) to the primed cells for an additional 6 h. 10 g of tissue or 5 g of macrophage RNA was analyzed with Cat1, Cat2, and glyceraldehyde-3-phosphate dehydrogenase probes (26). A 1.3-kilobase BstX1/XhoI fragment of Cat3 was isolated for random-prime labeling (26).…”

“…Prior to RNA preparation, macrophages were incubated in the presence or absence of 20 units ml Ϫ1 IFN␥ (Sigma) for 2 h followed by the addition of 100 ng ml Ϫ1 LPS (Escherichia coli 055:B5, Sigma) to the primed cells for an additional 6 h. 10 g of tissue or 5 g of macrophage RNA was analyzed with Cat1, Cat2, and glyceraldehyde-3-phosphate dehydrogenase probes (26). A 1.3-kilobase BstX1/XhoI fragment of Cat3 was isolated for random-prime labeling (26). The NOS2 cDNA probe was cloned by RT/PCR from mouse mammary tumor cDNA using the primers 5Ј-CAG TGC CCT GCT TTG TGC GAA GT-3Ј and 5Ј-AAC GTT TCT GGC TCT TGA GCT GGA A-3Ј; the product then was ligated into the vector pGEM-T Easy (Promega), and the sequence was verified using flanking T7 and SP6 primers.…”

“…The NOS2 cDNA probe was cloned by RT/PCR from mouse mammary tumor cDNA using the primers 5Ј-CAG TGC CCT GCT TTG TGC GAA GT-3Ј and 5Ј-AAC GTT TCT GGC TCT TGA GCT GGA A-3Ј; the product then was ligated into the vector pGEM-T Easy (Promega), and the sequence was verified using flanking T7 and SP6 primers. Densitometry was performed as described (26). Nucleotide size markers (Life Technologies, Inc.) determined the relative size of each message.…”