Altered growth and improved resistance of Arabidopsis against Pseudomonas syringae by overexpression of the basic amino acid transporter AtCAT1.

Amino acid transporters in plants are crucial for distributing amino acids between plant organs and cellular compartments. The H(+)-coupled plasma membrane transporter CAT1 (cationic amino acid transporter 1) facilitates the high-affinity uptake of basic amino acids. The uptake of lysine (Lys) via the roots was not altered in loss-of-function mutants, in accordance with the minor expression of CAT1 in roots, but plants ectopically overexpressing CAT1 incorporated Lys at higher rates. Exogenous Lys inhibited the primary root of Arabidopsis, whereas lateral roots were stimulated. These effects were augmented by the presence or absence of CAT1. Furthermore, the total biomass of soil-grown plants ectopically overexpressing CAT1 was reduced and the time to flowering was accelerated. These effects were accompanied by only minor changes in the overall amino acid profile. Interestingly, CAT1 belongs to a specific small cluster of nitrogen-containing metabolite transporter genes that are rapidly up-regulated upon infection with Pseudomonas syringae and that may participate in the systemic response of plants to pathogen attack. The overexpression of CAT1 indeed enhanced the resistance to the hemibiotrophic bacterial pathogen P. syringae via a constitutively activated salicylic acid (SA) pathway, which is consistent with the developmental defects and the resistance phenotype.

Sex differences of oxidative stress to cholestatic liver and kidney injury in young rats.

BACKGROUND: Sexual dimorphism plays a role in the liver and in renal injuries. However, whether sex is a risk factor in bile duct ligation (BDL) in young rats has never been examined. METHODS: Six male and six female rats treated with BDL were sacrificed 2 weeks after surgery and were designated as BDL-M and BDL-F groups. The other six male and six female rats that received sham ligation were designated as sham-M and sham-F groups. Plasma biochemistry and liver and kidney asymmetric dimethylarginine (ADMA)-related molecules were examined. RESULTS: Both BDL-M and BDL-F groups had elevated plasma aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, and transforming growth factor-ß1 levels. The BDL-F group had lower plasma AST and ALT levels than the BDL-M group. The BDL-M and BDL-F groups had elevated plasma ADMA levels. The cationic amino acid transporter 1 (CAT1) level was increased in the BDL-F group as compared to the sham-F group, whereas the CAT2 level was reduced in the both BDL-M and BDL-F groups. CONCLUSION: We found that young male rats were prone to higher degrees of biochemical liver and kidney injury to cholestasis. Sex differences in modulation of oxidative stress markers, such as ADMA, may play a role. Our results support careful monitoring and optimal treatment of cholestatic disease, especially in young male patients.

Cationic and neutral amino acid transporter transcript abundances are differentially expressed in the equine intestinal tract.

To test the hypothesis that AA transporter transcripts are present in the large intestine and similarly expressed along the intestinal tract, mRNA abundance of candidate AA transporter genes solute carrier (SLC) family 7, member 9 (SLC7A9), SLC7A1, SLC7A8, and SLC43A1 encoding for b(0,+)-type AA transporter (b(0,+)AT), cationic AA transporter-1 (CAT-1), L-type AA transporter-2 (LAT-2), and L-type AA transporter-3 (LAT-3), respectively, was determined in small and large intestinal segments of the horse. Mucosa was collected from the equine small (jejunum and ileum) and large intestine (cecum, left ventral colon, and left dorsal colon), flash frozen in liquid nitrogen, and stored at -80 degrees C. Messenger RNA was isolated from tissue samples, followed by manufacture of cDNA. Relative quantitative reverse transcription-PCR was conducted using the 2(-DeltaDeltaCT) method, with glyceraldehyde-3-phosphate dehydrogenase serving as the housekeeping gene. Compared with the jejunum, cationic and neutral AA transporter SLC7A9 mRNA abundance was similar in the ileum, cecum, and large intestinal segments. Compared with the jejunum, cationic AA transporter SLC7A1 mRNA abundance was similar in the ileum and decreased in the cecum, left ventral colon, and left dorsal colon (P < 0.001). Neutral AA transporter SLC7A8 mRNA abundance decreased from the cranial to caudal end of the intestinal tract (P < 0.001). Neutral AA transporter SLC43A1 mRNA abundance was similar in the ileum and left dorsal colon and increased in the cecum (P < 0.01) and left ventral colon (P < 0.1) compared with the jejunum. Cationic and neutral AA transporter SLC7A9 mRNA abundance was similarly expressed in the large compared with small intestine, whereas cationic AA transporter SLC7A1 was of low abundance in the large intestine; neutral AA transporters SLC7A8 and SLC43A1 were differentially expressed with decreased abundance of SLC7A8 and increased abundance of SLC43A1 in the large intestine. Results indicate that the large intestine might contribute to both cationic and neutral AA uptake and absorption predominantly via transporters LAT-3 and b(0,+)AT.

Arginine transport is augmented, through modulation of cationic amino acid transporter-1, in obstructive uropathy in rats.

BACKGROUND: The decrease in glomerular filtration rate (GFR), which is characteristic of obstructive uropathy, was suggested to be associated with attenuated nitric oxide (NO) generation. Since availability of L-arginine, the sole precursor for NO, governs NO synthesis, we aimed to determine the role of glomerular arginine transport in rats subjected to 24 h of bilateral ureteral ligation (BUO). METHODS: Glomerular arginine transport was measured by uptake of radiolabeled arginine ([(3)H]-L-arginine), cationic amino acid transporters (CAT)-1 and -2 and arginases I and II mRNA expression were determined using reverse transcription-polymerase chain reaction. CAT-1, arginase I, and arginase II protein contents were evaluated by Western blotting. RESULTS: L-Arginine transport by freshly harvested glomeruli from BUO rats was significantly augmented than in controls. The aforementioned findings were associated with a significant increase in glomerular CAT-1 mRNA expression, while CAT-2 mRNA was unchanged. Western blotting demonstrated a significant increase in CAT-1 abundance in BUO. Expression of both glomerular arginase I and II mRNA and protein content were significantly elevated in BUO. CONCLUSIONS: BUO induces an increase in glomerular arginine transport via upregulation of CAT-1, probably due to increase in arginine utilization by a non-NO pathway.

Identification and expression pattern of cationic amino acid transporter-1 mRNA in small intestinal epithelia of Angus steers at four production stages.

Although dietary supplementation of cationic AA (CAA), especially l-Lys, is known to be essential for optimal growth of beef cattle, the proteins responsible for absorption of CAA by bovine intestinal epithelia have not been described. Cationic AA transporter-1 (CAT-1) is a major intestinal CAA transporter, demonstrating a high-affinity (muM) transport activity for l-Lys in other mammals, and is widely expressed by small intestinal epithelia of nonruminants, but neither sequence nor expression pattern data exist for CAT-1 in cattle. Therefore, the goal of this research was to compare the relative expression (putative) of CAT-1 mRNA by duodenal, jejunal, or ileal small intestinal epithelia across and within commercially relevant beef cattle production and development stages. Twenty-four Angus steers were assigned randomly (n = 6) to 1 of 4 treatments (suckling, weanling, growing, and finishing) after all steers were born. Duodenal, jejunal, and ileal epithelia were scraped, and total RNA was extracted after the steers were killed at 32, 184, 248, or 423 d of age. Average daily gains of the steers did not differ (1.09 +/- 0.05 kg/d) among stages, whereas the small intestinal length relative to BW decreased (P < 0.01) with steer development. Using standard reverse transcription-PCR cloning techniques, we generated a partial-length bovine CAT-1 complementary DNA (695 bp; GenBank accession no. DQ399522) from jejunal mRNA samples, which possessed 89 and 87% identities to pig and human CAT-1 orthologs, respectively. On the basis of this bovine-specific genetic data, a real-time PCR-based assay of reverse-transcribed mRNA was developed and used to measure relative changes in bovine CAT-1 mRNA abundance in intestinal epithelia as steers developed. The CAT-1 mRNA was expressed by the duodenum, jejunum, and ileum of all 4 production stages. In contrast to expression by duodenal or ileal epithelium, jejunal expression of CAT-1 mRNA by growing steers was greater (P = 0.005) than that by suckling, weanling, or finishing steers. In terms of the expression of CAT-1 mRNA within production stage, jejunal expression was greater (P = 0.002) than that by duodenum or ileum for growing steers. In contrast, no intestinal site difference was found for suckling, weanling, or finishing steers. These data indicate that previously reported Na(+)-independent uptake of Lys by jejunal and ileal epithelia likely occurred by CAT-1, and that the potential capacity for CAT-1-mediated uptake of CAA for beef steers may be greatest for the "growing" phenotype.

Homocysteine induces endothelial dysfunction via inhibition of arginine transport.

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. High levels of plasma homocysteine (HCY) increase oxidative stress and reduce endothelial-dependent relaxation. We determined whether hyperhomocysteinemia-induced endothelial dysfunction is mediated through inhibition of cellular transport of L-arginine. In endothelial cells, HCY had a biphasic effect on arginine transport. HCY treatment for 6 hr increased L-arginine uptake by 34%; however, uptake was decreased by 25% after 24 h. HCY caused membrane hyperpolarization during both 6 and 24 h incubation periods, indicating that the negative charge facilitating arginine uptake was maintained. HCY significantly reduced expression of cellular arginine transporter protein (CAT-1) after 24 h treatment; whereas endothelial nitric oxide synthase (eNOS) protein levels and basal eNOS activity were not altered. Nevertheless, nitric oxide (NO) formation was significantly decreased. The antioxidant ascorbic acid prevented the effect of HCY on arginine transport. HCY induced formation of the peroxynitrite biomarker nitrotyrosine, which was blocked by supplemental L-arginine. HCY treatment of aortic rings caused decreased vasorelaxation to acetylcholine, which was prevented by supplemental arginine. In conclusion, HCY decreased NO formation and induced endothelial dysfunction without altering protein level or basal activity of eNOS, but through decreases in function and protein expression of the CAT-1 transporter. Reduced arginine supply may lead to eNOS uncoupling and generation of superoxide, contributing to HCY-induced oxidative stress.

Protein kinase C activation promotes the internalization of the human cationic amino acid transporter hCAT-1. A new regulatory mechanism for hCAT-1 activity.

The human cationic amino acid transporter hCAT-1 is almost ubiquitously expressed and probably the most important entity for supplying cells with extracellular arginine, lysine, and ornithine. We have previously shown that hCAT-1-mediated transport is decreased after protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) (Gräf, P., Forstermann, U., and Closs, E. I. (2001) Br. J. Pharmacol. 132, 1193-1200). In the present study, we examined the mechanism of this down-regulation. In both Xenopus laevis oocytes and U373MG glioblastoma cells, PMA treatment promoted the internalization of hCAT-1 (fused to the enhanced green fluorescence protein (EGFP)) as visualized by fluorescence microscopy. Biotinylation of cell surface proteins and subsequent Western blot analyses confirmed that the cell surface expression of hCAT-1.EGFP was significantly reduced upon PMA treatment. Pretreatment with the PKC inhibitor bisindolylmaleimide I prevented the reduction by PMA of both hCAT-1.EGFP-induced arginine transport and the internalization of the transporter. Similar results were obtained with hCAT-1 expressed endogenously in DLD-1 colon carcinoma cells. Inhibition of protein synthesis did not augment the PMA effect. In addition, the PMA effect was reverted in washout experiments without changing the hCAT-1 protein expression, suggesting that the PMA effect is reversible in these cells. PKC did not phosphorylate hCAT-1 directly as evidenced by in vivo phosphorylation experiments and mutational analysis, indicating an indirect action of PKC on hCAT-1.

Renal transcription of high-affinity type-2 cationic amino acid transporter is up-regulated in LPS-stimulated rodents.

OBJECTIVE: Sepsis stimulates renal nitric oxide (NO) biosynthesis through up-regulation of inducible NO synthase (iNOS) expression. Type-2 cationic amino acid transporter (CAT-2) mediation of trans-membrane L-arginine (L-Arg) transportation has been identified as one of the crucial regulatory mechanisms involved in the formation of NO by iNOS. We had previously shown that CAT-2B, a high-affinity alternative-spliced transcript of the CAT-2, is involved in induced NO biosynthesis by iNOS (Nitric Oxide, 2002). In this present study, we sought to assess the effects of sepsis on the expression of CAT-2B in lipopolysaccharide (LPS)-stimulated rat kidney. METHODS: Forty rats were randomized to either a normal saline (N/S)-treated group or a LPS-treated group. Renal NO production was determined using chemiluminescence. Semi-quantitative RT-PCR was used to determine the mRNA concentrations of iNOS and L-Arg transporters (CAT-1, CAT-2 and CAT-2B) in kidney. RESULTS: Lipopolysaccharide-coinduced iNOS, CAT-2 and CAT-2B mRNA expression in kidney and caused renal NO overproduction. A significant linear regression relationship was defined between renal NO concentrations and iNOS, CAT-2 and CAT-2B, respectively. On the contrary, CAT-1 expression was not affected by LPS-stimulation. CONCLUSIONS: We provide the first evidence to illustrate that sepsis/septic shock induces the transcription of high-affinity CAT-2B in renal tissues. Transcription of iNOS, CAT-2 and CAT-2B correlates well with renal NO biosynthesis. Regulation of L-Arg uptake by modulating the expression regulation of induced CAT-2 and CAT-2B might be a potential target for therapies against renal pathologic conditions related to NO overproduction.

In vivo distribution of receptor for ecotropic murine leukemia virus and binding of envelope protein of Friend Murine leukemia virus.

Ecotropic infection by Murine leukemia virus (MuLV) infection is initiated by the interaction between the receptor-binding domain of the viral surface glycoprotein (SU) and the cell-surface receptor, mCAT-1. To study the in vivo localization of viral binding site in mice, green fluorescence protein (GFP)-tagged Friend SU (F-SU/GFP) was incubated with tissue sections. Lymphohematopoietic organs and a part of the glandular tissues of C3H as well as C57BL/6 mice revealed positive signals for F-SU/GFP binding on the cell surface. In contrast, C4W mice, which is a partial congenic mouse strain carrying the Fv-4 (r) gene on a BALB/c genetic background, exhibited negative signals in most of the organs except for a very weak binding in the pancreas. The expression of mCAT-1 mRNA determined by reverse transcriptase (RT)-polymerase chain reaction (PCR) revealed a similar distribution in C3H, C57BL/6 and C4W mice. Most of the organs including lymphohematopoietic organs and glandular organs revealed significant expression of mRNA for mCAT-1 gene, while the liver, heart and muscle did not. The results from binding assay were consistent with the fact that Friend MuLV-induced pathogenesis was usually associated with lymphohematopoietic systems, although mRNA expression for mCAT-1 was rather ubiquitous. The discrepancy between F-SU/GFP binding and mRNA expression for mCAT-1 in lymphohematopoietic organs of C4W mice would support the receptor interference effect by the Fv-4 (r) gene causing the resistance of C4W mouse to Friend MuLV infection.