Two-way arginine transport in human endothelial cells: TNF-alpha stimulation is restricted to system y(+).
Human umbilical vein endothelial cells transport arginine through two Na(+)-independent systems. System y(+)L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of Na(+), and referable to the expression of SLC7A6/y(+)LAT2, SLC7A7/y(+)LAT1, and SLC3A2/4F2hc. System y(+) is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y(+). Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y(+)L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-alpha, while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-alpha or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production. (+info)
Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells.
To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1(b) cells with respect to infection though the ecotropic receptor pathway. (+info)
Garlic attenuates nitric oxide production in rat cardiac myocytes through inhibition of inducible nitric oxide synthase and the arginine transporter CAT-2 (cationic amino acid transporter-2).
It is now accepted that allicin, the main biologically active compound in garlic, exhibits antioxidant activity. The present study was designed to test the hypothesis that the antioxidant activity of garlic can be partially attributed to the inhibition of nitric oxide (NO) production by cytokine-induced NO synthase (iNOS). Cardiac myocytes cultured from neonatal Wistar rats were stimulated by lipopolysaccharide (LPS) and incubated for 24 h with various concentrations of allicin. This resulted in marked inhibition of nitrite production. Interestingly, a low concentration of allicin (10 microM) was significantly more potent in abrogating the effect of LPS on nitrite production than a higher concentration (40 microM). Allicin decreased steady-state iNOS mRNA levels, and this effect was maximal when a lower concentration was used (10 microM compared with 40 microM). In order to explore additional effects of allicin on NO generation that might counteract the effect on iNOS, we assessed the effects of higher allicin concentrations on arginine transport. Allicin inhibited the uptake of 1 mM extracellular arginine in a concentration-dependent manner. The expression of the two arginine transporters that are expressed in cardiac myocytes [CAT-1 (cationic amino acid transporter-1) and CAT-2] was studied using reverse transcription-PCR. A concentration of 200 microM allicin abolished the expression of CAT-2 mRNA, 100 microM significantly attenuated it, whereas 50 microM had no effect. Allicin had no effect on steady-state CAT-1 mRNA levels. Our results suggest that allicin inhibits iNOS activity through two different mechanisms: at lower concentrations it decreases iNOS mRNA levels, whereas at higher concentrations it inhibits arginine transport through down-regulation of CAT-2 mRNA. (+info)
Intrauterine growth retardation is associated with reduced activity and expression of the cationic amino acid transport systems y+/hCAT-1 and y+/hCAT-2B and lower activity of nitric oxide synthase in human umbilical vein endothelial cells.
Intrauterine growth retardation (IUGR) is associated with vascular complications leading to hypoxia and abnormal fetal development. The effect of IUGR on L-arginine transport and nitric oxide (NO) synthesis was investigated in cultures of human umbilical vein endothelial cells (HUVECs). IUGR was associated with membrane depolarization and reduced L-arginine transport (V(max)= 5.8+/-0.2 versus 3.3+/-0.1 pmol/microg protein per minute), with no significant changes in transport affinity (K(m)=159+/-15 versus 137+/-14 micromol/L). L-Arginine transport was trans-stimulated (8- to 9-fold) in cells from normal and IUGR pregnancies. IUGR was associated with reduced production of L-[3H]citrulline from L-[3H] arginine, lower nitrite and intracellular L-arginine, L-citrulline, and cGMP. IUGR decreased hCAT-1 and hCAT-2B mRNA, and increased eNOS mRNA and protein levels. IUGR-associated inhibition of L-arginine transport and NO synthesis, and membrane depolarization were reversed by the NO donor S-nitroso-N-acetyl-L,D-penicillamine. In summary, endothelium from fetuses with IUGR exhibit altered L-arginine transport and NO synthesis (L-arginine/NO pathway), reduced expression and activity of hCAT-1 and hCAT-2B and reduced eNOS activity. Alterations in L-arginine/NO pathway could be critical for the physiological processes involved in the etiology of IUGR in human pregnancies. (+info)
Nutritional control of mRNA stability is mediated by a conserved AU-rich element that binds the cytoplasmic shuttling protein HuR.
The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene increases during nutritional stress as part of the adaptive response to starvation. Amino acid limitation induces coordinate increases in stability and translation of the cat-1 mRNA, at a time when global protein synthesis decreases. It is shown here that increased cat-1 mRNA stability requires an 11 nucleotide AU-rich element within the distal 217 bases of the 3'-untranslated region. When this 217-nucleotide nutrient sensor AU-rich element (NS-ARE) is present in a chimeric mRNA it confers mRNA stabilization during amino acid starvation. HuR is a member of the ELAV family of RNA-binding proteins that has been implicated in regulating the stability of ARE-containing mRNAs. We show here that the cytoplasmic concentration of HuR increases during amino acid starvation, at a time when total cellular HuR levels decrease. In addition, RNA gel shift experiments in vitro demonstrated that HuR binds to the NS-ARE and binding was dependent on the 11 residue AU-rich element. Moreover, HuR binding to the NS-ARE in extracts from amino acid-starved cells increased in parallel with the accumulation of cytoplasmic HuR. It is proposed that an adaptive response of cells to nutritional stress results in increased mRNA stability mediated by HuR binding to the NS-ARE. (+info)
L-Arginine transport is augmented through up-regulation of tubular CAT-2 mRNA in ischemic acute renal failure in rats.
BACKGROUND: Ischemic acute renal failure (iARF) is associated with increased nitric oxide (NO) production during the reperfusion period, as endothelial nitric oxide synthase (eNOS) is maximally activated, and renal tubular inducible NOS (iNOS) is stimulated. Increased NO production leads to augmented tubular injury, probably through the formation of peroxynitrite. l-Arginine (l-Arg), the only precursor for NO, is transported into cells by cationic amino acid transporters, CAT-1 and CAT-2. We hypothesized that the increased NO production observed in iARF may result from increased l-Arg uptake, which would be reflected in the augmented expression of l-Arg transporter(s). METHODS: Ischemic acute renal failure was induced in rats by right nephrectomy + left renal artery clamping for 60 minutes. l-Arg uptake was examined in freshly harvested glomeruli and tubuli from control, sham operated, and animals subjected to 15, 30, and 60 minutes, and 24 hours of reperfusion, following 60 minutes of ischemia. Using RT-PCR, renal tissues were examined further for the expression of iNOS, CAT-1, CAT-2, arginase I and arginase II. RESULTS: Tubular expression of iNOS mRNA was initiated by ischemia, continued to increase after 60 minutes of reperfusion, and decreased after 24 hours. l-Arg transport into glomeruli was similar in all experimental groups. l-Arg uptake into tubuli was markedly augmented following the 60-minute reperfusion, while it moderately increased after 24 hours of reperfusion. This was accompanied by a parallel, preferential increase in tubular CAT-2 mRNA expression at 60 minutes of reperfusion. CAT-1 mRNA expression was unchanged, as detected by RT-PCR. In addition, the expression of arginase II and arginase I mRNA was attenuated by 30 minutes and one hour of reperfusion, and returned to baseline values after 24 hours of reperfusion. CONCLUSIONS: Ischemic ARF is associated with augmented tubular CAT-2 mRNA expression, which leads to enhanced l-Arg transport and increased NO production. This may contribute to the renal injury exhibited in iARF. (+info)
System y+ localizes to different membrane subdomains in the basolateral plasma membrane of epithelial cells.
We report here that the system y+ cationic amino acid transporter ATRC1 localized to clusters within the basolateral membrane of polarized Madin-Darby canine kidney and human embryonic kidney (HEK) cells, suggesting that the transporters are restricted to discrete membrane microdomains in epithelial cells. Based on solubility in nonionic detergents, two populations of ATRC1 molecules existed: approximately half of the total ATRC1 in HEK cells associated with the actin membrane cytoskeleton, whereas another one-fourth resided in detergent-resistant membranes (DRM). In agreement with these findings, cytochalasin D reduced the amount of ATRC1 associated with the actin membrane cytoskeleton. Although some ATRC1 clusters in HEK cells colocalized with caveolin, the majority of ATRC1 did not colocalize with this marker protein for a type of DRM called caveolae. This distribution of ATRC1 is somewhat different from that reported for pulmonary artery endothelial cells in which transporters cluster predominantly in caveolae, suggesting that differences in the proportion of ATRC1 in specific membrane microdomains correlate with differences in the physiological role of the transporter in polarized kidney epithelial vs. vascular endothelial cells. (+info)
Regulation of the S100B gene by alpha 1-adrenergic stimulation in cardiac myocytes.
We previously reported that S100B, a 20-kDa Ca(2+)-binding homodimer, inhibited the postinfarct myocardial hypertrophic response mediated by alpha(1)-adrenergic stimulation through the protein kinase C (PKC) signaling pathway. In the present study, we examined whether the same pathway induced the S100B gene, supporting the hypothesis that S100B is a feedback negative regulator of this pathway. We transfected cultured neonatal rat cardiac myocytes with a luciferase reporter gene driven by the maximal human S100B promoter and progressively shorter segments of this promoter sequentially deleted from the 5' end. We identified a basic promoter essential for transcription spanning 162 bp upstream of the transcription initiation site and positive (at -782/-162 and -6,689/-4,463) and negative (at -4,463/-782) myocyte-selective regulatory elements. We showed that the basic and maximal S100B promoters were activated specifically by alpha(1)-adrenergic agonists through the alpha(1A)-adrenergic receptor, but not by any other trophic hormonal stimuli. The activation of the S100B promoter was mediated through the PKC signaling pathway. Transcription enhancer factor-1 (TEF-1) and related to TEF-1 (RTEF-1) influenced transcription from the maximal, but not the basic, promoter implicating active MCAT elements upstream from the basic promoter. Acting in opposing fashions, TEF-1 transrepressed the S100B promoter and RTEF-1 transactivated the promoter. Our results suggest that alpha(1)-adrenergic stimulation induces the S100B gene after myocardial infarction through the PKC signaling pathway and that this induction is modulated by TEF-1 and RTEF-1. (+info)