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induced obesity, namely uncoupling of the endothelial nitric oxide (NO) synthase (eNOS) in PVAT. Materials and MethodsMaterials and Methods are available in the online-only Data Supplement.© 2015 American Heart Association, Inc. Objective-The present study was conducted to investigate the contribution of perivascular adipose tissue (PVAT) to vascular dysfunction in a mouse model of diet-induced obesity. Approach and Results-Obesity was induced in male C57BL/6J mice with a high-fat diet for 20 weeks, and vascular function was studied with myograph. In PVAT-free aortas isolated from obese mice, the endothelium-dependent, nitric oxide-mediated vasodilator response to acetylcholine remained normal. In contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when PVAT was left in place. Adipocytes in PVAT were clearly positive in endothelial nitric oxide synthase (eNOS) staining, and PVAT nitric oxide production was significantly reduced in obese mice. High-fat diet had no effect on eNOS expression but led to eNOS uncoupling, evidenced by diminished superoxide production in PVAT after eNOS inhibition. As mechanisms for eNOS uncoupling, arginase induction and l-arginine deficiency were observed in PVAT. Obesity-induced vascular dysfunction could be reversed by ex vivo l-arginine treatment and arginase inhibition. Conclusions-Diet-induced obesity leads to Results Role of PVAT in Vascular Dysfunction in Diet-Induced Obese MiceMale C57BL/6J mice were fed with either HFD or normal control diet (NCD) for 20 weeks starting at the age of 8 weeks.The HFD-treated mice developed obesity (Table). Vasodilator response to acetylcholine was studied using aortas with or without PVAT in a wire myograph system. In aortas without PVAT, no significant differences in endothelium-dependent, NO-mediated vasodilator response to acetylcholine were found between NCD and HFD groups ( Figure 1A). In contrast, a clear reduction in the vasodilator response to acetylcholine was observed in the aorta of obese animals compared with lean controls when PVAT was left intact ( Figure 1B). The acetylcholine-induced vasodilation in the mouse aorta was NO-dependent because it could be completely blocked by the NO synthase inhibitor N G -nitro-l-arginine methyl ester (l-NAME; Figure 1C). In endothelium-denuded aortic rings from control mice, acetylcholine induced a significant vasodilation if PVAT was left intact ( Figure I in the online-only Data Supplement). The PVAT-mediated vasodilation was preventable with l-NAME, indicating that PVAT-derived NO contributes to acetylcholine-induced vasodilation under normal conditions. Reduced NO Production in PVAT of Diet-Induced Obese MiceTo directly access NO production from PVAT, time-lapse fluorescence imaging was performed with aorta sections prepared from NCD or HFD-fed mice stained with the fluorescent NO probe 4,5-diaminofluorescein diacetate. As shown in Figure 1D, basal NO production could be detected in PVAT. The PVAT NO production was enhanced by ace...

The CAT proteins (CAT for cationic amino acid transporter) are amongst the first mammalian amino acid transporters identified on the molecular level and seem to be the major entry path for cationic amino acids in most cells. However, CAT proteins mediate also efflux of their substrates and thus may also deplete cells from cationic amino acids under certain circumstances. The CAT proteins form a subfamily of the solute carrier family 7 (SLC7) that consists of four confirmed transport proteins for cationic amino acids: CAT-1 (SLC7A1), CAT-2A (SLC7A2A), CAT-2B (SLC7A2B), and CAT-3 (SLC7A3). SLC7A4 and SLC7A14 are two related proteins with yet unknown function. One focus of this review lies on structural and functional differences between the different CAT isoforms. The expression of the CAT proteins is highly regulated on the level of transcription, mRNA stability, translation and subcellular localization. Recent advances toward a better understanding of these mechanisms provide a second focus of this review.

A crucial cause of the decreased bioactivity of nitric oxide (NO) in cardiovascular diseases is the uncoupling of the endothelial NO synthase (eNOS) caused by the oxidative stress-mediated deficiency of the NOS cofactor tetrahydrobiopterin (BH 4 ). The reversal of eNOS uncoupling might represent a novel therapeutic approach. The treatment of apolipoprotein E knockout (ApoE-KO) mice with resveratrol resulted in the up-regulation of superoxide dismutase (SOD) isoforms (SOD1-SOD3), glutathione peroxidase 1 (GPx1), and catalase and the down-regulation of NADPH oxidases NOX2 and NOX4 in the hearts of ApoE-KO mice. This was associated with reductions in superoxide, 3-nitrotyrosine, and malondialdehyde levels. In parallel, the cardiac expression of GTP cyclohydrolase 1 (GCH1), the rate-limiting enzyme in BH 4 biosynthesis, was enhanced by resveratrol. This enhancement was accompanied by an elevation in BH 4 levels. Superoxide production from ApoE-KO mice hearts was reduced by the NOS inhibitor L-N G -nitro-arginine methyl ester, indicating eNOS uncoupling in this pathological model. Resveratrol treatment resulted in a reversal of eNOS uncoupling. Treatment of human endothelial cells with resveratrol led to an up-regulation of SOD1, SOD2, SOD3, GPx1, catalase, and GCH1. Some of these effects were preventable with sirtinol, an inhibitor of the protein deacetylase sirtuin 1. In summary, resveratrol decreased superoxide production and enhanced the inactivation of reactive oxygen species. The resulting reduction in BH 4 oxidation, together with the enhanced biosynthesis of BH 4 by GCH1, probably was responsible for the reversal of eNOS uncoupling. This novel mechanism (reversal of eNOS uncoupling) might contribute to the protective effects of resveratrol.

Pharmacologic interventions that combine eNOS up-regulation and reversal of eNOS uncoupling can markedly increase bioactive NO in the vasculature and produce beneficial hemodynamic effects such as a reduction of blood pressure.

In this study, we aimed at analyzing the human homologues of the murine cationic amino acid transporters mCAT-1, mCAT-2A, and mCAT-2B. cDNAs encoding hCAT-1 had been previously reported by two independent groups [Albritton, L.M., et al. (1993) Genomics 12, 430; Yoshimoto, T., et al. (1991) Virology 185, 10]. We isolated cDNAs encoding hCAT-2A and hCAT-2B from a human liver cDNA library and from cDNA derived from the human hepatoma cell line HepG2, respectively. Analyses of the deduced amino acid sequences of both carriers demonstrated 90.9% identity with the respective murine proteins. In their functional domains (42 amino acids), both hCAT-2A and hCAT-2B differ only by one residue from the respective mouse proteins. Thus, CAT-2 proteins demonstrate a higher interspecies conservation than CAT-1 proteins that are overall 86.5% identical between mouse and human and differ by seven residues in the functional domain. The high degree of sequence conservation was reflected by the functional similarity of the human carriers with their mouse homologues. When expressed in Xenopus oocytes, hCAT-1 and hCAT-2B demonstrated transport properties consistent with y+. Unlike the mouse CAT-1 and CAT-2B, whose transport properties could hardly be distinguished, the transport properties of the human CAT-1 and CAT-2B isoforms showed clear differences: hCAT-1 had a 3-fold higher substrate affinity and was more sensitive to trans-stimulation than hCAT-2B. In contrast to the y+ carriers, hCAT-2A exhibited a 10-30-fold lower substrate affinity, a greater maximal velocity, and was much less sensitive to trans-stimulation at physiological substrate concentrations.

O-(2-fluoroethyl)-L-tyrosine (FET) labeled with fluorine-18 is an important and specific tracer for diagnostics of glioblastoma via positron emission tomography (PET). However, the mechanism of its quite specific accumulation in tumor tissue has not been understood so far. In this work we demonstrate that [(3)H]L-tyrosine is primarily transported by the system L transporter LAT1 in human LN229 glioblastoma cells. FET reduced tyrosine transport, suggesting that it shares the same uptake pathway. More importantly, accumulation of FET was significantly reduced after siRNA-mediated downregulation of LAT1. Xenopus laevis oocytes expressing human LAT1 together with the glycoprotein 4F2hc (necessary to pull LAT-1 to the plasma membrane) exhibited a similar accumulation of FET as observed in glioblastoma cells. In contrast, no accumulation was observed in control oocytes, not overexpressing an exogenous transporter. Because LAT1 works exclusively as an exchanger of amino acids, substrates at one side of the membrane stimulate exchange against substrates at the other side. Extracellular FET stimulated the efflux of intracellular [(3)H]L-leucine, demonstrating that FET is indeed an influx substrate for LAT1. However, FET injected into oocytes was not able to stimulate uptake of extracellular [(3)H]L-leucine, indicating that FET is not a good efflux substrate. Our data, therefore, suggest that FET is trapped within cells due to the asymmetry of its intra- and extracellular recognition by LAT1. If also found for other transporters in tumor cells, asymmetric substrate recognition may be further exploited for tumor-specific accumulation of PET-tracers and/or other tumor-related drugs.

Abstract-Endothelial dysfunction is often associated with a relative substrate deficiency of the endothelial nitric oxide synthase (eNOS) in spite of apparently high intracellular arginine concentrations. For a better understanding of the underlying pathophysiological mechanisms, we aimed to characterize the intracellular arginine sources of eNOS. Our previous studies in human endothelial EA.hy926 cells suggested the existence of two arginine pools: pool I can be depleted by extracellular lysine, whereas pool II is not freely exchangeable with the extracellular space, but accessible to eNOS. Key Words: endothelial nitric oxide synthase Ⅲ neutral amino acid transport Ⅲ system N Ⅲ intracellular arginine pool Ⅲ proteasome N O synthesized from arginine by endothelial nitric oxide synthase (eNOS) is a potent vasodilator and a critical modulator of blood flow and blood pressure. In addition, it mediates vasoprotective actions through inhibiting smooth muscle cell proliferation, platelet aggregation, and leukocyte adhesion. Under pathophysiological conditions associated with endothelial dysfunction, such as diabetes, hypertension, or hypercholesterolemia, supply of the substrate arginine seems to be limiting for NO synthesis. 1 This is in spite of intracellular arginine concentrations sufficiently high to saturate eNOS, a phenomenon termed the arginine paradox. 2 In order to understand this paradox, it seems important to elucidate the intracellular substrate sources for eNOS. Our previous studies in human endothelial EA.hy926 cells have demonstrated the existence of two arginine pools: pool I can be depleted by extracellular lysine through an exchange mechanism mediated by membrane transporters such as the cationic amino acid transporter 1 (CAT-1) or the system y ϩ L transporter 4F2hc/y ϩ LAT2. 3 Both transporters are expressed in endothelial cells and catalyze the exchange of cationic amino acids. 4,5 In addition, 4F2hc/y ϩ LAT2 mediates also the exchange of extracellular neutral amino acids against intracellular cationic amino acids. 6 In contrast to the arginine pool I, pool II is not freely exchangeable with extracellular lysine, but accessible to eNOS, thereby rendering eNOS independent of extracellular arginine. 3 The arginine paradox might therefore be explained by alterations in pool II or an impaired access of eNOS to pool II. Recent findings suggest that an increased production of the endogenous inhibitor asymmetrical dimethyl arginine (ADMA), derived from breakdown of proteins containing methylated arginine residues, might underlie the arginine paradox. 7-9 Plasma concentrations of ADMA are increased in patients suffering conditions associated with endothelial dysfunction. However, even the highest ADMA concentrations found in plasma from patients with renal failure are 5-to 10-fold lower than the plasma arginine concentrations. It is therefore tempting to speculate that ADMA might specifically accumulate in the arginine pool II of endothelial cells, thereby exerting a larger inhibitory action on eNOS than a...