Alice Habermeier

Structure and Function of Cationic Amino Acid Transporters (CATs)

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.

Resveratrol reverses endothelial nitric-oxide synthase uncoupling in apolipoprotein E knockout mice.

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 (BH4). 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 BH4 biosynthesis, was enhanced by resveratrol. This enhancement was accompanied by an elevation in BH4 levels. Superoxide production from ApoE-KO mice hearts was reduced by the NOS inhibitor l-NG-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 BH4 oxidation, together with the enhanced biosynthesis of BH4 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.

Role of Neutral Amino Acid Transport and Protein Breakdown for Substrate Supply of Nitric Oxide Synthase in Human Endothelial Cells

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. In this study, we demonstrate that the eNOS accessible pool II is also present in human umbilical vein endothelial cells (HUVECs), but not in ECV bladder carcinoma cells transfected with an expression plasmid for eNOS. In the endothelial cells, one part of pool II (referred to as pool IIA) consisted of recycling of citrulline to arginine. This part could be depleted by neutral amino acids that match the substrate profile of system N transporter 1 (SN1), presumably by the removal of intracellular citrulline. SN1 was expressed in EA.hy926 cells and HUVECs as shown by real-time RT-PCR. The second part of pool II (referred to as pool IIB) could not be depleted by any of the cationic or neutral amino acids tested. Our data demonstrate that pool IIB is nourished by protein breakdown and thus represents a substrate pool likely to accumulate protein-derived endogenous inhibitors of eNOS. Preferential use of the arginine pool IIB under pathophysiological conditions might therefore explain the arginine paradox.

Antiatherosclerotic Effects of Small-Molecular-Weight Compounds Enhancing Endothelial Nitric-Oxide Synthase (eNOS) Expression and Preventing eNOS Uncoupling

Many cardiovascular diseases are associated with reduced levels of bioactive nitric oxide (NO) and an uncoupling of oxygen reduction from NO synthesis in endothelial NO synthase (eNOS uncoupling). In human endothelial EA.hy 926 cells, two small-molecular-weight compounds with related structures, 4-fluoro-N-indan-2-yl-benzamide (CAS no. 291756-32-6; empirical formula C16H14FNO; AVE9488) and 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide (CAS no. 450348-85-3; empirical formula C17H13F2NO3; AVE3085), enhanced eNOS promoter activity in a concentration-dependent manner; with the responsible cis-element localized within the proximal 263 base pairs of the promoter region. RNA interference-mediated knockdown of the transcription factor Sp1 significantly reduced the basal activity of eNOS promoter, but it did not prevent the transcription activation by the compounds. Enhanced transcription of eNOS by AVE9488 in primary human umbilical vein endothelial cells was associated with increased levels of eNOS mRNA and protein expression, as well as increased bradykinin-stimulated NO production. In both wild-type C57BL/6J mice and apolipoprotein E-knockout (apoE-KO) mice, treatment with AVE9488 resulted in enhanced vascular eNOS expression. In apoE-KO mice, but not in eNOS-knockout mice, treatment with AVE9488 reduced cuff-induced neointima formation. A 12-week treatment with AVE9488 or AVE3085 reduced atherosclerotic plaque formation in apoE-KO mice, but not in apoE/eNOS-double knockout mice. Aortas from apoE-KO mice showed a significant generation of reactive oxygen species. This was partly prevented by nitric-oxide inhibitor Nω-nitro-l-arginine methyl ester, indicating eNOS uncoupling. Treatment of mice with AVE9488 enhanced vascular content of the essential eNOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin and reversed eNOS uncoupling. The combination of an up-regulated eNOS expression and a reversal of eNOS uncoupling is probably responsible for the observed vasoprotective properties of this new type of compounds.

Uncoupling of Endothelial Nitric Oxide Synthase in Perivascular Adipose Tissue of Diet-Induced Obese Mice

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 L-arginine deficiency and eNOS uncoupling in PVAT. The combination therapy with L-arginine and arginase inhibitors may represent a novel therapeutic strategy for obesity-induced vascular disease.

6-mercaptopurine and 9-(2-phosphonyl-methoxyethyl) adenine (PMEA) transport altered by two missense mutations in the drug transporter gene ABCC4.

Multiple drug resistance protein 4 (MRP4, ABCC4) belongs to the C subfamily of the ATP‐binding cassette (ABC) transporter superfamily and participates in the transport of diverse antiviral and chemotherapeutic agents such as 6‐mercaptopurine (6‐MP) and 9‐(2‐phosphonyl methoxyethyl) adenine (PMEA). We have undertaken a comprehensive functional characterization of protein variants of MRP4 found in Caucasians and other ethnicities. A total of 11 MRP4 missense genetic variants (nonsynonymous SNPs), fused to green fluorescent protein (GFP), were examined in Xenopus laevis oocytes for their effect on expression, localization, and function of the transporter. Radiolabeled 6‐MP and PMEA were chosen as transport substrates. All MRP4 protein variants were found to be expressed predominantly in the oocyte membrane. A total of four variants (Y556C, E757 K, V776I, and T1142 M) exhibited a 20% to 40% reduced expression level compared to the wild type. Efflux studies showed that 6‐MP is transported by MRP4 in unmodified form. Compared to wild‐type MRP4, the transmembrane variant V776I, revealed a significant lower activity in 6‐MP transport, while the amino acid exchange Y556C in the WalkerB motif displayed significantly higher transport of PMEA. The transport properties of the other variants were comparable to wild‐type MRP4. Our study shows that Xenopus oocytes are well suited to characterize MRP4 and its protein variants. Carriers of the rare MRP4 variants Y556C and V776I may have altered disposition of MRP4 substrates. Hum Mutat 29(5), 659–669, 2008.

Neuronal Nitric Oxide Synthase Modulates Maturation of Human Dendritic Cells

Dendritic cells (DCs) are the most potent APCs of the immune system. Understanding the intercellular and intracellular signaling processes that lead to DC maturation is critical for determining how these cells initiate T cell-mediated immune processes. NO synthesized by the inducible NO synthase (iNOS) is important for the function of murine DCs. In our study, we investigated the regulation of the arginine/NO-system in human monocyte-derived DCs. Maturation of DCs induced by inflammatory cytokines (IL-1β, TNF, IL-6, and PGE2) resulted in a pronounced expression of neuronal NOS (nNOS) but only minimal levels of iNOS and endothelial NOS were detected in human mature DCs. In addition, reporter cell assays revealed the production of NO by mature DCs. Specific inhibitors of NOS (N-nitro-l-arginine methyl ester) or of the NO target guanylyl cyclase (H-(1,2,4)-oxadiazolo [4,3-a] quinoxalin-1-one) prevented DC maturation (shown by decreased expression of MHC class II, costimulatory and CD83 molecules and reduced IL-12 production) and preserved an immature phenotype, indicating an autocrine effect of nNOS-derived NO on human DC maturation. Notably, inhibitor-treated DCs were incapable of inducing efficient T cell responses after primary culture and generated an anergic T cell phenotype. In conclusion, our results suggest that, in the human system, nNOS-, but not iNOS-derived NO, plays an important regulatory role for the maturation of DCs and, thus, the induction of pronounced T cell responses.

Arginine transport in human erythroid cells: discrimination of CAT1 and 4F2hc/y+LAT2 roles

Since arginine metabolites, such as nitric oxide and polyamines, influence the expression of genes involved in erythroid differentiation, the transport of the cationic amino acid may play an important role in erythroid cells. However, available data only concern the presence in these cells of CAT1 transporter (system y+), while no information exists on the role of the heterodimeric transporters of system y+L (4F2hc/y+LAT1 and 4F2hc/y+LAT2) which operates transmembrane arginine fluxes cis-inhibited by neutral amino acids in the presence of sodium. Using erythroleukemia K562 cells and normal erythroid precursors, we demonstrate here that arginine transport in human erythroid cells is due to the additive contributions of a leucine-sensitive and leucine-insensitive component. In both cell types, leucine inhibition of arginine influx is much less evident in the absence of sodium, a hallmark of system y+L. In K562 cells, N-ethylmaleimide, a known inhibitor of CAT transporters (system y+), suppresses only a fraction of arginine influx corresponding to leucine-insensitive uptake. Moreover, in Xenopus oocytes coexpressing 4F2hc and y+LAT2, leucine exerts a marked inhibition of arginine transport, partially dependent on sodium, while no inhibition is seen in oocytes expressing CAT1. Lastly, silencing of SLC7A6, the gene for y+LAT2, lowers arginine transport and doubles the intracellular content of the cationic amino acid in K562 cells. We conclude that arginine transport in human erythroid cells is due to both system y+ (CAT1 transporter) and system y+L (4F2hc/y+LAT2 isoform), which mainly contribute, respectively, to the influx and to the efflux of the cationic amino acid.

Relative contribution of different l-arginine sources to the substrate supply of endothelial nitric oxide synthase

In certain cases of endothelial dysfunction l-arginine becomes rate-limiting for NO synthesis in spite of sufficiently high plasma concentrations of the amino acid. To better understand this phenomenon, we investigated routes of substrate supply to endothelial nitric oxide synthase (eNOS). Our previous data with human umbilical vein (HUVEC) and EA.hy.926 endothelial cells demonstrated that eNOS can obtain its substrate from the conversion of l-citrulline to l-arginine and from protein breakdown. In the present study, we determined the quantitative contribution of proteasomal and lysosomal protein degradation and investigated to what extent extracellular peptides and l-citrulline can provide substrate to eNOS. The RFL-6 reporter cell assay was used to measure eNOS activity in human EA.hy926 endothelial cells. Individual proteasome and lysosome inhibition reduced eNOS activity in EA.hy926 cells only slightly. However, the combined inhibition had a pronounced reducing effect. eNOS activity was fully restored by supplementing either l-citrulline or l-arginine-containing dipeptides. Histidine prevented the restoration of eNOS activity by the dipeptide, suggesting that a transporter accepting both, peptides and histidine, mediates the uptake of the extracellular peptide. In fact, the peptide and histidine transporter PHT1 was expressed in EA.hy926 cells and HUVECs (qRT/PCR). Our study thus demonstrates that l-citrulline and l-arginine-containing peptides derived from either intracellular protein breakdown or from the extracellular space seem to be good substrate sources for eNOS.

Granulocyte functions are independent of arginine availability

Arginine depletion via myeloid cell arginase is critically involved in suppression of the adaptive immune system during cancer or chronic inflammation. On the other hand, arginine depletion is being developed as a novel anti‐tumor metabolic strategy to deprive arginine‐auxotrophic cancer cells of this amino acid. In human immune cells, arginase is mainly expressed constitutively in PMNs. We therefore purified human primary PMNs from healthy donors and analyzed PMN function as the main innate effector cell and arginase producer in the context of arginine deficiency. We demonstrate that human PMN viability, activation‐induced IL‐8 synthesis, chemotaxis, phagocytosis, generation of ROS, and fungicidal activity are not impaired by the absence of arginine in vitro. Also, profound pharmacological arginine depletion in vivo via ADI‐PEG20 did not inhibit PMN functions in a mouse model of pulmonary invasive aspergillosis; PMN invasion into the lung, activation, and successful PMN‐dependent clearance of Aspergillus fumigatus and survival of mice were not impaired. These novel findings add to a better understanding of immunity during inflammation‐associated arginine depletion and are also important for the development of therapeutic arginine depletion as anti‐metabolic tumor therapy.