l-Citrulline ameliorates pathophysiology in a rat model of superimposed preeclampsia.

BACKGROUND AND PURPOSE: Preeclampsia, characterized by hypertension, proteinuria and restriction of fetal growth, is one of the leading causes of maternal and perinatal mortality. So far, there is no effective pharmacological therapy for preeclampsia. The present study was conducted to investigate the effects of supplementation with l-citrulline in Dahl salt-sensitive rats, a model of superimposed preeclampsia. EXPERIMENTAL APPROACH: Parental Dahl salt-sensitive rats were treated with l-citrulline (2.5 g·L-1 in drinking water) from the day of mating to the end of lactation period. Blood pressure was monitored throughout pregnancy and markers of preeclampsia were assessed. Endothelial function of the pregnant Dahl salt-sensitive rats was assessed by wire myograph. KEY RESULTS: In Dahl salt-sensitive rats, l-citrulline supplementation significantly reduced maternal blood pressure, proteinuria and levels of circulating soluble fms-like tyrosine kinase 1. l-Citrulline improved maternal endothelial function by augmenting the production of nitric oxide in the aorta and improving endothelium-derived hyperpolarizing factor-mediated vasorelaxation in resistance arteries. l-Citrulline supplementation improved placental insufficiency and fetal growth, which were associated with an enhancement of angiogenesis and reduction of fibrosis and senescence in the placentas. In addition, l-citrulline down-regulated genes involved in the TLR4 and NF-κB signalling pathways. CONCLUSION AND IMPLICATIONS: This study shows that l-citrulline supplementation reduced gestational hypertension and improved placentation and fetal growth in a rat model of superimposed preeclampsia. l-Citrulline supplementation may provide an effective and safe therapeutic strategy for preeclampsia that benefits both the mother and the fetus.

B Lymphocyte-Deficiency in Mice Causes Vascular Dysfunction by Inducing Neutrophilia.

B lymphocytes have been implicated in the development of insulin resistance, atherosclerosis and certain types of hypertension. In contrast to these studies, which were performed under pathological conditions, the present study provides evidence for the protective effect of B lymphocytes in maintaining vascular homeostasis under physiological conditions. In young mice not exposed to any known risk factors, the lack of B cells led to massive endothelial dysfunction. The vascular dysfunction in B cell-deficient mice was associated with an increased number of neutrophils in the circulating blood. Neutrophil depletion in B cell-deficient mice resulted in the complete normalization of vascular function, indicating a causal role of neutrophilia. Moreover, vascular function in B cell-deficient mice could be restored by adoptive transfer of naive B-1 cells isolated from wild-type mice. Interestingly, B-1 cell transfer also reduced the number of neutrophils in the recipient mice, further supporting the involvement of neutrophils in the vascular pathology caused by B cell-deficiency. In conclusion, we report in the present study the hitherto undescribed role of B lymphocytes in regulating vascular function. B cell dysregulation may represent a crucial mechanism in vascular pathology.

[Asymmetric dimethylarginine (ADMA) in retinal vein occlusion-Results from the Gutenberg RVO study]. / Asymmetrisches Dimethylarginin (ADMA) bei retinalen Venenverschlüssen ­ Ergebnisse aus der Gutenberg-RVO-Studie.

BACKGROUND: Asymmetric dimethylarginine (ADMA) is considered an independent cardiovascular risk factor (cvRF) and thus represents a potential new biomarker for retinal vein occlusion (RVO). METHODS: Overall, 92 patients with RVO and the same number of matched controls were included in the Gutenberg RVO study. All patients underwent a standardized examination for cvRF at the study center of the population-based Gutenberg health study (GHS) as well as ophthalmological examinations and intensive laboratory tests. This article presents a substudy of patients (≤65 years old) and the controls in whom ADMA was additionally determined by high performance liquid chromatography (HPLC) at baseline and 4-6 weeks later. RESULTS: Out of 44 patients with RVO 22 had central retinal vein occlusion (CRVO), 15 had branch retinal vein occlusion (BRVO) and 7 had hemiretinal vein occlusion (hemi-RVO). The ADMA levels were 0.383 ± 0.094 µM (mean ± standard deviation) in RVO patients at baseline and 0.380 ± 0.093 µM (p = 0.514, initial vs. follow-up) after the follow-up period versus 0.360 ± 0.077 µM (p = 0.175, controls vs. RVO) in controls (n = 44). Arterial hypertension was the most prevalent risk factor in 22 (50%) of the patients and in 11 (25%) of the controls (odds ratio, OR 2.77, 95% confidence interval, CI 0.97-7.95; p = 0.058). The ADMA values above the 95th percentile (>0.530 µM) were detected in 4 patients with RVO (9.1%) but not in any of the controls (p = 0.041, RVO vs. controls). CONCLUSION: Hypertension is the most important risk factor for RVO. Due to the high number of hypertensive patients in the cohort, the relevance of ADMA as an independent risk factor could neither be confirmed nor disproved.

Human cationic amino acid transporters are not affected by direct nitros(yl)ation.

A direct inhibiting effect of NO on the function of CAT-1 and -2A has been postulated to occur via nitrosylation of cysteine residues in the transporters. Neither the NO donor SNAP nor a mixture of SIN-1 and Spermine NONOate, that generates the strong nitrosating agent N2O3, reduced CAT-mediated L-arginine transport. Direct nitros(yl)ation does either not occur in CATs or does not affect their transport function. A regulatory effect of NO or nitrosating agents on CAT-mediated transport under physiological conditions seems, therefore, unlikely.

Cationic Amino Acid Transporter-1-Mediated Arginine Uptake Is Essential for Chronic Lymphocytic Leukemia Cell Proliferation and Viability.

Interfering with tumor metabolism by specifically restricting the availability of extracellular nutrients is a rapidly emerging field of cancer research. A variety of tumor entities depend on the uptake of the amino acid arginine since they have lost the ability to synthesize it endogenously, that is they do not express the rate limiting enzyme for arginine synthesis, argininosuccinate synthase (ASS). Arginine transport through the plasma membrane of mammalian cells is mediated by eight different transporters that belong to two solute carrier (SLC) families. In the present study we found that the proliferation of primary as well as immortalized chronic lymphocytic leukemia (CLL) cells depends on the availability of extracellular arginine and that primary CLL cells do not express ASS and are therefore arginine-auxotrophic. The cationic amino acid transporter-1 (CAT-1) was the only arginine importer expressed in CLL cells. Lentiviral-mediated downregulation of the CAT-1 transporter in HG3 CLL cells significantly reduced arginine uptake, abolished cell proliferation and impaired cell viability. In a murine CLL xenograft model, tumor growth was significantly suppressed upon induced downregulation of CAT-1 in the CLL cells. Our results suggest that inhibition of CAT-1 is a promising new therapeutic approach for CLL.

Reconstitution of T Cell Proliferation under Arginine Limitation: Activated Human T Cells Take Up Citrulline via L-Type Amino Acid Transporter 1 and Use It to Regenerate Arginine after Induction of Argininosuccinate Synthase Expression.

In the tumor microenvironment, arginine is metabolized by arginase-expressing myeloid cells. This arginine depletion profoundly inhibits T cell functions and is crucially involved in tumor-induced immunosuppression. Reconstitution of adaptive immune functions in the context of arginase-mediated tumor immune escape is a promising therapeutic strategy to boost the immunological antitumor response. Arginine can be recycled in certain mammalian tissues from citrulline via argininosuccinate (ASA) in a two-step enzymatic process involving the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). Here, we demonstrate that anti-CD3/anti-CD28-activated human primary CD4+ and CD8+ T cells upregulate ASS expression in response to low extracellular arginine concentrations, while ASL is expressed constitutively. ASS expression peaked under moderate arginine restriction (20 µM), but no relevant induction was detectable in the complete absence of extracellular arginine. The upregulated ASS correlated with a reconstitution of T cell proliferation upon supplementation of citrulline, while the suppressed production of IFN-γ was refractory to citrulline substitution. In contrast, ASA reconstituted proliferation and cytokine synthesis even in the complete absence of arginine. By direct quantification of intracellular metabolites we show that activated primary human T cells import citrulline but only metabolize it further to ASA and arginine when ASS is expressed in the context of low amounts of extracellular arginine. We then clarify that citrulline transport is largely mediated by the L-type amino acid transporter 1 (LAT1), induced upon human T cell activation. Upon siRNA-mediated knockdown of LAT1, activated T cells lost the ability to import citrulline. These data underline the potential of citrulline substitution as a promising pharmacological way to treat immunosuppression in settings of arginine deprivation.

Asymmetric dimethylarginine is transported by the mitochondrial carrier SLC25A2.

Asymmetric dimethyl L-arginine (ADMA) is generated within cells and in mitochondria when proteins with dimethylated arginine residues are degraded. The aim of this study was to identify the carrier protein(s) that transport ADMA across the inner mitochondrial membrane. It was found that the recombinant, purified mitochondrial solute carrier SLC25A2 when reconstituted into liposomes efficiently transports ADMA in addition to its known substrates arginine, lysine, and ornithine and in contrast to the other known mitochondrial amino acid transporters SLC25A12, SLC25A13, SLC25A15, SLC25A18, SLC25A22, and SLC25A29. The widely expressed SLC25A2 transported ADMA across the liposomal membrane in both directions by both unidirectional transport and exchange against arginine or lysine. The SLC25A2-mediated ADMA transport followed first-order kinetics, was nearly as fast as the transport of the best SLC25A2 substrates known so far, and was highly specific as symmetric dimethylarginine (SDMA) was not transported at all. Furthermore, ADMA inhibited SLC25A2 activity with an inhibition constant of 0.38 ± 0.04 mM, whereas SDMA inhibited it poorly. We propose that a major function of SLC25A2 is to export ADMA from mitochondria missing the mitochondrial ADMA-metabolizing enzyme AGXT2. There is evidence that ADMA can also be imported into mitochondria, e.g., in kidney proximal tubulus cells, to be metabolized by AGXT2. SLC25A2 may also mediate this transport function.

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.

Induced arginine transport via cationic amino acid transporter-1 is necessary for human T-cell proliferation.

Availability of the semiessential amino acid arginine is fundamental for the efficient function of human T lymphocytes. Tumor-associated arginine deprivation, mainly induced by myeloid-derived suppressor cells, is a central mechanism of tumor immune escape from T-cell-mediated antitumor immune responses. We thus assumed that transmembranous transport of arginine must be crucial for T-cell function and studied which transporters are responsible for arginine influx into primary human T lymphocytes. Here, we show that activation via CD3 and CD28 induces arginine transport into primary human T cells. Both naïve and memory CD4(+) T cells as well as CD8(+) T cells specifically upregulated the human cationic amino acid transporter-1 (hCAT-1), with an enhanced and persistent expression under arginine starvation. When hCAT-1 induction was suppressed via siRNA transfection, arginine uptake, and cellular proliferation were impaired. In summary, our results demonstrate that hCAT-1 is a key component of efficient T-cell activation and a novel potential target structure to modulate adaptive immune responses in tumor immunity or inflammation.

Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase.

OBJECTIVE: To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). METHODS & RESULTS: A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4: BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. CONCLUSIONS: Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.

Hypomorphic variants of cationic amino acid transporter 3 in males with autism spectrum disorders.

Cationic amino acid transporters (CATs) mediate the entry of L-type cationic amino acids (arginine, ornithine and lysine) into the cells including neurons. CAT-3, encoded by the SLC7A3 gene on chromosome X, is one of the three CATs present in the human genome, with selective expression in brain. SLC7A3 is highly intolerant to variation in humans, as attested by the low frequency of deleterious variants in available databases, but the impact on variants in this gene in humans remains undefined. In this study, we identified a missense variant in SLC7A3, encoding the CAT-3 cationic amino acid transporter, on chromosome X by exome sequencing in two brothers with autism spectrum disorder (ASD). We then sequenced the SLC7A3 coding sequence in 148 male patients with ASD and identified three additional rare missense variants in unrelated patients. Functional analyses of the mutant transporters showed that two of the four identified variants cause severe or moderate loss of CAT-3 function due to altered protein stability or abnormal trafficking to the plasma membrane. The patient with the most deleterious SLC7A3 variant had high-functioning autism and epilepsy, and also carries a de novo 16p11.2 duplication possibly contributing to his phenotype. This study shows that rare hypomorphic variants of SLC7A3 exist in male individuals and suggest that SLC7A3 variants possibly contribute to the etiology of ASD in male subjects in association with other genetic factors.

System L amino acid transporter LAT1 accumulates O-(2-fluoroethyl)-L-tyrosine (FET).

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.

Metabolism via Arginase or Nitric Oxide Synthase: Two Competing Arginine Pathways in Macrophages.

Macrophages play a major role in the immune system, both as antimicrobial effector cells and as immunoregulatory cells, which induce, suppress or modulate adaptive immune responses. These key aspects of macrophage biology are fundamentally driven by the phenotype of macrophage arginine metabolism that is prevalent in an evolving or ongoing immune response. M1 macrophages express the enzyme nitric oxide synthase, which metabolizes arginine to nitric oxide (NO) and citrulline. NO can be metabolized to further downstream reactive nitrogen species, while citrulline might be reused for efficient NO synthesis via the citrulline-NO cycle. M2 macrophages are characterized by expression of the enzyme arginase, which hydrolyzes arginine to ornithine and urea. The arginase pathway limits arginine availability for NO synthesis and ornithine itself can further feed into the important downstream pathways of polyamine and proline syntheses, which are important for cellular proliferation and tissue repair. M1 versus M2 polarization leads to opposing outcomes of inflammatory reactions, but depending on the context, M1 and M2 macrophages can be both pro- and anti-inflammatory. Notably, M1/M2 macrophage polarization can be driven by microbial infection or innate danger signals without any influence of adaptive immune cells, secondarily driving the T helper (Th)1/Th2 polarization of the evolving adaptive immune response. Since both arginine metabolic pathways cross-inhibit each other on the level of the respective arginine break-down products and Th1 and Th2 lymphocytes can drive or amplify macrophage M1/M2 dichotomy via cytokine activation, this forms the basis of a self-sustaining M1/M2 polarization of the whole immune response. Understanding the arginine metabolism of M1/M2 macrophage phenotypes is therefore central to find new possibilities to manipulate immune responses in infection, autoimmune diseases, chronic inflammatory conditions, and cancer.

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.

Differential cystine and dibasic amino acid handling after loss of function of the amino acid transporter b0,+AT (Slc7a9) in mice.

Cystinuria is an autosomal recessive disease caused by mutations in SLC3A1 (rBAT) and SLC7A9 (b(0,+)AT). Gene targeting of the catalytic subunit (Slc7a9) in mice leads to excessive excretion of cystine, lysine, arginine, and ornithine. Here, we studied this non-type I cystinuria mouse model using gene expression analysis, Western blotting, clearance, and brush-border membrane vesicle (BBMV) uptake experiments to further characterize the renal and intestinal consequences of losing Slc7a9 function. The electrogenic and BBMV flux studies in the intestine suggested that arginine and ornithine are transported via other routes apart from system b(0,+). No remarkable gene expression changes were observed in other amino acid transporters and the peptide transporters in the intestine and kidney. Furthermore, the glomerular filtration rate (GFR) was reduced by 30% in knockout animals compared with wild-type animals. The fractional excretion of arginine was increased as expected (∼100%), but fractional excretions of lysine (∼35%), ornithine (∼16%), and cystine (∼11%) were less affected. Loss of function of b(0,+)AT reduced transport of cystine and arginine in renal BBMVs and completely abolished the exchanger activity of dibasic amino acids with neutral amino acids. In conclusion, loss of Slc7a9 function decreases the GFR and increases the excretion of several amino acids to a lesser extent than expected with no clear regulation at the mRNA and protein level of alternative transporters and no increased renal epithelial uptake. These observations indicate that transporters located in distal segments of the kidney and/or metabolic pathways may partially compensate for Slc7a9 loss of function.

Identification of cysteine residues in human cationic amino acid transporter hCAT-2A that are targets for inhibition by N-ethylmaleimide.

In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y(+)L (y(+)LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y(+) (most likely CAT-1), but not system y(+)L (Devés, R., Angelo, S., and Chávez, P. (1993) J. Physiol. 468, 753-766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y(+)LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y(+)LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced transporter expression in the plasma membrane, indicating that NEM reduces the intrinsic transporter activity. Individual mutation of each of the seven cysteine residues conserved in all CAT isoforms did not lead to NEM insensitivity of hCAT-2A. However, a cysteine-less mutant was no longer inhibited by NEM, suggesting that inhibition occurs through modification of more than one cysteine in hCAT-2A. Indeed, also the double mutant C33A/C273A was insensitive to NEM inhibition, whereas reintroduction of a cysteine at either position 33 or 273 in the cysteine-less mutant led to NEM sensitivity. We thus identified Cys-33 and Cys-273 in hCAT-2A as the targets of NEM inhibition. In addition, all proteins with Cys-33 mutations showed a pronounced reduction in transport activity, suggesting that, surprisingly, this residue, located in the cytoplasmic N terminus, is important for transporter function.

Decoding the substrate supply to human neuronal nitric oxide synthase.

Nitric oxide, produced by the neuronal nitric oxide synthase (nNOS) from L-arginine is an important second messenger molecule in the central nervous system: It influences the synthesis and release of neurotransmitters and plays an important role in long-term potentiation, long-term depression and neuroendocrine secretion. However, under certain pathological conditions such as Alzheimer's or Parkinson's disease, stroke and multiple sclerosis, excessive NO production can lead to tissue damage. It is thus desirable to control NO production in these situations. So far, little is known about the substrate supply to human nNOS as a determinant of its activity. Measuring bioactive NO via cGMP formation in reporter cells, we demonstrate here that nNOS in both, human A673 neuroepithelioma and TGW-nu-I neuroblastoma cells can be fast and efficiently nourished by extracellular arginine that enters the cells via membrane transporters (pool I that is freely exchangeable with the extracellular space). When this pool was depleted, NO synthesis was partially sustained by intracellular arginine sources not freely exchangeable with the extracellular space (pool II). Protein breakdown made up by far the largest part of pool II in both cell types. In contrast, citrulline to arginine conversion maintained NO synthesis only in TGW-nu-I neuroblastoma, but not A673 neuroepithelioma cells. Histidine mimicked the effect of protease inhibitors causing an almost complete nNOS inhibition in cells incubated additionally in lysine that depletes the exchangeable arginine pool. Our results identify new ways to modulate nNOS activity by modifying its substrate supply.

A chimera carrying the functional domain of the orphan protein SLC7A14 in the backbone of SLC7A2 mediates trans-stimulated arginine transport.

In human skin fibroblasts, a lysosomal transport system specific for cationic amino acids has been described and named system c. We asked if SLC7A14 (solute carrier family 7 member A14), an orphan protein assigned to the SLC7 subfamily of cationic amino acid transporters (CATs) due to sequence homology, may represent system c. Fusion proteins between SLC7A14 and enhanced GFP localized to intracellular vesicles, co-staining with the lysosomal marker LysoTracker(®). To perform transport studies, we first tried to redirect SLC7A14 to the plasma membrane (by mutating putative lysosomal targeting motifs) but without success. We then created a chimera carrying the backbone of human (h) CAT-2 and the protein domain of SLC7A14 corresponding to the so-called "functional domain" of the hCAT proteins, a protein stretch of 81 amino acids that determines the apparent substrate affinity, sensitivity to trans-stimulation, and (as revealed in this study) pH dependence. The chimera mediated arginine transport and exhibited characteristics similar but not identical to hCAT-2A (the low affinity hCAT-2 isoform). Western blot and microscopic analyses confirmed localization of the chimera in the plasma membrane of Xenopus laevis oocytes. Noticeably, arginine transport by the hCAT-2/SLC7A14 chimera was pH-dependent, trans-stimulated, and inhibited by α-trimethyl-L-lysine, properties assigned to lysosomal transport system c in human skin fibroblasts. Expression analysis showed strong expression of SLC7A14 mRNA in these cells. Taken together, these data strongly suggest that SLC7A14 is a lysosomal transporter for cationic amino acids.

Impairment of the extrusion transporter for asymmetric dimethyl-L-arginine: a novel mechanism underlying vasospastic angina.

A 37-year old male patient presented with frequent angina attacks (up to 40/day) largely resistant to classical vasodilator therapy. The patient showed severe coronary and peripheral endothelial dysfunction, increased platelet aggregation and increased platelet-derived superoxide production. The endothelial nitric oxide synthase (eNOS)-inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) reduced superoxide formation in platelets identifying "uncoupled" eNOS as a superoxide source. Oral L-arginine normalized coronary and peripheral endothelial dysfunction and reduced platelet aggregation and eNOS-derived superoxide production. Plasma concentrations of the endogenous NOS inhibitor asymmetric dimethyl-L-arginine (ADMA), representing an independent risk factor for cardiovascular disease, were normal in the patient. However, immediately after oral administration of cationic amino acid (CAA), plasma ADMA levels rose markedly, demonstrating increased ADMA efflux from intracellular stores. ADMA efflux from mononuclear cells of the patient was accelerated by CAA, but not neutral amino acids (NAA) demonstrating impairment of y(+)LAT (whose expression was found reduced in these cells). These data suggest that impairment of y(+)LAT may cause intracellular (endothelial) ADMA accumulation leading to systemic endothelial dysfunction. This may represent a novel mechanism underlying vasospastic angina and vascular dysfunction in general. Moreover, these new findings contribute to the understanding of the l-arginine paradox, the improvement of eNOS activity by oral L-arginine despite sufficient cellular l-arginine levels to ensure proper function of this enzyme.