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Removal of intracellular asymmetric dimethyl-l-arginine (ADMA) requires system y+L membrane transporter-despite significant activity of the metabolising enzyme dimethylarginine dimethylaminohydrolase (DDAH)
... This concept makes it also easier to understand the so-called L-arginine paradox which consists in the beneficial effect of oral L-arginine on vascular function in patient cohorts despite sufficient L-arginine plasma concentrations for adequate substrate supply to eNOS. According to the recent findings, the export of ADMA in endothelial cells is mediated by y + L amino acid transporters (y + LAT-1 and -2) [107,108]: These transporters exchange intracellular cationic amino acids against extracellular neutral amino acids and sodium cations and thus provide an active (energy-dependent) efflux pathway for ADMA. Down-regulation of the y + LAT isoforms in cultured endothelial cells by siRNA, lead to an intracellular ADMA accumulation [107,108]. ...
... According to the recent findings, the export of ADMA in endothelial cells is mediated by y + L amino acid transporters (y + LAT-1 and -2) [107,108]: These transporters exchange intracellular cationic amino acids against extracellular neutral amino acids and sodium cations and thus provide an active (energy-dependent) efflux pathway for ADMA. Down-regulation of the y + LAT isoforms in cultured endothelial cells by siRNA, lead to an intracellular ADMA accumulation [107,108]. Consequently, an attractive explanation for the beneficial effects of L-arginine is the export of ADMA via the cationic amino acid transporter (CAT-1) using cationic amino acids (e.g. L-arginine) in exchange in the setting of dysfunctional or down-regulated expression of y + LAT [106]. ...
Many cardiovascular diseases and drug-induced complications are associated with - or even based on - an imbalance between the formation of reactive oxygen and nitrogen species (RONS) and antioxidant enzymes catalyzing the break-down of these harmful oxidants. According to the "kindling radical" hypothesis, the formation of RONS may trigger in certain conditions the activation of additional sources of RONS. According to recent reports, vascular dysfunction in general and cardiovascular complications such as hypertension, atherosclerosis and coronary artery diseases may be connected to inflammatory processes. The present review is focusing on the uncoupling of endothelial nitric oxide synthase (eNOS) by different mechanisms involving so-called "redox switches". The oxidative depletion of tetrahydrobiopterin (BH4), oxidative disruption of the dimeric eNOS complex, S-glutathionylation and adverse phosphorylation as well as RONS-triggered increases in levels of asymmetric dimethylarginine (ADMA) will be discussed. But also new concepts of eNOS uncoupling and state of the art detection of this process will be described. Another part of this review article will address pharmaceutical interventions preventing or reversing eNOS uncoupling and thereby normalize vascular function in a given disease setting. We finally turn our attention to the inflammatory mechanisms that are also involved in the development of endothelial dysfunction and cardiovascular disease. Inflammatory cell and cytokine profiles as well as their interactions, which are among the kindling mechanisms for the development of vascular dysfunction will be discussed on the basis of the current literature.
L-Arginine is the physiological substrate for the nitric oxide synthase (NOS) family, which synthesises nitric oxide (NO) in endothelial and neuronal cells. NO synthesis can be inhibited by endogenous asymmetric dimethylarginine (ADMA). NO has explicit roles in cellular signalling and vasodilation. Impaired NO bioavailability represents the central feature of endothelial dysfunction associated with vascular diseases. Interestingly, dietary supplementation with L-arginine has been shown to alleviate endothelial dysfunctions caused by impaired NO synthesis. In this study the transport kinetics of [3H]-arginine and [3H]-ADMA into the central nervous system (CNS) were investigated using physicochemical assessment and the in situ brain/choroid plexus perfusion technique in anesthetized mice. Results indicated that L-arginine and ADMA are tripolar cationic amino acids and have a gross charge at pH 7.4 of 0.981. L-Arginine (0.00149 +/- 0.00016) has a lower lipophilicity than ADMA (0.00226 +/- 0.00006) as measured using octanol-saline partition coefficients. The in situ perfusion studies revealed that [3H]-arginine and [3H]-ADMA can cross the blood-brain barrier (BBB) and the blood-CSF barrier. [3H]-Arginine (11.6nM) and [3H]-ADMA (62.5nM) having unidirectional transfer constants (Kin) into the frontal cortex of 5.84 +/- 0.86 and 2.49 +/- 0.35 ml.min-1.g-1, respectively, and into the CSF of 1.08 +/- 0.24 and 2.70 +/- 0.90 ml.min-1.g-1, respectively. In addition, multiple-time uptake studies revealed the presence of CNS-to-blood efflux of ADMA. Self- and cross-inhibition studies indicated the presence of transporters at the BBB and the blood-CSF barriers for both amino acids, which were shared to some degree. Importantly, these results are the first to demonstrate: (i) saturable transport of [3H]-ADMA at the blood-CSF barrier (choroid plexus) and (ii) a significant CNS to blood efflux of [3H]-ADMA. Our results suggest that the arginine paradox, in other words the clinical observation that NO-deficient patients respond well to oral supplementation with L-arginine even though the plasma concentration is easily sufficient to saturate endothelial NOS, could be related to ADMA transport.
Many diseases and drug-induced complications are associated with – or even caused by – an imbalance between the formation of reactive oxygen and nitrogen species (RONS) and antioxidant enzymes catalyzing the breakdown of these harmful oxidants. According to the “kindling radical” hypothesis, initial formation of RONS may trigger the activation of additional sources of RONS in certain pathological conditions. This chapter will focus on the uncoupling of endothelial nitric oxide synthase (eNOS) by RONS and will focus on the different “redox switches” that are involved in the uncoupling process of eNOS. The oxidative depletion of tetrahydrobiopterin (BH4), oxidative disruption of the zinc-sulfur cluster in the binding region of the dimeric eNOS complex, and S-glutathionylation of the eNOS reductase domain will be discussed as potential pathways for eNOS uncoupling. In addition, protein kinase C (PKC)-dependent phosphorylation of threonine 495 in the reductase domain, protein tyrosine kinase-2 (PYK-2)-dependent phosphorylation of tyrosine 657 in the reductase domain, RONS-triggered increases in levels of asymmetric dimethylarginine (ADMA), and l-arginine depletion will be highlighted as alternative reasons for dysfunctional eNOS. Finally, the clinical perspectives of eNOS uncoupling (and dysfunction) for cardiovascular disease are presented.
Elevated plasma concentrations of endogenously formed asymmetric (ADMA) and symmetric dimethyl-l-arginine (SDMA) are associated with adverse clinical outcomes. Our aim was to investigate the cellular uptake properties of ADMA by the human cationic amino acid transporter 1 (CAT1; SLC7A1). Human embryonic kidney cells (HEK293) stably overexpressing CAT1 (HEK-CAT1) and vector-transfected control cells (HEK-VC) were established to determine cellular uptake of labeled [(3)H]ADMA and [(3)H]l-arginine. Uptake of ADMA and l-arginine were significantly (p<0.001) higher in HEK-CAT1 than in HEK-VC at all investigated concentrations. Apparent V(max) values of cellular ADMA and l-arginine uptake by CAT1 were 26.9 ± 0.8 and 11.0 ± 0.2 nmol mg protein(-1) min(-1), respectively. K(m) values were 183 ± 21 μmoll(-1) (ADMA) and 519 ± 36 μmoll(-1) (l-arginine). Uptake of ADMA was inhibited by l-arginine and SDMA with IC(50) values (95% CI) of 227 (69-742) μmoll(-1) and 273 (191-390) μmoll(-1), respectively. ADMA and SDMA inhibited CAT1-mediated uptake of l-arginine with IC(50) values of 758 (460-1251) μmoll(-1) and 789 (481-1295) μmoll(-1), respectively. Efflux of ADMA was significantly increased in HEK-CAT1 cells as compared to HEK-VC (p<0.05). CAT1 mediates the cellular uptake of ADMA. In its physiological concentration range ADMA is unlikely to impair CAT1-mediated transport of l-arginine. Conversely, high (but still physiological) concentrations of l-arginine can inhibit CAT1-mediated cellular uptake of ADMA.
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