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Asymmetric dimethylarginine (ADMA) is a product of the post-translational methylation of arginine residues within proteins, and the subsequent proteolysis of these arginine methylated proteins. These methylated arginine residues are competitive inhibitors of all nitric oxide synthases, competing with L-arginine to bind the active site of NOS. To date, they are the only known by-products of post- translational protein modification to have biological effects.
Protein arginine methylation is a common post-translational modification, and has been shown to coordinate cellular functions such as signal transduction, transcriptional regulation and protein-protein interactions79. ADMA is synthesized following the methylation of arginine residues in proteins by a group of methyltransferases that are termed protein arginine methyl‐transferases (PRMTs)80. To date, 11 mammalian PRMTs have been identified. The three methylated arginine products are N-monomethyl-L-arginine (L-NMMA), NN-symmetric dimethylarginine (SDMA) and ADMA. Only L-NMMA and ADMA are inhibitors of NOS and circulating levels of ADMA are considerably higher than L-NMMA, hence ADMA is considered the principal methylarginine inhibitor of NOS activity.
Methylarginines only appear in the cytosol as a result of protein degradation, and no direct synthetic route for the production of ADMA, SDMA and L‐NMMA from free arginine has yet been identified. Furthermore, the synthesis and degradation of methylated argenines are closely coupled with the synthesis and degradation of methylated proteins81. Thus, intracellular ADMA levels are governed by PRMT activity, protein turnover and clearance.
Intracellular levels of ADMA are in the low micromolar range, whereas intracellular arginine levels are 10-100 fold greater. However, despite this vast excess of arginine, supplementation of arginine can enhance endothelial function through increased NO generation82. This has been termed the ‘arginine paradox’. However, recent work in human endothelial cells has demonstrated that not all intracellular arginine is available for metabolism by membrane bound eNOS83. Moreover, enzyme kinetic studies have demonstrated that even with physiological concentrations of L-arginine, dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular ADMA concentrations as low 5uM84.Therefore, ADMA is likely to be a critical regulator of endothelial function at pathophysiological levels.
ADMA is removed from the body through predominantly by metabolism by the dimethylarginine dimethylaminohydrolase (DDAH) enzymes, although renal excretion and metabolism by the alternative AGX-2 pathway also occurs (figure 1.4)85, 86. There are 2 DDAH enzymes, DDAH-1 and DDAH-2, although DDAH-1 has far greater hydrolase activity than DDAH-2 and hence is the principal pathway of ADMA elimination87. Heterozygous deletion of DDAH-1 results in a 40-45% decrease in total DDAH activity in vivo, and also causes a phenotype of systemic hypertension and endothelial dysfunction88.
It follows, therefore, that pharmacological modulation of methylarginine levels is an attractive therapeutic strategy in conditions characterised by decreased NO bioavailability and endothelial dysfunction, and further that this could be achieved through targeting methylarginine synthesis by PRMTs or by targeting degradation through DDAHs. However, PRMTs may not be suitable targets for pharmacological manipulation since they are essential for many fundamental biological processes.
For example PRMT1 plays an important role in the regulation of histone function, and deletion of PRMT1 is lethal in utero89. Additionally, targeting PRMT activity is complicated by the presence of numerous PRMT isoforms that share substantial sequence homology90.
By contrast, hepatic DDAH-1 is an attractive target for therapy in cirrhosis and portal hypertension. The liver is a major site of DDAH-1 expression and DDAH activity91. Plasma levels of ADMA are elevated in cirrhosis, and are elevated further in ACLF precipitated by alcoholic hepatitis 72. Hepatic levels of ADMA correlate with HVPG in patients with ACLF, associated with decreased hepatic expression of DDAH-1. Moreover, DDAH-1 is sensitive to oxidative stress92, hence ROS production by activated KCs is hypothesized to decrease DDAH-1 expression and activity, and thereby increase levels of the eNOS inhibitor ADMA, thus decreasing local NO generation (figure 1.3).
A further evolving area of interest is ADMA-independent actions of DDAH-1, possibly mediated through direct protein-protein interaction. DDAH-1 has been shown to directly interact and regulate the phosphorylation of neurofibromin93. Additionally, DDAH-1 is thought to phosphorylate Akt independent of ADMA metabolizing activity94, as well as play a role in cell cycle regulation95. These observations, which require further study, have important implications for any off-target effects of therapeutic strategies to augment DDAH-1.
Unlike DDAH-1, DDAH-2 is expressed in immune cells and has been suggested to play a role in the regulation of iNOS-mediated NO generation in conditions of inflammation and infection96. The genetic location of DDAH-2 in the major histocompatibility complex III region of chromosome 6 lends support to this
hypothesis96, as does data demonstrating an association between human DDAH-2 promoter polymorphisms and outcome in sepsis97. Recently, Lambden et al characterized the phenotype of Ddah2-/- mice, which display unchanged basal blood pressure, unlike the Ddah1+/- mouse which displays systemic hypertension98. Moreover, the Ddah2-/- mouse displays increased sensitivity and mortality to polymicrobial sepsis compared to wild type, consistent with a role in immune regulation and function.
DDAH L-Cit DMA AGXT-2 DGV Urine
Figure 1.4: The DDAH-ADMA pathway - adapted from Leiper and Caplin99. L-arginine (L-Arg) is present in the circulation at >100 times the concentrations of the free endogenous methylarginines: ADMA and symmetric dimethylarginine (SDMA). ADMA but not SDMA inhibits all 3 isoforms of nitric oxide synthase (NOS), decreasing the production of nitric oxide. L-arginine and the free
methylarginines are thought to enter the cell (shown on the left) through the y+ transporter. ADMA and SDMA are generated intracellularly following the methylation, by protein-arginine
methyltransferases (PRMT), and subsequent proteolysis, of constituent protein arginine residues. ADMA but not SDMA is hydrolyzed by DDAH to form dimethylamine (DMA) and L-citrulline (L-Cit), which can be reincorporated into proteins. The major pathway of ADMA elimination is metabolism by DDAH-1 with the product DMA excreted in the urine. Both SDMA and ADMA are also substrates for alanine-glyoxylate aminotransferase-2 (AGXT2), leading to the formation of symmetrical and asymmetrical α-keto-δ-dimethylguanidino valeric acid (DGV) that is also excreted in the urine.