Pharmacological activation of dimethylarginine dimethylaminohydrolase (DDAH) activity by inorganic nitrate and DDAH inhibition by NG-hydroxy-l-arginine, Nω,Nω-dimethyl-l-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-l-citrulline: results and overview
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Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of nitric oxide (NO) synthase. SDMA is excreted in the urine without major metabolization. About 10% of daily produced ADMA are excreted unchanged in the urine. The major elimination route of ADMA (about 90%) involves its hydrolysis to dimethylamine (DMA) and l-citrulline by dimethylarginine dimethylaminohydrolase (DDAH) and excretion of DMA in the urine. High circulating and low excretory concentrations of ADMA are considered risk factors. Experimentally, DDAH activity can be inhibited by SH-specific agents such as inorganic and organic mercury compounds, and by S-nitrosothiols which block the SH group of a particular cysteine moiety of DDAH that is essential for its hydrolytic activity. Alternatively, DDAH activity can be inhibited by organic compounds that compete with the substrate ADMA for DDAH. Arginine analogs that contain substituents on guanidine nitrogen atom(s) (NG) represent a class of DDAH inhibitors. In the present study, we investigated the effects of physiological and natural amino acid derivatives of l-arginine and l-citrulline as well as of nitrate and nitrite, the major circulating and excretory metabolites of NO and NO donating drugs. Here, we report for the first time that the physiological NG-hydroxy-l-arginine, an isolable intermediate in NO synthesis, inhibits recombinant DDAH-1 activity (IC50 ≈ 100 µM). Two plant l-citrulline derivatives, i.e., Nω,Nω-dimethyl-l-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-l-citrulline (connatin), were found to inhibit almost completely hepatic DDAH activity in vitro in rat homogenate at a concentration of 100 µM each. At pharmacological concentrations (i.e., 1 mM), inorganic nitrate, but not inorganic nitrite, was found to increase rat liver DDAH activity. In urine of 18 patients with Becker’s muscular dystrophy, nitrate was found to correlate closely with DMA (Spearman, r = 0.73, p = 0.002). In summary, NG-hydroxy-l-arginine, Nω,Nω-dimethyl-l-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-l-citrulline are novel inhibitors of DDAH activity. This article provides an overview of amino acid-based DDAH inhibitors and discusses potential underlying inhibition mechanisms.
KeywordsActivation ADMA Connatin Dimethyl citrulline NG-Hydroxy-l-arginine Inhibition Nitrate
Becker’s muscular dystrophy
Nitric oxide synthase
Protein arginine methyltransferase
Drs. M. Nukina and C. Kimura (Course of the Science of Bioresources, the United Graduate School of Agricultural Sciences, Iwate University (Yamagata University), Yamagata, Japan) are sincerely thanked for providing connatin and dimethyl citrulline which were isolated, purified and characterized structurally in their laboratory.
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflicts of interest.
Rats received humane care and the study protocol complied with the institutional guidelines of the Hannover Medical School.
- Achan V, Broadhead M, Malaki M, Whitley G, Leiper J, MacAllister R, Vallance P (2003) Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol 23:1455–1459CrossRefPubMedGoogle Scholar
- Ashmore T, Fernandez BO, Branco-Price C, West JA, Cowburn AS, Heather LC, Griffin JL, Johnsen RS, Feelisch M, Murray AJ (2014) Dietary nitrate increasing arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart. J Physiol 592:4715–4731CrossRefPubMedPubMedCentralGoogle Scholar
- Boucher JL, Custot J, Vadon S, Delaforge M, Lepoivre M, Tenu JP, Yapo A, Mansuy D (1994) N omega-hydroxyl-l-arginine, an intermediate in the l-arginine to nitric oxide pathway, is a strong inhibitor of liver and macrophage arginase. Biochem Biophys Res Commun 203:1614–1621CrossRefPubMedGoogle Scholar
- Chobanyan K, Thum T, Suchy MT, Zhu B, Mitschke A, Gutzki FM, Beckmann B, Stichtenoth DO, Tsikas D (2007) GC-MS assay for hepatic DDAH activity in diabetic and non-diabetic rats by measuring dimethylamine (DMA) formed from asymmetric dimethylarginine (ADMA): evaluation of the importance of S-nitrosothiols as inhibitors of DDAH activity in vitro and in vivo in humans. J Chromatogr B 858:32–41CrossRefGoogle Scholar
- Hanff E, Hafner P, Bollenbach A, Bonati U, Kayacelebi AA, Fischer D, Tsikas D (2018) Effects of single and combined metformin and L-citrulline supplementation on l-arginine-related pathways in Becker muscular dystrophy patients: possible biochemical and clinical implications. Amino Acids 50:1391–1406CrossRefPubMedGoogle Scholar
- Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, Arghandawi S, Pearl V, Benjamin N, Loukogeorgakis S, Macallister R, Hobbs AJ, Webb AJ, Ahluwalia A (2010) Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO. Hypertension 56:274–281CrossRefPubMedGoogle Scholar
- Kimura C, Nukina M, Igarashi K, Sugawara Y (2005) beta-Hydroxyergothioneine, a new ergothioneine derivative from the mushroom Lyophyllum connatum, and its protective activity against carbon tetrachloride-induced injury in primary culture hepatocytes. Biosci Biotechnol Biochem 69:357–363CrossRefPubMedGoogle Scholar
- Leiper J, Murray-Rust J, McDonald N, Vallance P (2002) S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase. Proc Natl Acad Sci U S A 99:13527–13532CrossRefPubMedPubMedCentralGoogle Scholar
- Leiper J, Nandi M, Torondel B, Murray-Rust J, Malaki M, O’Hara B, Rossiter S, Anthony S, Madhani M, Selwood D, Smith C, Wojciak-Stothard B, Rudiger A, Stidwill R, McDonald NQ, Vallance P (2007) Disruption of methylarginine metabolism impairs vascular homeostasis. Nat Med 13:198–203CrossRefPubMedGoogle Scholar
- Murray-Rust J, Leiper J, McAlister M, Phelan J, Tilley S, Santa Maria J, Vallance P, McDonald N (2001) Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylaminohydrolase. Nat Struct Biol 8:679–683 (Erratum in: Nat Struct Biol 8:818) CrossRefPubMedGoogle Scholar
- Tsikas D, Schwedhelm KS, Surdacki A, Giustarini D, Rossi R, Kukoc-Modun L, Kedia G, Ückert S (2018b) S-Nitroso-N-acetyl-l-cysteine ethyl ester (SNACET) and N-acetyl-l-cysteine ethyl ester (NACET)-cysteine-based drug candidates with unique pharmacological profiles for oral use as NO, H2S and GSH suppliers and as antioxidants: results and overview. J Pharm Anal 8:1–9CrossRefPubMedGoogle Scholar
- Wójcicka G, Jamroz-Wiśniewska A, Czechowska G, Korolczuk A, Marciniak S, Bełtowski J (2016) The paraoxonase 1 (PON1), platelet-activating factor acetylohydrolase (PAF-AH) and dimethylarginine dimethylaminohydrolase (DDAH) activity in the metformin treated normal and diabetic rats. Eur J Pharmacol 789:187–194CrossRefPubMedGoogle Scholar