Pharmacological activation of dimethylarginine dimethylaminohydrolase (DDAH) activity by inorganic nitrate and DDAH inhibition by N^G-hydroxy-l-arginine, N^ω,N^ω-dimethyl-l-citrulline and N^ω,N^ω-dimethyl-N^δ-hydroxy-l-citrulline: results and overview

Abstract

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) (N^G) 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 N^G-hydroxy-l-arginine, an isolable intermediate in NO synthesis, inhibits recombinant DDAH-1 activity (IC₅₀ ≈ 100 µM). Two plant l-citrulline derivatives, i.e., N^ω,N^ω-dimethyl-l-citrulline and N^ω,N^ω-dimethyl-N^δ-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, N^G-hydroxy-l-arginine, N^ω,N^ω-dimethyl-l-citrulline and N^ω,N^ω-dimethyl-N^δ-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.