Abstract 5650: Interactions Between the Endogenous NO Synthase Inhibitor Asymmetric Dimethylarginine and the Leukocyte-derived Hemoprotein Myeloperoxidase

Abstract

Background: ADMA and the NO oxidizing leukocyte-derived hemoprotein MPO are associated with cardiovascular diseases. ADMA and MPO per se coincide with increased oxidative stress and induction of inflammatory responses. Furthermore, the enzymes responsible for ADMA homeostasis (i.e. dimethylarginine dimethylaminohydrolase, DDAH) are regulated in a redox-sensitive fashion. Therefore, the aim of the study was to investigate whether there is an interaction between ADMA and MPO resulting in increased leukocyte (PMN) activation and reduced NO bioavailability. Methods: To test a direct leukocyte activating effect of ADMA, isolated human PMN were subjected to ADMA, SDMA, and fMLP -an inductor of NADPH oxidase activity- to determine PMN degranulation (MPO activity, MPO and elastase levels) and leukocyte superoxide production. NO synthase and DDAH activity was measured by GC-MS and LC-MS/MS, respectively. In vivo, the effect of chronic ADMA (250 μ mol/kg/d; 28 days) treatment on MPO concentrations was induced by osmotic minipumps in C57 mice. To test the effect of MPO on DDAH1 and -2 gene expression, taqman rtPCR technique was performed in liver and kidney tissue of MPO knockout mice. The effect of MPO on DDAH activity in vitro was studied using a recombinant DDAH enzyme. Results: ADMA and fMLP incubation -but not SDMA- significantly increased superoxide production, PMN degranulation and MPO release. Furthermore, ADMA but not SDMA was able to impair NO synthesis of PMN and enhanced PMN’s adhesion to cultured HUVECs in vitro. In vivo, chronic treatment with ADMA resulted in a significant increase of plasma MPO concentrations in C57 mice in contrast to vehicle-treated controls. DDAH1 gene expression was markedly increased in liver and kidney tissue of MPO knockout mice compared to C57 controls. In vitro, MPO and its product HOCl were able to impair recombinant DDAH activity, whereas inactivated MPO was not. Summary and conclusions: ADMA profoundly activates PMN resulting in increased degranulation and MPO release, superoxide production, and impaired NO synthesis. MPO modulates DDAH expression and impairs DDAH activity. Our data revealed a two-way interaction between the ADMA and MPO pathway, which may open a new avenue for treatment of cardiovascular disease.