Kelly, P.D.; (2011) Elucidation of the biochemical and physiological role of DDAH2. Doctoral thesis , UCL (University College London).

Abstract

The asymmetric methylarginines monomethyl-L-arginine (L-NMMA) and asymmetric dimethylarginine (ADMA) are endogenously occurring inhibitors of the nitric oxide synthase (NOS) enzymes. Elevated plasma ADMA has been identified in a range of human cardiovascular disorders, some of which are associated with impaired NO signaling. The dimethylarginine dimethlyaminohydrolase (DDAH) enzymes are responsible for the degradation of asymmetric methylarginines in vivo. The physiological link between DDAH, ADMA, and NOS was demonstrated by a mouse genetic knockout of DDAH1. Ddah1+/- mice had elevated ADMA concentrations and impaired NOmediated vasoreactivity which resulted in increased systemic vascular resistance and increased blood pressure. In 1999, a second isoform of DDAH was identified, DDAH2. This thesis reports the biochemical characterization of a novel mouse genetic knockout of DDAH2. Although ADMA is considered the most prominent methylarginine species in vivo, this study finds significant concentrations of L-NMMA are also present. Previous work has hypothesized a possible immune function for DDAH2, independent of DDAH1. Mouse peritoneal macrophages were found to contain DDAH2 and exhibit DDAH enzymatic activity, but no DDAH1 was detected. NO generation by cytokine treated peritoneal macrophages from ddah2-/- mice was significantly less then from ddah2+/+ macrophages. Conversely, pharmacological inhibition of DDAH in macrophages did not result in indirect NOS inhibition; this may be due to isoform specificity of the previously developed DDAH inhibitors. To determine the functional significance of the decreased NO generation capacity of ddah2-/- macrophages, their in vivo and in vitro antibacterial properties were investigated. Ddah2 genotype did not have any effect on the ability of peritoneal macrophages to kill S. aureus in vitro. But, in a mouse model of polymicrobial sepsis, ddah2-/- mice showed a significant increase in mortality. Follow-up characterization of this model showed a decrease in bacterial clearance by ddah2-/- mice along with a significant increase in asymmetric methylarginine concentration and changes in the concentration of several cytokines.

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