Agmatine enhances the NADPH oxidase activity of neuronal NO synthase and leads to oxidative inactivation of the enzyme

It is established that agmatine, an endogenously formed decarboxylated argi nine, is a weak competitive inhibitor of neuronal nitric-oxide synthase (nN OS) with an apparent K-i value of 660 muM [Biochem J 316: 247-249, 1996]. A lthough agmatine is known to bind to alpha -adrenergic and imidazoline rece ptors, it has been suggested that some of the pharmacological actions of ag matine, such as the prevention of morphine tolerance, may be due to the inh ibition of nNOS. In the current study, we have discovered that agmatine, at concentrations much lower than the reported K-i value, leads to a time-, c oncentration-, NADPH-, and calmodulin-dependent irreversible inactivation o f nNOS. The kinetics of inactivation could be described by an apparent diss ociation constant for the initial reversible complex (K-i) and a pseudo fir st-order inactivation constant (k(inact)) of 29 muM and 0.01 min(-1), respe ctively. As determined by high-performance liquid chromatography analysis, the mechanism of inactivation involves alteration of the prosthetic heme mo iety of nNOS, in part to protein-bound products. Moreover, we discovered th at agmatine causes a 3-fold increase in the NADPH oxidase activity of nNOS leading to the production of H2O2 and is a likely cause for the inactivatio n of the enzyme. Both the inactivation of nNOS and the oxidative stress pro duced should now be considered in the pharmacological actions of agmatine a s well as provide insight into the potential biological effects of endogeno usly formed agmatine.