Agmatine Enhances the NADPH Oxidase Activity of Neuronal NO Synthase and Leads to Oxidative Inactivation of the Enzyme

Abstract

It is established that agmatine, an endogenously formed decarboxylated arginine, is a weak competitive inhibitor of neuronal nitric-oxide synthase (nNOS) with an apparent K_i value of 660 μM [Biochem J316:247–249, 1996]. Although agmatine is known to bind to α-adrenergic and imidazoline receptors, it has been suggested that some of the pharmacological actions of agmatine, such as the prevention of morphine tolerance, may be due to the inhibition 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-, concentration-, NADPH-, and calmodulin-dependent irreversible inactivation of nNOS. The kinetics of inactivation could be described by an apparent dissociation constant for the initial reversible complex (K_i) and a pseudo first-order inactivation constant (k_inact) of 29 μM and 0.01 min⁻¹, respectively. As determined by high-performance liquid chromatography analysis, the mechanism of inactivation involves alteration of the prosthetic heme moiety of nNOS, in part to protein-bound products. Moreover, we discovered that 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 inactivation of the enzyme. Both the inactivation of nNOS and the oxidative stress produced should now be considered in the pharmacological actions of agmatine as well as provide insight into the potential biological effects of endogenously formed agmatine.