Differential effects of substrate-analogue inhibitors on nitric oxide synthase dimerization

Nitric oxide synthase (NOS) isoforms are hemoenzymes that are only active as homodimers. We have examined the effect of the substrate-analogue inhibitors, N^G-monomethyl-l-arginine (l-NMA), N^G-nitro-l-arginine (l-NNA), N^G-nitro-l-arginine methyl ester (l-NAME), N⁵-(1-iminoethyl)-l-ornithine (l-NIO), and N⁶-(1-iminoethyl)-l-lysine (l-NIL), the guanidine-containing inhibitor aminoguanidine (AG), and the amidine moiety-containing iNOS-specific inhibitor 1400W, on the formation of NOS dimer. Of these inhibitors, l-NMA effectively not only inhibited iNOS dimerization, but also destabilized its dimeric form in RAW264.7 cells stimulated with lipopolysaccharide plus interferon-γ, but not eNOS dimerization in endothelial cells. Importantly, this inhibition was highly correlated with NO production. These inhibitory effects were significantly reversed by addition of l-arginine. However, l-NNA, l-NAME, and AG in part or significantly increased dimerization of iNOS and eNOS in intact cells, and the other inhibitors assessed did not alter dimerization of iNOS and eNOS. These data taken together suggest that substituted groups of an arginine guanidino moiety play an important role in NOS dimerization as well as its catalytic activity. Our results indicate that l-NMA can inhibit iNOS-dependent NO production by preventing iNOS dimerization and destabilizing its dimeric form.