AMINOGUANIDINE IS AN ISOFORM-SELECTIVE, MECHANISM-BASED INACTIVATOR OF NITRIC-OXIDE SYNTHASE

Aminoguanidine produces a time-dependent inactivation of the citrulline forming activity of all three nitric oxide synthase isoforms that is blocked by arginine. Aminoguanidine inactivates both the NADPH oxidase and citrulline forming activities of GH(3) pituitary constitutive nitric oxide synthase (cNOS) but does not alter its cytochrome c reductase activity, GH(3) pituitary cells contain an NOS isoform identical physically, kinetically, and immunologically to cerebellar neuronal NOS (Wolff and Datto, Biochemical J, (1992) 285, 201-206), The inactivation of GH(3) cNOS NADPH oxidase activity, as measured without added tetrahydrobiopterin cofactor, is saturable, is inhibited by arginine, and follows pseudo-first-order kinetics with an inactivation rate constant of 0.25 min(-1) and a K-i value of 0.83 mM aminoguanidine, The inactivation of the citrulline forming activity of GH(3) cNOS by aminoguanidine was not saturable by aminoguanidine, Aminoguanidine, at concentrations in the millimolar range, inhibited the citrulline forming activity of endothelial cNOS by an apparently nonsaturable mechanism, Aminoguanidine inactivates the citrulline forming activity of murine macrophage iNOS, The inactivation is saturable and follows pseudo-first-order kinetics with an inactivation rate constant of 0.46 min(-1) and a K-i value of 16 mu M. The inactivation of the constitutive isoforms of nitric oxide synthase by aminoguanidine required the concurrent presence of Ca2+, calmodulin, NADPH, tetrahydrobiopterin, and oxygen in preincubations and was not reversed either by dilution or dialysis, These observations support the assertion that aminoguanidine is a mechanism-based inactivator of the nitric oxide synthase isoforms and exhibits marked specificity for the inactivation of the inducible isoform. (C) 1995 Academic Press, Inc.