Formation of guanidinosuccinic acid, a stable nitric oxide mimic, from argininosuccinic acid and nitric oxide-derived free radicals

Guanidinosuccinic acid (GSA) is noted for its nitric oxide (NO) mimicking a ctions such as vasodilatation and activation of the N-methyl-D-aspartate (N MDA) receptor. We have reported that GSA is the product of argininosuccinat e (ASA) and some reactive oxygen species, mainly the hydroxyl radical. We t ested for GSA synthesis in the presence of NO donors. ASA (1 mM) was incuba ted with NOR-2, NOC-7 or 3-morpholinosydomine hydrochloride (SIN-1) at 37 d egrees C. GSA was determined by HPLC using a cationic resin for separation and phenanthrenequinone as an indicator. Neither NOR-2 or NOC-7 formed GSA. SIN-1, on the other hand, generates NO and the superoxide anion which, in hull, generated peroxynitrite which was then converted to the hydroxyl radi cal. Incubation of ASA with SIN-1 leads, via this route, to GSA. When ASA w as incubated with 1 mM SIN-1, the amount of GSA produced depended on the in cubation time and the concentration of ASA. Among the tested SIN-1 concentr ations, from 0.5 to 5 mM, GSA synthesis was maximum at 0.5 mM and decreased with increasing concentrations of SIN-1. Carboxy-PTIO, a NO scavenger, com pletely inhibited GSA synthesis. SOD, a superoxide scavenger, decreased GSA synthesis by 20%, and catalase inhibited CSA synthesis only by 12%; DMSO, a hydroxyl radical scavenger completely inhibited GSA synthesis in the pres ence of SIN-1. These data suggest that the hydroxyl radical derived from a combination of NO and the superoxide anion generates GSA, a stable NO mimic . Meanwhile, synthesis of GSA by NO produces reactive oxygen and activates the NMDA receptor that generates NO from GSA, suggesting a positive feed ba ck mechanism.