Inactivation and recovery of nitric oxide synthetic capability in cytokine-induced RAW 264.7 cells treated with ''irreversible'' NO synthase inhibitors

As measured at 100 mu M extracellular arginine, aminoguanidine produced a time- and concentration-dependent inactivation of nitric oxide (NO) synthesis by cytokine-induced RAW cells, Inactivation obeyed first-order kinetics and occurred at a maximal rate of 0.22 min(-1) with a half-maximal inactivation rate observed at a concentration of 670 mu M aminoguanidine (K-I value). Inactivation of NO synthetic activity in the presence of N-G-methyl-L-arginine similarly followed first-order kinetics with a maximal inactivation rate of 0.07 min(-1) and a K-I value of 170 mu M. Inactivation of NO synthetic activity in the presence of diphenyliodonium chloride occurred with a maximal inactivation rate of 0.24 min(-1) with a K-I value of 14 mu M. Diphenyliodonium chloride also produced a first-order rate of inactivation of cytokine-inducible nitric oxide synthase (iNOS) activity affinity purified from cytokine-induced RAW cells with a maximal inactivation rate of its cytochrome c reductase activity of 0.24 min(-1) with a K-I value of 18 mu M. Cytokine-induced RAW cells were treated with aminoguanidine, N-G-methyl-L-arginine, and diphenyliodonium chloride at concentrations and for a time sufficient to completely inactivate NO synthesis by the cells and were allowed to recover in drug-free medium. Despite the presence of cycloheximide, NO synthetic rate recovered from 70 to 90% of its pretreatment activity over 4 h in cells exposed to either aminoguanidine or N-G-methyl-L-arsnine but did not recover from exposure to diphenyliodonium chloride. Analysis by sucrose density gradient centrifugation of the cytochrome c reductase and citrulline-forming activities in extracts of cells recovered from aminoguanidine treatment revealed that recovery was accompa nied by a diminished population of iNOS monomers with an increased population of iNOS dimers. This observation is consistent with the hypothesis that for the mechanism-based inactivator aminoguanidine, functional dimers can be assembled from ''drug-undamaged'' monomers during the recovery period. (C) 1997 Academic Press.