Inactivation and Recovery of Nitric Oxide Synthetic Capability in Cytokine-Induced RAW 264.7 Cells Treated with “Irreversible” NO Synthase Inhibitors1

As measured at 100 μmextracellular 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−1with a half-maximal inactivation rate observed at a concentration of 670 μmaminoguanidine (KIvalue). Inactivation of NO synthetic activity in the presence ofNG-methyl-l-arginine similarly followed first-order kinetics with a maximal inactivation rate of 0.07 min−1and aKIvalue of 170 μm. Inactivation of NO synthetic activity in the presence of diphenyliodonium chloride occurred with a maximal inactivation rate of 0.24 min−1with aKIvalue of 14 μm. 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−1with aKIvalue of 18 μm. Cytokine-induced RAW cells were treated with aminoguanidine,NG-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 orNG-methyl-l-arginine 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 accompanied 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.