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 produce d a time- and concentration-dependent inactivation of nitric oxide (NO ) synthesis by cytokine-induced RAW cells, Inactivation obeyed first-o rder kinetics and occurred at a maximal rate of 0.22 min(-1) with a ha lf-maximal inactivation rate observed at a concentration of 670 mu M a minoguanidine (K-I value). Inactivation of NO synthetic activity in th e presence of N-G-methyl-L-arginine similarly followed first-order kin etics 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 o f 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. Cyto kine-induced RAW cells were treated with aminoguanidine, N-G-methyl-L- arginine, and diphenyliodonium chloride at concentrations and for a ti me sufficient to completely inactivate NO synthesis by the cells and w ere allowed to recover in drug-free medium. Despite the presence of cy cloheximide, NO synthetic rate recovered from 70 to 90% of its pretrea tment activity over 4 h in cells exposed to either aminoguanidine or N -G-methyl-L-arsnine but did not recover from exposure to diphenyliodon ium chloride. Analysis by sucrose density gradient centrifugation of t he cytochrome c reductase and citrulline-forming activities in extract s of cells recovered from aminoguanidine treatment revealed that recov ery 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 aminogua nidine, functional dimers can be assembled from ''drug-undamaged'' mon omers during the recovery period. (C) 1997 Academic Press.