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 microM 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 microM 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 microM. 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 microM. 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 microM. 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-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.
Wolff
D J
DJ
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854, USA.
Lubeskie
A
A
Li
C
C
D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006146:Guanidines / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D009861:Onium Compounds / Q000494:pharmacology; D011994:Recombinant Proteins; D019323:omega-N-Methylarginine / Q000494:pharmacology
D001713:Biphenyl Compounds; D016207:Cytokines; D004791:Enzyme Inhibitors; D006146:Guanidines; D008070:Lipopolysaccharides; D009861:Onium Compounds; D011994:Recombinant Proteins; C031291:diphenyliodonium; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; C004479:pimagedine