Reactive oxygen species production by monoamine oxidases in intact cells

Monoamine oxidase (MAO) A and B are mitochondrial enzymes involved in the o xidative deamination of endogenous and exogenous amines. At present, the pr oduction of H2O2 by MAO in intact cells and its functional consequences in cell function have not been extensively investigated. The aim of this study was to define whether, in intact cells, the metabolism of small amounts of MAO substrates was able to induce a detectable H2O2 production. Hydrogen p eroxide production was measured using a luminol-amplified chemiluminescence assay in three cell types, rat mesangial cells, rabbit proximal tubule cel ls and Hep-G2 cells, containing different MAO A/MAO B ratios. Our results s howed that cell incubation with tyramine (50 mu mol/l) led to a time-depend ent H2O2 generation which was fully inhibited by MAO A (clorgyline and RO 4 1-1049) and MAO B (selegiline and RO 19-6327) inhibitors. The extent of inh ibition of H2O2 production by selective inhibitors was in agreement with th e amount of MAO isoforms expressed in each cell type, as determined by West ern blot analysis and enzyme assay. Altogether, these findings show that, i n a normal cell environment, MAO can be a source of reactive oxygen species which could have a functional impact on cell functions. In addition, we pr opose the luminol-amplified chemiluminescence assay as a rapid and sensitiv e procedure to characterize the monoamine oxidase isoforms and their regula tion in intact cells.