Possible Involvement of NADPH Oxidase in Lanthanide Cation-Induced Superoxide Anion Generation in BY-2 Tobacco Cell Suspension Culture

A rapid and concentration-dependent generation of superoxide anion (·O2−), measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent, was observed when two lanthanide salts (LaCl3 and GdCl3) were added to tobacco (Nicotiana tabacum) cell suspension culture. Addition of superoxide dismutase (480 U·ml−1) and Tiron (5 μmol·L−1) to cell culture suspension decreases the level of lanthanide cation-induced ·O2− generation, suggesting that ·O2− generation is extra-cellular. Pretreatment of the cell culture suspension with diphenyleneiodonium (10 and 50 μmol·L−1), quinacrine (1 and 5 mmol·L−1) and imidazol (10 mmol· L−1), inhibitors of NADPH oxidase, notably inhibits the generation of superoxide induced by lanthanide cation, implying the possible involvement of activation of NADPH oxidase. In addition, addition of SHAM (1 and 5 mmol·L−1), azide (0.2 and 1 mmol·L−1), inhibitor of peroxidase, has no influence on ·O2− generation.