Superoxide generation system as a target for evaluation of antiallergic drugs

Rat peritoneal mast cells were stimulated in generate superoxide anion (O2) by the addition of compound 48/80 (48/80), a typical histamine releaser. Biphasic elevation of [Ca²]i was also induced by 48/80. The first phase was inhibited by TMB-8 and the second by diltiazem. And 48/80-induced O2 generation was inhibited by TMB-8, forskolin, isoprenaline, dibutyryl cAMP, KT 5926, a MLCK inhibitor, and manoalide, a phospholipase A2 inhibitor, and enhanced by KT-5720, an A-kinase inhibitor. Our data suggest the followings: (1) 48/80-induced O2 generation may be initiated by an increase of [Ca²⁺]i via cAMP-dependent protein phosphorylation such as MLCK phosphorylation; (2) the mechanisms of O2 generation in mast cells may be similar to that in neutrophils (NADPH-oxidase system). Epinastine, ketotifen, oxatomide and mequitazine dose-dependently prevented the N-formyl-Met-Leu-Phe- and phorbol 12-myristate 13-acetate-induced O2 generation from rat neutrophils, but cromolyn sodium did not prevent it. When membrane and cytosol fractions were incubated with each drug, epinastine, ketotifen and mequitazine prevented O2 generation. On the other hand, when only the membrane fraction was incubated with each drug, ketotifen and mequitazine prevented O2 generation, but epinastine did not. Epinastine may inhibit the NADPH oxidase system through the obstruction of NADPH oxidase-associated cytosol components. In conclusion, our studies suggest that the inhibitory effects on O2 generation may be useful as a new marker in the evaluation of innovative antiallergic drugs.