Dynamic changes in the state of actin polymerization in human alveolar cells exposed to the oxidant agent paraquat

To investigate the role of dynamic changes in actin cytoskeleton in cellular response to oxidative stress, we have analyzed the state of actin polymerization and synthesis in human alveolar cells exposed to paraquat, an oxidant agent. Cellular content of monomeric actin was assayed by DNase I inhibition. It decreased significantly in treated cells and depended on paraquat concentration. Paraquat treatment of cells caused an increase of the filamentous pool of actin and a parallel decrease of the monomeric one. Such shift was shown to be irreversible. SDS-PAGE of cytoskeletal fractions was performed under reducing and non-reducing conditions. No cross-linking of actin monomers to form large aggregates appeared to be related to the observed paraquat-induced increase of the filamentous actin pool. Morphological analyses by indirect immunofluorescence and ultrastructural examination confirmed the presence of microfilaments in treated cells. Conventional bundles of filaments were not observed, but numerous single filaments appeared dispersed within the cytoplasm. Pulse-chase experiments showed a strong increase of de novo synthesis of actin in treated cells, whereas actin degradation rate remained unaffected. In conclusion, the different approaches lead to a concordant picture of cellular response to oxidant stress at the level of the actin filament system. Actin pools are modified: the overall number of filaments increases, whereas the monomeric species decreases. As a result of the shift of actin from the monomeric pool to the filamentous one, the de novo synthesis of actin is increased.