抄録
Acridine orange (AO), a weakly basic fluorescent dye, is permeable to plasma and vesicle membranes and preferentially remains in intracellular acidic regions. Using fluorescence microscopy, we observed dynamic changes in AO-loaded cultured mouse cells during illumination with blue light. Immediately after the start of illumination, the successive disruption of vesicle membrane was observed as a flash of fluorescence, and shortly after that, blebs were formed on the plasma membrane regardless of the occurrence of vesicle disruption. Vesicle disruption was almost completely inhibited when cells were treated with the vacuolar H+-ATPase inhibitor bafilomycin A1 followed by staining with AO, but not when bafilomycin A1 was treated after AO staining. Thus, the filling of AO in the vesicle, which is driven by vacuolar H+-ATPase, is initially required for vesicle disruption. In contrast, bafilomycin A1 did not prevent plasma membrane blebbing, indicating that the blebs are formed independently of the vesicle disruption. Both the vesicle disruption and the formation of plasma membrane blebs were partially inhibited by removal of oxygen from the cell environment and by singlet oxygen scavengers, sodium azide, ascorbic acid, and L-histidine, but not by the hydroxyl radical scavenger dimethyl thiourea. Thus, both phenomena are likely caused at least in part by the generation of singlet oxygen. These photosensitive features of plasma and vesicle membranes may be based on the use of the photodynamic effect, such as cancer therapy. [J Physiol Sci. 2006;56 Suppl:S111]
