Abstract
Purpose::
In a previous study, we have demonstrated that ethambutol, an anti-tuberculosis agent, induces vacuoles and cell death in retinal cell culture. Since the origin of vacuoles was not identified, in the present study, we examined the possibility that ethambutol-induced vacuoles derive from organelles such as mitochondria or lysosomes.
Methods::
Retinal cell cultures were prepared from newborn rats as previously described. For staining of lysosomes and mitochondria, Lysotracker Red DND-99 and Mitotracker Red CM-H2XROS (Molecular Probes) were used, respectively. For zinc fluorescence, FluoZin-3 was used. After double staining, cells were observed under the confocal microscope (UltraView, Perkin Elmer). Immunocytochemistry to LAMP (Lysosomal membrane-associated glycoproteins) was done.
Results::
24 hr after exposure to 1 mM ethambutol, severe vacuolar changes were observed. Staining with lysotracker showed that most of vacuoloes were probably enlarged lysosomes. In contrast, mitotracker staining revealed no overlap between mitochondria and vacuoles. Since ethambutol-induced vacuole formation was dependent on endogenous zinc, we additionally looked for the presence of zinc in vacuoles. FluoZin-3 staining showed that almost all vacuoles contain high levels of labile zinc. The zinc fluorescence was quenched with TPEN, a cell-permeant zinc chelator. In contrast, Fluo-3, a fluorescent calcium dye, did not stain vacuoles, either. LAMP immunoreactivity was found on the membrane of vacuoles at early time points, which subsequently decreased.
Conclusions::
The present results suggest that ethambutol-induced vacuoles in retinal cells are likely enlarged lysosomes. Although the mechanism is unknown, these vacuoles contain high levels of zinc, and hence may be a form of "zincosomes". The possible link between zinc-containing vacuole formation and cell death seems to warrant future investigations.
Keywords: retina • apoptosis/cell death • retinal degenerations: cell biology