Heterogeneity of Clara cell glutathione - A possible basis for differencesin cellular responses to pulmonary cytotoxicants

Clara-cell populations show a high degree of variation in susceptibility to injury by bioactivated cytotoxicants. Because glutathione (GSH) is critica l for detoxification of electrophilic metabolites, heterogeneity in Clara c ell GSH levels may lead to a wide range of cytotoxic responses. This study was designed to define the distinct GSH pools within Clara cells, character ize heterogeneity within the population, and examine whether heterogeneity contributes to susceptibility. Using fluorescent imaging combined with high -performance liquid chromatography analysis, semiquantitative measurements were obtained by evaluation of GSH using monochlorobimane and monobromobima ne. In steady-state conditions, the GSH measured in isolated cells was in t he femtomole range, but varied 4-fold between individual cells. Clara cells analyzed in situ and in vitro confirmed this heterogeneity. The response o f these cells to compounds that modulate GSH was also variable. Diethylmale ate depleted GSH, whereas GSH monoethylester augmented it. However, both ac ted nonuniformly in isolated Clara cells. The depletion of intracellular GS H caused a striking decrease in cell viability upon incubation with naphtha lene (NA). The sulfhydryl-binding fluorochrome BODIPY, which colocalized wi th tetramethylrosamine, a mitochondrial dye, demonstrated by confocal micro scopy that cellular sulfhydryls are highest in the mitochondria, next-highe st in cytoplasm, and lowest in the nucleus, These pools responded different ly to modulators of GSH. We concluded that the steady-state intracellular G SH of Clara cells exists in distinct pools and is highly heterogeneous with in the population, and that the heterogeneity of GSH levels corresponds clo sely to the response of Clara cells to injury by NA.