Inhibitors of V-type ATPases, bafilomycin A1 and concanamycin A, protect against β-amyloid-mediated effects on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction

The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTI), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide-induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysosomal acidification in amyloid peptide-treated differentiated PC12 cell cultures using selective vacuolar-type (V-type) ATPase inhibitors, The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V-type ATPase, Treating nerve growth factor-differentiate PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of beta-amyloid peptides A beta(1-42), A beta(1-40), and A beta(25-35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit A beta uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing A beta-mediated MTT dye conversion also reverse lysosomal pH, These results suggest that lysosomal acidification is necessary for A beta effects on MTT dye conversion.