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

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Abstract
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 (MTT), 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 lysbsomal acidification in amyloid peptide-treated differentiated PC12 cell cultures using selective vacuolar-type (N-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-differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of β-amyloid peptides Aβ(1-42), Aβ(1-40), and Aβ(25-35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Aβ uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Aβ-mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Aβ effects on MTT dye conversion.