THE RAPID DECLINE OF MTT REDUCTION IS NOT A MARKER OF DEATH SIGNALING IN OUABAIN-TREATED CELLS

Decreased staining with dimethylthiazol diphenyltetrazolium (MTT) is widely used for cell death detection. This study examined MTT assay as a marker of the Na-i(+),K-i(+)-independent mode of cell death revealed in ouabain-treated C7-MDCK cells derived from distal tubule of the Madin-Darby canine kidney. The action of 3-mu M ouabain on MTT reduction in C7-MDCK cells exhibited bipartite kinetics with a rapid similar to 2-fold decline occurring in 30-120 min and a delayed similar to 8-10-fold decrease after 10 hr of ouabain addition. Treatment with ouabain for 18 hr led to 6-fold activation of caspase-3, 4-fold elevation of chromatin fragmentation, and massive cell detachment. Caspase-3 activation, chromatin fragmentation and cell detachment were completely abolished by acidification of the incubation medium from pH 7.2 to 6.7. In contrast, the 2-fold inhibition of MTT reduction seen in 5 hr of ouabain addition was not affected by medium acidification. Within the 5-hr time window, we did not observe any significant impact of ouabain on the cellular redox state estimated by the autofluorescence ratio of reduced pyridine nucleotides and oxidized flavoproteins. In rat aortic endothelial cells and primary astrocytes, exposure to 5-mM ouabain attenuated MTT reduction but did not affect cell survival. Thus, our results show that diminished staining with MTT in ouabain-treated cells is not sufficient proof of triggering of the cell death machinery. We speculate that altered endo-and exocytoses evoked by cardiotonic steroids contribute to decreased MTT reduction.