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

被引:17
作者
Akimova, O. A. [1 ]
Mongin, A. A. [2 ]
Hamet, P. [1 ]
Orlov, S. N. [1 ]
机构
[1] CHUM Hotel Dieu, Ctr Rech, Montreal, PQ H2W 1T7, Canada
[2] Albany Med Coll, Albany, NY 12208 USA
基金
加拿大健康研究院;
关键词
tetrazolium; ouabain; Na+/K+-ATPase; cell death; pH;
D O I
10.1170/T763
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:71 / 77
页数:7
相关论文
共 40 条
  • [31] Apoptosis in vascular smooth muscle cells: Role of cell shrinkage
    Orlov, SN
    Dam, TV
    Tremblay, J
    Hamet, P
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1996, 221 (03) : 708 - 715
  • [32] Activation of cAMP signaling transiently inhibits apoptosis in vascular smooth muscle cells in a site upstream of caspase-3
    Orlov, SN
    Thorin-Trescases, N
    Dulin, NO
    Dam, TV
    Fortuno, MA
    Tremblay, J
    Hamet, P
    [J]. CELL DEATH AND DIFFERENTIATION, 1999, 6 (07) : 661 - 672
  • [33] Inhibition of Na+,K+ pump affects nucleic acid synthesis and smooth muscle cell proliferation via elevation of the [Na+]i/[K+]i ratio:: possible implication in vascular remodelling
    Orlov, SN
    Taurin, S
    Tremblay, J
    Hamet, P
    [J]. JOURNAL OF HYPERTENSION, 2001, 19 (09) : 1559 - 1565
  • [34] Inversion of the intracellular Na+/K+ ratio blocks apoptosis in vascular smooth muscle cells by induction of RNA synthesis
    Orlov, SN
    Taurin, S
    Thorin-Trescases, N
    Dulin, NO
    Tremblay, J
    Hamet, P
    [J]. HYPERTENSION, 2000, 35 (05) : 1062 - 1068
  • [35] Inhibition of Na+,K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i/[K+]i ratio
    Pchejetski, D
    Taurin, S
    Sarkissian, SD
    Lopina, OD
    Pshezhetsky, AV
    Tremblay, J
    deBlois, D
    Hamet, P
    Orlov, SN
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2003, 301 (03) : 735 - 744
  • [36] Proteome analysis and functional expression identify mortalin as an antiapoptotic gene induced by elevation of [Na+]i/[K+]i ratio in cultured vascular smooth muscle cells
    Taurin, S
    Seyrantepe, V
    Orlov, SN
    Tremblay, TL
    Thibault, P
    Bennett, MR
    Hamet, P
    Pshezhetsky, AV
    [J]. CIRCULATION RESEARCH, 2002, 91 (10) : 915 - 922
  • [37] Antiapoptotic effect of ouabain on human umbilical vein endothelial cells
    Trevisi, L
    Visentin, B
    Cusinato, F
    Pighin, I
    Luciani, S
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2004, 321 (03) : 716 - 721
  • [38] TREVISI L, 2005, 4 WORLD C CELL MOL B, P122
  • [39] VISTICA DT, 1991, CANCER RES, V51, P2515
  • [40] Inhibition of Na,K-ATPase activates P13 kinase and inhibits apoptosis in LLC-PK1 cells
    Zhou, XM
    Jiang, GS
    Zhao, AP
    Bondeva, T
    Hirszel, P
    Balla, T
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2001, 285 (01) : 46 - 51