Cell death triggered by cardiotonic steroids: role of cell volume perturbations and a1-na +, k +-ATPase subunit

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10th international congress
& quot-CELL VOLUME REGULATION: NOVEL THERAPEUTIC TARGETS AND PHARMACOLOGICAL APPROACHES& quot-
y^K 576. 36. 053:577. 15:615. 015. 44
ABSTRACTS
CELL DEATH TRIGGERED BY CARDIOTONIC STEROIDS: ROLE OF CELL VOLUME PERTURBATIONS AND ai-NA+, K±ATPASE SUBUNIT
Akimova, O.A. 1'- 2, Platonova, A.A. 1'- 3, Koltsova, S.V. 1'- 3, Maksimov, G.V. 1, Grygorczyk, R. 3, Van Huysse, J.W. 4, and Orlov, S.N. 1−3
1 Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russian Federation
2 Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow, Russian Federation
3 Research Centre, University of Montreal Hospital, Montreal, Canada
4 Departments of Medicine and Biochemistry, University of Ottawa, Ottawa, Canada
This study examine the role of cell volume modulation and Na+, K±ATPase a-subunits in cell type-specific cytotoxic action of cardiotonic steroids (CTS). Long-term exposure to ouabain caused massive death of MDCK renal epithelial cells expressing a variant of the a1 isoform, CTS-sensitive a1S, documented by their detachment, chromatin cleavage and complete loss of lactate dehydro-genase (LDH) but had no impact on vascular smooth muscle cells (VSMC) from the rat expressing CTS-resistant a1R-Na+, K±ATPase. Neither MDCK nor VSMC were affected by Na+, K±ATPase inhibition in K±free medium. Unlike the distinct impact on cell survival, 2-hr exposure to ouabain and K±free medium led to the same elevation of the [Na+]i/[K+]i ratio in both cell types. 5−10 min before the detachment of ouabain-treated MDCK cells, their volume was augmented by ~30−40% whereas massive LDH release from hyposmotically-swollen cells was documented when their volume was increased by ~5-fold. In additional experments, MDCK cells were stably trans-fected with a cDNA encoding a1R- and a2R-Na+, K±
ATPase, whose expression was confirmed by RT-PCR. At concentration of 10M ouabain led to complete inhibition of 86Rb influx both in mock- and a2R-transfected cells, whereas maximal inhibition of 86Rb influx in a1R-transfectd cells was observed at 1,000M ouabain. In contrast to massive death of mock- and a2R-transfected cells exposed to 3M ouabain, a1R-cells survived after 24-hr incubation with 1000M ouabain. We did not observe any effect of extra and intracellular Ca2±chelators, [Ca2+]i-raising compounds, inhibitors of Ras signaling as well as activators and inhibitors of serine-threonine kinas-es on the death of ouabain-treated MDCK cells. Thus, our results showed that the rupture of plasma membranes in ouabain-treated MDCK cells was not directly caused by cell swelling mediated by Na+, K±ATPase inhibition and inversion of the [Na^i/[K+]i ratio. Downstream intermediates of [Na+]j/[K+]j-independent signaling triggered by interaction of CTS with a1S- but not with a1R-Na+, K±ATPase are currently under investigation.
24
Бюллетень сибирской медицины, 2013, том 12, № 4, с. 24−68

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