Search for intermediates of Na +,K +-ATPase-mediated [Na +] i/[K +] i-independent death signaling triggered by cardiotonic steroids

Abstract

Previously, we reported that ouabain and other cardiotonic steroids (CTS) kill renal epithelial and vascular endothelial cells via their interaction with the Na +,K +-ATPase α-subunit, but independently of elevation of the [Na +] i/[K +] i ratio. In distinct cell types, side-by-side with inhibition of Na +,K +-ATPase-mediated ion fluxes, CTS trigger [Ca 2+] i oscillation, activation of Ras, mitogen-activated protein kinases (MAPK), phosphoinositide-3 kinase (PI3K), and protein kinase C as well as the production of reactive oxygen species and cytoskeleton reorganization. This study examined the potential involvement of the above-listed intermediates in death signaling triggered by ouabain in C7-Madin–Darby canine kidney cells. In these cells, twofold decreased staining with dimethylthiazol diphenyltetrazolium (MTT) and detachment of up to 80% of dead cells were detected in 6 and 24 h of ouabain addition, respectively. We did not observe any effect of extra- (EGTA) and intracellular (BAPTA) Ca 2+-chelators, [Ca 2+] i-raising compounds (thapsigargin, ATP), inhibitors of Ras signaling (α-hydroxyfarnesyl-sulphosphoric acid), PI3K (wortmannin), MAPK ERK1/2 kinase (PD98059), tyrosine kinases (genistein) as well as activators (4β-PMA, 8-Br-cAMP, 8-Br-cGMP, forskolin) and inhibitors (calphostin) of serine–threonine kinases on MTT staining and death of ouabain-treated cells. Ouabain did not affect cellular redox state and the production of superoxide anion and hydroperoxide. Neither N-acetylcysteine nor reduced gluthatione suppressed the death of ouabain-treated cells. Thus, our results show that none of the above-listed signaling systems plays a major role in the development of N a i + , K i + -independent death machinery triggered by CTS interaction with the Na +,K +-ATPase α-subunit.