The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

Abstract

The mechanisms of cell death signaling triggered by cardiotonic steroids are poorly understood. Based on massive detachment of ouabain-treated Madin-Darby canine kidney (MDCK) cells, it may be proposed that the cytotoxic action of these compounds is mediated by anoikis, i.e. a particular mode of death occurring in cells lacking cell-to-extracellular matrix interactions. We tested this hypothesis. Six hour incubation of MDCK cells with ouabain, marinobufagenin or K+-free medium almost completely blocked Na+,K+-ATPase, increased Na (i) + content by approximately 10-fold and suppressed cell attachment to regular-plastic-plates by up to 5-fold. In contrast, the death of attached cells was observed after 24-h incubation with ouabain but not in the presence of marinobufagenin or K+-free medium. Cells treated with ouabain and undergoing anoikis on ultra-low attachment plates exhibited different cell volume behaviour, i.e. swelling and shrinkage, respectively. The pan-caspase inhibitor z-VAD.fmk and the protein kinase C activator PMA rescued MDCK cells from anoikis but did not influence the survival of ouabain-treated cells, whereas medium acidification from pH 7.2 to 6.7 almost completely abolished the cytotoxic action of ouabain, but did not significantly affect anoikis. Our results show that the Na (i) + ,K (i) + -independent mode of MDCK cell death evoked by ouabain is not mediated by anoikis.

Ouabain modulates epithelial cell tight junction

“…Furthermore, experiments measuring DNA degradation after 24 h of incubation with 10 nM of ouabain do not suggest stimulation of either apoptosis or necrosis [monolayer without ouabain 1.86 ± 0.24 (n = 3), monolayer with ouabain 1.98 ± 0.61 (n = 3)]. Ouabain at 3 μM increases caspase-3 in MDCK cells after 20 h (25). We observe that 10 nM ouabain does not increase the active form of caspase-3 within 2 d of treatment (Fig.…”

Swelling rather than shrinkage precedes apoptosis in serum-deprived vascular smooth muscle cells

“…Importantly, light scattering, refractometry and Coulter electronic size techniques are applicable to suspended cells only and cannot be used to analyze volume changes in cells undergoing shape transition, such as budding in apoptotic cells. It should be also noted that harsh trypsinization routinely employed for cell volume estimation in substrate-attached cells might perturb cell volume and lead to massive cell death (so-called anoikis) [32]. Moreover, Tastesen et al [24] demonstrated different volume behavior in adherent and non-adherent cancer cells undergoing apoptosis.…”

Endogenous Cardiotonic Steroids: Physiology, Pharmacology, and Novel Therapeutic Targets

“…Fascinating inquiries have been made into the relationship between signaling through the Na + /K + -ATPase and cell death (Masuda et al, 1995; Watabe et al, 1996; Orlov and Hamet, 2004; Akimova et al, 2008b; Mijatovic et al, 2008; Stoklosa et al, 2008; Uddin et al, 2008b). In particular, Orlov et al (2004) demonstrated that high concentrations of ouabain seems to induce a “death signal” in endothelial cells that can be readily dissociated from the effects of Na + /K + -ATPase inhibition by extracellular potassium depletion.…”