Na + /K + pump and endothelial cell survival: [Na + ] i /[K + ] i -independent necrosis triggered by ouabain, and protection against apoptosis mediated by elevation of [Na + ] i

Abstract

Recent studies have demonstrated the tissue-specific effect of Na+/K+ pump inhibition by ouabain and other cardiac glycosides on cell viability. The vascular endothelium is an initial target of cardiac glycosides employed for the management of congestive heart failure as well as circulating endogenous ouabain-like substances (EOLS), the production of which is augmented in volume-expanded hypertension. This study examined the role of the Na+/K+ pump in the survival of cultured porcine aortic endothelial cells (PAEC). Complete Na+/K+ pump inhibition with ouabain led to PAEC death, indicated by cell detachment and decreased staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Based on cell swelling and resistance to benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk) a pan-caspase inhibitor, this type of cell death was classified as necrosis. In contrast to ouabain, Na+/K+ pump inhibition in K+-free medium did not affect PAEC viability and sharply attenuated apoptosis triggered by 3H decay-induced DNA damage. Necrosis evoked by ouabain was preserved after dissipation of the transmembrane gradient of K+ and Na+, whereas dissipation of the Na+ gradient abolished the antiapoptotic action of K+-free medium. Comparative analysis of these results and modulation of intracellular Na+ and K+ content by the above-listed stimuli showed that interaction of ouabain with Na+/K+-ATPase triggered necrosis independently of inhibition of Na+/K+ pump-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio, whereas protection against apoptosis under Na+/K+ pump inhibition in K+-depleted medium was mediated by [Na+]i elevation. The role of Na+/K+ pump-mediated regulation of endothelial cell survival and vascular remodelling seen in hypertension should be investigated further in context of EOLS and chronic treatment with digitalis.

Cardiotonic Steroids Differentially Affect Intracellular Na+ and [Na+] /[K+] -independent Signaling in C7-MDCK Cells

“…Similarly to ouabain, 5 h of incubation in K ϩ -free medium resulted in ϳ6-fold elevation of [Na ϩ ] i content (Fig. 5B) and massive loss of intracellular K ϩ (20). However, neither decreased MTT staining nor cell detachment was detected in K ϩ -free medium (Fig.…”

Ouabain at pathological concentrations might induce damage vascular endothelial cells

“…The effect of ouabain as a sodium pump (Na [7] . The effect of ouabain on cell survival is tissue-specific [8] . Twentyfour hours of treatment with ouabain detaches and kills Ouabain has been known to act as a hormone and is possibly involved in the pathogenesis of hypertension [9] .…”

[Na+] i /[K+] i -independent death of ouabain-treated renal epithelial cells is not mediated by Na+,K+-ATPase internalization and de novo gene expression

“…Surprisingly, we found that in contrast to CTS [14], the 24-h inhibition of Na + ,K + -ATPase in a K + -free medium does not influence the survival of PAEC and MDCK cells [13,15]. Later on, this intriguing observation was confirmed by Contreras et al [16].…”

“…In previous studies, we observed that 6-8-h treatment with 3 μM ouabain did not affect the survival of C7-MDCK cells and massive accumulation of dead cells occurred in 10-12 h [13,15]. Keeping this in mind, we limited the incubation time to 6 h. Figure 2 shows that 6-h exposure to ouabain decreased the amount of biotinylated Na + ,K + -ATPase by approximately twofold but did not significantly affect the total Na + ,K + -ATPase content.…”

“…Thus, a 48-h treatment with high doses of ouabain did not alter the survival of renal epithelial cells from the Rhesus monkey [6,7], human lymphocytes [8], and smooth muscle cells from the rat aorta [9,10] but culminated in massive death of epithelial cells from the Madin-Darby canine kidney (MDCK) [11,12] and endothelial cells from the pig aorta (PAEC) [13]. Surprisingly, we found that in contrast to CTS [14], the 24-h inhibition of Na + ,K + -ATPase in a K + -free medium does not influence the survival of PAEC and MDCK cells [13,15].…”