Cardiotonic Steroids and the Sodium Potassium Pump

Abstract

Cardiotonic steroids (CTSs), such as digoxin, are used to treat heart failure as they inhibit the sodium potassium pump (NKP). However, sporadic studies report NKP stimulation by a CTS called ouabain. This thesis investigates such reports and explores mechanisms for stimulation. Rabbit cardiomyocytes were isolated and voltage clamped, and the NKP current (Ip) identified. Exposure of myocytes to 5 nM or 10 nM ouabain for ~1-minute significantly increased Ip to 0.69±0.09 pA/pF (N=6) and 0.64±0.05 pA/pF respectively vs. 0.46±0.03 pA/pF in 25 controls. Rostafuroxin, a putative ouabain antagonist, abolished this stimulation (Ip=0.40±0.04 pA/pF, N=7). Higher ouabain concentrations, or a longer ouabain exposure duration of 5 m, also abolished stimulation. 1 h incubation with 10 nM ouabain caused NKP inhibition. NKP activity is regulated by glutathionylation, a reversible oxidative modification; Cysless FXYD3 inclusion in pipette solutions, preventing de-glutathionylation, abolished ouabain-induced stimulation. Stimulation was strongly dependent on the presence of the rhamnose sugar moiety of ouabain. 10-500 nM ouabagenin (ouabain without a sugar moiety) did not increase Ip, nor did 5–30 nM digoxin which possesses a different steroid and sugar moiety. Dihydroouabain, differing from ouabain by possessing a saturated lactone, increased Ip at 1 nM to 0.65±0.02 pA/pF, N=6 (p<0.01). Effects of long-term CTS exposure were investigated using cell viability to indirectly measure NKP activity. 24 h low-dose ouabain exposure significantly increased viability in MCF-7 and MDA-MB-468 human breast cancer cells, albeit to a greater degree in MCF-7 cells which are comparatively FXYD-3 rich. This may implicate FXYD in the mechanism of stimulation. As ouabain was previously used therapeutically in humans, this study supports exploration of ouabain as a treatment for new onset atrial fibrillation, to lower high levels of intracellular sodium.