Isoform-specific Stimulation of Cardiac Na/K Pumps by Nanomolar Concentrations of Glycosides

It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current (I_P) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of I_P because of the following: (1) it was absent in 0 mM [K⁺]_o, as was I_P; (2) it was absent in 0 mM [Na⁺]_i, as was I_P; (3) at reduced [Na⁺]_i, the outward current was reduced in proportion to the reduction in I_P; (4) it was eliminated by intracellular vanadate, as was I_P. Our previous work suggested guinea pig ventricular myocytes coexpress the α₁- and α₂-isoforms of the Na/K pumps. The stimulation of I_P appears to be through stimulation of the high glycoside affinity α₂-isoform and not the α₁-isoform because of the following: (1) regulatory signals that specifically increased activity of the α₂-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the α₁-isoform did not affect the stimulation; (3) changes in [K⁺]_o that affected activity of the α₁-isoform, but not the α₂-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the α₁-isoform but not the α₂-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total I_P increased by 35 ± 10% (mean ± SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the α₂-isoform, then activity of the α₂-isoform increased by 107 ± 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the α₂-isoform, but both the stimulatory and inhibitory concentrations of ouabain were ∼10-fold lower than those for DHO. Stimulation of I_P by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the α₁- and α₃-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the α₃-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of I_P that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of I_P, and where the contributions of the high glycoside affinity α₂- and α₃-isoforms could be separated from that of the α₁-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I_P in heart by nanomolar concentrations of endogenous ouabain-like molecules.