The inotropic effect of cardioactive glycosides in ventricular myocytes requires Na super(+)-Ca super(2) super(+) exchanger function

Glycoside-induced cardiac inotropy has traditionally been attributed to direct Na super(+)-K super(+)-ATPase inhibition, causing increased intracellular [Na super(+)] and consequent Ca super(2+) gain via the Na super(+)-Ca super(2+) exchanger (NCX). However, recent studies suggested alternative mechanisms of glycoside-induced inotropy: (1) direct activation of sarcoplasmic reticulum Ca super(2+) release channels (ryanodine receptors; RyRs); (2) increased Ca super(2+) selectivity of Na super(+) channels (slip-mode conductance); and (3) other signal transduction pathways. None of these proposed mechanisms requires NCX or an altered [Na super(+)] gradient. Here we tested the ability of ouabain (OUA, 3 mu M), digoxin (DIG, 20 mu M) or acetylstrophanthidin (ACS, 4 mu M) to alter Ca super(2+) transients in completely Na super(+)-free conditions in intact ferret and cat ventricular myocytes. We also tested whether OUA directly activates RyRs in permeabilized cat myocytes (measuring Ca super(2+) sparks by confocal microscopy). In intact ferret myocytes (stimulated at 0.2 Hz), DIG and ACS enhanced Ca super(2+) transients and cell shortening during twitches, as expected. However, prior depletion of [Na super(+)] sub(i) (in Na super(+)-free, Ca super(2+)-free solution) and in Na super(+)-free solution (replaced by Li super(+)) the inotropic effects of DIG and ACS were completely prevented. In voltage-clamped cat myocytes, OUA increased Ca super(2+) transients by 48 plus or minus 4% but OUA had no effect in Na super(+)-depleted cells (replaced by N-methyl-D-glucamine). In permeabilized cat myocytes, OUA did not change Ca super(2+) spark frequency, amplitude or spatial spread (although spark duration was slightly prolonged). We conclude that the acute inotropic effects of DIG, ACS and OUA (and the effects on RyRs) depend on the presence of Na super(+) and a functional NCX in ferret and cat myocytes (rather than alternate Na super(+)-independent mechanisms).