Ouabain Increases Sarcoplasmic Reticulum Calcium Release in Cardiac Myocytes

Abstract

The inotropic and toxic effects of cardiac glycosides are thought to be related to their ability to inhibit the Na,K-ATPase. We examined the effects of ouabain and its analogs on sarcoplasmic reticulum (SR) Ca 2ϩ release in intact cat ventricular myocytes under Na ϩ -free conditions and in myocytes in which the sarcolemma was permeabilized using saponin so that cytoplasmic ionic composition was fixed by the bath solutions. We also compared ouabain actions in cat myocytes to those in rat myocytes because the latter is considered to be a glycoside-insensitive species. In intact cat myocytes (Na ϩ -free conditions), spontaneous Ca 2ϩ sparks were prolonged and frequency, amplitude and width were reduced by exposure to ouabain (3 M). Nearly identical results were obtained with its analogs dihydroouabain or ouabagenin (10 M). The frequency of spontaneous Ca 2ϩ waves was also reduced by ouabain. In contrast, ouabain (100 M) had negligible effects on sparks and waves in rat myocytes in Na ϩ -free conditions, consistent with the decreased sensitivity to cardiac glycosides observed in this species. In cat myocytes permeabilized with saponin (0.01%), ouabain (Ն50 nM) decreased spark frequency and increased background SR Ca 2ϩ leak only when the SR was well loaded (free [Ca 2ϩ ] ϭ 275 nM) and not when SR load was low (free [Ca 2ϩ ] ϭ 50 nM). Similar effects were observed in rat myocytes only when ouabain concentration was 1 M. These results suggest that the cellular actions of cardiac glycosides may include a direct effect on SR Ca 2ϩ release, possibly through activation of SR Ca 2ϩ release channels (ryanodine receptors). In addition, these results are consistent with the idea that direct activation of SR Ca 2ϩ release is dependent on the extent of SR Ca 2ϩ load, with elevated load increasing sensitivity of the channel release mechanism to activation by glycoside.

Involvement of the Na+/Ca2+ Exchanger in Ouabain-Induced Inotropy and Arrhythmogenesis in Guinea-Pig Myocardium as Revealed by SEA0400

“…Thus, the present results provide pharmacological evidence for a contribution of the reverse mode NCX activity in the cardiac glycoside-induced inotropy. However, other mechanisms such as stimulation of sarcoplasmic reticulum (SR) Ca 2+ release have also been postulated (23). The present results do not exclude such possibilities.…”

Digitalis: new actions for an old drug

“…Finally, channel activation does not occur in the purified RyR, suggesting that it may not be the RyR itself that serves as the binding site for glycoside but that ancillary proteins associated with RyRs [possibly including that recently identified by Fujino et al (35)] might bind the drug and regulate RyR activity in its native environment. This idea was to some extent reinforced by observations that, under Na ϩ -free conditions, there is an increase in an apparent SR Ca 2ϩ leak induced by glycosides as measured in the form of Ca 2ϩ sparks and waves in both intact and permeabilized cat cardiac myocytes (94). These observations were interpreted to suggest that a putative high-affinity glycoside binding site may involve a protein or proteins that exist as part of a complex with the RyR, thus providing the ability to fine-tune and amplify Ca 2ϩ release from the SR.…”

Cardiac Glycosides as Novel Inhibitors of Human Ether-a-go-go-Related Gene Channel Trafficking

“…Nevertheless, considerable evidence exists that cardiac glycosides may also target sites other than the Na ϩ /K ϩ -ATPase. For example, cardiac glycosides directly activate calcium release channels in the sarcoplasmic reticulum of the heart (Sagawa et al, 2002;Nishio et al, 2004) and modulate proteins in the nuclear factor-B signaling pathway (Srivastava et al, 2004). Given this multiplicity of effects, we designed two experiments to directly address possible off-target effects.…”