Ouabain increases sarcoplasmic reticulum calcium release in cardiac myocytes.

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(2+) release in intact cat ventricular myocytes under Na(+)-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(+)-free conditions), spontaneous Ca(2+) sparks were prolonged and frequency, amplitude and width were reduced by exposure to ouabain (3 microM). Nearly identical results were obtained with its analogs dihydroouabain or ouabagenin (10 microM). The frequency of spontaneous Ca(2+) waves was also reduced by ouabain. In contrast, ouabain (100 microM) had negligible effects on sparks and waves in rat myocytes in Na(+)-free conditions, consistent with the decreased sensitivity to cardiac glycosides observed in this species. In cat myocytes permeabilized with saponin (0.01%), ouabain (>or=50 nM) decreased spark frequency and increased background SR Ca(2+) leak only when the SR was well loaded (free [Ca(2+)] = 275 nM) and not when SR load was low (free [Ca(2+)] = 50 nM). Similar effects were observed in rat myocytes only when ouabain concentration was 1 microM. These results suggest that the cellular actions of cardiac glycosides may include a direct effect on SR Ca(2+) release, possibly through activation of SR Ca(2+) release channels (ryanodine receptors). In addition, these results are consistent with the idea that direct activation of SR Ca(2+) release is dependent on the extent of SR Ca(2+) load, with elevated load increasing sensitivity of the channel release mechanism to activation by glycoside.

Positive inotropic effects of ouabain in isolated cat ventricular myocytes in sodium-free conditions.

The inotropic and toxic effects of cardiac steroids are thought to result from Na(+)-K(+)-ATPase inhibition, with elevated intracellular Na(+)(Na)causing increased intracellular Ca(2+)(Ca) via Na-Ca exchange. We studied the effects of ouabain on cat ventricular myocytes in Na(+)-free conditions where the exchanger is inhibited. Cell shortening and Ca transients (with fluo 4-AM fluorescence) were measured under voltage clamp during exposure to Na(+)-free solutions [LiCl or N-methyl-D-glucamine (NMDG) replacement]. Ouabain enhanced contractility by 121 +/- 55% at 1 micromol/l (n = 11) and 476 +/- 159% at 3 micromol/l (n = 8) (means +/- SE). Ca transient amplitude was also increased. The inotropic effects of ouabain were retained even after pretreatment with saxitoxin (5 micromol/l) or changing the holding potential to -40 mV (to inactivate Na(+) current). Similar results were obtained with both Li(+) and NMDG replacement and in the absence of external K(+), indicating that ouabain produced positive inotropy in the absence of functional Na-Ca exchange and Na(+)-K(+)-ATPase activity. In contrast, ouabain had no inotropic response in rat ventricular myocytes (10-100 micromol/l). Finally, ouabain reversibly increased Ca(2+) overload toxicity by accelerating the rate of spontaneous aftercontractions (n = 13). These results suggest that the cellular effects of ouabain on the heart may include actions independent of Na(+)-K(+)-ATPase inhibition, Na-Ca exchange, and changes in Na.

Enhancement of L-type Ca(2+) current from neonatal mouse ventricular myocytes by constitutively active PKC-betaII.

The cardiac L-type calcium current (I(Ca)) can be modified by activation of protein kinase C (PKC). However, the effect of PKC activation on I(Ca) is still controversial. Some studies have shown a decrease in current, whereas other studies have reported a biphasic effect (an increase followed by a decrease in current or vice versa). A possible explanation for the conflicting results is that several isoforms of PKC with opposing effects on I(Ca) were activated simultaneously. Here, we examined the influence of a single PKC isoform (PKC-betaII) on L-type calcium channels in isolation from other cardiac isoforms, using a transgenic mouse that conditionally expresses PKC-betaII. Ventricular cardiac myocytes were isolated from newborn mice and examined for expression of the transgene using single cell RT-PCR after I(Ca) recording. Cells expressing PKC-betaII showed a twofold increase in nifedipine-sensitive I(Ca). The PKC-betaII antagonist LY-379196 returned I(Ca) amplitude to levels found in non-PKC-betaII-expressing myocytes. The increase in I(Ca) was independent of Ca(v)1.2-subunit mRNA levels as determined by quantitative RT-PCR. Thus these data demonstrate that PKC-beta is a potent modulator of cardiac L-type calcium channels and that this specific isoform increases I(Ca) in neonatal ventricular myocytes.