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

Abstract

The inotropic and toxic effects of cardiac steroids are thought to result from Na+-K+-ATPase inhibition, with elevated intracellular Na+(Na[Formula: see text])causing increased intracellular Ca2+(Ca[Formula: see text]) 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[Formula: see text] 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 μmol/l ( n = 11) and 476 ± 159% at 3 μmol/l ( n = 8) (means ± SE). Ca[Formula: see text] transient amplitude was also increased. The inotropic effects of ouabain were retained even after pretreatment with saxitoxin (5 μmol/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 μmol/l). Finally, ouabain reversibly increased Ca2+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[Formula: see text].

Effect of Cardiac Glycosides on Action Potential Characteristics and Contractility in Cat Ventricular Myocytes: Role of Calcium Overload

“…In the experiments in which I Ca was measured, a low concentration of Ba 2ϩ (0.2 mM) was sufficient to block background K ϩ current but had little effect on magnitude and kinetics of I Ca in the presence of 1.8 mM CaCl 2 . We have used this approach previously (Nishio et al, 2002) and found it to be a useful means to measure I Ca under conditions in which normal physiological conditions are otherwise maintained (physiological [Ca 2ϩ ], temperature, K ϩ gradient). Molecular Structures of the Six Cardiac Glycosides Studied.…”

Digitalis: new actions for an old drug

“…n ϭ 6 myocytes from 2 to 4 cats for each point. strom's group (95) reported that patch-clamped myocytes under Na-free conditions showed increased Ca 2ϩ transient magnitude and a positive inotropic effect in a glycoside-sensitive species (cat) but not in an insensitive species (rat). As a result of these observations made over the last nearly half century, it may still be necessary to consider that Na i ϩ accumulation may not be the only mechanism responsible for all of the cardiac actions of digitalis.…”

Ouabain Increases Sarcoplasmic Reticulum Calcium Release in Cardiac Myocytes

“…Myocytes were loaded with fluo-4AM (5 M for 30 min at room temperature) then washed twice in normal Tyrode's solution before being placed in the experimental chamber and allowed to settle to the laminin-coated coverslip floor. Superfusion was then initiated with Na ϩ -free solution (Li ϩ replacement) which had a low CaCl 2 to prevent cell death due to Ca 2ϩ overload (Nishio et al, 2002). It is not known whether these conditions are sufficient to ensure that cells are completely depleted of Na ϩ ; however, even if some Na ϩ remains in the cell, there is still no external source of Na ϩ that could allow the increase in internal Na ϩ necessary for Na ϩ pump inhibition to contribute to the cellular actions of ouabain.…”