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The use of combination drugs is very common in therapeutics as in the treatment of infectious diseases, cancer and heart failure but controversies about analysis of these interactions are frequent. The aim of the present work was to characterize the interaction between ouabain and 8-methoxy-3,9-dihydroxy coumestan (LQB93), a non-steroidal synthetic inhibitor of Na+,K+-ATPase, as well as the interaction between ouabain and ouabagenin, two cardiac glycosides sharing the same binding site. Inhibition of rat kidney Na+,K+-ATPase with increasing concentrations of the drugs alone or of mixtures of ouabain:ouabagenin and LQB93:ouabain in a fixed 1:4 ratio was performed. In other experiments, increasing concentrations of LQB93 (or ouabain) in the presence of a fixed concentration of ouabain (or ouabagenin) were used for determining the concentration pairs eliciting 50% inhibition in order to construct isobolograms. The mixture (experimental) curve for the ouabain:ouabagenin combination was superimposed on the additive (theoretical) curve indicating additivity, in accordance with the isobolographic analysis. On the other hand, the empirical curve for LQB93:ouabain (IC50=10.6 microM) was significantly shifted to the left in relation to the theoretical curve (IC50=30.7 microM) indicating synergism, further confirmed by the isobolographic analysis. As a conclusion, we show that the combination of a newly synthesized non-steroidal inhibitor and ouabain have a synergistic effect on Na+,K+-ATPase, further supporting a mechanism of inhibition different from ouabain. Present data also support the use of both the isobolograms and combination curves for the assessment of drug interactions occurring at the same molecular target, a situation poorly investigated.

Although cardiac steroids (CS) have long been used to treat cardiac insufficiency, the mechanism(s) of action of these agents remain open to question. While many results indicate that inhibition of Na+,K+-ATPase underlies both the therapeutic and toxic actions of CS, other studies suggest that actions on the SR membrane system may be important. We used two experimental approaches and measurements of left ventricular diastolic pressure (LVDP) in isolated guinea pig hearts to test whether CS had an intracellular site of action. In the first approach, we compared the inotropic effects of a hydrophilic CS, ouabain, and a hydrophobic CS, digitoxin, after the activity of the Na+ pump was reduced by perfusing hearts with solutions maintained at 5 degrees C. Under these conditions, exposure of hearts to 1 microM ouabain for 60 min did not increase LVDP above control levels. In contrast, an equi-effective concentration of digitoxin (0.3 microM) increased LVDP by 40 +/- 8.5% (p < 0.01) over pre-drug control levels. In the second experimental approach, we compared the inotropic effects of ouabain and digitoxin in the presence of rapid-cooling contractures (RCC), which result in the release of SR Ca2+. Hearts were perfused with Tyrode solution or Tyrode solution containing either digitoxin (0.3 microM) or ouabain (1 microM) for 180 sec, rapidly cooled and the RCC responses were analyzed. Compared to RCC elicited in Tyrode solution alone, or in Tyrode solution containing ouabain, RCC in the presence of digitoxin reached peak amplitudes more rapidly, but elicited reduced peak amplitude values. Based on these findings, we suggest that: 1) the ability of the hydrophobic CS, digitoxin, but not the hydrophilic CS, ouabain, to produce a positive inotropic effect at 5 degrees C, when the activity of the Na+ pump is markedly reduced, is consistent with a mechanism other than Na+ pump inhibition and involves an intracellular location; and 2) the diminished RCC observed in the presence of the hydrophobic CS, digitoxin, indicate that this alternative mechanism may involve effects on the SR Ca2+ release channel.

On the assumption that digoxin-like immunoreactivity may represent digitalis-like sodium pump inhibitors in the mammalian body, many investigators have used radioimmunoassay for digoxin to monitor such factors during the past decade. The presence of digoxin-like immunoreactivity has been confirmed by numerous studies using biochemical, immunological or morphological methods. Very recently, ouabain or a very similar substance, which did not cross-react with antidigoxin antibodies, was identified from the human plasma as the long-sought sodium pump inhibitor. However, it is yet to be determined whether sodium pump inhibitory activity in the circulation results from one substance or several. Some researchers still insist on the possible physiological roles of digoxin-like immunoreactivity which may or may not be related to the regulation of sodium pump. These issues are critically reviewed in this article.