Revisiting the binding kinetics and inhibitory potency of cardiac glycosides on Na+,K+-ATPase (α1β1): Methodological considerations

Ouabain and digoxin are classical inhibitors of the Na+,K+-ATPase. In addition to their conventional uses as therapeutic agents or experimental tools there is renewed interest due to evidence suggesting they could be endogenous hormones. Somewhat surprisingly, different publications show large discr...

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Bibliographic Details
Published in:Journal of pharmacological and toxicological methods 2018-11, Vol.94 (Pt 2), p.64-72
Main Authors: Noël, François, Azalim, Pedro, do Monte, Fernando M., Quintas, Luis Eduardo M., Katz, Adriana, Karlish, Steven J.D.
Format: Article
Language:English
Subjects:
Animals
Cardiac glycosides
Cardiac Glycosides - pharmacokinetics
Cardiac Glycosides - pharmacology
Digoxigenin - pharmacokinetics
Digoxin
Digoxin - pharmacokinetics
Enzyme
Humans
Inhibition
Kidney - enzymology
Kinetics
Methods
Na+,K+-ATPase
Ouabain
Ouabain - analogs & derivatives
Ouabain - pharmacokinetics
Protein Binding
Slow binding inhibitors
Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors
Sodium-Potassium-Exchanging ATPase - metabolism
Structure-Activity Relationship
Swine
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Summary:Ouabain and digoxin are classical inhibitors of the Na+,K+-ATPase. In addition to their conventional uses as therapeutic agents or experimental tools there is renewed interest due to evidence suggesting they could be endogenous hormones. Somewhat surprisingly, different publications show large discrepancies in potency for inhibiting Na+,K+-ATPase activity (IC50), particularly for the slow binding inhibitors, ouabain and digoxin. Using purified pig kidney Na+,K+-ATPase (α1β1FXYD2) and purified detergent-soluble recombinant human Na+,K+-ATPase (α1β1FXYD1) we have re-evaluated binding and inhibition kinetics and effects of K+ concentration for ouabain, digoxin, ouabagenin and digoxigenin. We demonstrate unequivocally that for slow binding inhibitors, ouabain and digoxin, long incubation times (≥60 min at 37 °C) are required to avoid under-estimation of potency and correctly determine inhibition (IC50 around 100–200 nM at 5 mM K+) contrary to what occurs when pre-incubation of the drugs without ATP is followed by a short incubation time. By contrast, for the rapidly bound inhibitors, ouabagenin and digoxigenin, short incubation times suffice (
ISSN:1056-8719
1873-488X
DOI:10.1016/j.vascn.2018.09.001