Revisiting the binding kinetics and inhibitory potency of cardiac glycosides on Na+,K+-ATPase (α1β1): Methodological considerations
| Autor: | Adriana Katz, Fernando M. do Monte, Pedro Azalim, Luis Eduardo M. Quintas, Steven J.D. Karlish, François Noël |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Pharmacology chemistry.chemical_classification 030102 biochemistry & molecular biology Digoxin Toxicology Receptor–ligand kinetics Ouabain 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Enzyme Biochemistry chemistry medicine Potency Digoxigenin Na+/K+-ATPase IC50 medicine.drug |
| Zdroj: | Journal of Pharmacological and Toxicological Methods. 94:64-72 |
| ISSN: | 1056-8719 |
| Popis: | Introduction 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. Methods 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. Results 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 ( Discussion The data resolve discrepancies in the literature attributable to sub-optimal assay conditions. Similar IC50 values are obtained for pig kidney and recombinant human Na+,K+-ATPase, showing that inhibitory potencies are not determined by the species difference (pig versus human) or environment (membrane-bound versus detergent-soluble) of the Na+,K+-ATPase. The present methodological considerations are especially relevant for drug development of slow binding inhibitors. |
| Databáze: | OpenAIRE |
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