title: Inhibition of the alpha1beta1 isoform of the Na, K-ATPase by 8-methoxycoumestrol without positive inotropic effect in human myocardium--novel aspects of cardiac glycoside pharmacology.
abstract: BACKGROUND
  The Na,K-ATPase (NKA) is necessary for maintaining the resting membrane potential by transporting Na and K ions across the cell membrane. Although its 3 isoforms expressed in human heart (alpha1beta1, alpha2beta1, and alpha3beta1) possess similar biochemical properties, their specific functions in human tissues remain unknown. In our search for an isoform-specific agent, which can serve to identify isoform-specific functions, we examined 8-methoxycoumestrol in its ability to inhibit the NKA and to produce inotropism in connection with the possibility to identify the NKA isoform-specific functions.
  
  
  RESULTS
  In radioligand binding experiments (membrane preparations of yeast expressing isoforms alpha1beta1, alpha2beta1, and alpha3beta1; backdoor phosphorylation; and [H]-ouabain, n = 3), 8-methoxycoumestrol (1-10 microM) produced no or only little inhibition of specific ouabain binding. However, when NKA activity of the alpha1beta1 isoform was measured in membrane preparations from human kidney (reduced form of nicotinamide adenine dinucleotide-coupled assay, n = 3), a concentration-dependent full inhibition of the activity was induced by 8-methoxycoumestrol (IC50: 90 +/- 97 nM), similar to that observed for classical cardiac glycosides digitoxin, digoxin, methyldigoxin, and beta-acetyldigoxin (IC50 = 287 +/- 190 nM, 409 +/- 171 nM, 282 +/- 482 nM, 587 +/- 135 nM, P > 0.05). However, unlike the classical cardiac glycosides, 8-methoxycoumestrol did not increase cardiac contractility of electrically stimulated human right atrial trabeculae.
  
  
  CONCLUSIONS
  These results indicate that 8-methoxycoumestrol inhibits the human alpha1beta1 NKA by a mechanism different to that of cardiac glycosides. In addition, the inhibition of the alpha1beta1 NKA activity seems not sufficient to evoke positive inotropy in human trabeculae, indicating that either the positive inotropic effect of cardiac glycosides is not mediated via the alpha1beta1 isoform or the specific glycoside binding to alpha1beta1 is needed for positive inotropy.
authors.affiliation: Laboratory of Muscle Research and Molecular Cardiology, Department of Internal Medicine III, University of Cologne, Köln 50924, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Potter
authors.forename: C
authors.initials: C
authors.lastname: Hauck
authors.forename: E S C
authors.initials: ES
authors.lastname: Pôças
authors.forename: H
authors.initials: H
authors.lastname: Diedrichs
authors.forename: C
authors.initials: C
authors.lastname: Adam
authors.forename: H
authors.initials: H
authors.lastname: Reuter
authors.forename: T
authors.initials: T
authors.lastname: Wittwer
authors.forename: T
authors.initials: T
authors.lastname: Wahlers
authors.forename: A J M
authors.initials: AJ
authors.lastname: da Silva
authors.forename: P R R
authors.initials: PR
authors.lastname: Costa
authors.forename: F
authors.initials: F
authors.lastname: Noël
authors.forename: J
authors.initials: J
authors.lastname: Müller-Ehmsen
mesh_terms: D000368:Aged; D002301:Cardiac Glycosides / Q000494:pharmacology*; D003375:Coumestrol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006325:Heart Atria / Q000187:drug effects / Q000503:physiopathology; D006801:Humans; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000378:metabolism / Q000601:surgery; D008297:Male; D008875:Middle Aged; D015394:Molecular Structure; D009119:Muscle Contraction / Q000502:physiology; D009206:Myocardium / Q000378:metabolism*; D009392:Nephrectomy; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
chemical_list: C545723:8-methoxycoumestrol; D002301:Cardiac Glycosides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D000254:Sodium-Potassium-Exchanging ATPase; D003375:Coumestrol