title: A sensitive [Na,K]ATPase assay specific for inhibitors acting through the digitalis-binding site.
abstract: Efforts to study the endogenous sodium pump inhibitor (ESPI) have been complicated by the limited specificity of available assays. We recently developed an assay of [Na,K]ATPase inhibition more sensitive than conventional assays. This enhancement reflects a prereaction step that increases binding affinity of digitalislike molecules to the digitalis receptor. We tested the possibility that this enhanced inhibition is limited to inhibitors acting specifically through the digitalis-binding site. Using both the standard and sensitive [Na,K]ATPase methods, known specific inhibitors of the sodium pump (ouabain, digoxin, bufalin) showed significant increases in the inhibition in the sensitive as compared with the standard [Na,K]ATPase assay (ouabain 34.4 +/- 7.3 vs. 8.3 +/- 0.5%, digoxin 43.2 +/- 9.1 vs. 7.2 +/- 3.1%, bufalin 46.9 +/- 5.0 vs. 22.6 +/- 1.6%). Some proposed candidates for the ESPI, acknowledged to be nonspecific inhibitors, showed no enhancement (oleic acid 36.0 +/- 4.5 vs. 34.8 +/- 5.6%, lysophosphatidyl choline 10.8 +/- 3.5 vs. 12.8 +/- 5.2%, and vanadate 34.3 +/- 8.8 vs. 33.8 +/- 1.4%). Other candidates, whose inhibitory specificity is unknown, including an ESPI from the peritoneal dialysate of patients with renal failure were also studied. The ESPI showed enhanced inhibition (24.1 +/- 4.9 vs. 5.3 +/- 2.0%). These studies suggest that the sensitive assay in conjunction with a standard [Na,K]ATPase assay may allow the early determination of candidates interacting specifically with the digitalis receptor to inhibit the sodium pump.
authors.affiliation: Department of Medicine Endocrine-Hypertension Division, Harvard Medical School, Brigham and Women's Hospital, Boston, MA 02115, USA.
authors.forename: Q F
authors.initials: QF
authors.lastname: Tao
authors.forename: P A
authors.initials: PA
authors.lastname: Soszynski
authors.forename: N K
authors.initials: NK
authors.lastname: Hollenberg
authors.forename: S W
authors.initials: SW
authors.lastname: Graves
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002018:Bufanolides / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D004070:Digitalis / Q000378:metabolism*; D004077:Digoxin / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000378:metabolism / Q000494:pharmacology; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism / Q000494:pharmacology; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology; D010946:Plants, Medicinal; D010947:Plants, Toxic; D012044:Regression Analysis; D012835:Silver Nitrate / Q000378:metabolism / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000187:drug effects* / Q000378:metabolism; D013256:Steroids / Q000378:metabolism / Q000494:pharmacology; D013629:Tamoxifen / Q000378:metabolism / Q000494:pharmacology; D014638:Vanadates / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D002018:Bufanolides; D004791:Enzyme Inhibitors; D008244:Lysophosphatidylcholines; D009829:Oleic Acids; D013256:Steroids; C027082:digitalis receptor; D013629:Tamoxifen; D019301:Oleic Acid; D014638:Vanadates; D010042:Ouabain; D004077:Digoxin; D012835:Silver Nitrate; D000254:Sodium-Potassium-Exchanging ATPase; C022777:bufalin