A sensitive [Na,K]ATPase assay specific for inhibitors acting through the digitalis-binding site

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.