Na-K pump site density and ouabain binding affinity in cultured chick heart cells.
The possible existence of multiple [3H]ouabain binding sites and the relationship between ouabain binding and Na-K pump inhibition in cardiac muscle were studied using cultured embryonic chick heart cells. [3H]ouabain bound to a single class of sites in 0.5 mM K (0.5 Ko) with an association rate constant (k+1) of 3.4 X 10(4) M-1.s-1 and a dissociation rate constant (k-1) of 0.0095 s-1 [corrected]. Maximal specific [3H]ouabain binding RT to myocyte-enriched cultures is 11.7 pmol/mg protein and Kd is 0.43 microM in 0.5 Ko, whereas Kd,apparent is 6.6 microM in 5.4 Ko. The number of binding sites per myocyte was calculated by correcting for the contribution of fibroblasts in myocyte-enriched cultures using data from homogeneous fibroblast cultures (RT = 3.3 pmol/mg protein; Kd = 0.19 microM in 0.5 Ko). Equivalence of [3H]ouabain binding sites and Na-K pumps was implied by agreement between maximal specific binding of [3H]ouabain and 125I-labeled monoclonal antibody directed against Na+-K+-ATPase (approximately 2 X 10(6) sites/cell). However, [3H]ouabain binding occurred at lower concentrations than inhibition of ouabain-sensitive 42K uptake in 0.5 Ko. Further studies in both 0.5 K and 5.4 Ko showed that ouabain caused cell Na content Nai to increase over the same range of concentrations that binding occurred, implying that increased Nai may stimulate unbound Na-K pumps and prevent a proportional decrease in 42K uptake rate. The results show that Na-K pump inhibition occurs as a functional consequence of specific ouabain binding and indicate that the Na-K pump is the cardiac glycoside receptor in cultured heart cells.
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002478:Cells, Cultured; D002642:Chick Embryo; D005455:Fluorescent Antibody Technique; D007457:Iodine Radioisotopes; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D010042:Ouabain / Q000378:metabolism* / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D011197:Potassium Radioisotopes; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology; D014316:Tritium
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D007473:Ion Channels; D011197:Potassium Radioisotopes; D014316:Tritium; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium