Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells.
[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D004285:Dogs; D004848:Epithelium / Q000378:metabolism* / Q000648:ultrastructure; D007668:Kidney; D007700:Kinetics; D008854:Microscopy, Electron; D010042:Ouabain / Q000378:metabolism*; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014316:Tritium
D014316:Tritium; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase