Characterization of the Na, K-ATPase pump in cultured bovine corneal endothelial cells.
Bovine corneal endothelial cells in culture possess Na, K-ATPase pump sites, as measured by [3H]ouabain binding, and demonstrated an active ouabain-sensitive 86Rb+ uptake. The binding of [3H]ouabain, a specific inhibitor of Na, K-ATPase, was used to quantitative the density of Na, K-ATPase pump sites in bovine corneal endothelial cell cultures. [3H]ouabain binding was time-dependent and reached saturation after 1-2 hr. The specific binding represented more than 99% of the total cell-associated [3H]ouabain, and about 85% of this binding was abolished in the presence of K+ ions. The binding was concentration-dependent and saturated at a ouabain concentration of 2 X 10(-8)M with a dissociation constant (Kd) of 1.0 X 10(-8)M. The number of [3H]ouabain binding sites was maximal in sparse, activity growing cultures and decreased accompanying the development of a confluent monolayer. A pump density of 2.2 X 10(6) pump sites cell-1 was estimated for sparse cultures, declining to 0.8 X 10(6) pump site cell-1 at confluence. The activity of the Na, K-ATPase pump in bovine corneal endothelial cell cultures was evaluated by measuring 86Rb+ influx. Sparse and confluent cultures demonstrated 86Rb+ ouabain-sensitive uptake at a rate of 4.2 nmol (10(6) cells)-1 min-1 and 1.5 nmol (10(6) cells)-1 min-1, respectively. The ouabain-sensitive 86Rb+ uptake was linear for at least 30 min, while the ouabain-insensitive 86Rb+ uptake was slower and declined during the 30-min time period.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004727:Endothelium / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011868:Radioisotopes; D012413:Rubidium; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D007473:Ion Channels; D011868:Radioisotopes; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium