Interaction of ouabain with the Na(+) pump in intact epithelial cells

Open Access
Publisher Website
Google Scholar
Abstract
To determine the specificity and efficacy of [3H]ouabain binding as a quantitative measure of the Na+ pump (Na+,K+-ATPase) and as a marker for the localization of pumps involved in transepithelial Na+-transport, the interaction of [3H]ouabain with its receptor in pig kidney epithelial (LLC-PK1) cells was analyzed. When these epithelial cells are depleted of Na+ and exposed to 2 .mu.M [3H]ouabain in a Na+-free medium, binding is reduced by 90%. When depleted for K+ and incubated in a K+-free medium, the ouabain binding rate is increased compared with that measured at 5 mM. This increase is only demonstrable when Na+ is present. The increased rate could be attributed to the predominance of the Na+-stimulated phosphorylated form of the pump, as K+ is not readily available to stimulate dephosphorylation. Some binding in the K+-free medium is attributable to pump turnover (and therefore, recycling of K+) because analysis of K+-washout kinetics demonstrated that addition of 2 .mu.M ouabain to K+-depleted cells increased the rate of K+ loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na+,K+-ATPase is operating in the Na+-pump mode or is phosphorylated in the presence of Na+. When LLC-PK1 cells were exposed to ouabain at 4.degree. C, binding was reduced by 97%. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37.degree. C. Even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na+ pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na+ pump is not operating.