Insulin increases the turnover rate of Na+-K+-ATPase in human fibroblasts

Insulin stimulates K+ transport by the Na+-K+-ATPase in human fibroblasts. In other cell systems, this action represents an automatic response to incr eased intracellular [Na+] or results from translocation of transporters fro m an intracellular site to the plasma membrane. Here we evaluate whether th ese mechanisms are operative in human fibroblasts. Human fibroblasts expres sed the alpha (1) but not the alpha (2) and alpha (3) isoforms of Na+-K+-AT Pase. Insulin increased the influx of Rb+, used to trace K+ entry, but did not modify the total intracellular content of K+, Rb+, and Na+ over a 3-h i ncubation period. Ouabain increased intracellular Na+ more rapidly in cells incubated with insulin, but this increase followed insulin stimulation of Rb+ transport. Bumetanide did not prevent the increased Na+ influx or stimu lation of Na+-K+-ATPase. Stimulation of the Na+-K+ ATPase by insulin did no t produce any measurable change in membrane potential. Insulin did not affe ct the affinity of the pump toward internal Na+ or the number of membrane-b ound Na+-K+-ATPases, as assessed by ouabain binding. By contrast, insulin s lightly increased the affinity of Na+-K+-ATPase toward ouabain. Phorbol est ers did not mimic insulin action on Na+-K+-ATPase and inhibited, rather tha n stimulated, Rb+ transport. These results indicate that insulin increases the turnover rate of Na+-K+-ATPases of human fibroblasts without affecting their number on the plasma membrane or modifying their dependence on intrac ellular [Na+].