Acute and chronic regulation of Na+/K+-ATPase transport activity in the RN22 Schwann cell line in response to stimulation of cyclic AMP production.

Na+/K+-ATPase-dependent Rb+ uptake of RN22 Schwann cells was stimulated by cholera toxin (0.25 microg/ml), forskolin (2 mM), or 8-bromo cAMP (1 mM). At 2 h Rb+ uptake was increased by 162+/-6% (cholera toxin), 151+/-14% (forskolin), and 207+/-15% (8-bromo cAMP). Cholera toxin or 8-bromo cAMP treatment for 12-24 h resulted in a second peak of Na+/K+-ATPase-dependent Rb+ transport activity of 186+/-12 and 265+/-9% of control, respectively. Cholera toxin also transiently stimulated the activity of the Na+, K+, 2Cl- -cotransporter with a peak at 2 h (179+/-9%), returning to basal levels by 24 h. Inhibition of the Na+,K+,2Cl- -cotransporter by bumetanide (0.1 mM) or by reduction of the Na+ gradient (10 mM veratridine treatment) prevented the early peak in ATPase activity but not the second peak. These results indicated that the early transient stimulation of Na+/K+ ATPase activity by cholera toxin was due to an increase in cellular Na+, secondary to stimulation of Na+,K+,2Cl -cotransport activity. Western blot analysis of cellular homogenates and purified membrane fractions showed that the second peak of Rb+ uptake activity was a result of translocation of transport protein from an intracellular microsomal pool to the plasma membrane. Rb+ uptake by dominant negative protein kinase A mutants of the RN22 cell was not stimulated by cholera toxin treatment (acute or chronic) confirming the cAMP/protein kinase A dependency of both acute and long-term regulation of transport activity. In the absence of a change in Michaelis constants or of an increase in total transport protein of cellular homogenates, neither a change in enzyme kinetics nor an increase in de novo synthesis of transport protein could account for the increase in transport activity.