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 stimulate d by cholera toxin (0.25 mu g/ml), forskolin (2 mM), or 8-bromo cAMP(1 mM). At 2 h Rb+ uptake was increased by 162 +/- 6% (cholera toxin), 1 51 +/- 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 i n ATPase activity but not the second peak. These results indicated tha t the early transient stimulation of Na+/K+ ATPase activity by cholera toxin was due to an increase in cellular Na+, secondary to stimulatio n of Na+ ,K+, 2Cl(-)-cotransport activity. Western blot analysis of ce llular homogenates and purified membrane fractions showed that the sec ond peak of Rb+ uptake activity was a result of translocation of trans port protein from an intracellular microsomal pool to the plasma membr ane. Rb+ uptake by dominant negative protein kinase A mutants of the R N22 cell was not stimulated by cholera toxin treatment (acute or chron ic) 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 o f cellular homogenates, neither a change in enzyme kinetics nor an inc rease in de novo synthesis of transport protein could account for the increase in transport activity. (C) 1998 Wiley-Liss, Inc.