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

Abstract

Na+/K+‐ATPase‐dependent Rb+ uptake of RN22 Schwann cells was stimulated by cholera toxin (0.25 μg/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. GLIA 23:349–360, 1998. © 1998 Wiley‐Liss, Inc.

Mechanisms of sodium pump regulation

“…Although direct phosphorylation of the Na ϩ -K ϩ -ATPase appears to correlate with the well-documented PKA-mediated stimulation of enzyme phosphorylation and ouabain-sensitive 86 Rb ϩ uptake in renal proximal tubules (63), there is evidence to support the notion that the activation is secondary to an increase in plasma membrane pumps (64). Perhaps related to this are the observed PKA-induced increases in plasma membrane pumps of MDCK (323) and Schwann cells (312). Although direct phosphorylation of the Na ϩ -K ϩ -ATPase by PKA is an attractive simple mechanism for PKA-mediated regulation of the enzyme and appears to apply to at least some systems, other more complex mechanisms have been observed.…”

“…Cantiello (61) showed that phosphorylation of monomeric actin by PKA prevented the actin-mediated stimulation of the sodium pump, whereas phosphorylation of polymeric actin promoted it. Finally, in some cases, PKA does not regulate the sodium pump directly but, rather, alters the function of other Na ϩ transporters, leading to changes in cytoplasmic Na ϩ concentration, which in turn alter Na ϩ -K ϩ -ATPase activity (164,312).…”