Acute and chronic regulation of Na+/K+-ATPase transport activity in the RN22 Schwann cell line in response to stimulation of cyclic AMP production
✍ Scribed by William C. Stewart; Phillip H. Pekala; Edward M. Lieberman
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 160 KB
- Volume
- 23
- Category
- Article
- ISSN
- 0894-1491
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✦ Synopsis
Na ϩ /K ϩ -ATPase-dependent Rb ϩ 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 ϩ uptake was increased by 162 Ϯ 6% (cholera toxin), 151 Ϯ 14% (forskolin), and 207 Ϯ 15% (8-bromo cAMP). Cholera toxin or 8-bromo cAMP treatment for 12-24 h resulted in a second peak of Na ϩ /K ϩ -ATPase-dependent Rb ϩ transport activity of 186 Ϯ 12 and 265 Ϯ 9% of control, respectively. Cholera toxin also transiently stimulated the activity of the Na ϩ , K ϩ , 2Cl Ϫ -cotransporter with a peak at 2 h (179 Ϯ 9%), returning to basal levels by 24 h. Inhibition of the Na ϩ ,K ϩ ,2Cl Ϫ -cotransporter by bumetanide (0.1 mM) or by reduction of the Na ϩ 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 ϩ /K ϩ ATPase activity by cholera toxin was due to an increase in cellular Na ϩ , secondary to stimulation of Na ϩ ,K ϩ ,2Cl Ϫ -cotransport activity. Western blot analysis of cellular homogenates and purified membrane fractions showed that the second peak of Rb ϩ uptake activity was a result of translocation of transport protein from an intracellular microsomal pool to the plasma membrane. Rb ϩ 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.