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Acute and chronic regulation of Na + /K + ‐ATPase transport activity in the RN22 Schwann cell line in response to stimulation of cyclic AMP production
Author(s) -
Stewart William C.,
Pekala Phillip H.,
Lieberman Edward M.
Publication year - 1998
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/(sici)1098-1136(199808)23:4<349::aid-glia7>3.0.co;2-y
Subject(s) - cholera toxin , cotransporter , forskolin , biology , stimulation , bumetanide , ion transporter , protein kinase a , microbiology and biotechnology , medicine , endocrinology , sodium , biochemistry , kinase , chemistry , membrane , organic chemistry
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.