Glutathione removal reveals kinases as common targets for k-cl cotransport
stimulation in sheep erythrocytes.
Lauf, Peter K., Norma C. Adragna, and Nihal S. Agar.
Departments of Physiology and Biophysics, and Pharmacology & Toxicology,
Wright State University, Dayton OH, U.S.A, and Physiology Department, The
University of New England, Armidale, NSW, Australia.
APStracts 2:0070C, 1995.
K-Cl cotransport is activated by swelling, lowering of cellular free Mg, Mgi,
and thiol modification of erythrocytes. Direct actions by thiol reagents on
the K-Cl cotransport complex were separated from indirect effects through
non-oxidative changes in cellular glutathione (GSH). We used CDNB (1-chloro,
2,4 dinitrobenzene) which, conjugated to GSH, is extruded from the
erythrocyte as a thioether. CDNB caused a small biphasic effect (inhibition
and stimulation) on K-Cl cotransport, and, at 1 mM, abolished its stimulation
by N-ethylmaleimide (NEM), diamide, and methylmethane thiolsulfonate, and by
staurosporine, a kinase inhibitor, independent of the order of treatment.
Hence NEM and other activating thiol reagents, and perhaps GSH removal
itself, target unidentified kinases involved in activation of K-Cl
cotransport. CDNB also abrogated K-Cl cotransport stimulation by Mgi
-depletion, independent of the order of treatment indicating inhibition at a
second site nearer to the transporter. Furthermore, CDNB-treatment elevated
and rendered K-Cl cotransport insensitive to osmotic shrinkage suggesting
uncoupling from the regulator.
Received 1 November 1994; accepted in final form 13 January 1995.
APS Manuscript Number C649-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 28 February 1995.