Pk. Lauf et al., GLUTATHIONE REMOVAL REVEALS KINASES AS COMMON TARGETS FOR K-CL COTRANSPORT STIMULATION IN SHEEP ERYTHROCYTES, American journal of physiology. Cell physiology, 38(1), 1995, pp. 234-241
K-Cl cotransport is activated by swelling, lowering of cellular free M
g (Mg-i), and thiol modification of erythrocytes. Direct actions by th
iol reagents on the K-Cl cotransport complex were separated from indir
ect effects through nonoxidative changes in cellular glutathione (GSH)
. We used 1-chloro-2,4-dinitrobenzene (CDNB), 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), diazene
dicarboxylic acid bis[N,N-dimethylamide], methyl methanethiosulfonate,
and staurosporine, a kinase inhibitor, independent of the order of tr
eatment. Hence, NEM and other activating-thiol reagents, and perhaps G
SH removal itself, target unidentified kinases involved in activation
of K-Cl cotransport. CDNB also abrogated K-Cl cotransport stimulation
by Mg-i depletion independent of the order of treatment, indicating in
hibition at a second site nearer to the transporter. Furthermore, CDNB
treatment elevated and rendered K-Cl cotransport insensitive to osmot
ic shrinkage, suggesting uncoupling from the regulator.