GLUTATHIONE REMOVAL REVEALS KINASES AS COMMON TARGETS FOR K-CL COTRANSPORT STIMULATION IN SHEEP ERYTHROCYTES

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.