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 Mg (Mgi), and thiol modification of erythrocytes. Direct actions by thiol reagents on the K-Cl cotransport complex were separated from indirect 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), diazenedicarboxylic acid bis[N,N-dimethylamide], methyl methanethiosulfonate, and 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.
Lauf
P K
PK
Department of Physiology, Wright State University, Dayton, Ohio 45401, USA.
Adragna
N C
NC
Agar
N S
NS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000097:blood*; D004137:Dinitrochlorobenzene / Q000494:pharmacology*; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D005978:Glutathione / Q000037:antagonists & inhibitors*; D008274:Magnesium / Q000378:metabolism; D009997:Osmotic Pressure; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012756:Sheep; D019311:Staurosporine; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D027981:Symporters; D000096922:K Cl- Cotransporters
D000255:Adenosine Triphosphate; D000470:Alkaloids; D002352:Carrier Proteins; D004137:Dinitrochlorobenzene; D005978:Glutathione; D008274:Magnesium; D010770:Phosphotransferases; D019311:Staurosporine; D013438:Sulfhydryl Compounds; D027981:Symporters; D000096922:K Cl- Cotransporters