Effect of 1-chloro-2,4-dinitrobenzene on K+ transport in normal and sickle human red blood cells.
1-Chloro-2,4-dinitrobenzene (CDNB), which causes oxidative stress through depletion of reduced glutathione (GSH), increases the passive K+ permeability of red cells. In this paper, we investigated the effects of CDNB (1 mM) on the activities of the K+-Cl- cotransporter (KCC; measured as Cl--dependent K+ influx) and the Gardos channel (taken as clotrimazole-sensitive K+ influx, 5 microM) in human red cells, using 86Rb+ as a K+ congener. 45Ca2+ was used to study passive Ca2+ entry and active Ca2+ efflux via the plasma membrane Ca2+ pump. Both the Gardos channel and KCC were stimulated in both normal and sickle red cells. In sickle cells, stimulation of KCC was similar in oxygenated and deoxygenated cells; that of the Gardos channel was greater in deoxygenated cells. In normal red cells, stimulation of both pathways was greater in oxygenated cells (by 4 +/- 1-fold; all means +/- S.E.M., n = 3). The effects on the Gardos channel were dependent on extracellular Ca2+ and were associated with inhibition of the plasma membrane Ca2+ pump (by 29 +/- 3 %, P < 0.01) and increased Ca2+ sensitivity of the channel (EC50 for [Ca2+]i reduced from 260 +/- 26 to 175 +/- 15 nM; P < 0.05). Cell volume, pHi, ATP levels and passive Ca2+ entry were not affected by CDNB. The effects on KCC were inhibited (93 +/- 6 %) by prior treatment with the protein phosphatase inhibitor calyculin A (100 nM) and were not additive with stimulation by N-ethylmaleimide (1 mM), regardless of the order of addition. These findings are therefore consistent with inhibition of a regulatory protein kinase, although stimulation of the conjugate protein phosphatase(s) may also occur. KCC stimulation was also Ca2+ dependent. These findings are important for understanding how GSH depletion alters membrane permeability and how to protect against red cell dehydration.
Muzyamba
M C
MC
Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
Gibson
J S
JS
D000755:Anemia, Sickle Cell / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004137:Dinitrochlorobenzene / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D007509:Irritants / Q000494:pharmacology*; D008706:Methemoglobin / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D004137:Dinitrochlorobenzene; D006451:Hemoglobin, Sickle; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D007509:Irritants; C496923:KCNN4 protein, human; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D008706:Methemoglobin; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione; D011188:Potassium; D010100:Oxygen; D002118:Calcium