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Effect of 1‐chloro‐2,4‐dinitrobenzene on K + transport in normal and sickle human red blood cells
Author(s) -
Muzyamba M. C.,
Gibson J. S.
Publication year - 2003
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2003.00903.x
Subject(s) - chemistry , medicine , biology , microbiology and biotechnology , endocrinology
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 m m ) 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 μ m ) in human red cells, using 86 Rb + as a K + congener. 45 Ca 2+ was used to study passive Ca 2+ entry and active Ca 2+ efflux via the plasma membrane Ca 2+ 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 Ca 2+ and were associated with inhibition of the plasma membrane Ca 2+ pump (by 29 ± 3 %, P < 0.01) and increased Ca 2+ sensitivity of the channel (EC 50 for [Ca 2+ ] i reduced from 260 ± 26 to 175 ± 15 n m ; P < 0.05). Cell volume, pH i , ATP levels and passive Ca 2+ 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 n m ) and were not additive with stimulation by N ‐ethylmaleimide (1 m m ), 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 Ca 2+ dependent. These findings are important for understanding how GSH depletion alters membrane permeability and how to protect against red cell dehydration.