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The Gardos channel is responsible for CDNB-induced dense sickle cell formation
A Shartava1, J McIntyre, A K Shah
1Department of Structural and Cellular Biology, University of South Alabama, College of Medicine, Mobile 36688, USA.
Insights
Sickle cell red blood cells (RBCs) density relates to glutathione (GSH) levels. 1-chloro-2,4-dinitrobenzene (CDNB) induces dense cells by damaging the Gardos channel, not K(+)-Cl(-) co-transport.
Area of Science:
- Hematology
- Biochemistry
- Cell Biology
Background:
- Sickle cell disease (SCD) is characterized by abnormal red blood cells (RBCs).
- RBC density in SCD patients is inversely proportional to intracellular reduced glutathione (GSH) levels.
- Previous studies implicated K(+) leakage in CDNB-induced dense cell formation in sickle cells.
Purpose of the Study:
- To investigate the mechanism of 1-chloro-2,4-dinitrobenzene (CDNB)-induced dense cell formation in sickle cells.
- To determine the role of the Gardos channel versus the K(+)-Cl(-) co-transport system in this process.
- To assess the effect of pH on CDNB-induced dense cell formation and the efficacy of specific inhibitors.
Main Methods:
- Incubation of low-density sickle cells (LDSS) with 1-chloro-2,4-dinitrobenzene (CDNB) at 4°C.
- Treatment with clotrimazole (Gardos channel inhibitor) and DIOA (K(+)-Cl(-) co-transport inhibitor) at different pH values (7.4, 7.1, and 6.8).
- Monitoring the shift from LDSS to high-density sickle cells (HDSS) and changes in GSH content.
Main Results:
- CDNB induced a shift to HDSS with decreased GSH in LDSS.
- Clotrimazole inhibited dense cell formation at pH 7.4, 7.1, and 6.8.
- DIOA did not inhibit dense cell formation at any tested pH, including the optimal pH (6.8) for K(+)-Cl(-) co-transport.
Conclusions:
- CDNB-induced dense cell formation in sickle cells is primarily mediated by damage to the Gardos channel.
- The K(+)-Cl(-) co-transport system is not responsible for this CDNB effect.
- These findings provide crucial insights into the pathophysiology of sickle cell disease and potential therapeutic targets.
Abstract:
The red blood cells (RBCs) derived from blood taken from homozygous sickle cell (SS) patients demonstrate densities that are inversely proportional to the intracellular reduced glutathione (GSH) content. Addition of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) to low-density sickle cells (LDSS), at 4 degrees C, results in a shift of LDSS erythrocytes to high-density sickle cells (HDSS), with corresponding decreases in GSH. We have previously demonstrated that this CDNB effect was due to increased K(+) leakage and that dense cell formation could be inhibited by clotrimazole (specific for the Gardos channel) but not DIOA (specific for the K(+)-Cl(-) co-transport system) at pH 7.4 (Shartava et al. Am. J. Hematol. 1999;62:19-24). Here we demonstrate that clotrimazole (10 microM) inhibits dense cell formation at pH 7.1 and 6.8, while DIOA (1 mM) has no effect. As pH 6.8 is the optimal pH for the K(+)-Cl(-) co-transport system, we can now reasonably conclude that damage to the Gardos channel is responsible for CDNB-induced dense cell formation.