Submitted April 25, 2006
Accepted September 26, 2006
Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes
Clinton H. Joiner*, R. Kirk Rettig, Maorong Jiang, Mary Risinger, and Robert S. Franco
Cincinnati Comprehensive Sickle Cell Center, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States
Division of Hematology/Oncology, Dept of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States
Division of Hematology/Oncology, Dept of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States
* Corresponding author; email: clint.joiner{at}cchmc.org.
KCl cotransport (KCC) activity contributes to pathological dehydration in sickle (SS) RBC. KCC activation by urea was measured in SS and normal (AA) RBC as Cl-dependent Rb influx. KCC-mediated volume reduction was assessed by measuring reticulocyte cellular hemoglobin concentration (CHC) cytometrically. Urea activated KCC fluxes in fresh RBC to levels seen in swollen cells, although SS RBC required lower urea concentrations than normal (AA) RBC. Little additional KCC stimulation by urea occurred in swollen AA or SS RBC. The pH dependence of KCC in "euvolemic" SS RBC treated with urea was similar to that in swollen cells. Urea triggered volume reduction in SS and AA reticulocytes, establishing a higher CHC. Volume reduction was Cl-dependent, and limited by the KCC inhibitor, di-indenyl-oxyalkanoic acid. Final CHC depended on urea concentration, but not on initial CHC. Under all activation conditions, volume reduction was exaggerated in SS reticulocytes and produced higher CHC than in AA reticulocytes. The sulfhydryl reducing agent, dithiothreitol, normalized the sensitivity of KCC activation to urea in SS RBC, and mitigated the urea-stimulated volume decrease in SS reticulocytes, suggesting that the dysfunctional activity of KCC in SS RBC was due in part to reversible sulfhydryl oxidation.
