Dehydration of transferrin receptor-positive sickle reticulocytes during continuous or cyclic deoxygenation: role of KCl cotransport and extracellular calcium

Abstract

The K+ efflux that mediates sickle-cell dehydration may occur through several pathways, including two with a high capacity for mediating rapid K+ loss, KCl cotransport and the Ca(2+)-dependent K+ channel [K(Ca2+)]. The rate and pathway of red blood cell (RBC) dehydration most likely depends on cell age and hemoglobin (Hb) composition, with the presence of HbF playing an important role. Oxygenated sickle RBCs have relatively stable cell volume during incubation in vitro, whereas deoxygenated cells become dehydrated, and therefore more dense, due to activation of one or more K+ efflux pathways. In this investigation, sickle RBCs were deoxygenated either continuously or in 15-minute cycles for 4 hours, and the density increases of very young, transferrin receptor-positive (TfR+) cells and the remaining TfR-cells were determined. The contribution of KCl cotransport was estimated by replacing Cl-with NO3-.K(Ca2+) was inhibited by removal of Ca2+ or addition of charybdotoxin (ChTX). For both continuous and cyclic deoxygenation, TfR+ cells had a greater density increase when compared with TfR-cells. The lower percentage of HbF found in the TfR+ population may contribute to this difference. With continuous deoxygenation, the density shift was decreased by inhibition of K(Ca2+), but not by inhibition of KCl cotransport. With cyclic deoxygenation, the density shift was decreased in an independent, additive manner by inhibition of both pathways. Thus, cyclic deoxygenation of sickle cells under these conditions appears to activate both K(Ca2+) and the KCl cotransporter.

O2dependence of K+transport in sickle cells: the effect of different cell populations and the substituted benzaldehyde 12C79

“…We confirm that Cl Ϫ -independent K ϩ influxes were stimulated as PO 2 was reduced, as expected for mediation via P sickle . In control HbS cells, O 2 -dependence was similar to that in a previous report that involved a different method (passive K ϩ and Na ϩ fluxes) to monitor P sickle (22). Upon addition of 12C79, the stimulation of Cl Ϫ -independent K ϩ influxes occurred at lower PO 2 s, so that in both control cells and those treated with 12C79, the PO 2 s required for half-maximal activation of Cl Ϫ -independent K ϩ influx were similar to those required for half-maximal saturation of HbS with O 2 and half-maximal sickling ( Table 1).…”

“…While this may appear to suggest a lack of deoxygenation-induced Ca 2ϩ permeability incompatible with the properties of P sickle , it is not a unique finding. Joiner and colleagues (22,62) have shown that measurable transport via the Gardos channel is not always observed in unfractionated HbS cells upon deoxygenation. They suggest that most activity resides in a small subpopulation of HbS cells (perhaps those with least Ca 2ϩ pump activity) or that experimental conditions (for example, duration of deoxygenation, which in some cases may be several hours) are critical to allow sufficient Ca 2ϩ entry via P sickle .…”

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes

“…Accordingly, diluting the cytosol by preswelling of erythrocytes enhances the Ca 2+ sensitivity of breakdown of membrane phospholipid asymmetry as induced by hypotonic haemolysis (Williamson et al, 1985). Along those lines, activation of Ca 2+ -sensitive K + channels participates in the cell shrinkage and deformation of deoxygenized sickle cells (Bookchin et al, 1987;Lew and Bookchin, 1991;Brugnara et al, 1993;Joiner, 1993;Franco et al, 1996), which have most recently been shown to be highly sensitive to apoptosis (Lang et al, 2002b).…”

Differential oxygen sensitivity of the K+‐Cl− cotransporter in normal and sickle human red blood cells

“…K + ‐Cl − cotransport activity can therefore contribute to the development of sickle cells, whose raised microviscosity and reduced deformability (Ellory et al 1989 b ) contribute to vascular occlusion (at least in animal models) and hence to sickle cell disease. The relative importance of K + ‐Cl − cotransport in these processes, compared with that of the Ca 2+ ‐activated K + channel activity (Gardos channel: Gardos, 1958), also abnormally high in sickle cells, has been the subject of considerable research (for example: Ohnishi et al 1986; Ellory et al 1989 b ; Bookchin et al 1991; Apovo et al 1994; Franco et al 1996).…”