valinomycin and Anemia--Sickle-Cell

The Ca(2+)-sensitive K(+) channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca(2+) loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age-activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K(+) permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age-density distribution pattern during dehydration. However, when Ca(2+) loads were used to induce maximal K(+) fluxes via Gardos channels in all RBCs (F(max)), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F(max) was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age-activity relation revealed a monotonic decline in F(max) with cell age, with a broad quasi-Gaussian F(max) distribution among the RBCs.

Topics: Aging; Anemia, Sickle Cell; Calcium; Cell Movement; Dehydration; Erythrocytes; Glycated Hemoglobin; Hemoglobins; Humans; In Vitro Techniques; Intermediate-Conductance Calcium-Activated Potassium Channels; Ionophores; Normal Distribution; Potassium; Reference Values; Valinomycin

We describe a population of sickle cell anemia red cells (SS RBCs) ( approximately 4%) and a smaller fraction of normal RBCs (<0.03%) that fail to dehydrate when permeabilized to K(+) with either valinomycin or elevated internal Ca(2+). The nonshrinking, valinomycin-resistant (val-res) fractions, first detected by flow cytometry of density-fractionated SS RBCs, constituted up to 60% of the lightest, reticulocyte-rich (R1) cell fraction, and progressively smaller portions of the slightly denser R2 cells and discocytes. R1 val-res RBCs had a mean cell hemoglobin concentration of approximately 21 g of Hb per dl, and many had an elongated shape like "irreversibly sickled cells," suggesting a dense SS cell origin. Of three possible explanations for val-res cells, failure of valinomycin to K(+)-permeabilize the cells, low co-ion permeability, or reduced driving K(+) gradient, the latter proved responsible: Both SS and normal val-res RBCs were consistently high-Na(+) and low-K(+), even when processed entirely in Na-free media. Ca(2+) + A23187-induced K(+)-permeabilization of SS R1 fractions revealed a similar fraction of cal-res cells, whose (86)Rb uptake showed both high Na/K pump and leak fluxes. val-res/cal-res RBCs might represent either a distinct erythroid genealogy, or an "end-stage" of normal and SS RBCs. This paper focuses on the discovery, basic characterization, and exclusion of artifactual origin of this RBC fraction. Many future studies will be needed to clarify their mechanism of generation and full pathophysiological significance.

Topics: Anemia, Sickle Cell; Bumetanide; Calcimycin; Calcium; Cell Membrane Permeability; Drug Resistance; Electron Probe Microanalysis; Erythrocytes; Ionophores; Ouabain; Potassium; Potassium Channels; Rubidium; Sodium; Spectrophotometry, Atomic; Valinomycin

Sickle red blood cells (RBCs) become depleted of potassium, leading to dehydration and abnormally elevated cellular density. The increased sickling that results is important for both hemolysis and vasocclusion. In this study, sickle cells were subjected to high-speed centrifugation, and the bottom 15% were isolated. This procedure removed light cells and to a variable degree enriched cells that were denser than normal to produce a high-density-enriched (HDE) population of sickle cells. Autologous HDE cells from 3 subjects were labeled with biotin and re-infused. The following determinations were performed: (1) the survival and density changes of HDE cells; (2) the amount of fetal hemoglobin (HbF) in labeled cells after magnetic isolation; (3) the percentage of labeled F cells; (4) the percentage of labeled cells displaying external phosphatidylserine (PS). For patients with 3.5%, 4.5%, and 24% HbF in the HDE RBCs, the circulation half-time was 40, 80, and 180 hours, respectively. The percentage of HbF (measured in all 3 subjects) and of F cells (measured in 2 subjects) in labeled RBCs increased with time after re-infusion, indicating that HDE F cells have longer in vivo survival than HDE non-F cells. The percentage of PS(+), biotin-labeled HDE cells showed no consistent increase or decrease with time after re-infusion. These data provide evidence that HDE sickle cells, especially those that do not contain HbF, have a very short in vivo survival, and that the percentage of PS(+) cells in a re-infused HDE population does not change in a consistent manner as these cells age in the circulation.

Topics: Anemia, Sickle Cell; Biotin; Biotinylation; Cell Membrane; Cell Separation; Cell Survival; Cellular Senescence; Erythrocytes; Fetal Hemoglobin; Flow Cytometry; Humans; Intracellular Fluid; Ionophores; Phosphatidylserines; Sodium; Time Factors; Valinomycin

The formation of irreversibly sickled red cells has been studied by inducing cell shrinkage, ion loss, Ca2+ accumulation and membrane loss either singly or in combination. Valinomycin, A23187+Ca2+ or hypertonic saline caused shrinkage of the cells with retention of the sickled form after reoxygenation. The cells which had retained the sickle shape after treatment with the ionophores and reoxygenation remained sickled after exposure to hypotonic media. These cells were also osmotically insensitive. Retention of the sickled form was not dependent upon membrane loss as induced by repeated sickle-unsickle cycles or by A23187+Ca2+ treatment although repetitive sickling did give rise to shorter, stubbier spicules. Sickled red cells, either the endogenous irreversibly sickled cells or the sickled cells induced by deoxygenation, did not lose membrane by vesicle or spicule loss as normal cells or oxygenated sickle red cells do. Cell water loss without cell membrane loss appears to be an important factor in the irreversible sickling of red cells.

Topics: Anemia, Sickle Cell; Anti-Bacterial Agents; Calcimycin; Calcium; Erythrocyte Membrane; Erythrocytes, Abnormal; Humans; Saline Solution, Hypertonic; Valinomycin