Variability of Intracellular pH within Individual Populations of SS and AA Erythrocytes

Kaperonis, A. A.; Bertles, John F.; Chien, Shu

doi:10.1111/j.1365-2141.1979.tb03766.x

Cited by 17 publications

(15 citation statements)

References 23 publications

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“…Although most of our experiments were carried out in pH 6.8 buffer, pentacoordinate HbS-NO was found to also dominate in all pH 7.2 Trizma buffer (78% pentacoordinate ␣-nitrosyl after 1 h of incubation at 37°C). The pH (6.8) used in most of our experiments is not as subphysiological as one may think since the pH in the sickle red cell has been found to be 7.1-7.2 (42,43). Most importantly, the pentacoordinate HbS-NO species has been shown to predominate in venous blood (40).…”

Section: Discussionmentioning

confidence: 96%

Effects of Iron Nitrosylation on Sickle Cell Hemoglobin Solubility

Lockamy

Chen

et al. 2002

Journal of Biological Chemistry

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One mechanism by which nitric oxide (NO) has been proposed to benefit patients with sickle cell disease is by reducing intracellular polymerization of sickle hemoglobin (HbS). In this study we have examined the ability of nitric oxide to inhibit polymerization by measuring the solubilizing effect of iron nitrosyl sickle hemoglobin (HbS-NO). Electron paramagnetic resonance spectroscopy was used to confirm that, as found in vivo, the primary type of NO ligation produced in our partially saturated NO samples is pentacoordinate ␣-nitrosyl. Linear dichroism spectroscopy and delay time measurements were used to confirm polymerization. Based on sedimentation studies we found that, although fully ligated (100% tetranitrosyl) HbS is very soluble, the physiologically relevant, partially ligated species do not provide a significant solubilizing effect. The average solubilizing effect of 26% NO saturation was 0.045; much less than the 0.15 calculated for the effect of 26% oxygen saturation. Given the small amounts of NO-ligated hemoglobin achievable through any kind of NO therapy, we conclude that NO therapy does not benefit patients through any direct solubilizing effect.Sickle cell disease is caused by a mutation in the sixth codon of the ␤-globin gene that results in the hydrophobic residue valine replacing hydrophilic glutamate in the hemoglobin (Hb)

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Section: Discussionmentioning

confidence: 96%

Effects of Iron Nitrosylation on Sickle Cell Hemoglobin Solubility

Lockamy

Chen

et al. 2002

Journal of Biological Chemistry

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“…Earlier measurements (Seakins, Gibbs, Milner & Bertles, 1973;Kaperonis, Bertles & Chien, 1979) of RBC 2,3-DPG in 'top, middle and bottom' thirds of centrifuged columns of SS and AA cells density fractions, indicated that the increase in the total RBC 2,3-DPG: haemoglobin ratio in the SS cells could be attributed to the-upper and middle fractions. The bottom third of SS red cells showed a low normal 2,3-DPG: haemoglobin ratio.…”

Section: Measurements Of 23-diphosphoglycerate Concentrations In Denmentioning

confidence: 99%

Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells.

Ortiz

Lew

Bookchin

1990

The Journal of Physiology

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SUMMARY1. Our findings of a low total magnesium content in the dense fraction (over 1418 g ml-1) of sickle cell anaemia (SS) red cells seemed inconsistent with the low Mg2+ permeability and outward Mg2+ gradient seen in normal red cells, and prompted studies of the Mg2+ permeability and equilibria in the SS cells.2 5. The above findings suggested that the documented low sodium pump fluxes in dense SS cells may result from an increased Mg2+:ATP ratio, which is known to inhibit Na+-K+ exchange fluxes through the sodium pump. If so, deoxygenation, by increasing the Mg2+: ATP ratio, should inhibit the pump further, whereas increasing ATP should relieve the inhibition. Experiments designed to test this possibility showed that in these dense SS cells, the ouabain-sensitive K(86Rb) influx was low in oxygenated cells, was reduced further by deoxygenation, but was substantially increased after treatment with inosine, pyruvate and phosphate to increase their organic phosphate pool. These results were thus consistent with such a mechanism for Na+ pump inhibition in the dense SS cells.

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“…Separation of Sickle Cells by Density-Sickle cells are heterogeneous with respect to total magnesium, DPG content, and pH (39,40), which vary with cell density as the cell sickles, therefore we separated sickle cells into three fractions using a discontinuous arabinogalactose density gradient before analysis, thereby allowing comparison with unfractionated whole sickle blood. Cells were fractionated on discontinuous density gradients of Stractan II (arabinogalactan) as described previously (41) with minor modifications (42).…”

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confidence: 99%

Simultaneous Determination of Low Free Mg2+ and pH in Human Sickle Cells using 31P NMR Spectroscopy

Willcocks

Mulquiney

Ellory

et al. 2002

Journal of Biological Chemistry

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Magnesium is the second most abundant intracellular cation after potassium and is important in the regulation of more than 300 enzymes (1-3) and ion transport across cell membranes (4, 5). Some diseases, such as hypertension, pre-eclampsia, and sickle cell anemia exhibit pathologies linked to low magnesium levels (6 -8). The genetic defect in sickle cell anemia results in the synthesis of an abnormal ␤ hemoglobin (Hb) subunit, which polymerizes upon deoxygenation. This polymerization produces the characteristic sickle deformation in red blood cells and leads to microcirculatory occlusion and consequent tissue ischemia. Sickling depends strongly on the intracellular Hb concentration (9) and is enhanced by dehydration of erythrocytes, a process involving potassium and sodium transport across cell membranes, which is in turn thought to be regulated by cytosolic free magnesium, [Mg 2ϩ ] free , 1 via the KCl cotransporter (KCC1) (5, 10). Thus dehydration of sickle cells, and thereby the process of sickling, may be controlled by changes in the magnesium content (8, 11).To assess its role in sickle cell anemia, an accurate and reproducible method was required for the measurement of [Mg 2ϩ ] free . Although several methods have been developed to determine [Mg 2ϩ ] free in red blood cells, such as null point titration with metallochromic dyes (12), magnesium-sensitive electrodes (13,14), use of divalent cation ionophores (15), and 31 P NMR spectroscopy (16,17), the reported values vary 3-fold, from 0.2 to 0.6 mM (18 -20). Of these methods, the one noninvasive technique that has been used extensively is that of 31 P NMR spectroscopy (16 -19, 21-25). However, there are a number of potential errors associated with using the observed NMR signal to determine [Mg 2ϩ ] free , including uncertainty in the binding constant between ATP and magnesium (26), the effect of intracellular ionic content on this interaction (27), and the effect of metabolite-Hb interactions (23). Gupta and co-workers (2), the first to use this method, addressed the first two problems by determining an apparent MgATP binding constant, K (app)bMgATP , from the difference between the end points of the chemical shifts of the magnesium bound and unbound ␣ and ␤ phosphorus nuclei of ATP, ␦ ␣␤-MgATP and ␦ ␣␤-ATP . This decreased the number of equilibria that had to be considered and overcame the need to rely on adjusted published values of K (app)b , which varied 100-fold (28). However, as with any simplification, problems are associated with this approach, which include the possible difference in ionic strength between solutions and in vivo conditions, the accuracy of the chemical shift end points required to determine K (app)b (29), and the assumption that ␦ ␣-ATP did not vary.Because the chemical shifts of 31 P NMR phosphorus peaks depend on many different factors (30), accurate analysis should take all factors into account, which has become possible with the increased capacity of computation. Previous analyses using phosphorus chemical shifts have be...

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Variability of Intracellular pH within Individual Populations of SS and AA Erythrocytes

Cited by 17 publications

References 23 publications

Effects of Iron Nitrosylation on Sickle Cell Hemoglobin Solubility

Effects of Iron Nitrosylation on Sickle Cell Hemoglobin Solubility

Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells.

Simultaneous Determination of Low Free Mg2+ and pH in Human Sickle Cells using 31P NMR Spectroscopy

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