Narla Mohandas

The effect of malonyldialdehyde, a product of lipid peroxidation, on the deformability, dehydration and 51Cr‐survival of erythrocytes

Summary. Erythrocyte membrane lipid peroxidation has been reported to occur in various haemolytic anaemias. In the present study, treatment of human erythrocytes with malonyldialdehyde (MDA). a product of fatty acid peroxidation, induced membrane rigidity, cellular dehydration and reduced whole cell deformability. These effects of MDA were blocked by histamine and fluorescamine, which can act as alternate substrates for MDA. Additionally, reduced deformability of MDA‐treated rabbit cells was associated with shortened 51Cr survival in vivo. These findings suggest a biochemical basis for decreased survival of erythrocytes undergoing peroxidative damage of the membrane.

Deformability of isolated red blood cell membranes.

We have used a laser diffraction method (ektacytometry) to directly measure the membrane component of red cell deformability, without contributions from either cell geometry or internal viscosity. This technique was validated by subjecting resealed erythrocyte ghosts to manipulations previously shown to increase the membrane shear modulus. Heating above 45 degrees C, pH greater than 9.0 and less than 5.0, and micromolar concentrations of the cross-linking agents, glutaraldehyde and diamide, all reduced the deformability of resealed erythrocyte ghosts. We have applied this assay to the study of reduced cellular deformability of calcium-loaded red cells, and have shown that, for physiological concentrations of calcium, the effect of calcium on the physical properties of the membrane may be negligible when compared to its effect of promoting cell dehydration and subsequent increased cytoplasmic viscosity.

The role of membrane protein sulfhydryl groups in hydrogen peroxide-mediated membrane damage in human erythrocytes

The formation of spectrin-hemoglobin complex following treatment of red cells with hydrogen peroxide (H2O2) has previously been shown to be associated with alterations in cell shape, decreased membrane deformability and increased recognition of modified cells by anti-IgM immunoglobulin in a phagocytic assay by monocytes. Prior treatment with carbon monoxide completely inhibited the H2O2-associated membrane changes, indicating a role for oxidized hemoglobin in the complex formation. Also, in a cell-free system, blockage of sulfhydryl (SH) groups on purified spectrin by N-ethylmaleimide significantly reduced the complex formation, suggesting a role for SH groups of spectrin in crosslinking process. The present study was undertaken to examine the role of SH blockade by N-ethylmaleimide on intact red cells undergoing oxidative damage. Pretreatment of erythrocytes with N-ethylmaleimide at concentrations ranging from 0.1 to 0.2 mM resulted in decreased lipid peroxidation and spectrin hemoglobin crosslinking. Moreover, pretreatment with N-ethylmaleimide resulted in less marked alterations in cell shape and membrane deformability as well as reduced recognition of peroxidized cells by antiglobulin serum. N-Ethylmaleimide treatment had no effect on methemoglobin formation. Studies with 14C-labeled N-ethylmaleimide showed that over 50% of N-ethylmaleimide was incorporated into spectrin. Pretreatment of cells with higher concentrations of N-ethylmaleimide (over 0.2 mM) was associated with membrane dysfunction independent of H2O2. These results imply that blocking of reactive SH groups leads to reduced interaction of spectrin with oxidized globin. These data, along with our prior observations, indicate that SH groups on spectrin play an important role in hemoglobin oxidation-induced formation of spectrin-hemoglobin complex and the resultant deleterious effects on membrane properties.

Cell Membrane and Volume Changes during Red Cell Development and Aging a

This paper provides a summary of our understanding of cell membrane and volume changes during red cell development and aging. Cytoskeletal structures which include microtubules and microfilaments appear to play key roles in the genesis of the anucleate reticulocyte from its nucleated precursor cell, as well as in the early stages of reticulocyte development. The maturation of reticulocyte into red cell is accompanied by marked changes in cell shape and extensive remodeling of the membrane skeleton, resulting in the mature red cell acquiring a highly deformable yet remarkably stable membrane. The volume and cell density heterogeneity seen for circulating red cells also appears to be the result of the membrane changes that occur during reticulocyte maturation. Following its genesis from reticulocyte, the mature red cell undergoes further membrane and volume changes during its life span of 120 days. While it is clear that surface area loss, decrease in cell volume and cell surface modifications leading to binding of immunoglobulins accompany red cell aging, the cardinal cellular modification responsible for the removal of senescent red cells from the circulation is yet to be defined.

Reduced transglutaminase-catalyzed cross-linking of exogenous amines to membrane proteins in sickle erythrocytes

In order to determine the capacity of sickle cells to undergo transglutaminase-catalyzed cross-linking of membrane proteins, human normal and sickle erythrocytes were incubated with [ring-2-14C]histamine in the presence of Ca2+ and ionophore A23187. The [14C]histamine incorporation into membrane components was observed in freshly prepared erythrocytes. Incorporation of radioactivity into spectrin and Band 3 membrane components was significantly (P less than 0.001) less in sickle erythrocytes than in normal cells. Transglutaminase deficiency was excluded by the finding of increased activity of this enzyme in sickle cells from patients with reticulocytosis. The incorporation of [3H]spermine into red cell membranes was also less in sickle erythrocytes than in normal cells under the same conditions of incubation used for [ring-2-14C]histamine. Sickle erythrocytes were more permeable to these amines than normal cells. It is proposed that the gamma-glutamyl sites of membrane proteins in sickle erythrocytes are less accessible for transglutaminase-catalyzed cross-linking to histamine and polyamines in vitro, perhaps due to prior in vivo activation of this enzyme by the increased calcium in sickle cells and/or shielding secondary to altered membrane organization.

Polyamines do not inhibit erythrocyte ATPase activities

To test whether physiologic elevation of red cell polyamine levels might explain Na pump inhibition in sickle cells or uremic red cells, we have studied the effect of putrescine, spermidine and spermine on red cell membrane ATPase and Na-K active transport. Measurement of the ouabain-sensitive influx of 86Rb into intact cells showed no effect of spermine. However, cells became depleted of ATP during incubation with spermine. By 48 h, the cells showed substantial potassium loss and moderate sodium gain. Because the low permeability of red cell membranes for polyamines might have obscured some direct effects on intracellular processes, we measured active transport of 22Na out of red cell ghosts that had been resealed in the presence of 5 mmol/l spermine. In addition, we measured the Na-K, Mg, and Ca ATPase activities of broken membrane preparations in the presence of spermine, spermidine and putrescine. Polyamines had no direct effect on cation transport in red cells, although possible adverse effects on red cell metabolism could have a secondary effect on cation regulation.