Incorporation of fatty acids into phosphatidylcholine is reduced during storage of human erythrocytes: Evidence for distinct lysophosphatidylcholine acyltransferases

The incorporation of [1-¹⁴C]palmitic or [1-¹⁴C]oleic acid into phosphatidylcholine and the effect on blood group antigen expression were examined in human erythrocytes stored at 4°C for 0-3 weeks. Blood drawn into EDTA was obtained by venepuncture from healthy volunteers. A 50% suspension of washed erythrocytes was incubated in buffer containing [1-¹⁴C]fatty acid for up to 60 min at 37°C with moderate shaking. Phosphatidylcholine was extracted and analyzed for uptake of radiolabelled fatty acid and phospholipid phosphorus content. Incorporation of [1-¹⁴C]palmitic or [1-¹⁴C]oleic acid into phosphatidylcholine was reduced during storage. The mechanism for the reduction in radiolabelled fatty acid incorporation into phosphatidylcholine was a 64% (p < 0.05) reduction in membrane phospholipase A2 activity. Although human erythrocyte membranes isolated from freshly drawn blood are capable of reacylating lysophosphatidylcholine to phosphatidylcholine, with storage, a markedly different substrate preference between palmitoyl-Coenzyme A and oleoyl-Coenzyme A was observed. Lysophosphatidylcholine acyltransferase activity assayed with oleoyl-Coenzyme A was unaltered with storage. In contrast, lysophosphatidylcholine acyltransferase activity assayed with palmitoyl-Coenzyme A was elevated 5.5-fold (p < 0.05). Despite these changes, storage of erythrocytes for up to 3 weeks did not result in altered expression of the various blood group antigens investigated. We conclude that the incorporation of palmitate and oleate into phosphatidylcholine is dramatically reduced during storage of human erythrocytes. The observed differential in vitro substrate utilization suggests that distinct acyltransferases are involved in the acylation of lysophosphatidylcholine to phosphatidylcholine in human erythrocytes.     

FUSION OF INTACT HUMAN ERYTHROCYTES AND ERYTHROCYTE GHOSTS

Sendai virus is able to induce the fusion of human erythrocytes. Bivalent cations or ATP are not essential for polyerythrocyte formation. High fusion indices were obtained when Sendai virus was added to cells incubated in the presence of both EDTA and iodoacetic acid. Human erythrocyte ghosts prepared by gradual hemolysis still retain the potential to undergo virus-induced fusion. Fusion of human red blood cells without the addition of viruses was obtained by incubation of erythrocytes at pH 10.5 in the presence of Ca⁺⁺ (40 mM) or by addition of phospholipase C Clostridium perfringens preparations to cells previously agglutinated or polylysine.     

Effect of phospholipase A on actions of cobra venom cardiotoxins on erythrocytes and skeletal muscle

The actions of two phospholipase-free cardiotoxins from the venom of the cobra Naja naja siamensis were compared to phospholipase-contaminated cardiotoxins in terms of their ability to lyse human erythrocytes and to depolarize and contract skeletal muscle. The presence of 3–5% (w/w) phospholipase caused a 20–30-fold increase in the haemolytic activity of the two cardiotoxins, the pure cardiotoxins being virtually without haemolytic activity at 10^?7-10^?6 M. Phospholipase contamination did not enhance the ability of the cardiotoxins to cause contracture of chick biventer cervicis muscles and it caused less than a 2-fold increase in the depolarizing activity of the cardiotoxins on cultured skeletal muscle. Phospholipase-free cardiotoxins were about 10–20-times more active on cultured skeletal muscle fibres than on erythrocytes. These results support the hypothesis that some cardiotoxins have more affinity for the membranes of excitable cells than for those of other cells such as erythrocytes.     

Possible relationship between membrane proteins and phospholipid asymmetry in the human erythrocyte membrane

After incubation of human erythrocytes at 37 °C in the absence of glucose (A) for 24 h, (B) for 4 h with 8 mM hexanol or (C) for 3 h with SH reagents, phosphatidylethanolamine becomes partly susceptible to hydrolysis by phospholipase A₂ from Naja naja. The presence of glucose during the pretreatments suppresses this effect, except in the case of SH reagents that inhibit glycolysis. After incubation with tetrathionate, up to 45% of the phosphatidylethanolamine is degraded by the enzyme, an amount considerably in excess of the 20% attacked in fresh erythrocytes.Pancreatic phospholipase A₂, an enzyme unable to hydrolyse the phospholipids of intact erythrocytes, partially degrades phosphatidylcholine and phosphatidylethanolamine of erythrocytes pretreated with hexanol or SH reagents. Reagents capable of oxidizing SH groups to disulfides (tetrathionate, $\text{o-}\text{iodosobenzoate}$ and hydroquinone) even render susceptible to pancreatic phospholipase A₂ phosphatidylserine, a phospholipid supposed to be entirely located in the inner lipid layer of the membrane. Alkylating or acylating SH reagents have no such effect. It is postulated that disulfide bond formation between membrane protein SH groups leads to an alteration in protein-phospholipid interactions and consequently induces a reorientation of phospholipids between the inner and the outer membrane lipid layer.     

A lipid requirement for the (Ca2+ + Mg2+)-activated ATPase of erythrocyte membranes.

The lipid requirement of the (Ca²⁺ + Mg²⁺)-stimulated ATPase of human erythrocytes has been studied. The enzyme activity was lost after removal of the phospholipids using phospholipase A₂ from Naja naja and serum albumin. Optimal restoration of the (Ca²⁺ + Mg²⁺)-ATPase activity in the partially lipid-depleted membranes was obtained with oleate. The reactivation was not due to the removal of a permeability barrier for ATP, since lysolecithin or cholate did not show latent activity. Reactivation was also obtained with several negatively charged phospholipids. Among the ones normally found in the erythrocyte membranes, only phosphatidyl serine reactivated significantly.     

The effects of neuraminidase on concanavalin a agglutination of erythrocytes: Evidence for adsorption of neuraminidase to erythrocyte membrane

Neuraminidase-treated human erythrocytes, but not untreated erythrocytes, were agglutinated by concanavalin A. The degree of concanavalin A agglutinability was not directly related to sialic acid removal by neuraminidase. While maximal sialic acid release was obtained with 5 units neuraminidase/2 × 10⁹ erythrocytes, maximal concanavalin A agglutination was only obtained after exposure to 20 units neuraminidase. Binding of ³H-concanavalin A by erythrocytes was 10-fold higher with rabbit compared to human red cells.Neuraminidase treatment of human erythrocytes caused a relative increase in ³H-concanavalin binding, but the absolute amount was still 10-fold less than that bound to rabbit erythrocytes. Specific adherence of neuraminidase to Con A-Agarose could not be demonstrated. There was no evidence for contamination of the neuraminidase preparation with proteases using a sensitive assay. These studies suggest that neuraminidase adsorbs to erythrocyte membranes and leads to concanavalin A agglutination of human erythrocytes by a mechanism other than removal of sialic acid.     

The relationship of membrane lipids to species specific hemolysis by hemolytic factors from Stomoxys calcitrans (L.) (Diptera: Muscidae)

Posterior-midgut homogenate from female stable flies prepared at 12 h after feeding hemolyzed erythrocytes from 6 different mammalian species more readily than homogenate prepared at 22 h. A significant correlation was obtained between the per cent sphingomyelin content of the erythrocyte membrane and the time required for lysis by the 12 h homogenate. Erythrocytes with low sphingomyelin content were more sensitive to lysis than cells with high sphingomyelin. No such correlation exists for hemolysis by 22 h homogenate. Mean corpuscular volume and osmotic fragilities of erythrocytes were not related to hemolysis either by 12 or 22 h homogenate. Determination of phospholipase C and sphingomyelinase activities showed that the hydrolysis rate of phospholipase C in homogenates prepared at 12–14 h was almost twice as much as sphingomyelinase activity. Whereas hydrolysis rates in 22–24 h homogenate were not different and markedly reduced compared to the 12–14 h homogenate. The times required for erythrocyte hemolysis related to the phospholipase C and sphingomyelinase activity profiles suggests that these enzyme activities participate in the in vitro hemolysis of red blood cells. Bovine and human erythrocytes change their biconcave contour into a spiculated spherical shape when they are exposed to midgut homogenate. This shape change is interpreted as a detergent induced modification of the red cell membrane which renders the erythrocytes more vulnerable to hemolysis.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and non-agglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. this was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination.Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg²⁺, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca²⁺ (0.2 mM) had little effect on agglutinability, although high Ca²⁺ (5 mM) inhibited agglutinability of the resealed membranes somewhat.Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells.The agglutination of erythrocytes was not affected by cytochalasin B (40 μg/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes.It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca²⁺ or Mg²⁺ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitive to vinblastine.     

Does diamide treatment of intact human erythrocytes cause a loss of phospholipid asymmetry?

Diamide-treated human erythrocytes have been compared with native red cells as to the accessibility of their amino phospholipids to both phospholipase A₂ hydrolysis and fluorescamine labeling. In agreement with observations by others (Haest, C.W.M., Plasa, G., Kamp, D. and Deuticke, B. (1978) Biochim. Biophys. Acta 509, 21–32), treatment of intact human erythrocytes with diamide resulted in considerably enhanced degradation of amino phospholipids upon subsequent incubation of the cells with bee venom phospholipase A₂. The hydrolysis of phosphatidylethanolamine (PE) in control cells reached a plateau value at 5% after 10 min. In diamide-treated cells, on the other hand, PE hydrolysis did not level off. Contrastingly, dose-response curves recorded for the labeling of PE with the very fast reacting NH₂-group-specific reagent, fluorescamine, showed identical results for both native and diamide-treated erythrocytes. In each of these two cases, a plateau was reached after approx. 15% of the PE had been labeled. These results strongly suggest that the enhanced phospholipase-A₂-induced hydrolysis of amino phospholipids in diamide-treated erythrocytes may reflect a destabilization of the lipid bilayer, rather than an in situ loss of phospholipid asymmetry.     

Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes

Summary Osmotic swelling of human and rat erythrocytes does not induce regulatory volume decrease. Regulatory volume increase was observed in shrunken erythrocytes of rats only. This reaction was blocked by the inhibitors of Na⁺/H⁺ exchange. Cytoplasmic acidification in erythrocytes of both species increases the amiloride-inhibited component of²²Na influx by five- to eight-fold. Both the osmotic and isosmotic shrinkage of rat erythrocytes results in the 10- to 30-fold increase of amiloride-inhibited²²Na influx and a two-fold increase of furosemide-inhibited⁸⁶Rb influx. We failed to indicate any significant changes of these ion transport systems in shrunken human erythrocytes. The shrinking of quin 2-loaded human and rat erythrocytes results in the two- to threefold increase of the rate of⁴⁵Ca influx, which is completely blocked by amiloride. The dependence of volume-induced²²Na influx in rat erythrocytes and⁴⁵Ca influx in human erythrocytes on amiloride concentration does not differ. The rate of⁴⁵Ca influx in resealed ghosts was reduced by one order of magnitude when intravesicular potassium and sodium were replaced by choline. It is assumed that the erythrocyte shrinkage increases the rate of a nonselective Ca _o ²⁺ (Na _i ⁺ , K _i ⁺ ) exchange. Erythrocyte shrinking does not induce significant phosphorylation of membrane protein but increases the³²P incorporation in diphosphoinositides. The effect of shrinkage on the³²P labeling of phosphoinositides is diminished after addition of amiloride. It is assumed that volume-induced phosphoinositide response plays an essential role in the mechanism of the activation of transmembrane ion movements.     

The mechanism of change in the rate of agglutination of human erythrocytes under the influence of adrenaline

The study of erythrocytes of 80 men showed that adrenaline (10^?10–10^?6 g/mL) and phenylephrine (10^?10–10^?6 g/mL) dose-dependently increase the rate of agglutination of erythrocytes, judging by the decrease in the start time of agglutination, whereas ginipral (10^?10–10^?7 g/mL), on the contrary, decreases it. The effect of adrenaline and phenylephrine is blocked by nicergoline (10^?6 g/mL), enhanced by obzidan (10^?6 g/mL), and is not changed by yohimbine (10^?6 g/mL) and atenolol (10^?6 g/mL). These data indicate that the rate of agglutination increases with the activation of α1-adrenergic receptor (AR) and decreases with the activation of β₂-AR, whereas the activation of α₂- and β₁-AR does not affect it. Trifluoperazine (10^?6 g/mL) as a calmodulin antagonist, barium chloride (10^?6 g/mL) as a Ca²⁺-dependent K⁺-channel blocker, and indomethacin (10^?6 g/mL) as an inhibitor of cyclooxygenase and phospholipase A₂ inhibit the ability of adrenaline to increase the rate of agglutination of erythrocytes. This suggests that this effect of adrenaline is caused by an increased Ca²⁺ entry into the erythrocyte, activation of calmodulin, cyclooxygenase, and phospholipase A₂, and subsequent K⁺ release from the erythrocytes through the Ca²⁺-dependent K⁺ channels, which is regarded as a manifestation of eryptosis. Indirectly, this means that the potentiation of activation of α₁-AR and β₂-AR, respectively, increases and, conversely, decreases the rate of eryptosis.     

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.     

Changes in lipid metabolism and cell morphology following attack by phospholipase C (Clostridium perfringens) on red cells or lymphocytes

When intact human erythrocytes were treated with phospholipase C (Clostridium perfringens), up to 30% of the membrane phospholipids were broken down without significant cell lysis. Only phosphatidylcholine and sphingomyelin were attacked. Ceramide (derived from sphingomyelin) accumulated, but 1,2-diacylglycerol (derived from phosphatidylcholine) was largely converted into phosphatidate. Up to 12% of the cell phospholipid could be converted into phosphatidate in this way. Pig erythrocytes and lymphocytes showed a similar but smaller synthesis of phosphatidate after phospholipase C attack.Phospholipase C also caused a marked morphological change in erythrocytes, giving rise to spherical cells containing internal membrane vesicles. This change appeared to be due to ceramide and diacylglycerol accumulation rather than to increased phosphatidate content of the cells.     

Phospholipid asymmetry in the membranes of intact human erythrocytes and in spectrin-free microvesicles derived from them

Phospholipase A₂ from bee venom and Naja naja has been used to study the orientation of phospholipids present in the membrane of intact human erythrocytes and in spectrin-free microvesicles derived from the cells by treatment with Ca²⁺ and A23187. Little difference between the cells and microvesicles was observed in the apparent accessibility of phospholipids to the enzyme, suggesting that the original lipid asymmetry was maintained in the absence of spectrin. However, incubation of the microvesicles for 16 h at 37°C did lead to partial loss of asymmetry in the transmembrane distribution of phosphatidylcholine and phosphatidylethanolamine but not of phosphatidylserine. Despite the similarity of lipid asymmetry in cells and fresh microvesicles, the latter were about 40-fold more sensitive to phospholipase treatment than were cells. Although they retained the lipid asymmetry of intact cells, the microvesicles resembled ghosts in their great sensitivity to phospholipase A₂ attack, suggesting that the lipid packing in microvesicles and ghosts was similar. This conclusion was supported by the results of experiments with a fluorescent probe Merocyanine 540.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

Fusion of chicken erythrocytes by phospholipase C (Clostridium perfringens): The requirement for hemolytic and hydrolytic factors for fusion

Phospholipase C (Clostridium perfringens) preparations are able to induce fusion of chicken erythrocytes only in the presence of low concentrations (0.6 mM) of Mn²⁺. Anti-phospholipase C serum inhibits hemolysis and fusion of the cells only when added during the first 20 min of the incubation time. Heated phospholipase C preparations which fail to hemolyze chicken erythrocytes are able to induce fusion only in the presence of another hemolytic reagent such as prymnesin (an hemolytic toxin from Prymnesium parvum).     

The P2X7 receptor mediates the uptake of organic cations in canine erythrocytes and mononuclear leukocytes: comparison to equivalent human cell types

We previously demonstrated that canine erythrocytes express the P2X₇ receptor, and that the function and expression of this receptor is greatly increased compared with human erythrocytes. Using ⁸⁶Rb⁺ (K⁺) and organic cation flux measurements, we further compared P2X₇ in erythrocytes and mononuclear leukocytes from these species. Concentration response curves of BzATP- and ATP-induced ⁸⁶Rb⁺ efflux demonstrated that canine P2X₇ was less sensitive to inhibition by extracellular Na⁺ ions compared to human P2X₇. In contrast, canine and human P2X₇ showed a similar sensitivity to the P2X₇ antagonists KN-62 and Mg²⁺. KN-62 and Mg²⁺ also inhibited ATP-induced choline⁺ uptake into canine and human erythrocytes. BzATP and ATP but not ADP or NAD induced ethidium⁺ uptake into canine monocytes, T- and B-cells. ATP-induced ethidium⁺ uptake was twofold greater in canine T-cells compared to canine B-cells and monocytes. KN-62 inhibited the ATP-induced ethidium⁺ uptake in each cell type. P2X₇-mediated uptake of organic cations was 40- and fivefold greater in canine erythrocytes and lymphocytes (T- and B-cells), respectively, compared to equivalent human cell types. In contrast, P2X₇ function was threefold lower in canine monocytes compared to human monocytes. Thus, P2X₇ activation can induce the uptake of organic cations into canine erythrocytes and mononuclear leukocytes, but the relative levels of P2X₇ function differ to that of equivalent human cell types.     

ATPase and phosphatase activities from human red cell membranes: II. The effects of phospholipases on Ca2+-dependent enzymic activities

Summary Treatment of human red cell membranes with pure phospholipase A₂ results in a progressive inactivation of both Ca²⁺-dependent and (Ca²⁺+K⁺)-dependent ATPase and phosphatase activities. When phospholipase C replaces phospholipase A₂, Ca²⁺-dependent ATPase activity and Ca²⁺-dependent phosphorylation of red cell membranes are lost, while Ca²⁺-dependent phosphatase activity is enhanced and its apparent affinity for Ca²⁺ is increased about 20-fold. Activation of Ca²⁺-dependent phosphatase following phospholipase C treatment was not observed in sarcoplasmic reticulum preparation. Phospholipase C increases the sensitivity of the phosphatase to N-ethylmaleimide but has little effect on the kinetic parameters relating the phosphatase activity to substrate and cofactors, suggesting that no extensive structural disarrangement of the Ca²⁺-ATPase system has occurred after incubation with phospholipase C.     

Human Erythrocyte Receptor of l-Fucose-specific Lectin Produced by Streptomyces sp.

L-Fucose-specific lectin produced by Streptomyces no. 16-3 (SFL 16-3) was labeled with N- succinimidyl-[2, 3-³H]-propionate to quantitatively investigate its binding to human erythrocytes. The binding inhibition by sugars was competitive, and 5mM L-fucose or 20 mM d-mannose completely inhibited the binding. Among plant lectins, Lotus tetragonolobus, Ulex europeus I, soybean and wheat germ lectin showed competitive inhibition. The association constant and the average number of binding sites for human blood group O erythrocytes were approximately 3 × 10⁷ M^-1 and 1 × 10⁶ cell^-1, respectively. Trypsinization of erythrocytes preferentially increased the number of binding sites for human A and B erythrocytes but not for O erythrocytes.

Membrane components were extracted from human B and O erythrocytes and their binding activity for SFL 16-3 was tested using the hemagglutination-inhibition assay. Poly(glycosyl)-ceramide was the predominant receptor and its fucosyl residue was essential for binding. The crude glycoprotein fraction showed only slight inhibition activity.