Interaction of carboplatin with carrier human erythrocytes.

The antineoplastic drug Carboplatin (CBDCA) was encapsulated in human erythrocytes by means of transient hypotonic hemolysis, followed by isotonic resealing. Up to 5 mg/ml of packed cells could be entrapped, with about 70% cell recovery. In vitro incubation of the CBDCA-loaded erythrocytes in autologous plasma caused a very slow release of the drug from the cells (12% approximately in 3 h). The encapsulation conditions, performed at a low hematocrit, in order to obtain high amounts of the drug inside the carriers, impaired the metabolic properties of the loaded erythrocytes significantly. In particular, an almost complete disappearance of GSH was observed. Analysis of the intraerythrocytic metabolism of CBDCA showed that, in spite of its relatively high stability in aqueous solutions, in hemolysates and in the loaded erythrocytes a significant percentage of CBDCA is rapidly converted to other species that still retain an antiproliferative activity in vitro. This fast conversion could be extensively inhibited by previous conversion of oxyhemoglobin to methemoglobin or carbomonoxyhemoglobin, suggesting an important role of heme iron in this process. Encapsulation of CBDCA in selectively targeted human erythrocytes may represent a therapeutic strategy for increasing the drug concentration in specific organs, notably liver.     

Interaction of parvovirus B19 with human erythrocytes alters virus structure and cell membrane integrity

The unique region of the capsid protein VP1 (VP1u) of B19 virus (B19V) elicits a dominant immune response and has a phospholipase A₂ (PLA₂) activity required for the infection. Despite these properties, we have observed that the VP1u-PLA₂ motif occupies an internal position in the capsid. However, brief exposure to increasing temperatures induced a progressive accessibility of the PLA₂ motif as well as a proportional increase of the PLA₂ activity. Similarly, upon binding on human red blood cells (RBCs), a proportion of the capsids externalized the VP1u-PLA₂ motif. Incubation of B19V with RBCs from 17 healthy donors resulted in extensive virus attachment ranging between 3,000 and 30,000 virions per cell. B19V empty capsids represent an important fraction of the viral particles circulating in the blood (30 to 40%) and bind to RBCs in the same way as full capsids. The extensive B19V binding to RBCs did not cause direct hemolysis but an increased osmotic fragility of the cells by a mechanism involving the PLA₂ activity of the exposed VP1u. Analysis of a blood sample from an individual with a recent B19V infection revealed that, at this particular moment of the infection, the virions circulating in the blood were mostly associated to the RBC fraction. However, the RBC-bound B19V was not able to infect susceptible cells. These observations indicate that RBCs play a significant role during B19V infection by triggering the exposure of the immunodominant VP1u including its PLA₂ constituent. On the other hand, the early exposure of VP1u might facilitate viral internalization and/or uncoating in target cells.     

Adrenergic stimulation of membrane protein phosphorylation in human erythrocytes.

Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased 32P incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an alpha-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.     

Adriamycin-plasma membrane interaction in human erythrocytes

A great body of data increasingly point to the cell membrane as an important target for adriamycin (ADR). However, the exact mechanism by which ADR exerts its cytotoxic action through the interaction with the plasma membrane is still unknown. In this study, the interaction of ADR with red blood cells from healthy donors was investigated by freeze-fracturing (FF) and scanning electron microscopy (SEM). The results obtained can be summarized as follows: a) a dose-dependent modification in the intramembrane particle (IMP) distribution was revealed by FF on both fracture faces of the plasma membrane of erythrocytes treated with 50 or 100 microM ADR; b) SEM observations allowed to reveal a discocyte-stomatocyte transition induced by 50 microM ADR and the formation of mottled cells at the higher dose; c) these morphological and ultrastructural changes were not related to lipid peroxidation as demonstrated by experiments with radical scavengers or strong oxidant substances; d) the analysis of IMP density seemed to rule out a segregation process of membrane proteins suggesting that ADR interacts with the plasma membrane by becoming incorporated within the lipid bilayer.     

Peroxide-induced membrane damage in human erythrocytes

Erythrocytes exposed to H2O2 or t-butyl hydroperoxide (tBHP) exhibited lipid peroxidation and increased passive cation permeability. In the case of tBHP a virtually complete inhibition of both processes was caused by butylated hydroxytoluene (BHT), whereas pretreatment of the cells with CO increased both lipid peroxidation and K+ leakage. In the experiments with H2O2, on the other hand, both BHT and CO strongly inhibited lipid peroxidation, without affecting the increased passive cation permeability. These observations indicate different mechanisms of oxidative damage, induced by H2O2 and tBHP, respectively. The SH-reagent diamide strongly inhibited H2O2-induced K+ leakage, indicating the involvement of SH oxidation in this process. With tBHP, on the contrary, K+ leakage was not significantly influenced by diamide. Thiourea inhibited tBHP-induced K+ leakage, without affecting lipid peroxidation. Together with other experimental evidence this contradicts a rigorous interdependence of tBHP-induced lipid peroxidation and K+ leakage.     

The osmotic response of human erythrocytes and the membrane cytoskeleton

The volumes of human erythrocytes suspended in solutions of varying concentrations of sodium chloride and sucrose were measured by a Coulter Channelyzer Model H4 with appropriate corrections. The cells showed greatly restricted volume changes at osmolarities between 200-700 mOsm. At osmolarities outside this limit, on the other hand, the cells showed nonrestricted volume changes following essentially the predictions of an ideal osmometer. This unexpected volume response was not spuriously due to changes in shape or to a changing orientation of the cells as they traversed the aperture. The restricted volume change observed was abolished when the cells had previously been treated with diamide or had been heated for 60 minutes at 50 degrees C, conditions that are known to disturb the spectrin-actin network. The possibility must be considered that the osmotic behavior of human erythrocytes may be nonideal and that this nonideal behavior is primarily due to mechanical restriction provided by the spectrin-actin network of the membrane cytoskeleton.     

Rhnull human erythrocytes have an abnormal membrane phospholipid organization.

Rhnull human erythrocytes lack the antigens of the Rhesus blood group system, have an abnormal shape and an increased osmotic fragility, and are associated with mild chronic haemolytic anaemia. Studies with phospholipase A2 and sphingomyelinase C show that the asymmetric distribution of phosphatidylethanolamine (PtdEtn) in the membrane of these cells differs from that found in control cells. The amount of PtdEtn which can be hydrolysed by phospholipase A2 in the presence of sphingomyelinase C in intact Rhnull cells is twice as high as that in normal erythrocytes. In intact Rhnull cells all of the phosphatidylcholine (PtdCho) present in the membrane can be readily exchanged with a PtdCho-specific exchange protein, whereas in control cells 75% is readily exchanged and 25% at a much lower rate. This indicates that PtdCho experiences a relatively fast transbilayer movement in the Rhnull cells. The observation that the loss of two membrane polypeptides in the Rhnull cells leads to abnormal shape, increased osmotic fragility, abnormal PtdEtn distribution and enhanced transbilayer mobility of PtdCho strongly suggests that one or both polypeptides are essential for the maintenance of a proper membrane-membrane skeleton interaction.     

Interaction of aldolase with actin-containing filaments. Structural studies.

Electron micrographs of the paracrystals formed when fructose bisphosphate aldolase (EC 4.1.2.13) is added to actin-containing filaments were analysed by computer methods so that ultrastructural changes could be correlated with the various stoicheiometries of binding determined in the preceding paper [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98]. Paracrystals formed with aldolase and either F-actin or F-actin-tropomyosin have a single light transverse band every 38 nm, which is due to aldolase molecules cross-linking the filaments. In contrast, the paracrystals formed between aldolase and F-actin-tropomyosin-troponin filaments show two transverse bands every 38 nm: a major band, interpreted as aldolase binding to troponin, and a minor band, interpreted as aldolase cross-linking the filaments. The intensity of the minor band varies with Ca2+ concentration, being greatest when the Ca2+ concentration is low. A model for the different paracrystal structures which relates the various patterns and binding stoicheiometries to structural changes in the actin-containing filaments is proposed.     

A calcium-activated polyphosphoinositide phosphodiesterase in the plasma membrane of human and rabbit erythrocytes.

Haemoglobin-free human erythrocyte ghosts that were prepared in the presence of EDTA and were then exposed to Ca2+ showed a substantial loss of phosphatidylinositol phosphate and phosphatidylinositol diphosphate, measured either chemically or by loss of 32P from the lipids of prelabelled membranes. At the same time there was, as reported previously (Allan, D. and Michell, R.H., (1976) Biochim. Biophys. Acta 455, 824--830), and approximately equivalent rise in the diacylglycerol content of the membranes. Analysis of the 32P-labelled water-soluble material released during this process showed that the major products were inositol diphosphate and inositol triphosphate. No change was seen in the phosphatidylinositol or phosphatidate content of the membranes, and there was no Ca2+-activated loss of 32P from the phosphatidate of prelabelled membranes: this suggests that Ca2+ did not activate phosphoinositide phosphomonoesterases or phosphatidate phosphomonoesterase in human erythrocyte membranes. It is concluded that human erythrocyte membranes contain at their cytoplasmic surface a Ca2+-activated phosphodiesterase that is active against both phosphatidylinositol phosphate and phosphatidylinositol diphosphate. Rabbit erythrocytes also contained this enzyme, but in these cells there was also evidence for the presence of a Ca2+-activated phosphatidate phosphomonoesterase.     

Interaction of dichlone with human erythrocytes. I. Changes in cell permeability

The uptake of the fungicide dichlone (2,3-dichloro-1,4-naphthoquinone) by human erythrocytes was extremely rapid, reaching a maximum within 5 min of treatment. Most of the dichlone taken up was present in the interior of the cell; only a small fraction of the pesticide (less than 5%) was bound to the cell membrane. Dichlone (3 · 10^?5M-10^?4M) induced a rapid loss of intracellular potassium from the erythrocytes; the leakage of K⁺ varied with the fungicide concentration as well as with cell concentration. Pretreatment of the cells with glutathione was able to reduce potassium loss. Cells exposed to dichlone showed increased osmotic fragility. Dichlone also inhibited Na⁺-K⁺ ATPase, which is associated with active ion transport. However, the leakage of potassium in dichlone-treated cells does not appear to be related to the interference with active ion transport. An extensive loss of potassium within a relatively short time after treatment suggests that dichlone produces its effect by increasing passive cation permeability, probably as a result of direct action on the membrane structure. Dichlone was able to induce hemolysis, but only at concentrations higher than those which resulted in K⁺ loss. The loss of hemoglobin appeared to be mainly due to osmotic swelling of the treated cells. Exposure of red cells to dichlone also resulted in a rapid and extensive formation of methemoglobin as well as a denaturation of hemoglobin. Thus, dichlone not only may be capable of lowering the capacity of erythrocytes to transport oxygen but also alters their permeability.     

Interaction of added amphiphilic lipids with the membrane of intact human erythrocytes to induce change in the cell shape

Addition of an appropriate amount of amphiphilic lipid, such as fatty acid, lysophospholipid and medium-chain phospholipid, into a suspension of human erythrocytes (pH 7.4) at 37 degree C resulted in their incorporation into the membrane and induction of a cell shape change of crenation (echinocyte-spherocyte) type without causing hemolysis. The extent of the shape change was dependent on the amount of the lipid incorporated and the crenation disappeared on removing the incorporated molecules from the membrane. The crenation induced by acidic lipids was further altered drastically by resuspending the treated cells in media of pH 6, 7, and 8, whereas that induced by choline-phospholipid or -lysophospholipid was not so pH-dependent. Based on these results, the mechanism of this shape change is discussed.     

Interaction of thermostable direct hemolysin of Vibrio parahaemolyticus with human erythrocytes.

The interaction between thermostable direct hemolysin produced by Vibrio parahaemolyticus WP-1 and human erythrocytes was studied. The lysis of human erythrocytes by the hemolysin was dependent of temperature and no hemolysis occurred at low temperature (0-4 C), but the hemolysin was adsorbed on human erythrocytes even at low temperature. No hemolysis was observed when antihemolysin antiserum was mixed with the hemolysin and human erythrocytes at zero time. On the other hand, lysis of the cells by hemolysin was not completely inhibited when the antiserum was added during the lag time and the inhibitory effect decreased with delay in the time of addition of antiserum. The inhibitory effect of the antiserum decreased with increase in the incubation temperature, increase in the concentration of divalent cations, and decrease in pH. These results suggest that lysis of human erythrocytes by the hemolysin is at least a two-step process consisting of adsorption of the hemolysin to human erythrocytes and the step(s) following adsorption.     

Interaction of rabbit muscle aldolase with phospholipid liposomes.

The interaction between rabbit muscle fructose diphosphate aldolase and phospholipid model membranes (liposomes) was studied by measurement of the tryptophan fluorescence of the enzyme. Interaction with liposomes decreases intrinsic fluorescence intensity of the enzyme and shifts the emission wavelength maximum to higher values. The effects appear to be strongly dependent on the nature of the phospholipid polar group and on ionic strength. Also, a reversible modification of specific activity of aldolase upon interaction with liposomes was found. It is postulated that aldolase binds to liposomes mainly by electrostatic interactions and that the binding causes a change in the conformation of the enzyme.     

A carbohydrate-deficient membrane glycoprotein in human erythrocytes of phenotype S-s-.

1. We investigated the membranes of human erythrocytes which completely lack the blood-group antigens S and s (denoted as S-s-) as part of a study of the structure and function of the surface glycoproteins of the human erythrocyte. 2. The S-s-erythrocyte-membrane glycoprotein PAS-3 band was much less intensely stained in comparison with that of the glycoprotein from normal erythrocyte membranes. The S-s-membrane glycoprotein PAS-4 band also showed decreased staining. 3. Examination with the lectins from Maclura aurantiaca (Osage orange) and Arachis hypogaea (groundnut) showed that the PAS-3 glycoprotein of S-s-erythrocyte membranes lacked the receptors for these lectins that are present on glycoprotein PAS-3 from normal erythrocytes. 4. Radioiodination with lactoperoxidase showed the presence of the polypeptide of glycoprotein PAS-3 in S-s-cells, although it was more weakly labelled than the protein in the normal erythrocyte. 5. Our results show that the PAS-3 glycoprotein of S-s-erythrocytes is deficient in some of the carbohydrates present in the protein from normal erythrocytes. Glycoprotein PAS-4 of normal erythrocytes is shown to be a complex containing both glycoproteins PAS-1 and PAS-3.