Recurrent decreases of erythrocyte electrophoretic mobility in rats exposed to 1.0 R whole-body x-radiation

Circulating erythrocytes from rats were examined up to 30 weeks post whole-body exposures of 1.0 R for alterations in the expression of net negative surface charge as measured by whole-cell microelectrophoresis in saline sorbitol. Erythrocyte electrophoretic mobility was determined in an apparatus composed of a horizontal transilluminated cylindrical chamber, equipped with a reversible, blacked platinum electrode, immersed in a water bath maintained at 25.0 +/- 1.0 degree C (Rank Brothers). In two separate experiments, recurrent decreases in the expression of net negative surface charge occurred at 10, 17, and 30 weeks post-irradiation. At these times distributional analyses of recorded erythrocyte electrophoretic mobility (EEPM) values revealed a skewing of the normally distributed EEPM population values to lower EEPM. Total sialic acid content released from hydrolyzed erythrocyte membrane preparations revealed no significant differences between erythrocytes from sham and irradiated animals. In vivo post-irradiation labeling of erythrocytes with diisopropyl-[32P] phosphorofluoridate at 4 and 33 weeks (separate experiments) indicated only a minor abbreviated erythrocyte life span at 33 weeks. Therefore, effects from low dose (1.0 R) whole-body irradiation would appear to include a recurrent defect in the expression of the net negative surface charge.     

Recurrent decreases of erythrocyte electrophoretic mobility in rats exposed to 1.0 R whole-body X-radiation

Circulating erythrocytes from rats were examined up to 30 weeks post whole-body exposures of 1.0 R for alterations in the expression of net negative surface charge as measured by whole-cell microelectrophoresis in saline sorbitol. Erythrocyte electrophoretic mobility was determined in an apparatus composed of a horizontal transilluminated cylindrical chamber, equipped with a reversible, blacked platinum electrode, immersed in a water bath maintained at 25.0±0.1°C (Rank Brothers). In two separate experiments, recurrent decreases in the expression of net negative surface charge occurred at 10, 17, and 30 weeks post-irradiation. At these times distribution analyses of recorded erythrocyte electrophoretic mobility (EEPM) values revealed a skewing of the normally distributed EEPM population values to lower EEPM. Total sialic acid content released from hydrolyzed erythrocyte membrane preparations revealed no significant differences between erythrocytes from sham and irradiated animals. In vivo post-irradiation labeling of erythrocytes with diisopropyl-[³²P] phosphorofluoridate at 4 and 33 weeks (separate experiments) indicated only a minor abbreviated erythrocyte life span at 33 weeks. Therefore, effects from low dose (1.0 R) whole-body irradiation would appear to include a recurrent defect in the expression of the net negative surface charge.     

The Effect of Fatal Carbon Monoxide Poisoning on the Surface Charge of Blood Cells

The objective of this investigation was to evaluate postmortem changes of electric charge of human erythrocytes and thrombocytes after fatal carbon monoxide (CO) poisoning. The surface charge density values were determined on the basis of the electrophoretic mobility measurements of the cells carried out at various pH values of electrolyte solution. The surface charge of erythrocyte membranes after fatal CO poisoning as well as after sudden unexpected death increased compared to the control group in the whole range of experimental pH values. Also, a slight shift of the isoelectric point of erythrocyte membranes to high pH values was observed. The surface charge of thrombocyte membranes after fatal CO poisoning decreased at low pH compared to the control group. However, at high pH, the values increased compared to the control group. The isoelectric point of thrombocyte membranes after fatal CO poisoning was considerably shifted toward low pH values compared to the control group. The observed changes are probably connected with the destruction of blood cell structure.     

Analysis of electrophoretic mobility data for human erythrocytes according to sublayer models

An attempt was made to analyze the electrophoretic mobility data of human erythrocytes in media of different pH values and ionic strengths through cell surface models in which the surface charge layer consists of several ion-penetrable sublayers with a uniform charge distribution in each sublayer. As a result, the three-sublayer model was found to explain the mobility data much better than the two-sublayer model in a wide range of ionic strength at all pH values studied.     

Effects of surface proteins of human erythrocyte membrane on the interaction with lipopolysaccharides from <Emphasis Type="Italic">Escherichia coli</Emphasis> O55:B5

The role of erythrocytic surface membrane proteins and membrane charge in the interactions of the erythrocytes with lipopolysaccharides (LPS) isolated from Escherichia coli O55:B5 (LPS _{E. coli} , S-form) has been examined by two independent methods, flow cytometry and cell electrophoresis. Treatment of erythrocytes with trypsin that modifies stereochemical properties of cell surface resulted in a 16% increase in the level of the erythrocyte fluorescence measured after their incubation with fluorescently labeled LPS _{E. coli} . Electrophoretic mobility (EM) of the trypsin-treated erythrocytes was reduced by 16%. The removal of sialic acids from the erythrocyte surface with neuraminidase had no considerable effect either on the relative EM values or fluorescence intensity after the incubation of cells with LPS. The results suggest that the major role in the incorporation of the S-form LPS into the membrane of human erythrocytes is played by stereochemical factors, whereas the cell surface charge is less significant.     

Effect of La(3+) on electrophoretic mobility and aggregation of intact human erythrocytes and those treated with low concentrations of glutaric aldehyde

It was shown that the treatment of erythrocytes with low concentrations of glutaraldehyde (0.01-0.1%) for 30-120 min to a variable extent the aggregation induced by 40-330 microM La3+. The effect of glutaraldehyde on the aggregation increased with concentration and time of fixation. La3+ ions decreased to a similar extent the electrophoretic mobility of intact erythrocytes and erythrocytes treated with 0.1% glutaraldehyde. No relationship was found between the change in the negative charge on the erythrocytes and the degree of their aggregation. Neuraminidase and trypsin were shown to decrease the surface charge on the erythrocytes and the aggregation of fixed erythrocytes.     

Theory of the electrokinetic behavior of human erythrocytes

We develop a theory of electrophoresis of human erythrocytes that predicts mobilities significantly smaller than those based on the classical Smoluchowski relation. In the classical treatment the charge is assumed to be spread uniformly on the hydrodynamic surface. The present model takes into account that most of the charge, due mainly to sialic acid, is contained in the glycocalyx. The glycocalyx is modeled as a permeable layer of polyelectrolyte molecules anchored to the cell membrane. The charge is assumed to be uniformly distributed throughout this layer. The fluid flow in the layer is treated as being dominated by Stokes friction arising from idealized polymer segments. The Navier-Stokes equations are solved to give the dependence of electroosomotic velocity with distance from the cell surface. An expression for the electrophoretic mobility is obtained which contains two parameters (a) the thickness of the glycocalyx and (b) the mean polymer segment radius. The best fit to experimental data is obtained if these are given the values 75 A and 7 A, respectively. Deviation from experimental data at low ionic strength (less than 0.05 M) occurs. However, this deviation is in the direction one would expect if at low ionic strength the polyelectrolyte layer expands slightly due to decreased charge shielding.     

Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria

This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-C1 cotransport, the frog erythrocytes were incubated in nitrate media at 20 °C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K⁺ influx measured as ⁸⁶Rb uptake for 60 min. The Na⁺-K⁺-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1–2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K⁺ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K⁺ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K⁺ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01–0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K⁺ influx into frog erythrocytes. These results indicate that about one-half of the Na⁺ -K⁺-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate “membrane-associated” ATP pool. Accepted: 12 July 1997     

"Spontaneous" oscillations of the cell surface in the frequency interval of 0.2-30 Hz

The local transverse displacements of the cell surface within the frequency range of 0.2-30 Hz occur on animal cells attached to cover glass: fibroblasts 3T6, primary culture of rat cardiomyocytes, mouse lymphocytes, human macrophages and erythrocytes, frog erythrocytes. The area of the cell rim, moving transversely, is no more than 0.5 X 0.5 microns. The maximum amplitude of the local transverse surface displacement is different in various cell types, being maximum (350-400 nm) in human erythrocytes and minimum in fibroblasts 3T6, mouse lymphocytes and human macrophages (20-30 nm). High-amplitude oscillations of human erythrocytes and low-amplitude oscillations of frog erythrocytes correlate with differences in the size of elastic shear modulus.     

Functional integrity of desensitized beta-adrenergic receptors

The adenylate cyclase-coupled beta 2-adrenergic receptor of the frog erythrocyte has served as a useful model system for elucidating the mechanisms of catecholamine-induced densensitization. In this system, it has been previously demonstrated that agonist-induced refractoriness is associated with sequestration of the beta-adrenergic receptors in vesicles away from the cell surface and from their effector unit, the adenylate cyclase system (Stadel, J.M., Strulovici, B., Nambi, P., Lavin, T.N., Briggs, M.M., Caron, M.G., and Lefkowitz, R.J. (1983) J. Biol. Chem. 258, 3032-3038). These internalized beta-adrenergic receptors appear to be structurally intact as assessed by photoaffinity labeling, but their functional status has previously been unknown. In the present studies, we sought to assess the functionality of the sequestered vesicular receptors by fusing them to Xenopus laevis erythrocytes. This cell is suitable for such studies, since it has almost no detectable beta-adrenergic receptor or catecholamine-sensitive adenylate cyclase, but contains prostaglandin E1-stimulable adenylate cyclase. Fusion of beta-adrenergic receptor-containing vesicles from desensitized frog erythrocytes with X. laevis erythrocytes results in a 30-fold stimulation of the hybrid adenylate cyclase by the beta-adrenergic agonist isoproterenol. This effect was entirely blocked by the beta-antagonist propranolol. The catecholamine-sensitive adenylate cyclase activity established in the vesicle-Xenopus hybrids showed the characteristic agonist potency series of the donor frog erythrocyte beta 2-adrenergic receptor. Fusion of vesicles from desensitized frog erythrocytes in which the beta-adrenergic receptors had been inactivated with the group specific reagent dicyclohexylcarbodiimide, or of vesicles derived from control frog erythrocytes, which contain low amounts of beta-adrenergic receptor, did not establish catecholamine-sensitive adenylate cyclase activity in the hybrids. These data demonstrate that beta-adrenergic receptors internalized during desensitization retain their functionality when recoupled to an adenylate cyclase system from a different source. The functional uncoupling of these receptors during desensitization is thus more likely due to their sequestration away from the other components of the adenylate cyclase than to any alterations in the receptors themselves.     

Surface charge of endothelial cells estimated from electrophoretic mobility

The surface charge density of endothelial cells was estimated from cell electrophoresis. Cultured endothelial cells from the bovine pulmonary artery were suspended in saline and placed in the lumen of a glass capillary. A voltage was applied across the capillary ends and the velocity imparted to the cells was measured with a microscope. Erythrocyte mobility was also measured. The mobility in (micron/s)/(V/cm) was 0.74 +/- 0.08 for endothelial cells and 1.03 +/- 0.15 for erythrocytes. Charge density in esu/cm2 was calculated as 2.62 x 10(4) and 0.91 x 10(4) for endothelial and red cells, respectively. Removal of sialic acid did not affect the mobility of endothelial cells, but it reduced that of red cells to near zero. Endothelial cell mobility decreased either with ionic strength or calcium concentration. Our results strongly suggest that the surface charge of endothelial cells is dependent on sulfated glycosaminoglycans.     

The surface charge of human glutaraldehyde-fixed erythrocytes

We measured the number of charged residues at the surface of fresh human erythrocytes after fixation with glutaraldehyde by polyelectrolyte titration using polycations of different chemical composition and various molecular weights. Independent of the reagents used, the number was (8.5 +/- 1.5) X 10(8) negatively charged residues per cell. The surface charge density of 6.3 e/nm2 is considerably higher than that calculated from the electrophoretic mobility for which the surface charge density is calculated to be 0.09 e/nm2.     

Detection of Differences in Surface-charge-associated Properties of Cells by Partition in Two-polymer Aqueous Phase Systems

WHEN aqueous solutions of two polymers are mixed in certain proportions they may form two-phase systems^1,2 which can be buffered and used to partition and separate cells, particles and macromolecules by countercurrent distribution (CCD). Partition generally depends on polymer composition and concentration, the ionic composition and the charge sign of the material being partitioned. Such systems have been used to separate erythrocytes from white cells and erythrocytes on the basis of age. Changes in the surface properties of cells resulting from enzyme treatment or storage have also been demonstrated by this means³. Higher cell partition often accompanies increasing electrophoretic mobility which suggests that surface charge may be an important factor in partitioning^4–6. An apparent exception to this is the increased partition of stored human erythrocytes as compared with fresh⁷, as opposed to the mean electrophoretic mobility of both cell populations which remain identical⁸.     

The Effect of Contrast Medium SonoVue<Superscript>®</Superscript> on the Electric Charge Density of Blood Cells

The effect of contrast medium SonoVue® on the electric charge density of blood cells (erythrocytes and thrombocytes) was measured using a microelectrophoretic method. We examined the effect of adsorbed H⁺ and OH^? ions on the surface charge of erythrocytes or thrombocytes. Surface charge density values were determined from electrophoretic mobility measurements of blood cells performed at various pH levels. The interaction between solution ions and the erythrocyte’s or thrombocyte’s surface was described by a four-component equilibrium model. The agreement between the experimental and theoretical charge variation curves of the erythrocytes and thrombocytes was good at pH 2–9. The deviation observed at a higher pH may be caused by disregarding interactions between the functional groups of blood cells.     

Electrophoretic mobility of erythrocytes exposed to accelerated helium nuclei or to gamma-rays, as modified by adeturone

The electrophoretic mobility of human erythrocytes exposed in whole blood or in cell suspensions to relatively low doses (0.7-2.5 Gy) of accelerated helium nuclei (4.6 GeV/nucleon) or of gamma-rays was found to diminish with increase in radiation dose, as measured at 3 h post-irradiation. Pre-treatment with a radioprotectant, adeturone (S-2-aminoethylisothiuronium adenosine-5'-triphosphate), allowed electrophoretic mobility to be maintained within normal limits, with more stable values in the case of alpha-irradiations. The experimental evidence is discussed in terms of the protectant's involvement in repair of radiation-induced conformational changes in surface membrane components.     

Surface charge of mammalian neurones as revealed by microelectrophoresis

Summary The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M^–1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK=4.2. Trypsin treatment in mild conditions markedly reduced the surface charge: however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzenesulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge. On the basis of data obtained it is suggested that the charge of the outer membrane surface of neurones studied is mainly determined by carboxylic groups of membrane proteins, and changes in intracellular cAMP concentration influence the synthesis and reconstruction of these membrane components.     

Cationic ferritin binding sites and surface charge densities of transformed cells

Bioelectric surface properties of the high and low tumor-producing cell lines, NCTC 2472 and NCTC 2555, respectively, were determined by cationic ferritin binding and the electrophoretic mobility of intact cells. Measurements of anionic sites were bases on the number of cationic ferritin particles per 0.01 mu 2 that were electronically tagged and counted by an image analyzer. The average particle count was 45 for the control "high" cells and 34 for the control "low" cells. The surface charge densities, expressed as electrostatic units per cm-2 x 10(-13) were 2.34 and 1.18 at 50 V (2 mA) for the "high" and "low" control cells, respectively. Enzymic cleavage of sialic acid and other carbohydrate moieties resulted in up to an 81% reduction in the charge densities and a 57% reduction of the anionic sites of the "high" cells. The electrophoretic mobility of cells with bound cationic ferritin showed that up to 50% of the exposed anionic sites fail to bind cationic ferritin. Preliminary findings on the particle size/distribution by image analysis showed wide ranges in both particle size and interparticle distances that may limit cationic ferritin binding.     

An experimental test of new theoretical models for the electrokinetic properties of biological membranes. The effect of UO2++ and tetracaine on the electrophoretic mobility of bilayer membranes and human erythrocytes

For a large smooth particle with charges at the surface, the electrophoretic mobility is proportional to the zeta potential, which is related to the charge density by the Gouy-Chapman theory of the diffuse double layer. This classical model adequately describes the dependence of the electrophoretic mobility of phospholipid vesicles on charge density and salt concentration, but it is not applicable to most biological cells, for which new theoretical models have been developed. We tested these new models experimentally by measuring the effect of UO2++ on the electrophoretic mobility of model membranes and human erythrocytes in 0.15 M NaCl at pH 5. We used UO2++ for these studies because it should adsorb specifically to the bilayer surface of the erythrocyte and should not change the density of fixed charges in the glycocalyx. Our experiments demonstrate that it forms high-affinity complexes with the phosphate groups of several phospholipids in a bilayer but does not bind significantly to sialic acid residues. As observed previously, UO2++ adsorbs strongly to egg phosphatidylcholine (PC) vesicles: 0.1 mM UO2++ changes the zeta potential of PC vesicles from 0 to +40 mV. It also has a large effect on the electrophoretic mobility of vesicles formed from mixtures of PC and the negative phospholipid phosphatidylserine (PS): 0.1 mM UO2++ changes the zeta potential of PC/PS vesicles (10 mol % PS) from -13 to +37 mV. In contrast, UO2++ has only a small effect on the electrophoretic mobility of either vesicles formed from mixtures of PC and the negative ganglioside GM1 or erythrocytes: 0.1 mM UO2++ changes the apparent zeta potential of PC/GM1 vesicles (17 mol % GM1) from -11 to +5 mV and the apparent zeta potential of erythrocytes from -12 to -4 mV. The new theoretical models suggest why UO2++ has a small effect on PC/GM1 vesicles and erythrocytes. First, large groups (e.g., sugar moieties) protruding from the surface of the PC/GM1 vesicles and erythrocytes exert hydrodynamic drag. Second, charges at the surface of a particle (e.g., adsorbed UO2++) exert a smaller effect on the mobility than charges located some distance from the surface (e.g., sialic acid residues).     

Interaction of the frog brain kainate receptor expressed in Chinese hamster ovary cells with a GTP-binding protein.

A protein that binds kainate with high affinity has been purified and cloned from frog brain (Rana pipiens) and has approximately 35% sequence homology with mammalian non-N-methyl-D-aspartate glutamate receptors, some of which have been shown to be ligand-gated ion channels. Frog brain membranes and membranes from Chinese hamster ovary (CHO) cells transfected with the cDNA coding for the frog kainate-binding protein (CHO-4 cells) bound kainate with essentially identical affinity (KD values of 1.9 and 2.1 nM, respectively). In both tissues, the affinity for kainate decreased 9-fold in the presence of 100 microM GTP gamma S (guanosine 5'-O-(3-thio)triphosphate). No specific kainate binding to nontransfected CHO cell membranes was observed. GTP gamma S and GDP were effective inhibitors of kainate binding, while cGMP and adenosine 5'-O-(3-thio)triphosphate had no effect in either frog brain membranes or CHO-4 membranes. Pretreatment of CHO-4 cell membranes with pertussis toxin led to a 34% decrease in kainate binding. Kainate increased the binding of [3H]5'-guanylyl imidodiphosphate by 61%, and the rate of GTP hydrolysis by up to 5-fold. These results indicate that the kainate receptor cloned from frog brain can interact functionally with a G protein present in CHO-4 cell membranes.     

The ionogenic nature of the secretory-granule membrane. Electrokinetic properties of isolated chromaffin granules

1. Chromaffin granules isolated from the bovine adrenal medulla possess an electrophoretic mobility of -1.12mum.s(-1).cm.V(-1), corresponding to a surface zeta potential of -14.4mV and surface charge density of 1.38x10(-6)C.cm(-2). 2. The mobility of chromaffin granules is pH-dependent, indicating an amphoteric surface with an isoelectric point at pH3.0 and acidic groups with a pK(a) of 3.11. 3. Addition of bi- and ter-valent cations decreased the mobility of chromaffin granules in a dose-dependent fashion with a relative potency of La(3+)>Mn(2+)>Ca(2+) >Sr(2+)>Mg(2+)>Ba(2+). 4. Treatment with neuraminidase decreased the mobility of erythrocytes by 84%, whereas chromaffin-granule mobility was decreased by only 14%. This correlates well with the small complement of neuraminic acid present in the granule membrane. 5. The nature, origin and significance of the anionic surface charge of the chromaffin granule is discussed. It is concluded that the net negative charge at the surface of shear derives chiefly from a single type of chemical group, namely -CO(2) (-), contributed by the alpha-carboxyl group of constituent proteins, the phospholipid phosphatidylserine and, to a lesser extent, the sialic acid component of glycoproteins.