Bioelectrochemistry (v.62, #2)

Bioelectrochemistry of red blood cells by I Bernhardt; S Thomas (105).

The cooperative role of membrane skeleton and bilayer in the mechanical behaviour of red blood cells by Saša Svetina; Drago Kuzman; Richard E. Waugh; Primož Ziherl; Boštjan Žekš (107-113).
Red blood cell (RBC) shape, behaviour and deformability can be consistently accounted for by a model for the elastic properties of the RBC membrane that includes the elasticity of the membrane skeleton in dilation and shear, and the local and nonlocal resistance of the bilayer to bending. The role of the corresponding energy terms in different RBC shape and deformation situations is analyzed. RBC shape transformations are compared to the shape transformations of phospholipid vesicles that are driven by the difference between the equilibrium areas of the bilayer leaflets (ΔA 0). It is deduced that the skeleton energy contributions play a crucial role in the formation of an echinocyte. The effect of a transformation of the natural biconcave RBC shape into an echinocyte on its resistance to entry into capillary-sized cylindrical tubes is analyzed. It is shown that, during the aspiration of an echinocyte into a pipette, there are two competing skeleton deformation effects, which arise due to skeleton density changes, one due to spicule formation and the other due to deformation induced by micropipette aspiration. Furthermore, the shift of the observed dependence of the projection length on the aspiration pressure of more crenated cells towards higher aspiration pressures can be accounted for by an increase of the equilibrium area difference ΔA 0 and consequent modification of the nonlocal contribution to the cell elastic energy.
Keywords: Red blood cells; Deformability; Shapes; Membrane elastic properties; Echinocyte; Micropipette aspiration;

Erythrocyte membrane permeability for a series of diols by O.I. Gordiyenko; T.P. Linnik; E.O. Gordiyenko (115-118).
Erythrocyte membrane permeability coefficients for a series of diols have been defined by the method developed. The method is based on the physical and mathematical modeling of hypotonic hemolysis process. There have been also determined membrane permeability coefficients for erythrocytes treated with p-chloromercuribenzenesulfonic acid monosodium salt (pCMBS), which is known to block aqueous protein channels. Permeating process is shown to be conditioned both by hydrophilic/hydrophobic properties of the molecules and their geometrical parameters. The obtained results propose that, when exceeding the molecules diameter over a value of 4 Å, the permeability coefficient reduces due to decreasing of flow through the aqueous protein pores of a constant size. Permeability coefficients for comparatively hydrophobic molecules are almost directly proportional to the coefficients of partition between hydrophobic and hydrophilic phases, by pointing to a lipid way of permeation of these molecules through erythrocyte membranes.
Keywords: Erythrocyte membrane; Permeability; Diols;

Estimation of erythrocyte population state by the spherical index distribution by O.I. Gordiyenko; Yu.E. Gordiyenko; V.O. Makedonska (119-122).
The densities of cell distributions by spherical index (SI) in erythrocyte populations from healthy adults and donors with endocrine pathologies were determined via the developed method. The investigation shows that this characteristic varies for different donors, thereby reflecting the erythrocyte population state of an individual donor. Individual distribution curves obtained from healthy donors are close to Gaussian and are characterized by smooth curve plot with one maximum. Cells distribution by SI in donors with endocrine pathologies has a polymodal character. Our research shows that the developed method for determining erythrocyte distribution density by SI is a sensitive and informative test for quantitative evaluation of an erythrocyte population state. Moreover, this characteristic has clear physical and physiological significance, because an erythrocyte shape is strongly conditioned by the cell age and influences the ability to pass through microcapillaries in blood circulation.
Keywords: Erythrocytes; Spherical index; Endocrine pathologies;

Recent research on erythrocytes as model cells for photodynamic therapy showed differing behaviour of certain photosensitisers in erythrocytes compared to other cells. Differences of dye accumulation in the cell membrane were proposed to be the reason for the distinct photodynamic effects. Using pheophorbide a as an example, the combination of erythrocyte ghosts as models to follow the dye accumulation in the cell membrane and intact erythrocytes as model cells to show the photodynamic damage is provided. Evidence for the correctness of the combination of erythrocyte ghosts and intact erythrocytes as a functioning model system in photodynamic cell research is provided using the confocal laser scanning microscopy on intact, pheophorbide a loaded erythrocytes.
Keywords: Human erythrocytes; Erythrocyte ghosts; Photodynamic therapy; Pheophorbide a; Confocal microscopy;

Human red blood cells (RBCs) were loaded with the Ca2+-sensitive fluorescent dye fura-2 to investigate the effects of media ionic strength and prostaglandin E2 (PGE2) on the intracellular free Ca2+ concentration ([Ca2+]i). [Ca2+]i of intact RBCs in a Ca2+-containing physiological (high) ionic strength (HIS) solution was 75.1±8.3 nM after 5 min incubation, increasing to 114.9±9.6 nM after 1 h. In Ca2+-containing low ionic strength (LIS) solutions, [Ca2+]i was significantly lower than in the Ca2+-containing HIS solution (p=0.041 or 0.0385 for LIS solutions containing 200 or 250 mM sucrose, respectively), but, as in HIS solution, an increase of [Ca2+]i was seen after 1 h. In Ca2+-free (0 Ca2+ plus 15 μM EGTA) media, [Ca2+]i decreased (ranging from 15 to 21 nM), but were not significantly different in HIS or LIS, and did not change following 1 h incubation. The effect of the ionic strength and PGE2 on passive Ca2+ influx was investigated on ATP-depleted RBCs. Ca2+ influx was faster during the initial 10 min in comparison with the subsequent time period (10−45 min), both in HIS and LIS media, decreasing from 20.3±1.9 to 12.9±1.3 μmol/(lcells×h) in HIS, and from 36.7±5.3 to 8.6±1.2 μmol/(lcells×h) in LIS. Prostaglandin E2 (PGE2; 10−7–10−11 M), dissolved in deionised water or in ethanol, did not affect [Ca2+]i in either normal or in ATP-depleted RBCs suspended in Ca2+-containing HIS medium. Finally, the addition of carbachol (100 μM) did not affect [Ca2+]i. The present findings suggest that stimulation of the Ca2+-activated K+ channel by PGE2, reported in [J. Biol. Chem. 271 (1996) 18651], cannot be mediated via increased [Ca2+]i.
Keywords: Red blood cells; Ca2+ concentration; Ca2+ influx; Ionic strength; Prostaglandin;

Inhibitors of the K+(Na+)/H+ exchanger of human red blood cells by Erwin Weiss; Hans-Jochen Lang; Ingolf Bernhardt (135-140).
The effect of substances as possible inhibitors of the K+(Na+)/H+ exchanger in the human red cell membrane has been tested on the (ouabain+bumetanide+EGTA)-resistant K+ influx in both physiological (HIS) and low ionic strength (LIS) solution with tracer kinetic methods. It is demonstrated that high concentrations of quinacrine (1 mM) and chloroquine (2 mM) inhibit the residual K+ influx in LIS solution to 60% and 85%, respectively, but activate it in HIS solution. Thus, chloroquine suppressed the 10-fold LIS-induced activation of the flux nearly completely. Amiloride derivatives were able to inhibit the K+ influx in both HIS and LIS solution. EIPA (75 μM) reduced the flux by about 20% and 55% in HIS and LIS solution, respectively. Newly developed drugs (HOE 642, 1 mM; HOE 694, 0.5 mM) designed to inhibit Na+/H+ exchanger isoforms showed an inhibition of the residual K+ influx of 40% and 33% in HIS and 65% and 44% in LIS solution, respectively, without haemolysis. The inhibitory effect of HOE 642 persisted in HIS (24%) and LIS (48%) solutions when Cl was replaced by CH3SO4 . The K+–Cl cotransport inhibitor DIOA (100 μM) stimulated the residual K+ influx in both solutions. It is, therefore, concluded that the K+–Cl cotransporter does not contribute to the residual K+ influx both in HIS and LIS media. Okadaic acid decreased the residual K+ influx by 40% and 25% in HIS and LIS solution, respectively, showing that the residual K+ influx is affected by phosphatases like other ion transport pathways. The results show that the residual K+ influx can be decreased further by inhibiting the K+(Na+)/H+ exchanger. It remains still unclear to what extent the K+(Na+)/H+ exchanger is inhibited by the different substances used. However, the ground state membrane permeability for K+ is much smaller than assumed so far.
Keywords: Red blood cells; K+(Na+)/H+ exchanger; Ion transport inhibitors; K+ leak; Membrane permeability;

Oxygen dependence of K+–Cl cotransport in human red cell ghosts and sickle cells by Asif I. Khan; Clare Drew; Sarah E. Ball; Vicky Ball; J.Clive Ellory; John S. Gibson (141-146).
KCC activity in normal human red cells (containing haemoglobin A, HbA, and termed HbA cells) is O2-dependent, being active in oxygenated cells but inactive in deoxygenated ones. The mechanism for O2 dependence is unknown but a role for Hb has been suggested. In this paper, we address two main questions. First, do membrane ghosts prepared from HbA cells retain an O2-sensitive KCC activity? Second, how is the response of KCC to changes in O2 tension altered in sickle cell patients heterozygous for HbS and HbC? We found that substantial Cl-dependent K+ influx, indicative of KCC activity, was present in both pink (5–10% normal Hb complement) and white (no measurable Hb) ghosts when equilibrated with air. KCC responded to deoxygenation in pink ghosts only (86±10% inhibition, mean±S.E.M., n=3), whilst KCC activity in white ghosts remained high (23±8% inhibition). Results indicate that pink ghosts retain an O2-dependent KCC activity but that this is lost in white ghosts. Second, HbSC-containing red cells showed sickling (88±3%) when deoxygenated, together with activation of the deoxygenation-induced cation pathway (Psickle) and the Gardos channel. KCC activity, however, was elevated in oxygenated HbSC cells, but inhibited by deoxygenation. Thus Hb polymerisation and sickling could be dissociated from the abnormal response of KCC to deoxygenation observed in HbS-containing red cells. These preparations provide a useful system with which to study the components involved in O2-sensitive membrane transport and why it is perturbed in certain pathological conditions (such as sickle cell disease and oxidant toxicity).
Keywords: O2; KCl cotransport; Ghost; Haemoglobinopathy; Red cell;

We compare the effects of 1-chloro-2,4-dinitrobenzene (CDNB) and phenazine methosulphate (PMS) on Gardos channel activity in normal human red cells. Both stimulate channel activity, both are dependent on the presence of extracellular Ca2+, and neither is affected by inhibitors of protein (de)phosphorylation. Of the two, PMS has a considerably greater effect. In addition, a major difference is that whilst CDNB has a greater stimulatory effect in oxygenated cells, by contrast, PMS is more effective in deoxygenated cells. These actions are correlated with ca. 30% inhibition of the plasma membrane Ca2+ pump (PMCA) and an increased sensitivity of the Gardos channel to Ca2+ (EC50 falling to about 150 nM). These findings are important in understanding how oxidants alter red cell cation permeability and may be relevant to the abnormal permeability phenotype shown by deoxygenated sickle cells.
Keywords: Gardos channel; 1-Chloro-2,4-dinitrobenzene; Phenazine methosulphate; Oxygen;

Oxygen sensitivity of red cell membrane transporters revisited by Clare Drew; Vicky Ball; Hannah Robinson; J Clive Ellory; John S Gibson (153-158).
In this paper, we provide an update on O2-dependent membrane transport in red cells. O2-sensitive membrane transport was compared in nucleated (chicken) and enucleated (human) red cells, to investigate effects on organic (glucose transporter [GLUT]) and inorganic (K+–Cl cotransporter [KCC]/Na+–K+–2Cl cotransporter [NKCC]) transporters, to study the response of so-called “housekeeping” transporters (Na+/K+ pump and anion exchanger [AE]) and, finally, to compare O2 sensitivity in normal human red cells with those from sickle cell patients. The Na+/K+ pump showed no change in activity between oxygenated and deoxygenated cells in any of the samples. KCC in normal human red cells had the greatest O2 sensitivity, being stimulated some 20-fold on oxygenation. It was more modestly stimulated by O2 in chicken red cells and HbS cells. By contrast, NKCC was stimulated by deoxygenation in all cases. GLUT showed little response to O2 tension, other than a small stimulation in deoxygenated chicken red cells. Finally, AE1 was stimulated by oxygenation in HbA cells, but this stimulation by O2 was absent in HbS cells and pink ghosts prepared from HbA cells. The significance of these findings is discussed.
Keywords: O2; GLUT; KCC; NKCC; Na+/K+ pump; AE;

Spherocyte shape transformation and release of tubular nanovesicles in human erythrocytes by Aleš Iglič; Peter Veranič; Kristijan Jezernik; Miha Fošnarič; Boris Kamin; Henry Hägerstrand; Veronika Kralj-Iglič (159-161).
We have studied dodecylmaltoside-induced echinocyte–spheroechincyte–spherocyte shape transformation and membrane vesiculation using transmission electron microscopy (TEM) on freeze-fracture replicas. It is indicated that spherical erythrocyte shape at higher dodecylmaltoside concentration is formed due to loss of membrane in the process where small, mostly tubular nanovesicles are released predominantly from the top of echinocyte and spheroechinocyte spicules.
Keywords: Dodecylmaltoside; Nanovesicles; Erythrocyte membrane; Erythrocyte shape; Membrane shear energy;

Effect of alkyl-β,d-glucopyranosides on hypertonic haemolysis of erythrocytes by Olga P. Synchykova; Natalia M. Shpakova; Valerij A. Bondarenko (163-167).
The effect of temperature (5–20 °C) and treatment with phenylhydrazine on the hypertonic lysis of erythrocytes in the presence of alkyl-β,d-glucopyranosides was studied. The results highlight an important role for the cytoskeleton-membrane complex, which allows the cells to both withstand hypertonic stress within the temperature range studied and facilitate the protective effect of alkylglucopyranosides at low temperatures (5 °C).
Keywords: Erythrocyte; Hypertonic haemolysis; Alkyl-β,d-glucopyranosides; Temperature; Phenylhydrazine;

The phosphorylation of tyrosine residues of human red blood cell (RBC) band 3 is regulated in vivo by constitutively active tyrosine-kinases (PTKs) and phosphotyrosine-phosphatases (PTPs), identified so far as, respectively, p72syk and p56/53lyn, and PTP1B and SHPTP-2. Tyr-phosphorylation of band 3 increases upon reduction of cell volume as in hypertonic media or during Ca2+-induced membrane vesiculation. We show here that old RBCs display higher Tyr-phosphorylation levels of band 3 than younger cells under hypertonic conditions, at least in part due to the reduced cell volume of old RBCs, a condition of lowered threshold for activation of volume-sensitive PTKs. We have also analysed the membrane-bound PTP activity and the relative abundance of PTP1B (as the main membrane-associated PTP) in RBCs of different age. Immunodetection of PTP1B in purified ghost membranes revealed that the catalytic, N-terminal domain of the PTP is partially cleaved, in an age-dependent manner, from the membrane-bound domain, and it is lost during the preparation of ghost membranes. This suggests that erythrocytes may undergo in vivo activation of the Ca2+-dependent calpain system that proteolytically regulates PTP1B activity, as already documented for other cell types. On the other hand, the assay of the PTP activity that remains associated with the membranes of RBCs of different age indicated that the PTP undergoes oxidative inactivation that can be further differentiated into reversible and irreversible components.
Keywords: Red blood cell ageing; PTP1B; Protein 4.1; Band 3; Vesiculation; Tyr-kinase;

Experiences in the measurement of RBC-bound IgG as markers of cell age by Renata Paleari; Ferruccio Ceriotti; Franco Azzario; Liliana Maccioni; Renzo Galanello; Andrea Mosca (175-179).
An immunologically mediated pathway has been largely accepted to be one of the mechanisms involved in the clearance of senescent or prematurely damaged RBC. According to this pathway, RBC removal is mediated by binding of naturally occurring IgG to clustered integral membrane proteins, followed by complement deposition. The validation of an immunoenzymatic method for the detection of RBC-bound autologous IgG is presented. The use of RBC-bound IgG as an index related to red cell age was evaluated by measuring IgG binding in RBC treated with the clustering agent ZnCl2, in density fractionated RBC and in a selected group of patients expected to have an altered RBC life span. The immunoenzymatic method for IgG detection resulted to be reproducible (CV=3.4%). IgG binding to in vitro clustered RBC was found to be enhanced to a very great extent, about 20 times higher with respect to untreated RBC. A slight but significant increase (about 1.8-fold) in membrane-bound IgG was observed in the highest density fraction of normal RBC, which constituted 1% of the total cells. A significantly greater number of RBC-bound IgG was measured in splenectomized β-thalassemia intermedia patients and in subjects with secondary decreases in the C3 complement fraction concentration.
Keywords: Naturally occurring antibodies; Erythrocytes; Aging; β-Thalassemia intermedia; Complement;

Voltage activation and hysteresis of the non-selective voltage-dependent channel in the intact human red cell by Poul Bennekou; Trine L. Barksmann; Lars R. Jensen; Berit I. Kristensen; Palle Christophersen (181-185).
Suspension of intact human red cells in media with low chloride and sodium concentrations (isotonic sucrose substitution) results in strongly inside positive membrane potentials, which activate the voltage-dependent non-selective cation (NSVDC) channel. By systematic variation of the initial Nernst potentials for chloride (degree of ion substitution) as well as the chloride conductance (block by NS1652), and by exploiting the interplay between the Ca2+-permeable NSVDC channel, the Ca2+-activated K+ channel (the Gárdos channel) and the Ca2+-pump, a graded activation of the NSVDC channel was achieved. Under these conditions, it was shown that the NSVDC channels exist in two states of activation depending on the initial conditions for the activation. The hysteretic behaviour, which in patch clamp experiments has been found for the individual channel unit, is thus retained at the cellular level and can be demonstrated with red cells in suspension.
Keywords: Human red cells; Non-selective voltage-dependent cation channel; Ca2+-transient; Hysteresis;

Temperature-dependent changes in erythrocytes' cytosol state during natural and artificial hypobiosis by S.V. Repina; O.A. Nardid; V.S. Marchenko; A.V. Shilo (187-190).
At present, the question of how the structural state of the erythrocyte cytosol is arranged to maintain essential permeabilities successfully both at normal temperature and during periods with a significant body temperature reduction during hypobiosis remains unanswered.In the present work, we performed comparative investigations of temperature-dependent changes in the cytosol state of erythrocytes from animals subjected to natural (winter hibernating ground squirrels) or artificial hypobiosis. The cytosol state was evaluated by the ESR method of spin probes (TEMPON) within the temperature range of 0–50 °C. Erythrocyte resistance to acid hemolysis, which is limited by the permeability of membranes for protons and the state of the anion channel, were determined using the method described by Terskov and Getelson [Biofizika 2 (1957) 259]. A change in cytosol microviscosity of erythrocytes was found as well as a temperature-dependent increase in acid resistance of erythrocytes.Our investigations allow us to conclude that physiological changes occurring in a mammalian organism during natural and artificial hypobiosis are accompanied by structural modifications of the erythrocyte cytosol.The temperature range where these modifications are observed (8, 15, 40 °C) suggests that the most probable modifying link is spectrin and/or the sites of its interaction with membrane. The interaction of cytoskeletal components with the cell membrane plays a key role in regulation of membrane permeability, suggesting an important role of this interaction in the adaptive reactions of erythrocytes.
Keywords: Erythrocyte; Permeability; Cytosol; Adaptation;

The activity of membrane-bound NADH-methemoglobin reductase in erythrocytes and the physical state of lipids in erythrocyte membranes under oxidative stress in cells were studied. A decrease of the activity of membrane-bound NADH-methemoglobin reductase and a change of physical state of the lipid bilayer of membranes under oxidative stress were found in erythrocytes in vivo and in vitro.
Keywords: NADH-methemoglobin reductase; Erythrocytes; Oxidative stress;

Effects of anti-GLUT antibodies on glucose transport into human erythrocyte ghosts by I. Afzal; J.A. Browning; C. Drew; J.C. Ellory; R.J. Naftalin; R.J. Wilkins (195-198).
We have studied the effects of anti-GLUT1 antibodies on the uptake of glucose into erythrocytes. Glucose transport into human erythrocyte ghosts was measured directly using 3H-2-deoxy-glucose, or indirectly by monitoring associated volume changes using light scattering. The uptake of glucose was significantly inhibited in ghosts resealed in solutions containing specific antibodies against GLUT1. Such an effect was not observed when an antibody against the oestrogen receptor, lacking specificity towards GLUT1, was employed instead. The antibodies were also without effect on the efflux of preloaded glucose from erythrocyte ghosts. The demonstration that anti-GLUT antibodies can inhibit glucose uptake is support for the hypothesis that they exaggerate the cytoplasmic barrier to glucose uptake created by endofacial segments of GLUT1.
Keywords: GLUT1; Erythrocyte; Ghost; Antibody;