Uniform ionophore A23187 distribution and cytoplasmic calcium buffering in intact human red cells

The divalent cation-selective ionophore A23187 has been used to characterize cytoplasmic Ca and Mg buffering, Ca2+-pump parameters and the properties of a Ca2+-activated K+-channel in intact red cells. A critical assumption in these studies has been that the ionophore causes a uniform increase in divalent cation-permeability in all the cells. This has now been tested directly in ATP-depleted human red cells by analysing the kinetics of ionophore-induced 45Ca-tracer and net Ca2+ fluxes. The experimental curves were all adequately fitted by single-exponentials at all ionophore concentrations tested. Moreover, statistical analysis of 61 individual tracer influx curves and of pooled data showed no trend towards fast second exponential components. These results demonstrate uniformity of ionophore distribution, ionophore-induced Ca2+-permeability, and cytoplasmic Ca-buffering among all the cells. Experiments involving mixing of cell suspensions with high and low original ionophore content, and involving ionophore extraction by albumin, demonstrate a rapid redistribution of ionophore among the cells, indicating that homogeneity of ionophoric effects is achieved through dynamic ionophore redistribution.     

Effect of carbonyl compounds on red blood cells deformability

The effect of Maillard reaction on red blood cells (RBC) deformability was investigated. Exposure of RBC to carbonyl compounds (dl-glyceraldehyde, glyoxal, glycolaldehyde, 3-deoxyglucosone, and d-glucose) leading to Maillard reaction caused a marked decrease in RBC deformability even at 1 mM level. The decrease rate depended on the kind of carbonyl compounds, in which both dl-glyceraldehyde and glyoxal significantly decreased the RBC deformability (p < 0.05). In addition, the decrease rate also differed among volunteers tested, indicating that the sensitivity against carbonyl compounds varies among them. In order to elucidate the mechanism of the decrease in RBC deformability, RBC was exposed to carbonyl compounds in the presence of aminoguanidine which is the inhibitor of AGE formation in Maillard reactions. Aminoguanidine inhibited the decrease in RBC deformability by dl-glyceraldehyde and glyoxal. When Hb which has a high reactivity with carbonyl compounds was incubated with those carbonyl compounds, dl-glyceraldehyde and glyoxal showed the high reactivity with Hb compared with other carbonyl compounds. These results indicate that Maillard reaction between RBC proteins and carbonyl compounds leads to the decrease in RBC deformability. On the other hand, generated by carbonyl compounds involved in lowering the deformability only to a negligible level.     

Role of calcium in volume regulation by dog red blood cells

Dog red blood cells (RBC) are shown to regulate their volume in anisosmotic media. Extrusion of water from osmotically swollen cells requires external calcium and is associated with net outward sodium movement. Accumulation of water by osmotically shrunken cells is not calcium dependent and is associated with net sodium uptake. Net movements of calcium are influenced by several variables including cell volume, pH, medium sodium concentration, and cellular sodium concentration. Osmotic swelling of cells increases calcium permeability, and this effect is diminished at acid pH. Net calcium flux in either direction between cells and medium is facilitated when the sodium concentrations is low in the compartment from which calcium moves and/or high in the compartment to which calcium moves. The hypothesis is advanced that energy for active sodium extrusion in dog RBC comes from passive, inward flow of calcium through a countertransport mechanism.     

Effects of ionophore A23187 on calcium fluxes from cultured adrenal cells

The effect of the calcium ionophore A23128 on calcium fluxes from Y-1 adrenal cortical cells was investigated. Conditions were chosen which are known to result in an inhibition of steroidogenesis (6 . 10(-6) M ionophore and 3 . 10(-4) M extracellular calcium). Calcium efflux from Y-1 cells exhibited two distinct phases. A fast phase which was insensitive to the mitochondrial poison sodium azide and a slow, azide-sensitive phase. The ionophore brought about a rapid increase in the rate of calcium efflux and an 84% reduction in the size of the calcium pool which was associated with the slow efflux phase as well as a reduction in its rate constant. A decrease in the size of the rapidly exchanging calcium pool was also detected. Ethanol, the solvent which was used for the ionophore, slightly increased the rate constant of the rapidly exchanging pool. Conditions which resulted in diminished steroidogenic capacity also brought about a reduction in the size of an energy dependent, intracellular pool. The data is interpreted as being consistent with a hypothesis that the ionophore-induced inhibition of steroidogenesis may be causatively related to the loss of intracellular calcium or to the mechanism which brings about the loss.     

Effect of changes in the rate of ionophore A23187-induced calcium influx on the pump-leak steady-state distribution of calcium in inosine-fed human red cells

We studied the effect of varying the rate of ionophore A23187-induced calcium influx on the mean calcium content of inosine-fed human red cells in pump-leak steady state. Slow calcium infusion caused only a marginal reduction in the mean calcium content of cells in the steady state relative to their content after sudden calcium addition.     

Properties of the basal calcium influx in human red blood cells

The basal ⁴⁵Ca²⁺ influx in human red blood cells (RBC) into intact RBC was measured. ⁴⁵Ca²⁺ was equilibrated with cells with t_1/2=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5±0.2 μmol/l_{packed cells} (n=6) at 37 °C. The average value of the Ca²⁺ influx rate was 43.2±8.9 μmol/l_{packed cells} hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 °C. The basal Ca²⁺ influx was saturable with Ca²⁺ up to 5 mmol/l but at higher extracellular Ca²⁺ concentrations caused further increase of basal Ca²⁺ influx. The ⁴⁵Ca²⁺ influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3′,4′,5-tetrachloro-salicylanilide (TCS) 10⁻⁶-10⁻⁵ mol/l) inhibited in part the Ca²⁺ influx. The results show that the basal Ca²⁺ influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca²⁺ influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca²⁺ are coupled via the RBC H⁺ homeostasis.     

Properties of the basal calcium influx in human red blood cells

The basal (45)Ca(2+) influx in human red blood cells (RBC) into intact RBC was measured. (45)Ca(2+) was equilibrated with cells with t(1/2)=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5+/-0.2 micromol/l(packed cells) (n=6) at 37 degrees C. The average value of the Ca(2+) influx rate was 43.2+/-8.9 micromol/l(packed cells) hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 degrees C. The basal Ca(2+) influx was saturable with Ca(2+) up to 5 mmol/l but at higher extracellular Ca(2+) concentrations caused further increase of basal Ca(2+) influx. The (45)Ca(2+) influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3',4',5-tetrachloro-salicylanilide (TCS) 10(-6)-10(-5) mol/l) inhibited in part the Ca(2+) influx. The results show that the basal Ca(2+) influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca(2+) influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca(2+) are coupled via the RBC H(+) homeostasis.     

Effects of ruthenium red, A23187 and D-600 on steroidogenesis in Y-1 cells.

The effects of the calcium antagonists ruthenium red and D-600 and the cation ionophore A23187 on steroidogenesis were investigated. Steroidogenesis triggered by corticotrophin and cyclic AMP was inhibited by each of the agents. Incubation of Y-1 cells with an excess of ethyleneglycol-bis-(beta-amino-ethylether)-N,N'-tetraacetic acid (EGTA) abolished the steroidogenic response to corticotrophin while the response to cyclic AMP was unaffected. The ability of ruthenium red and D-600 (1 . 10(-5) M), and A23187 (6 . 10(-6 M) to inhibit a response which does not require the presence of extracellular calcium (cyclic AMP induced steroidogenesis) suggests that they are altering intracellular calcium. Neither of the calcium antagonists nor the cation ionophore inhibited the steroidogenic response to exogenous pregnenolone, thereby suggesting that the cells were still viable. Only when A23187 was used in the presence of a 15-fold increase in extracellular calcium (4.8 mM) was the response to pregnenolone diminished. The data are interpreted as a further indication that, in intact cells, intracellular calcium plays a role in the steroidogenic pathway.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 induced calcium transients in human red cells

A transient increase of cellular calcium was induced by addition of the divalent cation ionophore A23187 to human red cells in the absence or presence of drugs. The peak height of the calcium transient was increased about five times at pH 6.9 and up to eighteen times at pH 7.4 by trifluoperazine (0.30 mM), and two to three times at pH 6.9 by compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). The time-dependent changes of cellular calcium were analysed by the aid of a pump-leak model based partly on the calcium dependent parameters obtained from calcium ATPase experiments, partly on the A23187 induced calcium fluxes determined in experiments with ATP depleted cells. The transient increase of cellular calcium induced within few minutes after the addition of ionophore A23187 could be explained satisfactorily by the model both in the absence and presence of the four drugs, whereas the final level of cellular calcium in the drug experiments was more difficult to predict from the pump-leak model. Comparison of experimental and model calcium transients suggested that trifluoperazine and TMB-8 affected both pump and leak, whereas compound 48/80, probably due to low membrane-permeability, mainly affected the leak and verapamil affected the pump only.     

Effects of halides and bicarbonate on chloride transport in human red blood cells

Chloride self-exchange was determined by measuring the rate of 36Cl efflux from human red blood cells at pH 7.2 (0 degrees C) in the presence of fluoride, bromide, iodide, and bicarbonate. The chloride concentration was varied between 10--400 mM and the concentration of other halides and bicarbonate between 10--300 mM. Chloride equilibrium flux showed saturation kinetics. The half-saturation constant increased and the maximum flux decreased in the presence of halides and bicarbonate: the inhibition kinetics were both competitive and noncompetitive. The competitive and the noncompetitive effects increased proportionately in the sequence: fluoride less than bromide less than iodide. The inhibitory action of bicarbonate was predominantly competitive. The noncompetitive effect of chloride (chloride self-inhibition) on chloride transport was less dominant at high inhibitor concentrations. Similarly, the noncompetitive action of the inhibitors was less dominant at high chloride concentrations. The results can be described by a carrier model with two anion binding sites: a transport site, and a second site which modifies the maximum transport rate. Binding to both types of sites increases proportionately in the sequence: fluoride less than chloride less than bromide less than iodide.     

Effect of high osmotic media on blood viscosity and red blood cell deformability

Effects of high osmotic media on the shape and deformability of RBC were examined for determining increasing factors of blood viscosity. Dog blood and Urographin (a hypertonic contrast medium) were used; the plasma osmolality was changed by Urografin suspended in blood. The viscosity was measured for normal RBC and glutaraldehyde-treated RBC suspensions with a cell volume concentration. The RBC deformability was evaluated from the difference in viscosity between the two suspensions. It was shown that normal RBC suspension increased the viscosity with increase in osmolality at high shear rate; hardened RBC suspension decreased the viscosity with increase in osmolality. It was concluded that the RBC deformability decreased with increasing osmolality.     

Assessment of oxidant susceptibility of red blood cells in various species based on cell deformability

The present study was designed to investigate the oxidant susceptibility of red blood cells (RBC) from four species (echidna, human, koala, Tasmanian devil) based on changes in cellular deformability. These species were specifically chosen based on differences in lifestyle and/or biology associated with varied levels of oxidative stress. The major focus was the influence of superoxide radicals generated within the cell (phenazine methosulfate, PMS, 50 μM) or in the extracellular medium (xanthine oxidase-hypoxanthine, XO-HX, 0.1 U/ml XO) on RBC deformability at various shear stresses (SS). RBC deformability was assessed by laser-diffraction analysis using a "slit-flow ektacytometer". Both superoxide-generating treatments resulted in significant increases of methemoglobin for all species (p < 0.01), with Tasmanian devil RBC demonstrating the most sensitivity to either treatment. PMS caused impaired RBC deformability for all species, but vast interspecies variations were observed: human and koala cells exhibited a similar sigmoid-like response to SS, short-beaked echidna values were markedly lower and only increased slightly with SS, while Tasmanian devil RBC were extremely rigid. The effect of XO-HX on RBC deformability was less when compared with PMS (i.e., smaller increase in rigidity) with the exception of Tasmanian devil RBC which exhibited essentially no deformation even at the highest SS; Tasmanian devil RBC response to XO-HX was thus comparable to that observed with PMS. Our findings indicate that ektacytometry can be used to determine the oxidant susceptibility of RBC from different species which varies significantly among mammals representing diverse lifestyles and evolutionary histories. These differences in susceptibility are consistent with species-specific discrepancies between observed and allometrically-predicted life spans and are compatible with the oxidant theory of aging.     

Effects of nitric oxide and prostacyclin on deformability and aggregability of red blood cells of rats ex vivo and in vitro.

Although many diseases of the heart and circulatory system have been linked with insufficient deformability and increased aggregability of red blood cells, there are only a few drugs which can modulate these biological functions of erythrocytes. Here, we show evidences that iloprost, stable prostacyclin analogue and SIN-1, active metabolite of molsidomine which spontaneously releases NO, may be sufficient pharmacological tools for modulating red blood cell deformability and aggregability. Deformability of red blood cells was measured by shear stress laser diffractometer (Rheodyn SSD) and expressed in percent of red blood cell deformability index (DI). MA-1 (Myrenne) erythrocyte aggregometer was used for photometric measurements of aggregability in arbitrary units (MEA) of mean extent of aggregation. Experiments were carried out on rats ex vivo and in vitro using whole rat blood or isolated erythrocytes. Ex vivo SIN-1 (infusion 2 mg/kg/min i.v.) and iloprost (bolus injection 10 microg/kg i.v.) significantly improved erythrocyte deformability and aggregability at 5-15 min after administration. L-NAME (10 mg/kg i.v.)- inhibitor of nitric oxide synthase, and aspirin (1 mg/kg i.v.) caused worsening of deformability of erythrocytes in experiments ex vivo. Studies in vitro also revealed improvement of red blood cell deformability and aggregability by SIN-1 (3 microM, 15 min incubation at 22 degrees C) or iloprost (1 microM, 15 min incubation at 22 degrees C) and this phenomenon appeared not only in whole blood but also in isolated red cells. It is concluded that NO- and prostacyclin-induced improvement of red blood cell deformability and aggregability results from direct action of these compounds on erythrocytes. NO-donors and iloprost could be useful in the treatment of disorders of blood fluidity.     

Abnormal blood flow and red blood cell deformability in severe malaria

Obstruction of the microcirculation plays a central role in the pathophysiology of severe malaria. Here, Arjen Dondorp and colleagues describe the various contributors to impaired microcirculatory flow in falciparum malaria: sequestration, rosetting and recent findings regarding impaired red blood cell deformability. The correlation with clinical findings and possible therapeutic consequences are discussed.     

Sodium and calcium movements in dog red blood cells

Determinants of 45Ca influx, 45Ca efflux, and 22Na efflux were examined in dog red blood cells. 45Ca influx is strongly influenced by the Na concentration on either side of the membrane, being stimulated by intracellular Na and inhibited by extracellular Na. A saturation curve is obtained when Ca influx is plotted as a function of medium Ca concentration. The maximum Ca influx is a function of pH (increasing with greater alkalinity) and cell volume (increasing with cell swelling). Quinidine strongly inhibits Ca influx. Efflux of 45Ca is stimulated by increasing concentrations of extracellular Na. 22Na efflux is stimulated by either Ca or Na in the medium, and the effects of the two ions are mutually exclusive rather than additive. Quinidine inhibits Ca-activated 22Na efflux. The results are considered in terms of a model for Ca-Na exchange, and it is concluded that the system shows many features of such a coupled ion transport system. However, the stoichiometric ratio between Ca influx and Ca-dependent Na efflux is highly variable under different experimental conditions. Because the Ca fluxes may reflect a combination of ATP-dependent, outward transport and Na-linked passive movements, the true stoichiometry of an exchanger may not be ascertainable in the absence of a specific Ca pump inhibitor. The meaning of these observations for Ca-dependent volume regulation by dog red blood cells is discussed.     

Measurement of biophysical properties of red blood cells by resistive pulse spectroscopy: volume, shape, surface area, and deformability

This paper presents a simple, new approach to the determination of size, shape, surface area, and deformability information for cells, notably red blood cells. The results are obtained by combining experimental measurements from resistive pulse spectroscopy (an extension of electronic cell-sizing methodology) with theoretical calculations for model cell systems. Assuming constancy of surface area and approximating red cell shapes by both prolate and oblate ellipsoids of revolution, values are determined for cell shape factor and volume under a variety of conditions. For red blood cells under low-stress conditions, shape factor, volume, and surface area results are found to be consistent with those available from the literature, when the oblate model is used. The applicability of this approach for determination of red cell properties under altered conditions is demonstrated by results for cell volume, at varying osmotic pressure and mechanical shear (tensile) stress. By quantitating the change in cell shape with stress, a new numerical scale for measuring cell deformability is also obtained, and data are presented on its variation for red cells at different osmolalities, over the range of 140 to 500 mOsm.