Suspensions Of Human Erythrocytes Research Articles

Purine and pyrimidine transport and permeation in human erythrocytes

Time courses of the uptake of radiolabeled hypoxanthine, adenine and uracil were measured by rapid kinetic techniques over substrate ranges from 0.02 to 5000 μM in suspensions of human erythrocytes at 25 or 30°C. At concentrations above 25 μM, the rate of intracellular phosphoribosylation of hypoxanthine and adenine was insignificant relative to their rates of entry into the cell and time courses of transmembrane equilibration of the substrates could be measured and analyzed by integrated rate analysis. Hypoxanthine and uracil are transported by simple facilitated carriers with directional symmetry, high capacity and Michaelis-Menten constants of about 0.2 and 5 mM, respectively. Adenine is probably transported by a carrier with similar properties but no saturability was detectable up to a concentration of 5 mM. Cytosine entered the cells much more slowly than the other three nucleobases, and its entry seems not to be mediated by a carrier. The hypoxanthine transporter resembles that of one group of mammalian cell lines, which does not exhibit any overlap with the nucleoside transporter and is resistant to inhibitors of nucleoside transport. Results from studies on the effects of the nucleobases on the ifnlux and countertransport of each other were complex and did not allow unequivocal conclusions as to the number of independent carriers involved. At concentrations below 5 μM, radiolabel from adenine and hypoxanthine accumulated intracellularly to higher than equilibrium levels. Part of this accumulation reflected metabolic trapping, especially when the medium contained 50 mM phosphate. But part was due to an apparent concentrative accumulation of free adenine and hypoxanthine up to 3-fold at medium concentrations 1 μM and when cells were incubated in phosphate-free medium. This concentrative accumulation could be due to the functioning of additional high-affinity, low-capacity, active transport systems for adenine and hypoxanthine, but other factors could be responsible, such as saturable binding to intracellular components.

No evidence for bradykinin hydrolysis in human erythrocyte suspensions: 1H NMR studies.

In view of their permeability to small peptides, it has been postulated that human erythrocytes may play a role in terminating the action of some circulating peptide hormones. Work using classical paper chromatographic techniques for detecting free amino acids indicated that the octapeptide, des-(Arg9)-bradykinin, enters these cells and its amino-terminal arginine residue is released by cytosolic aminopeptidase-P. We have used 1H NMR to monitor directly the release of arginine from bradykinin. The hydrolytic reaction rate in hemolysates, with an initial peptide concentration of 13.0 mmol l-1 was 6.5 mmol (1 packed red cell)-1 h-1. But no reaction was evident after a 4.5-h incubation with intact cells, thus contradicting the earlier suggestion that erythrocytes are involved in the primary inactivation of this hormone. This is consistent with our previous findings that the pentapeptide leu-enkephalin fails to enter human erythrocytes but that its lower-order degradation products may do so.

The rates of fast reactions of carbon dioxide and bicarbonate in human erythrocytes measured by carbon-13 NMR

The application of carbon-13 nuclear magnetic resonance spectroscopy to the study of the kinetics of millisecond timescale reactions of CO 2 in human erythrocyte suspensions is described. The rates of intracellular enzyme catalyzed CO 2 hydration and HCO 3 − dehydration were quantitatively determined, as well as the rates of CO 2 diffusion into and out of the erythrocytes. The method also provides an accurate measure of the intracellular pH in the range of pH 6.0 to pH 7.0. A temperature dependence study was used to determine the thermodynamic functions for the intracellular hydration-dehydration reaction.

Thromboxane induced red blood cell lysis

The two thromboxane A 2 mimetics, carbocyclic thromboxane A 2 (CTA 2) and U-46619 (9,11-methanoepoxy PGH 2) at concentrations of 400 ng/ml significantly enhanced the release of hemoglobin from both feline and human erythrocyte suspensions. This effect was significantly attenuated by the thromboxane receptor antagonist BM-13,505 indicating that the membrane leakiness is in some way receptor mediated. The effects also appear to be concentration-dependent over the range of 100–400 ng/ml. The membrane labilizing effect of thromboxane analogs is not due to a non-specific eicosanoid effect since iloprost, the stable prostacyclin analog, actually stabilized erythrocyte membranes. Moreover, synthetic thromboxane A 2 exerted similar effects to that of the two TxA 2-mimetics. This membrane labilizing action of thromboxanes may be important in propagating the other pathophysiologic effects of thromboxane A 2 in cardiovascular disease states.

Equilibrium exchange of dimethyl methylphosphonate across the human red cell membrane measured using NMR spin transfer

The 31P NMR spectrum of dimethyl methylphosphonate, in a suspension of human erythrocytes in a hypertonic medium, is characterized by separate intra- and extracellular resonances. The compound crosses the red cell membrane too rapidly for its transport to be monitored using conventional NMR time-course techniques. In the present work we adapted the saturation transfer method to measure the unidirectional flux of dimethyl methylphosphonate into the cell at equilibrium and thereby gained an estimate of its permeability coefficient. Repeated measurements on low hematocrit cell suspensions showed no significant variation in the permeability coefficients for cells from five different donors. Saturation transfer measurements conducted over a range of hematocrits demonstrated the hematocrit dependence of the unidirectional rate constant for dimethyl methylphosphonate influx. The calculated permeability coefficient was independent of hematocrit.

Human Erythrocyte Membrane Permeability and Nitroxyl Spin-Label Reduction

Nitroxyl spin labels are paramagnetic compounds that have demonstrated utility as contrast enhancing agents in proton magnetic resonance imaging. The time-course of contrast enhancement depends on distribution and elimination of these agents. Reduction, resulting in formation of the diamagnetic hydroxylamine, is the major metabolic pathway observed in vivo. This bioreduction has implications for the design of contrast agents and for understanding their imaging behavior. Bioreduction has been shown to occur, at least in part, intracellularly. As such, cell membrane permeability to nitroxyl spin labels may influence their bioreduction. In this study, this influence was examined using eight nitroxyl derivatives and the human erythrocyte suspension as a model biomembrane system. lonizable weak acids and bases were found to equilibrate rapidly across the erythrocyte membrane with half-times of equilibration ranging from <10s to 1.6min. These derivatives had low octanol:buffer distribution coefficients and were extensively ionized at the pH of the system (7.0). A strong acid, a phosphate ester, and a quaternary amine derivative were excluded by the cell membrane. Reduction of nitroxyl spin labels by the erythrocyte was shown to occur intracellularly. Except for the impermeable probes, the reduction rate was slow in comparison with the membrane penetration rate. The structural dependence of reduction rate was unrelated to penetration rate but correlated well with that observed in other reducing systems, namely ascrobic acid solution and rat tissue 10 homogenates.

Fatty fish-induced changes in membrane lipid composition and viscosity of human erythrocyte suspensions.

The effect of a daily supplement of 75-100 g fatty fish, providing 3 g of omega 3 fatty acids, on the lipid composition and deformability of erythrocytes was studied in six healthy male subjects. After 4 weeks of the fish supplementation the mean degree of unsaturation, expressed as double bond index, of erythrocyte phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) had increased significantly by 10% and 4%, respectively. The mean cholesterol/phospholipid ratio (C/P ratio) in the membranes had increased by 28% (p less than 0.001). No change was seen in the distribution of the four major phospholipid classes. After withdrawal of the supplement for 4 weeks the changes in PC were reversed, but the changes in PE persisted and the mean C/P ratio still was higher than basal by 16% (p less than 0.01). The effect on the C/P ratio by the fatty fish is in contrast with the previously observed lack in effect of cod-liver oil. The viscosity of erythrocyte suspensions with a packed cell volume fraction of 0.80 and measured at shear rates less than or equal to 8.11 s-1 was decreased after the fish. We suggest that the fall in the viscosity of erythrocyte suspensions, reflecting increased cell deformability, is probably due to the change in the fatty acid composition of erythrocyte PC.

Augmented transport of extracellular solutes in concentrated erythrocyte suspensions in couette flow

Local motions of suspended particles or droplets can augment molecular diffusion and govern the transport of solutes in flowing dispersions. An electrochemical technique was developed for measuring the rates of augmented transport of solutes in a direction normal to the bulk flow through erythrocyte suspensions in Couette flow. Human, bovine, and rabbit erythrocytes in the form of normal, hardened, and ghost cells at volume fractions from 0.1 to 0.4 were studied at shear rates up to 3680 sec −1. Augmentation is defined as ( D e D s − 1) × 100% , where D e and D s are, respectively, the effective diffusivities in flowing and stationary suspensions. The augmentation is correlated with a characteristic Péclet number, a 2 γ/ D sF, where a is the effective radius of the particle, γ is the shear rate, and D sF is the Fricke's diffusion coefficient. The data are well described by A = αPe β in the Pe range from 4 to 100. The values of α and β depend on particle volume fraction φ and rigidity. The augmentation, however, does not change significantly between φ = 0.2 and 0.4. For suspensions of normal human erythrocytes at a volume fraction of 0.4, the augmentation is well described by A(%) = 6.1 Pe 0.89. This correlation agrees with all the reported experimental evidence of transport of heat, urea, albumin, and platelets in flowing blood. The correlations for hardened erythrocyte suspensions are also in agreement with the results on transport of heat, helium, and sodium chloride in flowing suspensions of rigid microspheres. Particle deformability does not affect the augmentation significantly at φ = 0.1 or less. For more concentrated suspensions, normalized augmentation increases with φ for suspensions of hardened erythrocytes, whereas it decreases with φ for suspensions of normal and ghost erythrocytes. It is inferred from this result that at φ > 0.1, multiplet collisions are important for augmentation for suspensions of rigid particles, whereas membrane rotation and deformation is the dominant mechanism for suspensions of deformable particles. The results also indicate that transport of macromolecules is significantly enhanced in flowing blood. For example, in flowing blood at a shear rate of 500 sec −1, the augmented transport rate of lipoproteins with a D s = 10 −8 cm 2/sec is about 100-fold of the Brownian diffusion rate.

Direct observation of the NAD glycohydrolase reaction in human erythrocytes using NMR spectroscopy.

The hydrolysis of NAD by the extracellular membrane-associated enzyme NAD glycohydrolase was shown to be readily followed in concentrated suspensions of human erythrocytes using 1H spin-echo nuclear magnetic resonance spectroscopy (NMR). The maximal rate of the reaction was determined and the inhibitory effect of nicotinamide was confirmed by direct NMR observation. In addition, arginine, ergothioneine and iodoacetate did not influence the reaction rate. 31P NMR analyses of reaction media from whole cells showed that no extraneous degradation of NAD occurred and the only phosphate-containing product was ADP-ribose.

A multinuclear NMR study of 2,3-bisphosphoglycerate metabolism in the human erythrocyte

Resonances from 13C, 31P and 1H have been detected simultaneously in suspensions of human erythrocytes using a modified NMR spectrometer equipped with a probe tuned to four different frequencies. The utility of multinuclear NMR in the study of cellular metabolism is demonstrated with an investigation of 13C label flux through the 2,3-bisphosphoglycerate bypass in human erythrocytes. In a single experiment, the respective contributions of this bypass and the pentose-phosphate shunt were found to be 27 and 10% of the total glycolytic rate.

Cell volume dependence of 1H spin-echo NMR signals in human erythrocyte suspensions : The influence of in situ field gradients

The 1H spin-echo NMR signal amplitudes and intensities of low molecular weight solutes in the cytoplasm and extracellular fluid of suspensions of human erythrocytes were shown to depend on the osmotic pressure of the media. At low osmotic pressure (220 mosM/kg) freeze-thaw lysis of the cells resulted in signal enhancement which was greatest for extracellular molecules, but both intra- and extracellular species were almost equally enhanced at 580 mosM/kg. This effect is due to field gradients formed at cell boundaries as a result of differences in magnetic susceptibility between the medium and the cytoplasm. T 2 values measured using the Carr-Purcell-Meiboom-Gill pulse sequence, with τ = 0.0003 s, depended little on cell volume and absolute changes in volume magnetic susceptibility were also small. The mean field gradients, calculated from data obtained on cell suspensions at different osmotic pressures, were in the range 0.25–1.98 G/cm and 0.89–2.09 G/cm for intra- and extracellular compartments, respectively. The maintenance of isotonicity of the extracellular fluid during metabolic studies of cell suspensions is important in order to avoid artefacts in the determination of metabolite concentrations when using the spin-echo technique. Conversely it may be possible to perform transport measurements using spin-echo NMR to monitor the cell volume changes which occur during the transmembrane migration of molecules.

Intra-erythrocyte microviscosity and diffusion of specifically labelled [glycyl-alpha-13C]glutathione by using 13C n.m.r.

[alpha-13C]Glycine was incubated with suspensions of human erythrocytes under special buffer conditions to enrich specifically intracellular glutathione with 13C. The metabolically active cells were then subjected to 13C n.m.r. spectroscopy in which the longitudinal relaxation time(s) (T1) and nuclear Overhauser enhancement(s) of the free glycine and glutathione were measured. With the appropriate analysis, assuming the molecules to be isotropic rotors, intracellular rotational correlation times were calculated. Using these data together with the Stokes-Einstein equation, viscosity and translational diffusion coefficients were calculated. The results were compared with those from cell lysates and extracts. The cytosolic microviscosity probed by glutathione was only 1.9 +/- 0.3 times that of saline, suggesting, therefore, that most enzyme reactions involving this solute are not likely to be diffusion-controlled inside the erythrocyte.

Action of hydroxyethyl starch on the flow properties of human erythrocyte suspensions.

Hydroxyethyl starch (HES) has often been used as a plasma expander, but questions still remain concerning the mechanisms by which it produces changes in the rheological properties of blood and erythrocyte (RBC) suspensions under various flow conditions. The present investigation has shown that the dynamic viscosity of HES (232,000 and 565,000 daltons) solutions rises in a nonlinear fashion with increasing HES concentration, and for a given concentration of HES exhibits Newtonian behavior at shear rates between 0.15 to 124 sec-1. At low (less than 0.9 sec-1) shear rates the apparent viscosity of a 40% RBC suspension increases with lower concentrations of HES because of RBC aggregation. At higher concentrations of HES, increases in suspension viscosity are due to an increase in the viscosity of the continuous phase since the RBC are largely disaggregated. At high (greater than 36 sec-1) shear rates the relative viscosity (eta/eta O) of RBC suspensions slowly decreases with increasing HES concentration. At low shear rates eta/eta O increases and then decreases with increasing HES concentration. Evidence of the concentration-dependent effects of HES on RBC aggregation is provided not only by the viscometric analysis but also from measurements of erythrocyte sedimentation rate (ESR) and the zeta sedimentation ratio (ZSR). HES is a more potent aggregating agent in phosphate buffered saline (PBS) than it is in plasma. Polymer size has only a slight effect on the extent of RBC aggregation produced, but does have a significant effect on the concentration of polymer at which maximum aggregation occurs. The viscosity-corrected electrophoretic mobility of RBC in HES rises monotonically with the concentration of HES in the suspending medium. Decreases in the extent of RBC aggregation with increasing polymer concentrations probably result from an increase in the electrostatic repulsive forces between the cells.

Further investigations of ATP release from human erythrocytes exposed to ultrasonically activated gas-filled pores.

Suspensions of human erythrocytes in a firefly-extract solution were exposed to 2.0 MHz ultrasound in the presence of 3.4 microns dia. gas-filled pores. Lysis of the cells during ultrasonic exposure resulted in the release of ATP which produced light upon interaction with the firefly extract. The response was approximately proportional to the square of the SPTA intensity I for both continuous and pulsed exposures (1 mu s pulses, 1:9 duty cycle) within the range 5-100 mW/cm2. One method of graphical data presentation yielded an apparent threshold for the effect at 0.31 bar pressure amplitude; however, a response could probably be obtained at any nonzero exposure level with a sufficiently sensitive apparatus. The response was also proportional to the exposure duration t for the range 0.03-3 s. The product I2 t may have some significance as a dosage parameter for this system in an, as yet, undefined "low-dose" range. Measurements for exposures with higher intensities, longer durations, lower duty factors and with pulses longer than 300 mu s deviated from these trends.

Incorporation of labelled glycine into reduced glutathione of intact human erythrocytes by enzyme-catalysed exchange. A nuclear-magnetic-resonance study.

1H, 2H and 15N n.m.r. spectroscopy was used to monitor the incorporation of free glycine into the glycine residue of reduced glutathione (GSH) in suspensions of intact human erythrocytes. The following results were obtained. (i) By using 1H spin-echo n.m.r. the exchange reaction between [2H5]glycine and the protonated glycine residue of GSH was studied at various [2H5]glycine concentrations, thus enabling the calculation of an apparent Michaelis constant (Km) and maximal velocity (Vmax.) for the process. (ii) The reaction is catalysed by glutathione synthetase and proceeds most rapidly in the absence of glucose, which is the main physiological energy source of the erythrocyte. (iii) 15N n.m.r. spectroscopy, with a one-pulse sequence, and 2H n.m.r. spectroscopy, with an inversion recovery method, enabled demonstration of the incorporation of labelled glycine into an intra-erythrocyte peptide, consistent with incorporation into GSH. (iv) The exchange reaction, although inhibited by glucose, appeared not to be dependent on low ATP or 2,3-bisphosphoglycerate concentrations.

Decreased viscosity of human erythrocyte suspension due to drug-induced spherostomatocytosis.

The effect of two cationic drugs (chlorpromazine and isoxsuprine) on the suspension viscosity of human erythrocytes were examined, comparing with the effect of anionic drugs. (1) As increasing the drug concentrations, the cationic drugs transformed the erythrocytes to stomatocytes, then to spherostomatocytes, while trinitrobenzene sulfonate, dehydroepiandrosterone sulfate and lysolecithin induced echinocytes, as well known. (2) The suspension viscosity decreased in parallel with the appearance of spherostomatocytes, but it increased in echinocytosis. (3) The membrane fluidity, measured by spin label method, was not a major determinant for the suspension viscosity in these cases, because of no systematic correlation. (4) The rheoscopic observation under shear force demonstrated that the spherostomatocytes deformed easily to ellipsoid with smooth cell surface, while the echinocytes less easily deformed to ellipsoid on which the small spikes persisted at higher shear. These distinct difference in deformed shape under high shear force could be related to the decreased suspension viscosity of spherostomatocytes. (5) In addition, the transformation to spherostomatocytes, thus the decreased viscosity, was primarily determined by the intramembraneous drug concentration.

Use of a hemadsorption technique to evaluate the stability of the hemagglutination reaction of Escherichia coli cultures possessing human colonization factor antigens.

Two strains of Escherichia coli, producing different colonization factor antigens (CFA), were monitored for the population density of CFA-producing bacteria after repeated subculture. The production of CFA was estimated by flooding agar plates containing isolated colonies with suspensions of human or bovine erythrocytes. The erythrocytes were suspended in a low-ionic-strength buffer and were fixed to CFA-positive colonies with a 1.0% tannic acid solution. Strain H-10407, possessing CFA/I fimbriae, showed a rapid loss of the hemagglutinin when subcultured, whereas strain CL-9699, producing CFA/II, was very stable. By using the hemadsorption assay, we could rapidly and easily distinguish CFA- positive colonies from the CFA-negative variants. A survey of additional E. coli strains demonstrated the utility and specificity of the hemadsorption technique used.

A 1H n.m.r. study of isotope exchange catalysed by glycolytic enzymes in the human erythrocyte.

The exchange of hydrogen and deuterium atoms between the C-2 position of lactate and solvent was monitored in suspensions of human erythrocytes by using a non-invasive spin-echo p.m.r. method that permits continuous assessment of the rate and the extent of exchange. Exchange rates were measured in cells suspended in buffers made in 2H2O and 1H2O after the addition of L-[2-1H]lactate and L-[2-2H]lactate respectively. The rate of exchange is dependent on the activities of four glycolytic enzymes (fructose bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase and lactate dehydrogenase) and on the concentrations of their substrates. The dependence of the exchange on the following substrates was studied: (1) lactate, (2) the triose phosphates and fructose 1,6-bisphosphate and (3) pyruvate. Observation of the exchange in vitro, in a system produced by mixing the isolated enzymes, permits determination of the individual isotope-exchange equilibrium velocities of the enzymes. The dependence of the equilibrium velocity of human erythrocyte lactate dehydrogenase on NAD+ + NADH concentration was measured. Possible applications of these methods are discussed.

Erythrocyte glycolysis: Stimulation by nalidixic acid

Nalidixic acid, the urinary antiseptic, stimulates by 40% the rate of lactate production in suspensions of human erythrocytes in Krebs bicarbonate solution at pH 7.34 and 37°C. The stimulation is evident with the other glycolytic substrates, inosine and dihydroxyacetone. Stimulation was also seen with the much poorer substrate ribose. The concentrations of ATP and 2,3-DPG in cells incubated with nalidixic acid and ouabain were consistent with changes in ATPase activity that would result in enhanced and inhibited glycolysis, respectively. Nalidixic acid, however, did not stimulate further maximally activated erythrocyte ghost ATPase whereas ouabain inhibited it. Furthermore, nalidixic acid did not overcome the ouabain inhibition. Nalidixic acid failed to stimulate glycolysis in whole erythrocyte lysates and only marginally stimulated glycolysis in erythrocytes in a medium where choline chloride replaced NaCl and KHCO 3 replaced NaHCO 3. The rate enhancement of nalidixic acid on glycolysis was interpreted as being due to an increased sodium permeability of the erythrocyte membrane in a manner similar to the effect of dinitriphenol on mitochondrial membranes.

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.