Hypotonic Hemolysis Research Articles

Dark effect of 8-methoxypsoralen on human erythrocytes.

Furanocoumarin 8-methoxypsoralen (8-MOP) (1-100 micrograms mL-1) in the dark showed a protective affect against hypotonic haemolysis of the erythrocyte membrane. However, the effect against heat-induced haemolysis was dependent on the concentration of 8-MOP; lower concentrations of 8-MOP showed an inhibiting effect, whereas higher concentrations caused acceleration of haemolysis. 8-MOP was not able to induce haemolysis in isotonic solution at 20 or 37 degrees C. Reaction of erythrocytes with 8-MOP in the dark resulted in a shrinkage of the cells and alterations of their shapes. We conclude that modification of erythrocyte membrane by 8-MOP proceeds via reaction with membrane lipids and proteins. This indicates that the effect on the cell membrane plays an important role in the mechanism of the action of 8-MOP on the cells.

Free ADP-ribose in human erythrocytes: pathways of intra-erythrocytic conversion and non-enzymic binding to membrane proteins

We have previously identified free ADP-ribose (ADPR) as a normal metabolite in mature human erythrocytes. In this study the metabolic transformations of ADPR were investigated in both supernatants from erythrocyte lysates and intact erythrocytes, loaded with ADPR by means of a procedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophosphatase-catalysed hydrolysis to yield AMP, which was readily converted into the adenylic and inosinic nucleotide pools. To a lesser extent, ADPR underwent conversion into a compound that was identified as ADP-ribulose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic analysis. ADPRu was also susceptible to degradation by the dinucleotide pyrophosphatase, which was partially purified from erythrocyte lysates and characterized with respect to its substrate specificity. Isomerization of ADPR to ADPRu was markedly enhanced by ATP. Incubation of unsealed haemoglobin-free erythrocyte membranes with labelled ADPR did not cause any transformation of this nucleotide and resulted in its trichloroacetic acid- and formic acid-resistant binding to a number of membrane cytoskeletal proteins. These proteins include spectrin, glyceraldehyde 3-phosphate dehydrogenase (Ga3PDH), three proteins of molecular masses 98, 79 and 72 kDa, which apparently comigrate with bands 3, 4.1 and 4.2 respectively, and two additional proteins of molecular masses 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, to Ga3PDH was confirmed for the enzyme purified from human erythrocytes.

Inactivation of glutathione peroxidase following entrapment of purified α or β hemoglobin chains in human erythrocytes

Inactivation of glutathione peroxidase correlates with the rate of hemoglobin chain oxidation. The enzyme inactivation is mainly present in those conditions where the autoxidation of the oxygenated chains is followed by transformation of the oxidized molecule into a hemichrome. Free hemoglobin chains have been encapsulated in human red blood cells by a dialysis technique that involves transient hypotonic hemolysis followed by isotonic resealing. Chain-loaded erythrocytes represent a good in vitro model of thalassemia. The presence of free human chains in the cell alters the intraerythrocytic glutathione peroxidase activity (α chains are more effective in the inactivation of the enzyme with respect to the β chains).

Participation of band 3 protein in hypotonic hemolysis of human erythrocytes.

The study was designed to show whether band 3, anion exchange protein in human erythrocytes, is responsible for hemolysis in hypotonic solution. From ESR spectra of the spin labeled erythrocytes, it was found that the change of the osmotic condition had no effect on membrane fluidity at pH 7.4. At pH 6.0, however, the fluidity at the central part of the lipid bilayer was dependent on osmotic stress. In this case, the fluidity change as a function of KCl concentration was similar to the hemolysis curve. From circular dichroism (CD) spectra of erythrocyte ghosts, it was found that the conformational and/or associative changes of the membrane proteins correlated with the hypotonic hemolysis. In contrast to the osmotic stress, sodium dodecyl sulfate (SDS) changed the fluidity of the membranes but not the conformation of the membrane proteins regardless of the medium pH. In this case, the changes in the fluidity corresponded to the hemolysis curves in SDS solutions. Further, by using anion transport inhibitors, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, eosin 5-isothiocyanate and eosin-5-maleimide, a correlation between the effect on the hypotonic hemolysis and the conformational and/or associative change of band 3 was suggested. These results demonstrate that band 3 is responsible for the hypotonic hemolysis.

Effects of disinfectants on erythrocytes and isolated hepatocytes from rats and surface tension

The effects of formaldehyde (F), m-cresol (C), guaiacol (G), ethanol (E) and their mixture (FC, FCE, FG, FGE) on erythrocytes and isolated hepatocytes from rats and surface tension in water were examined. Hypotonic hemolysis of erythrocytes was inhibited by m-cresol, while guaiacol, formaldehyde and ethanol accelerated the hemolysis. Lower concentrations of the mixture inhibited hypotonic hemolysis, but higher concentrations accelerated hemolysis. Formaldehyde caused a decrease of transaminase (GOT, GPT) in the medium and hepatocytes. GOT and GPT in the medium were increased by m-cresol, but those in the hepatocyte were decreased by this agent. FC and FCE at 10 mM increased GOT in the medium, but FG and FGE decreased GOT. All mixtures decreased GOT and GPT in hepatocytes and GPT in the medium. All mixtures and formaldehyde inhibited GOT and GPT activity. Formaldehyde and m-cresol decreased hepatocyte viability. In the all mixtures-added hepatocytes, the viability was markedly lowered. Formaldehyde, m-cresol, guaiacol and ethanol caused a depression of surface tension, but the depressive effects of FG and FGE were weaker than that of guaiacol. These results suggest that the observed effects of the drug mixtures on erythrocytes and hepatocytes were the additive effects of the component drugs.

Mechanism of hypotonic hemolysis of human erythrocytes.

A mechanism of hemolytic hole formation during rapid hemolysis in a hypotonic medium has been investigated using eosin-5-maleimide (EMI) as a probe. The EMI-labeled erythrocytes revealed a distinct cluster and/or ring of intense fluorescence staining in a hypotonic 5 mM Hepes buffer (pH 7.4), but not in an isotonic buffer containing 150 mM KCl. This EMI cluster indicates an association of band 3 proteins, which correspond to a hemolytic hole. The hole was confirmed by an atomic force microscopy image. The erythrocytes showed a single large hole in the membrane. By the use of EMI-labeled ghosts, it was observed that the lateral clustering of band 3 was accompanied by a biphasic change of fluorescence intensity of EMI. This biphasic change is interpreted as the hemolytic hole formation by band 3, followed by a disappearance of the hole accompanied by band 3 diffusion or distribution within membrane. The latter event corresponds to a spontaneous membrane seal. When a cytoplasmic domain of band 3 was digested with trypsin, or when SH groups in the cytoplasm-facing components of the membrane were also labeled by EMI, no fluorescence change was observed. These results suggest that the association and/or dissociation of band 3 proteins in a hypotonic medium are strongly influenced by cytoplasmic domains. The apparent biphasic change of the fluorescence intensity in the hypotonic medium was well explained by assuming three events: swelling, clustering of band 3, and sealing accompanied by band 3 redistribution.

Entrapment of protein protease inhibitors in red blood cells

Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.

Morphological characterization of exovesicles and endovesicles released from human erythrocytes following treatment with amphiphiles

In order to morphologically characterize exo- and endovesicles released during treatment of erythrocytes with amphiphiles and to look for possible amphiphile-specific effects on the vesiculation pattern, human erythrocytes were treated at 37°C with amphiphiles at concentrations where they exhibit maximum protection against hypotonic haemolysis (cAH max). Released exo- and endovesicles and treated cells were studied by means of transmission (TEM) and scanning (SEM) electron microscopy. All sphero-echinocytogenic amphiphiles induced a release of both spherical and tubular exovesicles. Dodecyl maltoside, a nonionic amphiphile with a bulky polar head, induced a release of predominantly tubular exovesicles, while all other sphero-echinocytogenic amphiphiles induced a release of predominantly spherical exovesicles. Some branched tubular exovesicles were released by a double-chained cationic amphiphile. Tail- and tongue-like structures were often seen on the exovesicles. Spherical exovesicles were frequently invaginated. Stomatocytogenic amphiphiles induced endovesiculation. In erythrocytes treated with most of the stomatocytogenic amphiphiles the endovesicles were clustered, but with some amphiphiles the endovesicles were randomly distributed. Large ringformed endovesicles (octaethyleneglycol alkyl ethers) and endovesicles in chains (octyl and decyl glucopyranoside) also occurred. The endovesicle membrane was often budding into the lumen of the vesicle and in some cases this could ultimately lead to a vesicle inside the endovesicle. We conclude that amphiphiles do not only trigger vesiculation, but may also specifically affect the vesiculation processes.

Transmembrane redistribution of phospholipids of the human red cell membrane during hypotonic hemolysis

The transmembrane distribution of spin-labeled phospholipids was measured in human erythrocytes before and after hypotonic hemolysis by electron paramagnetic resonance. With a first series of partially water soluble probes a complete randomization of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyclin analogues was achieved when cells were resealed in the absence of Mg-ATP or when the aminophospholipid translocase was inhibited by vanadate or calcium. If the ghosts were resealed with Mg-ATP inside, the transmembrane asymmetry of the aminophospholipids was reestablished. With long chain insoluble spin-labeled lipids complete randomization was obtained with the phosphatidylcholine analogue but even in the presence of vanadate only a small percentage (approx. 15%) of the spin-labeled phosphatidylserine flopped to the outer monolayer and comparable percentage of the spin-labeled sphingomyclin flipped to the inner monolayer, indicating a hierarchy in the phospholipid redistribution for these water insoluble lipids during hemolysis. The mechanism by which a selective randomization takes place is not known. It may involve phosphatidylserine-protein interactions in the inner leaflet and sphingomyelin-cholesterol or sphingomyelin-sphingomyelin interaction in the outer leaflet.

The metabolic role of glucose 1,6-P2 in human erythrocytes studied by encapsulation procedures.

Red blood cells (RBC) possess many desirable properties of a suitable carrier of exogenous agents to modify the plasma environment or to be organ targeted within the reticuloendothelial system.1-4 Among the agents that can be encapsulated are: enzymes to correct inborn errors of metabolisms5, antineoplastic molecules in cancer6, chelators for transfusion iron overload.7,8 A different, non therapeutic, application of loaded RBC can be that of studying the metabolism of a certain substance transferred inside the cell or to observe its effect on the normal cellular metabolism. In this second view, we have applied the encapsulation procedures based on hypotonic hemolysis, isotonic resealing and reannealing, described by Ropars et al.9, to overloaded human RBC with glucose 1,6bisphosphate (G1c1,6P2) in order to investigate the metabolic role of the phosphorylated sugar in the whole cell. This study is of interest since Glc1,6P2 in RBC, besides being the coenzyme of phosphoglucomutase, appears to affect the three key enzymes of glycolysis, namely it inhibits hexokinase, and activates phosphofructokinase and pyruvate kinase.10 Intracellular Glc1,6P2 concentration has been increased by loading human RBC with the commercial product or with the enzyme Glc1,6P2- synthase purified from rabbit skeletal muscle11. In both experimental conditions the augmentation of the Glc1,6P2 cellular level is accompanied by a reduced glucose utilization. The significance of the results, with reference to the site(s) of glucose metabolism regulation by the bisphosphate, is discussed.

Protective effect of ulinastatin against liver injury caused by ischemia-reperfusion in rats.

The effects of ulinastatin (ULN), a human urinary protease inhibitor, on liver injury caused by ischemia-reperfusion were studied in rats. In the liver ischemia-reperfusion model, ULN suppressed the elevation of serum transaminase levels and tissue lipid peroxide levels in the liver. ULN did not exhibit a radical-trapping action on the superoxide and hydroxyl radicals as measured by electron spin resonance (ESR). ULN suppressed formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA)-induced superoxide production from polymorphonuclear leukocytes (PMNs) as measured by the cytochrome c assay. ULN did not inhibit either xanthine oxidase (XO) activity or the conversion of xanthine dehydrogenase (XDH) to XO during the ischemic period. ULN also strongly protected against the hypotonic hemolysis of rat erythrocytes. These results suggest that ULN's membrane stabilizing action and suppressive effect against PMNs superoxide production might be attributed to its suppressive effect on the liver's lipid peroxidation caused by ischemia-reperfusion.

Protective Effect of Ulinastatin against Liver Injury Caused by Ischemia-Reperfusion in Rats

The effects of ulinastatin (ULN), a human urinary pro tease inhibitor, on liver injury caused by ischemia-reperfusion were studied in rats. In the liver ischemia-reperfusion model, ULN suppressed the elevation of serum transaminase levels and tissue lipid peroxide levels in the liver. ULN did not exhibit a radical-trapping action on the superoxide and hydroxyl radicals as measured by electron spin resonance (ESR). ULN suppressed formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA)-induced superoxide production from polymorphonuclear leukocytes (PMNs) as measured by the cytochrome c assay. ULN did not inhibit either xanthine oxidase (XO) activity or the conversion of xanthine dehydrogenase (XDH) to XO during the ischemic period. ULN also strongly protected against the hypotonic hemolysis of rat erythrocytes. These results suggest that ULN’s membrane stabilizing action and suppressive effect against PMNs superoxide production might be attributed to its suppressive effect on the liver’s lipid peroxidation caused by ischemia-reperfusion.

Red blood cells as advanced drug delivery systems for antiviral nucleoside analogues.

A broad family of nucleoside analogues are able to inhibit the infectivity and replication of several HIV-1 strains in vitro.1,2 The most popular of these compounds, AZT (3’-azido-3’-deoxythymidine), has been approved in the U.S.A. as a prescription drug since 1987. Although AZT reduces morbidity and mortality associated with severe HIV infection, several toxic effects have limited its long-term administration. 2’,3’-Dideoxycytidine (ddCyd) was subsequently tested clinically3 as potential therapeutic agent. Again ddCyd has shown immunological and virologic improvement in AIDS patients but most individuals developed dose-limiting toxic effects. The same applies to 2’,3’-dideoxyinosine (ddl).4,5 In an attempt to overcome the problem of nucleoside analogue toxicity and to increase their half-life in circulation we have previously synthesized 2’,3’-dideoxycytidine-5’-phosphate (ddCMP) as a prodrug, encapsulated it in human erythrocytes by a procedure of hypotonic hemolysis and resealing, and found that it is dephosphorylated by endogenous pyrimidine nucleotidases and subsequently released by the cells as ddCyd.6 By this way the prodrug is confined within the erythrocytes so that its toxicity is strongly reduced and its delivery is slow enough to ensure a long-lasting plasma concentration.? Unfortunately this drug delivery system does not solve several other problems of nucleoside analogue efficacy. In fact, to be pharmacologically useful dideoxynucleosides must be phosphorylated to 5’-triphosphate by cellular kinases.1,2 Different cell types within the same species have different abilities to phosphorylate these compounds, furthermore, other drugs can potentiate or reduce the activity of these nucleoside analogues by interfering with their metabolism directly or indirectly.8 Macrophages, which are known to be important hosts for HIV-1 with a key role in the propagation of virus as well as in the pathogenesis of AIDS, has low phosphorylating activities.

Red and green low-powered He-Ne lasers protect human erythrocytes from hypotonic hemolysis.

The effect of low-powered He-Ne lasers (red beam, 632.8 nm, 10.2 mW, and green beam, 543.5 nm, 0.44 mW) on hypotonic hemolysis in human red cells was investigated. Red cell solutions (0.9% NaCl, pH 7.00) were centrifuged and the sediments were irradiated with the red or the green beam for 0 (control), 1, 3, 5, 7, 10, 15 and 30 minutes. Each of the 40 irradiated samples were transferred into hypotonic solution (0.405% NaCl, pH 7.00). After leaving the samples at room temperature for 10 minutes, optical density of every suspension was measured spectrophotomechanically at a wavelength of 545 nm to determine hemoglobin content. Hemolysis percentages of the control samples in hypotonic solution were 52.2 +/- 10.7% (mean +/- SD, N = 20) in the red group and 55.0 +/- 12.0% (N = 20) in the green group. Relative values of hemolysis in the hypotonic solution after irradiation with the red or the green beam decreased significantly between the 7- and 30-minute irradiation. These results demonstrate that low-powered He-Ne lasers have a protective effect on red cell membranes from hypotonic hemolysis and stabilize the cell membranes.

Effects of dehydroabietic acid on the erythrocyte membrane

The effects of dehydroabietic acid (DHAA), a dominant resin acid in pulp and paper mill effluents, on membrane-connected events were studied in human erythrocytes. Fifty percent haemolysis was achieved by 252 μM DHAA after 1 h of incubation at +37°C. At sublytic concentrations, DHAA protected erythrocytes against hypotonic haemolysis, with maximum protection occurring at 125 μM. In the lower range of sublytic concentrations, DHAA induced a slight echinocytosis; at higher sublytic concentrations erythrocytes were transformed to sphero-echinocytes and a release of acetylcholinesterase (exovesicles) occurred. Furthermore, at sublytic concentrations DHAA increased potassium efflux and passive potassium influx, while active potassium influx ((Na +-K +)-pump activity) and phosphate efflux were decreased. Our study indicates that DHAA acts on human erythrocytes in a way typical for amphiphilic compounds. It is proposed that DHAA by intercalating into the lipid bilayer of the membrane, affects the dynamics of the bilayer which in turn alters the permeability of the bilayer and the function of ion transporting membrane proteins.

Amphiphile-induced antihaemolysis is not causally related to shape changes and vesiculation

A wide variety of structurally different antihaemolytic amphiphiles were tested for their ability to induce exovesiculation (acetylcholinesterase (AChE) release, transmission electron microscopic (TEM) studies), endovesiculation (fluorescein isothiocyanate conjugated dextran (FITC-dextran) internalization, TEM studies) and shape changes in human erythrocytes at concentrations where they exert maximum protection against hypotonic haemolysis. The results show that vesiculation is a common phenomenon induced by amphiphiles in erythrocytes. Sphero-echinocytogenic amphiphiles induced exovesiculation, whereas stomatocytogenic amphiphiles induced endovesiculation. The antihaemolytic potency of the amphiphiles was not related to their ability to induce exo- or endovesiculation, or to the type or extent of shape changes induced, and it could not be ascribed to any molecular feature of the amphiphiles or to their charge. It is proposed that amphiphiles, when intercalated into the lipid bilayer of the membrane, rapidly induce rearrangements within the bilayer and that these rearrangements are associated with an increase in the permeability of the membrane; it is suggested that a rapid efflux of ions decreases the difference in osmotic pressure between cell interior and hypotonic buffer, thereby protecting cells from being lysed.

Effects of Calcium Antagonists on the Erythrocyte Membrane

The effects of dihydropyridine compounds nimodipine, nicardipine and NB818 (isopropyl methyl-6-carbamoyloxymethyl-4-(2,3-dichlorophenyl)-1,4-dihydro-2-methyl- 3,5- pyridine-dicarboxylate) on erythrocyte membranes have been studied. These compounds showed protective effects against hypotonic haemolysis, but not against heat-induced haemolysis. An increase in deformability of erythrocytes by these calcium antagonists was observed using a capillary tube centrifugal method. The erythrocytes showed slight stomatocytosis after 30 min of incubation with calcium antagonists, but did not show significant changes in mean corpuscular volume and ATP levels.

ＫＢ‐２７９６のＩｎ ｖｉｔｒｏおよびＩｎ ｖｉｖｏにおける抗血小板作用

The anti-platelet activity of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4- (2,3,4-trimethoxybenzyl) piperazine dihydrochloride, was studied in guinea pigs and mice. When guinea pig platelet-rich plasma (PRP) was employed, platelet function was inhibited at high doses of KB-2796. The IC50 value for [3H]5-HT release was 940 microM, and the IC50 values for collagen- and ADP-induced platelet aggregation were 210 and 390 microM, respectively. Oral administration of KB-2796 at 10-100 mg/kg dose-dependently inhibited the transient thrombocytopenia induced by collagen, but not that caused by ADP. KB-2796 protected mice from death after intravenous injection of collagen plus epinephrine, with an ED50 value of 9.5 mg/kg, p.o. Oral administration of KB-2796 at 10-100 mg/kg dose-dependently reduced guinea pig platelet retention in glass bead columns and reduced the leakage of ADP and ATP from erythrocytes passing through similar columns. KB-2796, at a concentration of 1-10 microM, produced a stabilizing effect on guinea pig erythrocytes against hypotonic hemolysis. These results suggest that KB-2796 is an inhibitor of platelet function and that its inhibition is related mainly to the inhibition of leakage of ADP and ATP from erythrocytes.

Effects of l-[(2-Thiazolin-2-yl)amino]acetyl-4-(l,3-dithiol-2-ylidene)-2,3,4,5-tetrahydro-1H-1-benzazepin-3,5-dione Hydrochloride (KF-14363) on Active Oxygen Production

The effects of KF-14363 on active oxygen production and membrane stabilization were studied. KF-14363 did not affect hypotonic hemolysis (10% and 70%) and did not inhibit lipid peroxide production induced by t-butyl hydroperoxide at concentrations of less than 100 microM. KF-14363 significantly inhibited active oxygen production in peritoneal exudate cells (PEEC) stimulated with arachidonic acid, A23187 and carbon tetrachloride (CCl4) at concentrations over 10 microM, 100 microM and 1 microM, respectively. It tended to inhibit formyl-methionyl-leucyl-phenylalanine-stimulated production of active oxygen in PEEC at concentrations over 10 microM, but there was no significant difference owing to large dispersion. Superoxide dismutase (SOD, 10(4) U/ml) significantly inhibited CCl4-stimulated production of active oxygen in PEEC. KF-14363 inhibited the radical production from CCl4 in the presence of a 9000 x g supernatant fraction of the rat liver which was administered with enzyme induction compounds (S9 mix). SOD (10(4) U/ml) was not effective in this system. In conclusion, KF-14363 inhibited active oxygen production in PEEC induced by various stimulants and also the radical formation from CCl4 in the presence of S9 mix solution.

Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase

Human erythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded erythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the beta anomer of glucose was more rapidly utilized than the alpha anomer in intact and unloaded erythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are alpha- and beta-preferential, respectively, in glucose phosphorylation.