The effect of chlorpromazine on the temperature and osmotic sensitivity of erythrocytes]

The effect of chlorpromazine on the development of cold shock in erythrocytes exposed to sodium chloride was shown to depend on the tonicity of the medium in which the cells were cooled from 37 degrees C down to 0 degrees C as well as on the amphipate concentration. After cooling of erythrocytes in a NaCl (0.75-1.5 M)-containing medium with chlorpromazine (7 x 10(-5) M, 2.1 x 10(-4) M and 3.5 x 10(-4) M) the hypertonic cold shock was inhibited, the protective effect of the amphipate being less pronounced at its increasing concentrations. After cooling of cells under conditions of moderate hypertonicity (0.3-0.6 M NaCl) no modifying effect of chlorpromazine on the sensitivity of erythrocytes to the temperature decrease from 37 degrees C down to 0 degrees C was manifested. However, under iso- and hypertonic conditions chlorpromazine used at 2.1 x 10(-4) M and 3.5 x 10(-4) M stimulated the cold shock development in erythrocytes. A sharp increase in the medium tonicity (up to 1.8-3.0 M and higher) the cells underwent isothermal hemolysis which was more expressed at 0 degrees C than at 37 degrees C. These data suggest that chlorpromazine significantly activates the hemolytic process at low temperatures.     

Hypertonic cryohemolysis of human red blood cells

Summary Hypertonic cryohemolysis of human erythrocytes is caused by incubation of the cells in hypertonic medium at a temperature of 20–50°C (stage 1), with subsequent cooling to 0°C (stage 2). In 0.86m sucrose hemolysis increases, with increasing stage 1 temperature, whereas in 1m NaCl cryohemolysis has a temperature optimum at a stage 1 temperature of about 30°C.Cryohemolysis is inhibited by preceding ATP depletion of the cells and bypreincubation of the cells in hypertonic medium at 0°C. In general, anesthetics inhibit cryohemolysis strongly. Only in 1m NaCl at stage 1 temperatures in the range of 40–50°C is cryohemolysis stimulated by these drugs, if present during the entire incubation period. This effect is abolished, however, when the anesthetic is added after piror incubation of the cells at 40–50°C in 1m NaCl.Ghost-bound ANS fluorescence indicates complicated conformation changes in the membrane structure during the various experimental stages leading to cryohemolysis.Some of the experimental results can be considered as examples of molecular hysteresis, thus indicating several different metastable structures of the membrane, under various experimental conditions.The described results support the working hypothesis of Green and Jung that the experimental procedure results in membrane protein damage, preventing normal adaptation of the membrane during cooling.     

Role of membrane thermotropic properties on hypotonic hemolysis and hypertonic cryohemolysis of human red blood cells

The hypothesis of a correlation between the effects of temperature on red blood cells hypotonic hemolysis and hypertonic cryohemolysis and two thermotropic structural transitions evidenced by EPR studies has been tested. Hypertonic cryohemolysis of red blood cells shows critical temperatures at 7 degrees C and 19 degrees C. In hypotonic solution, the osmotic resistance increases near 10 degrees C and levels off above 20 degrees C. EPR studies of red blood cell membrane of a 16-dinyloxyl stearic acid spin label show, in the 0-50 degrees C range, the presence of three thermotropic transitions at 8, 20, and 40 degrees C. Treatments of red blood cells with acidic or alkaline pH, glutaraldehyde, and chlorpromazine abolish hypertonic cryohemolysis and reduce the effect of temperature on hypotonic hemolysis. 16-Dinyloxyl stearic acid spectra of red blood cells treated with glutaraldehyde and chlorpromazine show the disappearance of the 8 degrees C transition. Both the 8 degrees C and the 20 degrees C transitions were abolished by acidic pH treatment. The correlation between the temperature dependence of red blood cell lysis and thermotropic breaks might be indicative of the presence of structural transitions producing areas of mismatching between differently ordered membrane components where the osmotic resistance is decreased.     

Hypertonic cryohemolysis and the cytoskeletal system

Hypertonic cryohemolysis is defined as the lysis of erythrocytes in a hypertonic environment when the temperature is lowered from above 15–18°C to below that temperature. This has been found to be a general phenomenon (that is, whether the solute is charged or not), to exhibit interesting temperature characteristics and to be preventable by agents such as valimomycin which tend to dissipate the concentration gradient across the cell membrane. As yet, no clear information is available to translate this phenomenon to the molecular level and to relate it to current structure/function concepts in the erythrocyte membrane. In this study, data are presented which would indicate on the basis of two entirely separate methodologies that the spectrin-actin cytoskeletal framework is involved in this phenomenon. The first of these methodologies is based on radiation-induced ablation of cryohemolysis and indicates that an intact macromolecular complex of an order of 16 000 000 daltons is required for cryohemolysis with hypertonic NaCl. The second methodology is based on selective cross-linking of spectrin and actin in the agent diamide, which resulted in concentration-dependent suppression of cryohemolysis. Polyacrylamide gel electrophoresis of the erythrocyte from diamide-treated cells showed intense protein aggregation with loss of spectrin-actin and bands 4.1, 4.2. We conclude that the spectrin-actin cytoskeletal system possibly including its interaction with phospholipids is the key to the phenomenon of hypertonic cryohemolysis.     

Hypertonic cryohemolysis: Ionophore and pH effects

Human erythrocytes suspended at 37 degrees C in hypertonic solution of either electrolytes or nonelectrolytes undergo hemolysis when the temperature is lowered toward 0 degrees C (Green, F.A., Jung, C.Y. 1977 J. Membrane Biol. 33:249). In the present studies this hypertonic cryohemolysis was profoundly affected by the pH of incubation, and was completely abolished at ph 5. In hypertonic NaCl, there was an apparent pH optimum at 6--6.5. In hypertonic sucrose, on the other hand, hemolysis increased progressively with increasing pH between 6 and 9. Amphotericin B inhibited hypertonic cryohemolysis in NaCl or KCl solution. No inhibiting effect of amphotericin B was observed when hypertonicity was due to sodium sulfate or sucrose. Valinomycin also inhibited hypertonic cryohemolysis in KCl, but did not affect the process in NaCl or sucrose solution. SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) and phloretin interfered with this valinomycin effect, whereas phlorizin did not. These results indicate that dissipation of an osmotic gradient across membranes may be responsible for the inhibition of the hemolysis by these inophores. Iso-osmotic cell shrinkage induced by valinomycin in 150 mM NaCl solution did not result in cryohemolysis.     

Induction of ATP depletion, intramembrane particle aggregation and exposure of membrane phospholipids in chicken erythrocytes by local anesthetics and tranquilizers

Incubation of chicken erythrocytes with 1 mM tetracaine, 10 mM lidocaine and 0.24–0.48 mM chlorpromazine significantly reduced the ATP content of the cells, while procaine even at concentrations as high as 10 mM had only a slight effect. When chlorpromazine was used, it was found that the final level of the ATP was dependent on the drug concentration, which at 0.48 mM depletes the cells to about 10% of the initial ATP content. The ATP depletion of chicken erythrocytes was accompanied by dephosphorylation of certain membrane proteins which were identified by acrylamide gel electrophoresis as an 180 000 dalton protein band and peptides with molecular weight of 60 000–100 000. Treatment of chicken and rat erythrocytes with 0.5 mM tetracaine and 1 mM lidocaine or with 0.48 mM chlorpromazine induced significant aggregation of intramembrane particles as revealed by the freeze-etching technique. Procaine (10 mM) had no effect. Incubation of chicken erythrocytes with the above-mentioned drugs induced also exposure of the masked membrane phospholipids to the action of phospholipase-C (Bacillus cereus) and to phospholipase A₂ (bee venom). Negligible amounts of phospholipids were hydrolyzed in the untreated cells, while about 40% of the membrane phosphatidylethanolamine and 50% of the phosphatidylcholine were hydrolyzed by phospholipase A₂ in chicken erythrocytes treated with 0.48 mM chlorpromazine.Treatment of chicken and rat erythrocytes with 0.48 mM chlorpromazine resulted also in an increase in the amount of the phospholipid fraction which could be extracted by dry ether. About 41% and 60% of phospholipids were respectively, as compared to 25% and 35% of phospholipids extracted from the same untreated cells.     

The influence of cyclic heating and cooling on <Emphasis Type="Italic">Escherichia coli</Emphasis> survival

Repeated heating and cooling in lethal (2–52°C) and nonlethal (2–37°C) temperature ranges resulted in cell death of Escherichia coli B/r and E. coli B_S?1 suspended in 0.01 M phosphate buffer, pH 7.0 at varying osmotic pressure, but not in cow’s milk. The lethal effect increased with the rate of heating and cooling and with increasing suspension media tonicity; it may be caused by the temperature destabilization of cellular osmotic homeostasis.     

The influence of chlorpromazine on the potential-induced shape change of human erythrocyte

The effect of chlorpromazine (CPZ) on the shape of human erythrocytes with different values of transmembrane potential (TMP) was investigated. The shape of red blood cells with negative values of the TMP remained unchanged after the formation of stomatocytes by chlorpromazine, while cells with positive TMP showed a characteristic time course of shape change during the incubation with CPZ. Experiments with vanadate show that this might be due to a difference in the activity of the phospholipid-translocase at different values of TMP.     

The role of choline phospholipids in hypertonic cryohemolysis

Hemolysis resulting from a warm-to-cold temperature shift in a Hypertonie environment (hypertonic cryohemolysis) is studied with the use of phospholipases as membrane probes of the phospholipids of the outer leaflet of the bilayer. Bee venom phospholipase A₂ which attacks only phosphatidylcholine (PC) in the intact erythrocyte results in inhibition of cryohemolysis produced by both hypertonic sodium chloride and sucrose. In both cases, about 25% of the loss of PC occurs before any such inhibition, suggesting the possibility of functionally separate domains of PC in the outer leaflet of the bilayer. Sphingomyelinase also attacks only sphingomyelin in the intact erythrocyte and results in inhibition of cryohemolysis due to hypertonic sodium chloride but not of that due to sucrose. In each case, inhibition of the enzymatic hydrolysis by EDTA abolished the effect on cryohemolysis. It is postulated that cryohemolysis is inhibited when phosphylipid interaction with membrane (cytoskeletal) proteins are abolished, but present knowledge of membrane structure does not permit a detailed mechanism to be proposed.     

Binding of lectins to "young" and "old" human erythrocytes

"Old" human erythrocytes showed a 21.2% decrease in cell surface area and a 2% decrease in the number of WGA receptor sites, but a 27% increase in the distribution density of the WGA (lectin) receptor site, when compared with "young" human erythrocytes. For a list of lectin abbreviations, see Materials and methods). Both "young" and "old" erythrocytes exhibited very weak binding activity for 125I-labeled PNA, but there was no difference in binding activity for PNA between "young" erythrocytes and "old" ones. Compared with "young" erythrocytes, decreases in the number and distribution density of receptor sites for five lectins including LPA, Con A, RCA-II, SBA and BPA on the cell surface were observed in aged erythrocytes. "Old" erythrocytes also showed a decrease in the number of PHA-E receptor sites, while the distribution density of the same receptor site remained unchanged. In view of these and other observations, it is thought that human erythrocyte aging is accompanied by elimination of some glycoconjugates which have affinity for six lectins, LPA, Con A, RCA-II, PHA-E, SBA and BPA, whereas no WGA receptor-containing glycoconjugates are released from erythrocyte membranes. Elimination of the glycoconjugates results in shrinkage of erythrocytes to reduce their cell surface areas.     

Shape determinants of McLeod acanthocytes

Summary We have sought to elucidate the spiculated shape of McLeod erythrocytes. Red cells from a normal donor and from a McLeod patient were incubated in phosphate-buffered saline containing 0, 0.05, or 0.1mm chlorpromazine at 0°C for 5 min. then glutaraldehyde-fixed, and examined by scanning electron microscopy. The normal red cells were biconcave disks in which chlorpromazine induced inward (negative) curvature: deep cupping (stomatocytosis) and multiple invaginations. The McLeod cells were mostly spiculated. Chlorpromazine at lower concentration converted them into biconcave disks and, at higher concentration, into stomatocytes. These results support the hypothesis that the spiculation of McLeod cells is the result of an imbalance of surface area between the two lipid leaflets of the membrane; that is, a bilayer couple effect.We determined the numerical density of intramembrane particles (IMP) in replicas of both fracture faces of red cells subjected to freeze fracture and rotary shadowing. These values were as follows (expressed per m² of membrane ±sd): the normal protoplasmic fracture face had 2200±306 and the McLeod had 2300±250. The normal exoplasmic fracture face had 388±75 and the McLeod had 330±59. We conclude that there is no evidence for derangement of band 3, the principal protein in theIMP, in McLeod erythrocytes.     

Single cell volume measurement by quantitative phase microscopy (QPM): a case study of erythrocyte morphology.

The measurement of the volume of intact, viable cells presents challenging problems in many areas of experimental and diagnostic science involved in the evaluation of cellular morphology, growth and function. This investigation details the implementation of a recently developed quantitative phase microscopy (QPM) method to measure the volume of erythrocytes under a range of osmotic conditions. QPM is a computational approach which utilizes simple bright field optics to generate cell phase maps which, together with knowledge of the cellular refractive index, may be used to measure cellular volume. Rat erythrocytes incubated in imidazole-buffered solutions (22 degrees C) of graded tonicity were analysed using QPM (n=10 cells/group, x63, 0.8 NA objective). Erythrocyte refractive index (1.367) was measured using a combination of phase and morphological data obtained from cells adopting spherical geometry under hypotonic conditions. Phase-computed volume increased with decreasing solution osmolality: 42.8 +/- 2.4, 48.7 +/- 2.3, 62.6 +/- 2.3, 90.8 +/- 7.7 microm3 in solutions of 540, 400, 240, and 170 mosmol/kg respectively. These volume changes were associated with crenated, bi-concave and spherical morphological states associated with increasing tonicity. This investigation demonstrates that QPM is a valid, simple and non-destructive approach for measuring cellular phase properties and volume. QPM cell volume analysis represents a significant advance in viable cell experimental capability and provides for acquisition of 'real-time' data - an option not previously available using other approaches.     

Action of glycosyl transferases upon "Bombay" (Oh) erythrocytes. Conversion to cells showing blood-group H and A specificities.

Individuals of the rare "Bombay" (Oh) blood-group phenotype lacking, due to a genetic defect, the alpha(1-2)fucosyl transferase, which is responsible for converting blood-group H precursor substances to H-specific structures. Treatment with GDP-fucose and alpha(1-2)fucosyl transferase prepared from gastric mucosa of O individuals to transform native or ficin-treated "Bombay" erythrocytes into cells phenotypically resembling O cells. The transformation was achieved, however, after prior incubation of the "Bombay" erythrocytes with neuraminidase, indicating that blood-group H precursor molecules on the surface of these cells are masked by sialyl residues. Blood-group A specificity was conferred upon neuraminidase-treated "Bombay" cells by enzymatic transfer of alpha-N-acetylgalactosamine residues, in addition to alpha-fucose residues.     

Requirements of drug-induced endocytosis by intact human erythrocytes.

Study of drug-induced endocytosis in intact human erythrocytes continues to provide an opportunity for correlating membrane functions such as invagination and fusion with erythrocytic energetics and other determinants of plasma membrane function like Ca++. The studies reported indicate that high concentrations of vinblastine and chlorpromazine can produce endocytic vacuoles, albeit in reduced amounts, even in severely ATP depleted erythrocytes. In contrast, primaquine-induced endocytosis seems definitely dependent upon persistence of erythrocytic ATP stores. The ionophore mediated entry of Ca++ into erythrocytes potentiates primaquine endocytosis, inhibits vinblastine endocytosis, and has no regular effect on chlorpromazine endocytosis. Sodium lactate enhances primaquine endocytosis, probably by causing an increase in the entry of primaquine into erythrocytes. Cytochalasin B neither enhances nor inhibits erythrocytic endocytosis, thereby suggesting that microfibrils or analogues of microfibrils in erythrocytes are not involved in endocytosis. Cyclic nucleotide inhibition of endocytosis is confined to a very high concentration range of nucleotides in the medium. Primaquine and chlorpromazine endocytosis are inhibited by cyclic nucleotides as is vinblastine endocytosis.     

Modifying effect of concanavalin A and diamide on erythrocyte sensitivity to cold shock]

The temperature (0 degrees C and 37 degrees C) and the medium tonicity (0.15-1.20 M NaCl) were shown to affect erythrocyte agglutination by concanavalin A. Treatment of cells with lectin caused no significant decrease in the erythrocyte hemolysis upon cooling. Diamide, unlike concanavalin A used at concentrations above 2.0 M decreases the cell sensitivity to the cold shock. The changes in the erythrocyte susceptibility to cooling within the temperature range of 37-0 degrees C correlate with changes in the electrophoretic spectrum of membrane proteins. The progressive decrease in the spectrin bands intensity with a simultaneous formation of high molecular weight protein aggregates not included in the gel composition was observed after diamide treatment. The diamide effect depends on the medium tonicity, at which the treatment was performed, being especially well pronounced in hypertonic media with 0.8-1.2 M NaCl concentrations, the maximal spectrin aggregation being observed under these conditions. It is suggested that the main factor of the mechanism underlying the erythrocyte hypertonic cold shock is the increase in the association of peripheral cytoskeleton proteins with plasma membrane in osmotically dehydrated cells which limits the ability of lipids to adapt during cooling and results in the stabilization of defects in the membrane structure at low temperatures. Diamide eliminates these unfavourable changes eventually resulting in the dissociation of peripheral proteins from the cytoplasmic surface of the membrane on the protein aggregation.     

"Ultramicroinjection" of macromolecules or small particles into animal cells. A new technique based on virus-induced cell fusion.

A new method is described for the introduction of macromolecules and small particles into animal cells. The first step in this procedure is the trapping of particles in ghosts of human erythrocytes. This is achieved by the gradual hemolysis of erythrocytes in the presence of the particles to be trapped. The second step is the Sendai virus-induced fusion of the ghosts containing the particles with cells. By this method, ferritin and latex spheres (diameter 0.1 mum) have been "injected" into cells.     

On the mechanism of shrinkage-induced potassium influx in rat and human erythrocytes.

The rates of 86Rb influx into human and rat erythrocytes were studied in media of various tonicity. At sucrose concentrations below 0.3 mol/l, the ouabain-insensitive, furosemide-inhibited component of influx increased in rat but not in human erythrocytes; this may be explained by a rise in the rate of Na+, K+, Cl-- and/or K+, Cl-cotransport. An increase in osmolarity resulted in a reduction of this as well as of the ouabain and furosemide-insensitive component in rat erythrocytes. At the same conditions a drastic inhibition of Na+, K(+)-pump occurred both in rat and human erythrocytes. We failed to observe a lag-phase in the activation of the cotransport in rat erythrocytes; i. e. the process of activation parallels the shrinkage of cells. In rat erythrocyte ghosts, the shrinkage-induced stimulation of the cotransport was lost, and the direction of their osmotic reaction (inhibition of transport pathways) was similar to that in human erythrocyte ghosts. It is suggested that the mechanism of volume regulation of ion transport in intact cells involves a step of physical amplification via a change in interactions between the protein carcass and the lipid bilayer.     

The interaction of 1-fluoro-2,4-dinitrobenzene with amino-phospholipids in membranes of intact erythrocytes,modified erythrocytes,and erythrocytes ghosts

Summary 1-Fluoro-2,4-dinitrobenzene (FDNB) has been used to study the availability of amino-containing phospholipids in erythrocyte membranes and ghosts in an aqueous, isotonic medium. It was found that the addition of bovine serum albumin (BSA) to the medium protects the cells from cation leak and protects some of the amino-phospholipids from reacting with the probe. In isotonic medium without BSA, 46% of the phosphatidylethanolamine and 12% of the phosphatidylserine of erythrocytes and 73% and 21% of these respective lipids of ghosts react with the probe. In the presence of 70 m BSA, 31% of phosphatidylethanolamine and 6.5% of phosphatidylserine of erythrocytes and 59% and 16% of these respective lipids of ghosts react with the probe. The labeling of these lipids does not change under conditions of varying tonicity, or after treatment of erythrocytes with pronase or lysolecithin. The data suggest that 46% of phosphatidylethanolamine and 12% of phosphatidylserine of the erythrocyte membrane are free in a lipid bilayer; 27% and 9% of these respective lipids are loosely bound to proteins which are lost during the preparation of ghosts and 27% of the phosphatidylethanolamine and 79% of the phosphatidylserine are tightly bound to core proteins which remain part of the erythrocyte membrane even after hemolysis.     

Proteolysis of ankyrin and of band 3 protein in chemically induced cell fusion. Ca2+ is not mandatory for fusion.

Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide a general mechanism for the fusion of lipid bilayers in biomembrane fusion reactions.     

Tonic shock induces detachment of Giardia lamblia

Background

The parasite Giardia lamblia must remain attached to the host small intestine in order to proliferate and subsequently cause disease. However, little is known about the factors that may cause detachment in vivo, such as changes in the aqueous environment. Osmolality within the proximal small intestine can vary by nearly an order of magnitude between host fed and fasted states, while pH can vary by several orders of magnitude. Giardia cells are known to regulate their volume when exposed to changes in osmolality, but the short-timescale effects of osmolality and pH on parasite attachment are not known.

Methodology and Principal Findings

We used a closed flow chamber assay to test the effects of rapid changes in media osmolality, tonicity, and pH on Giardia attachment to both glass and C2_Bbe-1 intestinal cell monolayer surfaces. We found that Giardia detach from both surfaces in a tonicity-dependent manner, where tonicity is the effective osmolality experienced by the cell. Detachment occurs with a characteristic time constant of 25 seconds (SD = 10 sec, n = 17) in both hypo- and hypertonic media but is otherwise insensitive to physiologically relevant changes in media composition and pH. Interestingly, cells that remain attached are able to adapt to moderate changes in tonicity. By exposing cells to a timed pattern of tonicity variations and adjustment periods, we found that it is possible to maximize the tonicity change experienced by the cells, overcoming the adaptive response and resulting in extensive detachment.

Conclusions and Significance

These results, conducted with human-infecting Giardia on human intestinal epithelial monolayers, highlight the ability of Giardia to adapt to the changing intestinal environment and suggest new possibilities for treatment of giardiasis by manipulation of tonicity in the intestinal lumen.