Cytosolic protein concentration is the primary volume signal in dog red cells

It is not known whether the activation of Na/H exchange by shrinkage in dog red cells is due to the packing of cell contents or a change in cell configuration. To make this distinction we prepared resealed ghosts that resembled intact cells in hemoglobin concentration and surface area, but had one-third their volume. A shrinkage-induced, amiloride-sensitive Na flux in the ghosts was activated at a much smaller volume in the ghosts than in the intact cells, but at the same concentration (by weight) of dry solids in both preparations. Na/H exchange in ghosts containing a mixture of 40% albumin and 60% hemoglobin (weight/weight) was activated by osmotic shrinkage at a dry solid concentration similar to that of intact cells or of ghosts containing only hemoglobin. We conclude that the process of Na/H exchange activation by cell shrinkage originates with an increase in the concentration of intracellular protein and not with a change in membrane configuration or tension. The macromolecular crowding that accompanies the reduction in cell volume probably alters the activities of key enzymes that in turn modulate the Na/H exchanger.     

The Active Transport of Sodium by Ghosts of Human Red Blood Cells

The outflux of Na²⁴ from prelabeled ghosts was measured under various conditions. Prelabeling was accomplished by hypotonic hemolysis of intact human cells in the presence of tracer Na²⁴. The resultant ghosts when subsequently washed were found to retain 10 to 20 per cent of the initial Na²⁴. Separate experiments indicated that this trapped amount resides in only a portion of ghosts comprising the total population. The characteristics of the outflux of this residual Na²⁴ indicated that the ghost system closely resembles intact red cells. The outflux of Na from ghosts could be divided into three components: active and passive transport and exchange diffusion. The active transport system, necessarily driven by metabolism, required the presence of K in the extracellular phase and was blocked by strophanthidin. The concentration dependence of the Na pump flux on the external K and internal Na appeared the same in ghosts as in intact cells. Certain other features of this ghost system are also discussed.     

Macromolecular crowding and volume perception in dog red cells

To differentiate whether the primary volume signal in dog red cells arises from a change in cell configuration or the concentration and dilution of cell contents, we prepared resealed ghosts that had the same surface area and hemoglobin concentration as intact cells but less than 1/3 their volume. Shrinkage of both intact cells and resealed ghosts triggered Na/H exchange. Activation of this transporter in the two preparations correlated closely with cytosolic protein concentration but not at all with volume. The Na/H exchanger was more sensitive to shrinkage in albumin-loaded resealed ghosts than in intact cells or ghosts containing only hemoglobin. Similar results were obtained for the swelling-induced [K-Cl] cotransporter. We believe perception of cell volume originates with changes in cytoplasmic protein concentration. We think the kinases and phosphatases that control the activation of membrane transporters in response to cell swelling or shrinkage are regulated by the mechanism of macromolecular crowding.     

Enzyme loading of electrically homogeneous human red blood cell ghosts prepared by dielelctric breakdown.

Human red blood cell ghosts were prepared by electrical haemolysis at 0 degrees C in isotonic solutions using a discharge chamber which was part of a high voltage circuit. The size distribution of the ghosts was normally distributed, the modal (=mean) volume was approx. 115 mum3, performing the electrical haemolysis in the following solution: 105 mM KCI, 20 mM NaCL, 4mM MgCl2, 7.6 mM Na2HPO4, 2.94 mM NaH2PO4, 10 mM glucose, pH 7.2. Resealing was carried out at o degrees C for 10 min (after the haemolytic step) and then for further 20 min at 37 degrees C. The mean volume of the ghost preparation could be changed by variation of the phosphate concentration in the above solution replacing a part of NaCl by phosphate (5 mM phosphate: 94 mum3, 15 mM phosphate: 135 mum3). The breakdown voltage of the ghost cell membranes measured with a hydrodynamic focusing Coulter Counter depends on the mean volume (94 mum3 = 1.04 V, 134 mum3 = 1.36 V). On the other hand, the breakdown voltage is constant throughout each size distribution pointing to an "electrically homogeneous" ghost preparation. The sensitiviity of the Coulter Counter to detect electrical inhomogeneities in the membranes of a ghost population is demonstrated by dielectric breakdown measurements of an apparently normally distributed ghost preparation containing two different "electrically homogeneous" ghost population i.e. with two different breakdown voltages. The ghost cells obtained by electrical haemolysis in the above solution containing 10mM phosphate were fairly impermeable to sucrose and behave like an ideal osometer. It is further demonstrated that ghost cells can be loaded with enzymes (e.g. urease) and drugs using this technique and that these loaded ghost cells can be used as bioactive capsules for clinical application.     

Effects of preparative procedures on the volume and content of resealed red cell ghosts

The effects of variations in preparative procedures on the volume and content of resealed red cell ghosts have been investigated. Following hypotonic lysis at 0 degrees C, and after a variable delay time (td), concentrated buffer was added to restore isotonicity; resealing was then induced by incubation at 37 degrees C for one hour. Using this procedure, both the resealed ghost volume and the residual hemoglobin (Hb) content decreased for increasing td. If ghosts were maintained at 0 degree C (i.e., no 37 degrees C incubation), they remained nearly spherical until isotonicity was restored. Their volume then fell abruptly, but subsequently increased toward an intermediate level. The fall in volume was greater and the final level achieved was smaller for longer delay times. At 0 degree C, return to isotonicity also halted the otherwise gradual loss of residual Hb from unsealed ghosts. In addition, ghosts with internal osmolality of 40 to 300 mosmol/kg were prepared by adding different amounts of concentrated buffer before resealing for one hour at 37 degrees C. Under these conditions, the final ghost volume was inversely related to the resealing osmolality (i.e., lower osmolality yielded a larger volume). Ghost volume also increased, along with Hb content, if the quantity or concentration of the red cell suspension added to the lysing medium was increased. We conclude that resealed ghost volume is influenced by the ratio of lysate to resealing medium osmolality and by the colloid osmotic pressure of the residual ghost Hb. These data indicate methods by which ghosts with desired characteristics can be prepared, and have potential application for studies of ghost mechanical and biophysical behavior.     

ATP-dependent sugar transport complexity in human erythrocytes

Human erythrocyte glucose sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions ([sugar](intracellular) = [sugar](extracellular), where brackets indicate concentration). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Biphasic exchange at 20 mM 3MG eliminates the possibility that the rapid exchange phase represents ATP-dependent 3MG binding to the glucose transport protein (GLUT1; cellular [GLUT1] of 相似文献   

Asymmetry of the Na+/H+ antiport of dog red cell ghosts. Sidedness of inhibition by amiloride

Amiloride is a potent inhibitor of the Na+/H+ antiport. Inhibition is generally competitive with extracellular Na+ and therefore believed to result from binding to the outward-facing transport site. It is not known whether amiloride can interact with the internal aspect of the antiport. This question was addressed by trapping the drug inside resealed dog red cell ghosts. The antiport, which is quiescent in resting ghosts, was activated by acid-loading the cytoplasm. This was accomplished by exchanging extracellular Cl- for internal HCO-3 through capnophorin, the endogenous anion exchanger. The activity of the Na+/H+ antiport was detected as an increase in cell volume, resulting from the net osmotic gain associated with coupled Na+/H+ and Cl-/HCO-3 exchange, or as the uptake of 22Na+. Intracellular amiloride, at concentrations in excess of 100 microM, failed to inhibit Na+/H+ exchange. This is approximately 10 times higher than the concentration required for half-maximal inhibition when amiloride is added externally. Independent experiments demonstrated that failure of internal amiloride to inhibit exchange was not due to leakage of the inhibitor, to differences in pH, or to binding or inactivation of amiloride by the soluble contents. It was concluded that the antiport is functionally asymmetric with respect to amiloride. This implies that the transport site undergoes a conformational change upon translocation across the membrane or, alternatively, that a second site required for amiloride binding is only accessible from the outside.     

Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells.

Chloride-dependent K transport ([K-Cl] cotransport) in dog red cells is activated by cell swelling. Whether the volume signal is generated by a change in cell configuration or by the dilution of some cytosolic constituent is not known. To differentiate between these two alternatives we prepared resealed ghosts that, compared with intact red cells, had the same surface area and similar hemoglobin concentration, but a greatly diminished volume. Swelling-induced [K-Cl] cotransport was activated in the ghosts at a volume (20 fl) well below the activation volume for intact cells (70 fl), but at a similar hemoglobin concentration (30-35 g dry solids per 100 g wet weight). Ghosts made to contain 40% albumin and 60% hemoglobin showed activation of [K-Cl] cotransport at a concentration of cell solids similar to intact cells or ghosts containing only hemoglobin. [K-Cl] cotransport in the resealed ghosts became quiescent at a dry solid concentration close to that at which shrinkage-induced Na/H exchange became activated. These results support the notion that the primary volume sensor in dog red cells is cytosolic protein concentration. We speculate that macromolecular crowding is the mechanism by which cells initiate responses to volume perturbation.     

A simulation study of the anomalous osmotic behavior of red cells

The factors responsible for movements of water across cell membranes were described mathematically and incorporated into a model which simulates water balance in the cell. Included in the model are a variable charge and osmotic coefficient of hemoglobin, a Na/K pump whose rate varies with ionic concentrations, and the standard electroneutrality and osmotic equilibrium assumptions. The model was used to investigate the phenomena whereby human red cells placed in media of varying tonicities exhibit steady state volume changes less than those predicted by van't Hoff's Law. The model results showed that this anomalous osmotic behavior was primarily due to changes in the osmotic coefficient of hemoglobin as its concentration in the cell varied. A second factor accounting for a part of this behavior was the alteration in the rate of the Na/K pump due to intracellular ionic concentration changes as cell volume varied. The effect of variable electrical charge on the hemoglobin molecule was found to be in the wrong direction to account for the observed osmotic behavior. Also, this effect was seen to produce relatively large changes in cell membrane potential, a result inconsistent with experimental data. It was concluded from the model results that the anomalous osmotic behavior of human red cells is primarily due to the variation in the osmotic coefficient of hemoglobin as the cell volume changes, and that the variable charge effect on the hemoglobin molecule, if it exists, does not play a role in this response.     

Role of the bilayer in the shape of the isolated erythrocyte membrane

Summary The determinants of cell shape were explored in a study of the crenation (spiculation) of the isolated erythrocyte membrane. Standard ghosts prepared in 5mm NaP_i (pH 8) were plump, dimpled disks even when prepared from echinocytic (spiculated) red cells. These ghosts became crenated in the presence of isotonic saline, millimolar levels of divalent cations, 1mm 2,4-dinitrophenol or 0.1mm lysolecithin. Crenation was suppressed in ghosts generated under conditions of minimal osmotic stress, in ghosts from red cells partially depleted of cholesterol, and, paradoxically, in ghosts from red cells crenated by lysolecithin. The susceptibility of ghosts to crenation was lost with time; this process was potentiated by elevated temperature, low ionic strength, and traces of detergents or chlorpromazine.In that ghost shape was influenced by a variety of amphipaths, our results favor the premise that the bilayer and not the subjacent protein reticulum drives ghost crenation. The data also suggest that vigorous osmotic hemolysis induces a redistribution of lipids between the two leaflets of the bilayer which affects membrane contour through a bilayer couple mechanism. Subsequent relaxation of that metastable distribution could account for the observed loss of crenatability.     

The Response of Duck Erythrocytes to Nonhemolytic Hypotonic Media : Evidence for a volume-controlling mechanism

Duck erythrocytes were incubated in hypotonic media at tonicities which do not produce hemolysis. The cells'' response can be divided into two phases: an initial rapid phase of osmotic swelling and a second more prolonged phase (volume regulatory phase) in which the cells shrink until they approach their initial isotonic volume. Shrinkage associated with the volume regulatory phase is the consequence of a nearly isosmotic loss of KCl and water from the cell. The potassium loss results from a transient increase in K efflux. There is also a small reduction in Na permeability. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain although this concentration of ouabain does change the cellular cation content. The over-all response of duck erythrocytes is considered as an example of "isosmotic intracellular regulation," a term used to describe a form of volume regulation common to euryhaline invertebrates which is achieved by adjusting the number of effective intracellular osmotic particles. The volume regulatory phase is discussed as the product of a membrane mechanism which is sensitive to some parameter associated with cell volume and is capable of regulating the loss of potassium from the cell. This mechanism is able to regulate cell size when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.     

The exchange of erythrocyte membrane phospholipids with rat liver extracts in vitro

Intact rat or human erythrocytes and their isolated (ghost) membranes were incubated with the high speed supernatant fraction of homogenates derived from ³²P-labeled rat livers. Phospholipid molecules were transferred between the red cell membranes and the liver extracts, as reflected by the convergence of their specific radioactivities with time. Whereas ghosts usually approached isotopic equilibrium with the liver supernatant fraction during a few hours of incubation at 37° C, the exchange of phospholipids by intact cells was no more than one-half, even after 18 hr. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin were all exchanged in both intact cells and ghosts, albeit to different extents. (A control experiment, incubating ³²P-labeled rat erythrocytes or ghosts with unlabeled rat liver extracts, also demonstrated the exchange of all four major phospholipids.) These data may signify that the phospholipids on the cytoplasmic side of the membrane of intact erythrocytes do not exchange with the phospholipids in exogenous liver extracts. If so, all four major phospholipid classes would appear to be present to some extent at both membrane surfaces. The first inference is in agreement with several other studies on this membrane, while the second inference is not.     

The influenza virus-induced fusion of erythrocyte ghosts does not depend on osmotic forces

The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.     

Membrane and intracellular modes of sugar-dependent increments in red cell stability.

Sugar-dependent increments in red cell stability under osmotic stress can be ascribed to changes either in the membrane or in the intracellular matrix. These two possible modes of action have been tested and characterized. Rheological investigation of membrane-free haemoglobin solutions has shown that D-glucose, but not D-fructose, promotes the formation of a visco-plastic gel structure. Gel strength is a function of glucose concentration, haemoglobin concentration and temperature. The ability of various sugars to promote gel formation correlates with their solution properties. The existence of gel structure reduces K+ and haemoglobin leak from red cells whose membranes were partially destroyed by gamma-radiation. Reduced osmotic swelling in the presence of glucose is also due to gel formation since the glucose effect is lost in resealed red cell ghosts. D-Fructose does not protect red cells against radiation damage; its mode of action in increasing red cell stability under osmotic stress is a membrane effect. Cell sizing using the Coulter Counter has shown that fructose, but not glucose, can increase the maximal volume at lysis. At 50 mM, D-fructose expands the red cell ghost volume by 11.2%; this represents a 7.2% increase in membrane area. Ghost expansion by fructose is fructose concentration dependent (0-100 mM) and is insensitive to temperature variation (0-37 degrees C).     

Cell volume and osmotic properties of erythrocytes after complement lysis measured by flow cytometry

The changes of volume distribution curves of erythrocytes during and after lysis by complement or nystatin or in hypotonic buffers were measured by flow cytometry. Biconcave and spheroidal ghosts were observed after complement lysis and spheroidal ghosts were seen only after nystatin and hypotonic lysis. The spheroidal ghosts derived from red cells lysed by complement or nystatin were permeable to sucrose; those from hypotonic lysis were sucrose-impermeable. Spheroidal ghosts after complement lysis remained permeable for sucrose whereas spheroidal ghosts after nystatin lysis resealed after removal of the drug by washing. Biconcave ghosts produced by complement lysis were almost impermeable to sucrose initially and therefore responded to osmotic changes, but they became sucrose-permeable upon prolonged incubation at 37 degrees C. The rate of sucrose equilibration increased as the stability of the biconcave shape diminished with increasing numbers of C5b-9 complexes. At 850 C5b-9 complexes/ghost, the biconcave shape and impermeability for sucrose were completely lost. The results support the hypothesis that complement C5b-9 complexes, in addition to the interaction with the lipid bilayer, may interact with the cytoskeleton of the erythrocyte membrane.     

Steady-state volumes and metabolism-independent osmotic adaptation in mammalian erythrocytes

Erythrocytes of various mammalian species -- including human -- maintain osmotic balance with the blood plasma (osmotic activity 270-310 mosmol). However, their intracellular levels of osmotically active ions (potassium, sodium, chloride, and hydrogencarbonate), water content and osmotic resistance deviate significantly. In the present report we study the relationship among intracellular water, potassium and sodium levels of the erythrocytes of various mammalian species and in the developing calf. In addition, the osmotic resistance, K(+) (Rb(+)) uptake and the DPH fluorescence anisotropy of various erythrocytes and erythrocyte ghost membranes were correlated. The results show no statistically significant relationship between erythrocyte water content and [K(+)+Na(+)] levels or K(+)/Na(+) ratios. The reversal of erythrocyte K(+)/Na(+) ratios coincides with the decrease of steady-state ATP levels in the developing calf. The mobility of lipids within the hydrophobic inner layer of the plasma membrane relates closely to passive K(+) (Rb(+)) uptake, and plays a significant role in regulatory volume changes.     

Monitoring volume changes in red cell ghosts by means of a trapped fluorescent probe

A method is described for monitoring changes in the volume of red cell ghosts by means of a fluorescent probe trapped inside them.. The fluorophore, 8-hydroxy-1,3,6-pyrenetrisulfonate, is partially quenched by the residual hemoglobin in the ghosts. When the ghosts swell, the hemoglobin concentration is reduced, the quenching is somewhat relieved and the fluorescence output increases. Opposite changes occur when the ghosts shrink. Fluorescence intensity is linearly related to ghost volume for both swelling and shrinking, but there is a larger change in fluorescence for shrinking from the isotonic volume than for an equivalent swelling. This method has been used to follow the swelling phase of dye-loaded ghosts suspended in a solution of a penetrating nonelectrolyte in isotonic saline.     

Amino acid transport by resealed ghosts from pigeon erythrocytes.

Resealed ghosts from pigeon erythrocytes were shown to haemolyse during incubation in isotonic media with pH values greater than about 7 and high concentrations of Na+ inside the ghosts seemed to enhance this effect. At lower pH values the ghosts were stable but still highly permeable to Na+ and K+, and moderately permeable to sucrose. Under the latter conditions the ghosts transported amino acids in a way qualitatively but not quantitatively similar to intact erythrocytes. The Na+-dependent transport of serine and alanine by the ghosts consisted essentially of an exchange of extracellular for intracellular amino acids, with no significant net flux. In contrast, net fluxes of glycine in the direction of the Na+-concentration gradient across the ghost membrane were demonstrated. However, under one condition a small net influx of glycine occurred against the prevailing Na+-concentration gradient. Unlike Na+-dependent glycine uptake, the uptake of six other amino acids by intact pigeon erythrocytes was not influenced by the nature of the anion present. The significance of these findings in relation to previous work on the Na+-gradient hypothesis of membrane transport is discussed.     

Water-phase palmitate concentrations in equilibrium with albumin-bound palmitate in a biological system.

The palmitate (PA) binding and transport capacity of human and bovine red cell membranes enables us to establish, in a biological system, the existence of a well-defined monomer concentration in equilibrium with PA bound to bovine serum albumin (BSA, 30 microM) inside the resealed red cell ghosts. Supernatants of suspensions of the [3H]PA-labeled ghosts contain a tiny quantity of dissolved binding capacities besides the monomer PA. This is demonstrated by linear regression of supernatant tracer concentrations versus ghost concentrations in a dilution series. The extrapolated value, corresponding to zero ghost concentration, is the monomer PA concentration in equilibrium with PA bound to BSA within the ghosts in molar ratio (nu). Measurements have been carried out for nu between 0.1 and 1.5 and at 0 degrees C, 10 degrees C, 23 degrees C and 38 degrees C. The important nu-dependent binding of PA to the ghost membrane itself enables us to use preparations of BSA-free ghosts in the same way, whereas this is impossible in the case of arachidonic acid. Within the physiological range of nu the PA monomer concentrations are accounted for by an apparent dissociation equilibrium constant (Kd) 3.4 10(-8) M at 38 degrees C calculated on basis of three equivalent binding sites per mol BSA. Kd depends on temperature with a well-defined enthalpy of 38.4 kJ/mol.     

Palmitate binding to and efflux kinetics from human erythrocyte ghost

At 0 degrees C, pH 7.3, palmitate (PA) binds to human erythrocyte ghosts suspended in 0.2% bovine serum albumin (BSA) solution with molar ratios of PA to BSA, v, between 0.2 and 1.3. The binding depends on the water phase PA concentration, measured in equilibrium experiments, using BSA-filled ghosts as semipermeable bags. The saturable binding has a capacity of 19.4 +/- 7.5 nmol g-1 packed ghosts (7.2 x 10(9) cells) and Kd = 13.5 +/- 5 nM. PA exchange efflux kinetics to 0.2% BSA is recorded from ghosts without and with 0.2% BSA with a resolution time of about 1 s. Data are analyzed in terms of compartmental models. Using BSA-free ghosts the kinetics is essentially monoexponential. The rate constant is 0.0287 +/- 0.0022 s-1. Using ghosts with BSA, the kinetics is biexponential with widely different rate constants. Extrapolated zero-time values reflect, according to additional investigations, 'instantaneous' release of PA from the outer surface of the ghosts. Analyses of the biexponential curve up to about 55% tracer efflux assign unequivocally values to three model parameters. (1) k1, the dissociation rate constant of the PA-BSA complex is (1.47 +/- 0.03) x 10(-3) s-1 and (2.56 +/- 0.08) x 10(-3) s-1 and (4.08 +/- 0.13) x 10(-3) s-1 at v = 0.2, 0.6 and 1.4, respectively. (2) k3*, the overall rate constant of PA transport from the inside of the ghost membrane to the medium is 0.0269 +/- 0.0020 s-1 independent of v. (3) Qkin, the ratio of PA on the inside of the membrane to PA on BSA within the ghosts is v dependent and smaller than a corresponding ratio Qeq measured in equilibrium by a value corresponding to PA on the outer surface. This fraction is released with a rate constant, k5, which is of the order of 1 s-1. The data suggest a maximum PA transport capacity, Jmax, of 2 pmol min-1 cm-2, 0 degrees C, pH 7.3.