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

Human red blood cell ghosts were prepared by electrical haemolysis at 0 °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 ($\text{=}\text{mean}$) volume was approx. 115 μm³, performing the electrical haemolysis in the following solution: 105 mM KCl, 20 mM NaCl, 4 mM MgCl₂, 7.6 mM Na₂H₂PO₄, 2.4 mM Na₂HPO₄, 10 mM glucose, pH 7.2.Resealing was carried out at 0 °C for 10 min (after the haemolytic step) and then for futher 20 min at 37 °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 μm³, 15 mM phosphate: 134 μm³).The breakdown voltage of the ghost cell membranes measured with a hydrodynamic focusing Coulter Counter depends on the mean volume ($\text{94 μ}\text{m}{}^3\text{= 1.04 V, 134 μ}\text{m}{}^3\text{= 1.36}\text{V}$). On the other hand, the breakdown voltage is constant throughout each size distribution pointing to an “electrically homogeneous” ghost preparation. The sensitivity 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 populations i.e. with two different breakdown voltages. The ghost cells obtained by electrical haemolysis in the above solution containing 10 mM phosphate were fairly impermeable to sucrose and behave like an ideal osmometer.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.     

Physiological characteristics of human red blood cell ghosts

The properties of ghosts prepared by hypotonic hemolysis at various ratios of cells (C) to hemolyzing solution (H) have been studied. At all ratios, hemoglobin (Hb) was found to be distributed equally between the ghost and supernatant compartments. Techniques employing Fe(59)-labelled Hb showed that during hemolysis all of the Hb is exchangeable and that following hemolysis the ghost is impermeable to Hb. Ghosts containing defined fractions of their original Hb were prepared by appropriately altering the ratio C/H. When washed and suspended in 0.17 M NaCl-PO(4)-buffered media, the ghosts returned to their initial volume, recovered normal shape, and behaved as osmometers. The rate of rehemolysis of these reconstituted ghosts was observed to be proportional to the concentration of Hb in the ghosts. The rate of rehemolysis was accelerated by the addition of n-butyl alcohol (BA). For a given concentration of BA, temperature, and Hb content the rate of rehemolysis was minimal around the isoelectric point of Hb. Rehemolysis by BA was inhibited by the addition of sucrose to the medium. K influx and outflux were measured and found to be increased by the addition of BA and not influenced by the presence of sucrose. These results on the rehemolytic characteristics of ghosts are consistent with and support the colloid-osmotic theory of hemolysis.     

Transport of sugars into human red blood cell ghosts

It has been found that human red cell ghosts react differently in the presence of various sugars in the medium. The stability of spheric ghosts is preserved in solutions of sugars entering red cells by means of the common carrier. In media of other sugars the ghosts' shapes change to shrunken, crenated forms and between the microscope slides to discoid ones. Under the conditions employed it was further observed that the incubation of fructose- or rhamnose-containing ghosts in solutions of sugars sharing the carriers led to an equilibration of sugars between the medium and the ghosts. The impermeability of ghosts for sugars not sharing the carriers was supported by the finding that fructose could be washed out to a much less extent than glucose. These results suggest that sugars without affinity to the carriers may move in the erythrocyte membrane through nonspecific sites (pores, channels).     

Demonstration of adenylate cyclase activity in human red blood cell ghosts.

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine 3',5'-monophosphate (cyclic AMP) - 2 h-1 - mg-1 protein. This enzymatic activity is strongly stimulated by NaF and 5'-guanylimidodiphosphate, is slightly stimulated by epinephrine, norepinephrine, isoproterenol, and prostaglandin E1 and is inhibited by calcium. The hormone stimulation is not potentiated by 5'-guanylylimidodiphosphate.     

Membrane transport of L-triiodthyronine by human red cell ghosts.

L-T₃ transport has been investigated in human red cell ghosts. Determination of initial T₃ uptake revealed two separate saturable uptake systems, one with a K_m of 1.6 × 10^?8M, the other with a K_m of 3.3 × 10^?6M. Binding experiments resulted in two dissociation constants, 1.4 × 10^?7M.and 2.6 × 10^?6M. Uptake was dependent on the ghost volume, indicating an intravesicular location of T₃. The T₃ was concentrated 6 times by the ghosts. Ouabain reduced the uptake by the low K_m system, but was without effect on the high K_m system. Thus evidence is provided both of binding of T₃ to the ghost membrane and of its uphill transport across the membrane.     

Unmasking of a potassium leak in resealed human red blood cell ghosts.

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0 degrees C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.     

Demonstration of adenylate cyclase activity in human red blood cell ghosts

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine ′,5′-monophosphatase (cyclic AMP) · 2 h^?1 · mg^?1 protein. This enzymatic activity is strongly stimulated by NaF and 5′-guanylimidodiphosphate, is slightly stimulated by epinephrine, norephrine, soproterenol, and prostaglandin E, and is inhibited by calcium. The hormone stimulation is not potentiated by 5′-guanylylimidodiphosphate.     

Kinetic characteristics of the sulfate self-exchange in human red blood cells and red blood cell ghosts

Summary The sulfate and the chloride self-exchange fluxes were determined by measuring the rate of the tracer efflux from radioactively labeled human red blood cells and red blood cell ghosts. The concentration dependence and the pH-dependence of the sulfate self-exchange flux were studied. In addition, the effects of some monovalent and divalent anions on the sulfate and the chloride self-exchange fluxes were investigated.The sulfate self-exchange fluxes saturate, exhibiting a concentration maximum at sulfate concentrations between 100 and 300mm (25°C). The position of the concentration maximum depends upon pH. At high sulfate concentrations a self-inhibition of the flux becomes apparent. The apparent half-saturation constant and the apparent self-inhibition constant at pH 7.2 were 30mm and 400mm respectively. Within the pH range of 6.3–8.5, both constants decreased with increasing pH. No saturation of the sulfate self-exchange flux was observed if the sulfate concentration was raised by substituting sulfate for isoosmotic amounts of a second salt (NaCl, NaNO₃, Na-acetate, Na-lactate, Na-succinate or Na₂HPO₄). Red blood cells and red blood cell ghosts display the same pattern of concentration responsiveness.The sulfate self-exchange flux exhibits a pH-maximum at about pH 6.2 (37°C). The location of the pH-maximum is little affected by variations of the sulfate concentration. The logarithmic plots (log vs. pH) revealed that the flux/pH relation can be approximated by two straight lines. The slopes of the alkaline branches of the flux/pH curves range from –0.55 to –0.86, the slopes of the branches of the curves range from 0.08 to 1.14 and were strongly affected by changes of the sulfate concentrations. The apparent pK's obtained from the alkaline and from the acidic branches of the flux/pH curves were about 7.0 and 6.0, respectively. Intact red blood cells and red blood cell ghosts display the same type of pH-dependency of the sulfate self-exchange flux.The sulfate self-exchange flux is competitively inhibited by nitrate, chloride, acetate, oxalate and phosphate. The chloride self-exchange flux is competitively inhibited by thiocyanate, nitrate, sulfate and phosphate. The inhibition constants for the various anion species increase in the given sequence.The results of our studies indicate that the sulfate self-exchange flux is mediated by a two-site transport mechanism consisting either of a mobile carrier or a two-site pore. The experiments reported in this paper do not permit distinguishing between both transport mechanisms. The similarities of the sulfate and the chloride self-exchange flux and the mutual competition between sulfate and chloride point to a common transport system for both anion species.     

Osmotic deformation of red blood cell ghosts induced by carbohydrates

The osmotic response of bovine red blood cell ghosts to a series of sugars is studied by light scattering. The sealed and right-side-out ghosts are prepared by the procedure of Steck and Kant (Steck, T.L. and Kant, J.A. (1974) Methods Enzymol. 31, 172–180), swollen in a hypotonic phosphate-buffered saline solution and their size and shape determined by elastic and quasielastic light scattering. Different carbohydrates are then added to the suspending medium in order to examine the osmotic responses, and the osmotic deformation of ghosts is shown to be spherically symmetric. Having thus established the deformation behavior, we then rank the osmotic activity of a carbohydrate relative to a standard, i.e., raffinose. It is found that the osmotic response of the ghosts to sucrose is about the same as that to raffinose, and the response to the smaller carbohydrates simply follows the number of carbons in various sugars; glucose and fractose are about 1.7 times less effective than raffinose, and pentaerythritol and meso-erythritol are 2.3 times less effective. Glyceraldehyde, which is 3.6 times less effective than raffinose, is the least effective sugar analog among those that we have tested.     

Unmasking of a potassium leak in resealed human red blood cell ghosts

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0° C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.     

The birefringence of the human red cell ghosts

The type of birefringence described by Mitchison, which extends some 0.5 µ in from the surface of the human red cell ghost in glycerol and which shows a maximum retardation of about 7 A, is only found in ghosts which are sufficiently well hemoglobinised to be seen with the ordinary microscope. Ghosts from which all hemoglobin has been lost are not visible with the ordinary microscope and are not birefringent, although they are clearly visible with phase contrast. About 90 per cent of the ghosts in glycerol preparations are of the latter type, the exact percentage being a function of time. Mitchison's measurements of birefringence, although reproducible, accordingly apply only to ghosts in which some hemoglobin still remains complexed with the lipoprotein layers of the red cell ultrastructure, and do not enable one to draw conclusions as to the thickness and orientation of the lipoprotein surface layers.     

Reconstitution of the basal calcium transport in resealed human red blood cell ghosts

The (45)Ca(2+) influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The (45)Ca(2+) equilibration occurred with t(1/2)=2.5 min and the steady-state was reached after 17 min with the level of 22+/-2 micromol/L(packed cells) at 37 degrees C. The rate of the influx was 97+/-17 micromol/L(packed cells)h. The (45)Ca(2+) influx was saturated with [Ca(2+)](0) at 4 mmol/L and was optimal at pH 6.5 and 30 degrees C. Divalent cations (10(-4)-10(-6)mol/L), nifedipine (10(-5)-10(-4)mol/L), DIDS (up to 10(-4)mol/L), and quinidine (10(-4)-10(-3)mol/L), inhibited the (45)Ca(2+) influx while uncoupler (10(-6)-10(-5)mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the (45)Ca(2+) influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca(2+) influx in intact RBC. The results show that the Ca(2+) influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca(2+) are coupled via the H(+) homeostasis similarly as in intact RBC but with modified mechanism.     

Volume-sensitive K influx in human red cell ghosts

K influx into resealed human red cell ghosts increases when the ghosts are swollen. The influx demonstrates properties similar to volume-sensitive K fluxes present in other cells. The influx is, for the most part, insensitive to the nature of the major intracellular cation and therefore is not a K-K exchange. The influx is much greater when the major anion is Cl than when the major anion is NO3; Cl stimulates the flux and, at constant Cl, NO3 inhibits it. Increase in the influx rate is rapid when shrunken ghosts are swollen or when NO3 is replaced by Cl. The volume-sensitive K influx requires intracellular MgATP at low concentrations, and ATP cannot be replaced by nonhydrolyzable ATP analogues. The volume-sensitive influx is inhibited by Mg2+ and by high concentrations of vanadate, but is stimulated by low concentrations of vanadate. It is not modified by cAMP, the removal of Ca2+ by EGTA, substances that activate protein kinase C, or by inhibition of phosphatidylinositol kinase. The influx is inhibited by neomycin and by trifluoperazine.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.     

Passive Ca transport in human red blood cell ghosts measured with entrapped arsenazo III

The rate of Ca influx into ghosts containing arsenazo III changes with time, being most rapid during the first 5 min after Ca is added to the outside and declining thereafter. The rate of Ca influx is a nonlinear function of extracellular Ca and plateaus as the latter is increased above 1 mM. The rate of Ca influx was measured as a function of the transmembrane gradients of Na and K and changes in the permeability of the membrane to K and Cl produced by valinomycin and SITS (4-acetamido-4'-isothiocyano-stilbene-2-2'-disulfonic acid), respectively. Changes in the rate of Ca influx are consistent with expected effects of these treatments on the membrane potential. Oligomycin (10 micrograms/ml) and quinidine (1 mM) inhibit the rate of Ca uptake by inhibiting Ca-induced changes in the K permeability. At constant membrane potential, furosemide produced a slight (15%) consistent increase in Ca uptake. Other experiments show that resealed ghosts are heterogeneous in their passive permeability to Ca and that A23187 can be used to effectively eliminate such differences. The results of this paper show that resealed human red cell ghosts containing arsenazo III can be used to continuously monitor intracellular free Ca and to study the factors that influence the permeability of the red cell membrane to Ca.