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.     

The effects of Ca2+ and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

We have studied beta-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5-8 nmol/min per ml ghosts and remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (Ka) for (+/-)-isoprenaline from 0.1 to 0.6 microM. THe apparent dissociation constant for propranolol (0.01 microM) remained unchanged. The Ka values for isoprenaline in native reticulocytes and in resealed ghosts were identical. The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal beta-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca2+ concentrations ranging between 0.1 and 1 microM. GTP stimulated isoprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3-5 but did not change the Ka value for isoprenaline. Ka values for the guanylnucleotides in different experiments varied between 0.3 and 2 microM. Ca2+ concentrations up to 4.6 microM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their Ka values. Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native beta-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are not observed in isolated membranes.     

Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. III. Permeability of spectrin-depleted inside-out membrane vesicles to hydrophilic nonelectrolytes. Formation of leaks by chemical or enzymatic modification of membrane proteins.

Spectrin-depleted inside-out vesicles (IOV's) prepared from human erythrocyte membranes were characterized in terms of size, ground permeability to hydrophilic nonelectrolytes and their sensitivity to modification by SH reagents, DIDS and trypsin. IOV's proved to have the same permeability of their lipid domain to erythritol as native erythrocytes, in contrast to resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)), which have a residual leak. On the other hand, IOV's have a slightly elevated permeability for mannitol and sucrose, nonelectrolytes which are almost (mannitol) or fully (sucrose) impermeant in the native membrane. These increased fluxes, which have a high activation energy and can be stimulated by phloretin, are, however, also much smaller than the corresponding leak fluxes observed in resealed ghosts. In view of these differences, formation of IOV's can be concluded to go along with partial annealing of barrier defects persisting in the erythrocyte membrane after preparation of resealed ghosts. Oxidation of SH groups of the IOV membrane by diamide produces an enhancement of permeability for hydrophilic nonelectrolytes which is much less pronounced than that induced by a similar treatment of erythrocytes or ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)). Moreover, proteolytic treatment of the vesicle membrane, although leading to a marked digestion of integral membrane proteins, only induces a minor, saturating increase of permeability, much lower than that in trypsinized resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 137-142 (Part II of this series)). Since absence of the cytoskeletal proteins, spectrin and actin, is the major difference between IOV's and resealed ghosts, these results may be taken as further evidence for a dependence of the barrier properties of the erythrocyte membrane bilayer domain on its interaction with cytoskeletal elements. In contrast, these barrier properties seem to be rather insensitive to perturbations of integral proteins.     

Membrane effects of imidoesters in hereditary stomatocytosis

The marked increase in cation (Na⁺, K⁺) permeability that results in swollen, cup-shaped red cells in the hereditary stomatocytosis syndrome can be corrected in vitro with a bifunctional crosslinking reagent, dimethyl adipimidate (DMA). ⁴⁵Ca influx in intact RBC, ⁴⁵Ca efflux in red ghosts, and ⁴⁵Ca retention in red ghosts are normal and not influenced by DMA. Endocytosis in resealed red ghosts is strikingly impaired but becomes normal if cells are first treated with 2 mM DMA. Protein kinase mediated phosphorylation of membrane proteins by AT³²P–only 20–40% of normal control values in both short-term (5 min) and more extended (60 min) incubations–is not improved by DMA. After reaction of ¹⁴C-DMA with stomatocytes, radiolabel is found associated with phosphatidyl serine and phosphatidyl ethanolamine and is also widely distributed among membrane proteins. Cation permeability of stomatocytes is corrected at DMA concentrations (1 mM) that result in barely detectable crosslinking of aminophospholipids or proteins, suggesting that either crosslinking of a minor component present in only small quantities or intramolecular (rather than intermolecular) crosslinking is responsible for the permeability effects. DMA, whose maximal crosslinking dimension is 7.3–9 Å, is the most effective bifunctional imidoester of those tested. Shorter (dimethyl malonimidate) or longer (dimethylsuberimidate) reagents are either less effective than DMA or totally without effect.     

Water exchange through erythrocyte membranes: Nuclear magnetic resonance studies on resealed ghosts compared to human erythrocytes

Summary The water diffusion across human erythrocyte membrane has been studied on intact cells and resealed ghosts by a doping NMR technique. Although the water exchange time of ghosts was longer than that of erythrocytes, no significant differences in their diffusional permeability were noticed for temperatures in the range 2–43°C. Contrary to what was previously noticed in erythrocytes, no significant increase in the water exchange time of ghosts in the acid range of pH occurred.     

Preparation and characterization of hormone-sensitive, resealed erythrocyte ghosts.

A method for preparing resealed turkey erythrocyte ghosts is described which utilizes hypotonic lysis and resealing following restoration of isotonicity. The resealed ghosts are isolated above 55% sucrose. The resealed ghosts are shown to be capable of maintaining high intracellular K+ concentrations in the presence of a low K+ extracellular environment. When ATP and an ATP-regenerating system are included during the resealing stage, (R)-(-)-epinephrine- and NaF-stimulated cyclic AMP accumulation, which is linear for 20 min, can be demonstrated. The concentration of (R)-(-)-epinephrine producing a half-maximal response in resealed ghosts is 1.0 +/- 0.4 X 10(-6) M. This is the same as that for (R)-(-)-epinephrine in the intact erythrocyte. The resealed ghosts are impermeable to Ca2+, but Ca2+ inhibition of cyclic AMP accumulation is noted if the divalent cation ionophore. A-23187, is present or if Ca2+ is included during the resealing stage.     

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the cation permeability of human red cell ghosts. Characterization and biological significance of two membrane sites with high affinities for Ca.

Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant ap pH 7.2 for Ca (K'p1) of 3-5 X 10(-7) M, for Sr of 7 X 10(-6) M. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes. K'D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK' values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mM. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1-140 mM) or K (0.1-6 mM) concentrations had little effect and did not change K'p1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K'p2) varied between 6 X 10(-7) and 4 X 10(-6) M at pH 7.2 Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca greater than Sr greater than Ba greater than Mg. K'p2 was independent on the pH value in the range between 6.0 and 8.0 Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill cofficients were affected neither by the ion composition nor by the Ph values of the intra-and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific "channel" has properties similar to the K channel in excitable tissues.     

Role of membrane-bound Ca in ghost permeability to Na and K

Summary The permeability of red cell ghosts to K is determined by the amount of membrane-bound Mg which, in turn, depends on internal Mg. Contrasting with such effect, an increase in cellular Ca raises K permeability. To test whether this, action is due to a competitive displacement of membrane Mg, the free Ca content of human red cell ghosts was altered by means of Ca-EGTA buffers. Net Na and K movements as well as Ca and Mg bindings, were assessed after incubation in a Na-medium at 37°C. Raising Ca from 3×10^–7 to 1×10^–2M caused a large K efflux with very little Na gain. Under similar conditions, Ca binding was increased without affecting membranebound Mg. Both Ca binding and K loss were markedly diminished by either adding ATP to the hemolytic medium or increasing internal Mg at a fixed Ca concentration. A Scatchard analysis showed three Ca binding sites, two of them having high affinity. It is concluded that Ca action does not arise from a displacement of membrane-bound Mg but from binding to different sites in the membrane. Presumably, high affinity sites are involved in the control of K permeability.     

Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction

Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins.     

Effect of L-carnitine and acetyl-L-carnitine on the human erythrocyte membrane stability and deformability

In this study we examined the effect of carnitine and acetylcarnitine on the human erythrocyte membrane stability and membrane deformability. Since erythrocyte membranes are impermeable to these compounds, we resealed erythrocyte ghosts in the presence of different concentrations of carnitine or acetylcarnitine. Resealed ghosts can be adequately studied in their cellular deformability and membrane stability properties by means of ektacytometry. Both carnitine and acetylcarnitine alter the membrane stability but not membrane deformability of the red cell membrane. Resealed ghosts containing 20, 50, 150, and 300 microM carnitine had 1.1, 1.6, 0.9, and 0.7 times the normal stability. While resealed ghosts containing 20, 50, 150, and 300 microM acetylcarnitine had 1.1, 1.5, 1.3, and 1.2 times the normal stability. Such changes were found to be reversible. We also conducted SDS PAGE of cytoskeletal membrane proteins from membrane fragments and residual membranes produced during membrane stability analysis, and unsheared resealed membranes in those samples where we observed an increase or a decrease of membrane stability. No changes in the cytoskeletal membrane proteins were noticed, even when the samples, prior SDS PAGE analysis, were treated with or without dithiothreitol. In addition, fluorescence steady state anisotropy of DPH in the erythrocyte membrane treated with carnitine or acetylcarnitine shows no modification of the lipid order parameter. Our results would suggest that both carnitine and its acetyl-ester, at physiological concentrations, may increase membrane stability in mature erythrocytes, most likely via a specific interaction with one or more cytoskeletal proteins, and that this effect would manifest when the erythrocytes are subjected to high shear stress.     

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbations of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.     

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.     

A flow EPR study of deformation and orientation characteristics of erythrocyte ghosts: A possible effect of an altered state of cytoskeletal network

Summary Using the flow EPR technique, we investigated the resealed ghost deformability in shear flow and the effects of the altered state of cytoskeletal network induced by hypotonic incubation of ghosts. Isotonically resealed ghosts in the presence of Mg-ATP, in which alteration of cytoskeletal network is not effected, have smooth biconcave discoid shapes, and show a flow orientation and deformation behavior similar to that of erythrocytes, except that higher viscosities are required to induce the same degrees of deformation and orientation as in erythrocytes. The flow behavior of resealed ghosts is Mg-ATP dependent, and the shape of the ghosts resealed without Mg-ATP is echinocytic. In contrast, the ghosts resealed by hypotonic incubation show a markedly reduced deformability even with Mg-ATP present. Nonreducing, nondenaturing polyacrylamide gel electrophoresis (PAGE) of the low ionic strength extracts from hypotonically resealed ghosts reveals a shift of the spectrin tetramer-dimer equilibrium toward the dimers. In the maleimide spin-labeled ghosts, the ratios of the weakly immobilized to the strongly immobilized EPR intensities are larger in hypotonically resealed ghosts than in the isotonically resealed ghosts, indicating an enhanced mobility in the spectrin structure in the former. Photomicrographs of hypotonically resealed ghosts show slightly stomatocytic transformations. These data suggest that the shape and the deformability loss in hypotonically resealed ghosts are related to an alteration of the spectrin tetramer-dimer equilibrium in the membrane. Thus, the shift of the equilibrium is likely to affect the regulation of the membrane deformability both in normal and pathological cells such as hereditary elliptocytes.     

A method for estimating free Ca within human red blood cells,with an application to the study of their Ca-dependent K permeability

Summary Murphy, Coll, Rich and Williamson (J. Biol. Chem. 255:6600–6608, 1980) described a null-point method for estimating intracellular free Ca in liver cells. They used digitonin to lyse the cells in solutions of varying Ca concentration. This method has been adapted for use with human red cells. The values found are about 0.4 m Ca in fresh cells, and from 0.4 to 0.7 m Ca in blood-bank cells, at pH 7.2 and 37°C. These are likely to be overestimates, and the errors and limitations of the method are discussed. Red cells may be loaded with Ca by metabolic depletion in Ca-containing solutions. Such cells have an elevated K permeability, and the relationships between free Ca, total Ca and K permeability were investigated, using⁸⁶Rb as a tracer for K.⁸⁶Rb flux studies show that the affinity of the K channel for Ca is the same in cells as in resealed ghosts where intracellular Ca can be controlled with Ca buffers, but the rate of tracer equilibration is 3–6 times faster in ghosts than in cells.     

Ultrastructure of hemoglobin-depleted human erythrocyte resealed ghosts

Both negative-stain and freeze-fracture electron microscopic techniques revealed that the ultrastructure of resealed white ghosts prepared at high dilution during the hemolysis step is very different from that of resealed ghosts prepared at low or moderate dilution (pink ghosts). The negative-stained resealed white ghosts showed light halo substructures on membrane surfaces and protrusions at the edge of the ghosts. Freeze-fracturing of these ghosts showed that membrane blebbing had occurred and that fragments of the membranes resealed to form small right-side-out vesicles ranging from 0.1 to 0.3 μm in diameter.     

Involvement of spectrin and ATP in infection of resealed erythrocyte ghosts by the human malarial parasite, Plasmodium falciparum

Resealed erythrocyte ghosts were prepared under different experimental conditions and were tested in vitro for susceptibility to infection with the human malarial parasite, Plasmodium falciparum. Resealed ghosts, prepared by dialyzing erythrocytes in narrow membrane tubing against low ionic strength buffer that was supplemented with magnesium ATP, were as susceptible to parasite infection as were normal erythrocytes. There was a direct correlation between intraerythrocytic ATP content and susceptibility to parasite infection. Neither MgCl2 nor sodium ATP could be substituted for magnesium ATP in maintaining high intraerythrocytic ATP concentration. When resealed ghosts were loaded with antispectrin IgG, malaria merozoite invasion was inhibited. At an average intracellular antispectrin IgG concentration of 3.5 micrograms/10(8) cells, there was a 35% inhibition of parasite invasion. This inhibition was due to spectrin crosslinking within the resealed ghosts, since the monovalent, Fab' fragments of antispectrin IgG had no inhibitory effect on invasion. These results indicate that the cytoskeleton plays a role in the complex process of merozoite entry into the host erythrocyte.     

The effects of Ca2+ and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

We have studied β-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5–8 nmol/min per ml ghosts are remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (K_a) for (±)-isoprenaline from 0.1 to 0.6 μM. The apparent dissociation constant for propranolol (0.01 μM) remained unchanged. The K_a values for isoprenaline in native reticulocytes and in resealed ghosts were identi The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal β-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca²⁺ concentrations ranging between 0.1 and 1 μM. GTP stimulated iosprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3–5 but did not change the K_a value for isoprenaline. K_a values for the guanylnucleotides in different experiments varied between 0.3 and 2 μM. Ca²⁺ concentrations up to 4.6 μM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their K_a values. Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native β-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are not observed in isolated membranes.     

Effects of temperature on water diffusion in human erythrocytes and ghosts--nuclear magnetic resonance studies

The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 degrees C up to 24 h or at 37 degrees C for 30 min did not change the water exchange time significantly, even if Mn2+ was present in the medium. This indicates that no significant penetration of Mn2+ is taking place under such conditions. (2) In case of cells previously incubated at 37 degrees C for longer than 30 min with concentrations of p-chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn2+ for more than 10 min at 37 degrees C. (3) When the Arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permeability and the activation energy of water diffusion (Ea,d) were noticed. The mean value of Ea,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of Ea,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for Ea,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37 degrees C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the values of inhibition being 46% and 21% respectively.     

钙离子通过降低细胞膜透性提高自絮凝颗粒酵母耐酒精能力

生长阶段和冲击阶段均添加 1 6 4mmol LCa2 能显著提高自絮凝颗粒酵母于 30℃在 2 0 % (V V)酒精冲击下的存活率 ,经过 9h冲击 ,对照组的存活率为 0 ,而添加Ca2 试验组的存活率为 5 0 0 % ,表明添加适当浓度的Ca2 能显著提高菌体的耐酒精能力。通过考察Ca2 对菌体于 30℃在 15 % (V V)酒精冲击下细胞膜透性的影响发现 ,生长阶段和冲击阶段均添加 1 6 4mmol LCa2 的试验组的菌体胞外核苷酸平衡浓度和细胞膜透性系数 (P′)分别仅为对照组水平的 5 0 0 %和 2 9 3% ,表明添加适当浓度的Ca2 能显著降低受冲击菌体的细胞膜透性 ;而且 ,添加Ca2 提高存活率与添加Ca2 降低胞外核苷酸浓度和P′存在直接的对应关系。因此 ,Ca2 提高自絮凝颗粒酵母耐酒精能力是与其降低受冲击菌体的细胞膜透性密切相关的。