Membrane stress increases cation permeability in red cells.

The human red cell is known to increase its cation permeability when deformed by mechanical forces. Light-scattering measurements were used to quantitate the cell deformation, as ellipticity under shear. Permeability to sodium and potassium was not proportional to the cell deformation. An ellipticity of 0.75 was required to increase the permeability of the membrane to cations, and flux thereafter increased rapidly as the limits of cell extension were reached. Induction of membrane curvature by chemical agents also did not increase cation permeability. These results indicate that membrane deformation per se does not increase permeability, and that membrane tension is the effector for increased cation permeability. This may be relevant to some cation permeabilities observed by patch clamping.     

Phospholipid asymmetry during erythrocyte deformation: maintenance of the unit membrane.

To assess the red blood cell (RBC) membrane's ability to maintain normal phospholipid orientation in the face of deforming stress, we examined RBC subjected to elliptical, tank-treading deformation. As determined by accessibility to phospholipase digestion and by labelling with fluorescamine, normal RBC are able to fully preserve their phospholipid asymmetry despite attaining over 96% of their maximal possible deformation. Phospholipid orientation is unchanged during deformation even for RBC that are ATP-depleted or vanadate-treated and for RBC that already have destabilized phospholipids due to treatment with t-butyl hydroperoxide. These data indicate that maintenance of phospholipid organization during marked deforming stress and tank-treading motion of the membrane is ascribable predominantly to the passive stabilizing effect of membrane proteins. This provides additional evidence for the concept of a unit membrane characterized by intimate associations between lipid and protein.     

Oscillatory deformation of human erythrocytes in sinusoidally modulated shear flow

The red cell deformation under oscillatory shear stress was studied. Shear stress was sinusoidally modulated between 8 and 32 dyn/cm2, thus, the extent of cellular deformation altered sinusoidally. At a low modulation frequency (less than 1.8 Hz), intact red cells perfectly responded to the shear stress applied on cells, and they could deform as much as the deformation in stationary shear flow. Above 2 Hz, the cellular deformation could not follow changes in shear stress along up-phase in the shear stress cycle. As decreasing the intracellular hemoglobin concentration, the cellular response to oscillatory shear stress became better. Treatment of cells with low concentrations of diamide impaired the response of intact cells to oscillatory shear stress, but unaffected the response of partially hemolyzed cells. These data suggest that the cellular response to oscillatory shear stress is determined by the cytoskeletal structure and the intracellular viscosity.     

Elastic area compressibility modulus of red cell membrane.

Micropipette measurements of isotropic tension vs. area expansion in pre-swollen single human red cells gave a value of 288 +/- 50 SD dyn/cm for the elastic, area compressibility modulus of the total membrane at 25 degrees C. This elastic constant, characterizing the resistance to area expansion or compression, is about 4 X 10(4) times greater than the elastic modulus for shear rigidity; therefore, in situations where deformation of the membrane does not require large isotropic tensions (e.g., in passage through normal capillaries), the membrane can be treated by a simple constitutive relation for a two-dimensionally, incompressible material (i.e. fixed area). The tension was found to be linear and reversible for the range of area changes observed (within the experimental system resolution of 10%). The maximum fractional area expansion required to produce lysis was uniformly distributed between 2 and 4% with 3% average and 0.7% SD. By heating the cells to 50 degrees C, it appears that the structural matrix (responsible for the shear rigidity and most of the strength in isotropic tension) is disrupted and primarily the lipid bilayer resists lysis. Therefore, the relative contributions of the structural matrix and lipid bilayer to the elastic, area compressibility could be estimated. The maximum isotropic tension at 25 degrees C is 10-12 dyn/cm and at 50 degrees C is between 3 and 4 dyn/cm. From this data, the respective compressibilities are estimated at 193 dyn/cm and 95 dyn/cm for structural network and bilayer. The latter value correlates well with data on in vitro, monolayer surface pressure versus area curves at oil-water interfaces.     

Reversibility of the low-salt transition of chromatin core particles.

The low-salt transition of chromatin core particles is reversible if the monovalent cation concentration is kept above 0.2 mM. Exposure of the particles to salt concentrations below this value results in a nonreversible secondary transition. The nonreversible changes are relatively slow with a half-time of about 15 minutes. Once exposed to such low ionic strength, the particles then begin to refold with increasing salt in at least two steps over a much higher ionic strength range than is required for the usual low-salt transition. The refolding is very fast, with a half-time less than a minute. Small differences between particles which had or had not been exposed to very low salt persist even when the particles are returned to near physiological ionic strengths.     

Electro-mechanical permeabilization of lipid vesicles. Role of membrane tension and compressibility.

A simple micropipet technique was used to determine the critical electric field strength for membrane breakdown as a function of the applied membrane tension for three different reconstituted membranes: stearoyloleoylphosphatidylcholine (SOPC), red blood cell (RBC) lipid extract, and SOPC cholesterol (CHOL), 1:1. For these membranes the elastic area expansivity modulus increases from approximately 200 to 600 dyn/cm, and the tension at lysis increases from 5.7 to 13.2 dyn/cm, i.e., the membranes become more cohesive with increasing cholesterol content. The critical membrane voltage, Vc, required for breakdown was also found to increase with increasing cholesterol from 1.1 to 1.8 V at zero membrane tension. We have modeled the behavior in terms of the bilayer expansivity. Membrane area can be increased by either tensile or electrocompressive stresses. Both can store elastic energy in the membrane and eventually cause breakdown at a critical area dilation or critical energy. The model predicts a relation between tension and voltage at breakdown and this relation is verified experimentally for the three reconstituted membrane systems studied here.     

Hypothyroidism in rats decreases mitochondrial inner membrane cation permeability

We investigated the cation permeability of liver mitochondria isolated from hypothyroid or euthyroid rats by measuring the rate of swelling of respiring mitochondria in acetate salts as a function of membrane potential. Mitochondria from hypothyroid rats have a decreased permeability of roughly 3-fold in the presence of monovalent cations K and tetramethylammonium at any (measured) membrane potential. Since the monovalent cation leak and the proton leak are known to respond similarly to membrane potential our results support the theory that the difference in non-phosphorylating respiration rate between mitochondria from hypothyroid and euthyroid rats is due to a difference in proton leak.     

Effects of pressure on red blood cell geometry during micropipette aspiration

Micropipette aspiration is a potentially useful and accurate technique to measure red blood cell (RBC) geometry. Individual RBCs are partially aspirated and from the resulting sphere diameter, total cell length, and pipette diameter, membrane area and cell volume can be calculated. In this study we have focused on possible shape artifacts associated with the aspirated portion of RBC. We observed that the apparent RBC geometry (calculated area and volume) changed markedly (P < 0.001) with the applied aspiration pressure; for normal human RBC the area increased by 5.6 +/- 0.6% and volume decreased by 4.7 +/- 0.6% when the aspiration pressure was increased from 20 to 100 mm water. The calculated membrane area dilation modulus was 7.4 dyn/ cm, which is far below the expected value, and microscopic observations revealed a membrane folding artifact as a possible artifact. These assumptions were strengthened by using a short-duration (3 s) pressure peak of 20-100-20 mm water. The folding then disappeared permanently, but a small (0.31 +/- 0.09%; P < 0.001) area decrease was detected which yields a realistic dilation modulus of 215 dyn/cm. We conclude that membrane folding can critically affect RBC micropipette measurements and that a transient pressure peak can unfold the RBC membrane, thus allowing accurate measurements of RBC geometry.     

An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. II. Second site revertants of the thiodigalactoside-dependent proton leak by the Val177/Asn319 permease

The double mutant of the lactose permease containing Val177/Asn319 exhibits proton leakiness by two pathways (see Brooker, R. J. (1991) J. Biol Chem. 266, 4131-4138). One type of H+ leakiness involves the uncoupled influx of H+ (leak A pathway) while a second type involves the coupled influx of H+ and galactosides in conjunction with uncoupled galactoside efflux (leak B pathway). In the current study, 14 independent lactose permease mutants were isolated from the Val177/Asn319 parent which were resistant to thiodigalactoside growth inhibition but retained the ability to transport maltose. All of these mutants contained a third mutation (besides Val177/Asn319) at one of two sites. Eight of the mutants had Ile303 changed to Phe, while six of the mutants had Tyr236 changed to Asn or His. Each type of triple mutant was characterized with regard to sugar transport, H+ leakiness, and sugar specificity. Like the parental strain, all three types of triple mutant showed moderate rates of downhill lactose transport and were defective in the uphill accumulation of sugars. However, with regard to proton leakiness, the triple mutants fell into two distinct categories. The mutant containing Phe303 was generally less H+ leaky than the parent either via the leak A or leak B pathway. In contrast, the triple mutants containing position 236 substitutions (Asn or His) were actually more H+ leaky via the leak A pathway and exhibited similar H+ leakiness via the leak B pathway at high thiodigalactoside concentrations. The ability of the position 236 mutants to grow better than the parent in the presence of low concentrations of thiodigalactoside appears to be due to a decrease in affinity for this particular sugar rather than a generalized defect in H+ leakiness. Finally, the triple mutants showed a sugar specificity profile which was different from either the Val177/Asn319 parent, the single Val177 mutant, or the wild-type strain. These results are discussed with regard to the effects of mutations on both the sugar and H+ transport pathways.     

Effects of p-chloromercuribenzene sulfonate on water transport across the marsupial erythrocyte membrane

The effects of exposure of red blood cells (RBC) of three species of marsupial to a mercury-containing sulfhydryl-modifying reagent, p-chloromercuribenzene sulfonate (PCMBS), on the water diffusional permeability ( P (d)) of their membranes were monitored by using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. For koala ( Phascolarctos cinereus), RBC the maximal inhibition was reached at 37 degrees C in 60 min with 1 mmol.l(-1) PCMBS or in 15-30 min with 2 mmol. l(-1) PCMBS. In contrast, in the case of red kangaroo ( Macropus rufus) or swamp wallaby ( Wallabia bicolor) RBC, maximal inhibition required an incubation of 90 min at 37 degrees C with 2 mmol.l(-1) PCMBS. For the RBC of all three species the value of maximal inhibition was very high, being 50-70% when measured at 25 degrees C, 60-80% at 30 degrees C and 60-70% at 37 degrees C. The lowest values of P (d) appeared to be around 2 x 10(-3)-3 x 10(-3) cm.s(-1) in the temperature range of 25-37 degrees C. The mean value of the activation energy of water diffusion ( E (a,d)) was approximately 20-25 kJ.mol(-1) for control and approximately 40 kJ.mol(-1) for PCMBS-inhibited RBCs. These results show that marsupial RBC have a basal permeability to water similar to that previously reported for human RBC, but a higher value of the PCMBS-inhibitable water permeability. This indicates that the higher water permeability of marsupial RBC compared with human RBC is associated with a higher fraction of protein-mediated water permeability.     

Protection of erythrocytes from sub-hemolytic mechanical damage by nitric oxide mediated inhibition of potassium leakage

The effects of mechanical stress on red blood cell (RBC) deformability were evaluated by subjecting cells to a uniform fluid shear stress of 120 Pa for 15-120 seconds at 37 degrees C. This level of stress induced significant impairment of RBC deformability as assessed by ektacytometry, with the degree of impairment independent of extracellular calcium concentration. Inhibition of RBC nitric oxide (NO) synthesis by a competitive inhibitor of NO synthases (N-omega-nitro-L-arginine methyl ester, L-NAME) had no effect on deformability after exposure to mechanical stress. The NO donor sodium nitroprusside (SNP) prevented the deterioration of RBC deformability in a dose-dependent manner with 10(-4) M being the most effective concentration. A similar protective effect by the non-selective potassium channel blocker, tetraethylammonium chloride (TEA) suggests that the effect of NO might be mediated by the inhibition of potassium leakage from RBC. These results suggest that NO may prevent mechanical deterioration of RBC exposed to high shear stresses. While RBC are not exposed to such high levels of shear stresses for prolonged periods under normal circulatory conditions, comparable levels of mechanical stress can be encountered under certain situations (i.e., artificial organs, extracorporeal circulation) and may result in subhemolytic damage and hemorheological alterations.     

Osmotic behavior of red blood cell as seen with an ultrasonic method

We have measured the density and ultrasonic velocity (usv) of swine red blood cell (RBC) suspensions in the wide osmolarity range from 300 mOsm to 1400 mOsm in saline solution. The cellular density and compressibility of RBC at each osmolarity were obtained using the fact that the density and the compressibility are additive by volume. The osmolarity dependence of hematocrit was also measured at a constant number concentration of RBC in the range of 300 mOsm to 1700 mOsm. The cellular density and the cellular compressibility of RBC as well as the inverse of hematocrit were expressed well into one unique exponential type equation f (pi) = a [1 - b exp (-c pi)] with a common value for the coefficient c = 0.0025 against the osmolarity pi. The results were analyzed with a simple consideration based only upon the contribution of free water inside the erythrocyte through the volume concentration phi of the free water in it. According to this theoretical analysis, the density and the compressibility of the free water were found to be 0.990 g/cm3 and 4.59 x 10(-11) cm2/dyne which agree closely with 0.998 g/cm3 and 4.59 x 10(-11) cm2/dyn of pure water at 20 degrees C within the experimental error.     

Potassium transport system of Rhodopseudomonas capsulata

Rhodopseudomonas capsulata required potassium (or rubidium or cesium as analogs of potassium) for growth. These cations were actively accumulated by the cells by a process following Michaelis-Menten saturation kinetics. The monovalent cation transport system had Km's of 0.2 mM K+, 0.5 mM Rb+, and 2.6 mM Cs+. The rates of uptake of substrates by the potassium transport system varied with the age of the culture, although the affinity constant for the substrates remained constant. The maximal velocity of uptake of K+ was lower in aerobically grown cells than in photosynthetically grown cells, although the Km's for K+ and for Rb+ were about the same.     

Elastic deformation and failure of lipid bilayer membranes containing cholesterol.

Giant bilayer vesicles were reconstituted from several lipids and lipid/cholesterol (CHOL) mixtures: stearolyloleoylphosphatidylcholine (SOPC), bovine sphingomyelin (BSM), diarachidonylphosphatidylcholine (DAPC), SOPC/CHOL, BSM/CHOL, DAPC/CHOL, and extracted red blood cell (RBC) lipids with native cholesterol. Single-walled vesicles were manipulated by micropipette suction and several membrane material properties were determined. The properties measured were the elastic area compressibility modulus K, the critical areal strain alpha c, and the tensile strength tau lys, from which the failure energy or membrane toughness Tf was calculated. The elastic area expansion moduli for these lipid and lipid/cholesterol bilayers ranged from 57 dyn/cm for DAPC to 1,734 dyn/cm for BSM/CHOL. The SOPC/CHOL series and RBC lipids had intermediate values. The results indicated that the presence of cholesterol is the single most influential factor in increasing bilayer cohesion, but only for lipids where both chains are saturated, or mono- or diunsaturated. Multiple unsaturation in both lipid chains inhibits the condensing effect of cholesterol in bilayers. The SOPC/CHOL system was studied in more detail. The area expansion modulus showed a nonlinear increase with increasing cholesterol concentration up to a constant plateau, indicating a saturation limit for cholesterol in the bilayer phase of approximately 55 mol% CHOL. The membrane compressibility was modeled by a property-averaging composite theory involving two bilayer components, namely, uncomplexed lipid and a lipid/cholesterol complex of stoichiometry 1/1.22. The area expansion modulus of this molecular composite membrane was evaluated by a combination of the expansion moduli of each component scaled by their area fractions in the bilayer. Bilayer toughness, which is the energy stored in the bilayer at failure, showed a maximum value at approximately 40 mol% CHOL. This breakdown energy was found to be only a fraction of the available thermal energy, implying that many molecules (approximately 50-100) may be involved in forming the defect structure that leads to failure. The area expansion modulus of extracted RBC lipids with native cholesterol was compared with recent measurements of intact RBC membrane compressibility. The natural membrane was also modeled as a simple composite made up to a compressible lipid/cholesterol matrix containing relatively incompressible transmembrane proteins. It appears that the interaction of incompressible proteins with surrounding lipid confers enhanced compressibility on the composite structure.     

A sensitive measure of surface stress in the resting neutrophil.

The simplest parameterized model of the "passive" or "resting receptive" neutrophil views the cell as being composed of an outer cortex surrounding an essentially liquid-like highly viscous cytoplasm. This cortex has been measured to maintain a small persistent tension of approximately 0.035 dyn/cm (Evans and Yeung. 1989. Biophys. J. 56:151-160) and is responsible for recovering the spherical shape of the cell after large deformation. The origin of the cortical tension is at present unknown, but speculations are that it may be an active process related to the sensitivity of a given cell to external stimulation and the "passive-active" transition. In order to characterize further this feature of the neutrophil we have used a new micropipet manipulation method to give a sensitive measure of the surface stress as a function of the surface area dilation of the highly ruffled cellular membrane. In the experiment, a single cell is driven down a tapered pipet in a series equilibrium deformation positions. Each equilibrium position represents a balance between the stress in the membrane and the pressure drop across the cell. For most cells that seemed to be "passive," as judged by their spherical appearance and lack of pseudopod activity, area dilations of approximately 30% were accompanied by only a small increase in the membrane tension, indicative of a very small apparent elastic area expansion modulus (approximately 0.04 dyn/cm). Extrapolations back to zero area dilation gave a value for the tension in the resting membrane of 0.024 +/- 0.003 dyn/cm, in close agreement with earlier measures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Smooth muscle cells contract in response to fluid flow via a Ca2+-independent signaling mechanism.

Smooth muscle cells (SMC) are exposed to fluid shear stress because of transmural (interstitial) flow across the arterial wall. This shear stress may play a role in the myogenic response and flow-mediated vasomotion. We, therefore, examined the effects of fluid flow on contraction of rat aortic SMC. SMC that had been serum-starved to induce a contractile phenotype were plated on quartz slides and exposed to controlled shear stress levels in a flow chamber. The area of the cells was quantified, and reduction in the cell area was reported as contraction. At 25 dyn/cm(2), significant area reduction was apparent 3 min after the onset of flow and exceeded 30% at 30 min. At 1 dyn/cm(2), significant contraction was not observed at 30 min. The threshold for significant shear-induced contraction appeared to be 11 dyn/cm(2). The signal transduction mechanism was studied at 25 dyn/cm(2). Intracellular calcium was imaged by using the calcium-sensitive fluorescent dye fura 2-AM. There was no detectable change in intracellular calcium during 10 min of exposure to shear stress, even though the cells displayed a significant calcium response to thapsigargin, calcium ionophore, and KCl. Further studies using pathway inhibitors provided evidence that the most important signal transduction pathway mediating calcium-independent contraction in response to fluid flow is the Rho-kinase pathway, although there was a suggestion that protein kinase C plays a secondary role.     

Inhibition of dynamin‐2 confers endothelial barrier dysfunctions by attenuating nitric oxide production

Hypoxia induces barrier dysfunctions in endothelial cells. Nitric oxide is an autacoid signalling molecule that confers protection against hypoxia‐mediated barrier dysfunctions. Dyn‐2 (dynamin‐2), a large GTPase and a positive modulator of eNOS (endothelial nitric oxide synthase), plays an important role in maintaining vascular homeostasis. The present study aims to elucidate the role of dyn‐2 in hypoxia‐mediated leakiness of the endothelial monolayer in relation to redox milieu. Inhibition of dyn‐2 by transfecting the cells with K44A, a dominant negative construct of dyn‐2, elevated leakiness of the endothelial monolayer under hypoxia. Sodium nitroprusside (nitric oxide donor) and uric acid (peroxynitrite quencher) were used to evaluate the role of nitric oxide and peroxynitrite in maintaining endothelial barrier functions under hypoxia. Administration of nitric oxide and uric acid recovered hypoxia‐mediated leakiness of K44A‐overexpressed endothelial monolayer. Our study confirms that inhibition of dyn‐2 induces leakiness in the endothelial monolayer by increasing the load of peroxynitrite under hypoxia.     

Thermoelasticity of large lecithin bilayer vesicles.

Micromechanical experiments on large lecithin bilayer vesicles as a function of temperature have demonstrated an essential feature of bilayer vesicles as closed systems: the bilayer can exist in a tension-free state (within the limits of experimental resolution, i.e., less than 10(-2) dyn/cm). Furthermore, because of the fixed internal volume, there is a critical temperature at which the vesicle becomes a tension-free sphere. Below this temperature, thermoelastic tension builds up in the membrane and the vesicle's internal pressure increases while the surface area remains constant. Above this temperature, the vesicle's surface area increases while the tension and internal pressure are negligible. Without mechanical support, the vesicles fragment into small vesicles because they have insufficient surface rigidity. In the upper temperature range we have measured the increase of surface area with temperature. These data established the thermal area expansivity to be 2.4 X 10(-3)/degrees C. At constant temperature, we used either pipet aspiration with suction pressures up to 10(4) dyn/cm2 or compression against a flat surface with forces up to 10(-2) dyn to produce area dilation of the vesicle surface on the order of 1%. The rate of increase of membrane tension with area dilation was calculated, which established the elastic area compressibility modulus to be 140 dyn/cm. The tension limit that produced lysis was observed to be 3-4 dyn/cm (equivalent to 2-3% area increase). The product of the elastic area compressibility modulus, the thermal area expansivity, and the temperature gives the reversible heat of expansion at constant temperature for the bilayer. This value is 100 ergs/cm2 at 25 degrees C, or approximately 5 kcal/mol of lecithin. Similarly, the product of the thermal area expansivity multiplied by the area compressibility modulus determines the rate of increase of thermoelastic tension with decrease in temperature when the area is held constant, i.e., -0.34 dyn/cm/degrees C.     

Lithium's inhibition of erythrocyte cation countertransport involves a slow process in the erythrocyte

Chronic administration of lithium (Li+) to human subjects results in reduction of Li+/Na+ countertransport in their erythrocytes (RBC). The time course of development of inhibition is much slower than one would expect for an immediate effect of Li+ on the RBC membrane. Possible explanations include pharmacokinetic delays, a mediating humoral agent, and a slow process in the RBC. To discriminate among these possibilities, we incubated human RBC in sterile culture by the method of Freedman (Freedman, J.C. 1983. J. Membrane Biol. 75:225--231), which permits much longer incubations than other methods. As gauged by eight measures, the incubated RBC remain viable for two weeks. Small changes in intracellular concentrations with time during incubation are in the same direction as the changes associated with natural aging of RBC in vivo, except for a rise in ATP and related cation shifts during the first few days of incubation. Treatment of incubated RBC with 2 mM Li+ inhibits countertransport by 48% without affecting Li+ leak efflux. The inhibition develops slowly: it is half-maximal after 1--2 days and maximal by 4--7 days. Differences between in vivo results and our incubated cells in the time course of inhibition are as expected from the pharmacokinetic delays operating in vivo. The inhibition is reversible on removing Li+. Li+ inhibits countertransport similarly slowly and to a similar degree from inside the RBC and from outside. Hence the slow time course of inhibition in vivo is not due to a humoral factor or to the time required for intracellular Li+ accumulation and is only partly due to pharmacokinetic delays. The delay must involve an unidentified slow process at the level of the RBC.     

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.