Clostridium perfringens epsilon toxin binds to erythrocyte MAL receptors and triggers phosphatidylserine exposure

Epsilon toxin (ETX) is a 33‐kDa pore‐forming toxin produced by type B and D strains of Clostridium perfringens. We previously found that ETX caused haemolysis of human red blood cells, but not of erythrocytes from other species. The cellular and molecular mechanisms of ETX‐mediated haemolysis are not well understood. Here, we investigated the effects of ETX on erythrocyte volume and the role of the putative myelin and lymphocyte (MAL) receptors in ETX‐mediated haemolysis. We observed that ETX initially decreased erythrocyte size, followed by a gradual increase in volume until lysis. Moreover, ETX triggered phosphatidylserine (PS) exposure and enhanced ceramide abundance in erythrocytes. Cell shrinkage, PS exposure and enhanced ceramide abundance were preceded by increases in intracellular Ca²⁺ concentration. Interestingly, lentivirus‐mediated RNA interference studies in the human erythroleukaemia cell line (HEL) cells confirmed that MAL contributes to ETX‐induced cytotoxicity. Additionally, ETX was shown to bind to MAL in vitro. The results of this study recommend that ETX‐mediated haemolysis is associated with MAL receptor activation in human erythrocytes. These data imply that interventions affecting local MAL‐mediated autocrine and paracrine signalling may prevent ETX‐mediated erythrocyte damage.     

Dielectric breakdown measurements of human and bovine erythrocyte membranes using benzyl alcohol as a probe molecule

Dielectric breakdown of intact erythrocytes and subsequent haemolysis in the presence of increasing concentrations of benzyl alcohol were investigated by means of an electrolytical discharge chamber and a hydrodynamic focusing Coulter Counter.Low concentrations of the drug stabilized human and bovine erythrocytes against haemolysis induced by dielectric breakdown of the cell membrane in isotonic solutions, while high concentrations caused lysis similar to hypotonic and mechanical haemolysis. The stabilizing effect of the drug on electrically induced haemolysis depends on the pulse length of the applied electric field. The critical dielectric breakdown voltage of the membranes of intact cells decreases progressively with increasing benzyl alcohol concentrations, at which the membrane is also more stabilized against electrical and osmotic haemolysis. Occasionally, an increase in the dielectric breakdown voltage is observed at drug concentrations at which lysis occurs. A similar dependence of the breakdown voltage on drug concentration was found for human erythrocyte ghost cells prepared by dielectric breakdown.The results are consistent with the electromechanical model suggested for the dielectric breakdown mechanism and with the assumption of Metcalfe, using NMR and ESR techniques, that the fluidity of the membrane increases with increasing benzyl alcohol concentration.     

Leukotoxin from Aggregatibacter actinomycetemcomitans causes shrinkage and P2X receptor‐dependent lysis of human erythrocytes

Leukotoxin (LtxA) is a virulence factor secreted by the bacterium Aggregatibacter actinomycetemcomitans, which can cause localized aggressive periodontitis and endocarditis. LtxA belongs to the repeat‐in‐toxin (RTX) family of exotoxins of which other members inflict lysis by formation of membrane pores. Recently, we documented that the haemolytic process induced by another RTX toxin [α‐haemolysin (HlyA) from Escherichia coli] requires P2X receptor activation and consists of sequential cell shrinkage and swelling. In contrast, the cellular and molecular mechanisms of LtxA‐mediated haemolysis are not fully understood. Here, we investigate the effect of LtxA on erythrocyte volume and whether P2 receptors also play a part in LtxA‐mediated haemolysis. We observed that LtxA initially decreases the cell size, followed by a gradual rise in volume until the cell finally lyses. Moreover, LtxA triggers phosphatidylserine (PS) exposure in the erythrocyte membrane and both the shrinkage and the PS‐exposure is preceded by increments in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Interestingly, LtxA‐mediated haemolysis is significantly potentiated by ATP release and P2X receptor activation in human erythrocytes. Furthermore, the LtxA‐induced [Ca²⁺]_i increase and following volume changes partially depend on P2 receptor activation. Theseobservations imply that intervention against local P2‐mediated auto‐ and paracrine signalling may prevent LtxA‐mediated cell damage.     

Theoretical Analysis of Net Tracer Flux Due to Volume Circulation in a Membrane with Pores of Different Sizes : Relation to solute drag model

When osmotic pressure across an artificial membrane, produced by a permeable electrically neutral solute on one side of it, is balanced by an external pressure difference so that there is no net volume flow across the membrane, it has been found that there will be a net flux of a second electrically neutral tracer solute, present at equal concentrations on either side of the membrane, in the direction that the "osmotic" solute diffuses. This has been ascribed to solute-solute interaction or drag between the tracer and the osmotic solutes. An alternative model, presented here, considers the membrane to have pores of different sizes. Under general assumptions, this "heteroporous" model will account for both the direction of net tracer flux and the observed linear dependence of unidirectional tracer fluxes on the concentration of the osmotic solute. The expressions for the fluxes of solutes and solvent are mathematically identical under the two models. An inequality is derived which must be valid if the solute interaction model and/or the heteroporous model can account for the data. If the inequality does not hold, then the heteroporous model alone cannot explain the data. It was found that the inequality holds for most published observations except when dextran is the osmotic solute.     

The origin and consequences of concentration dependence in gel chromatography

Concentration dependence of elution volume was determined for Blue Dextran 2000, Dextran 500, Dextran sulphate 500 and bovine serum albumin on columns of Sephadex G-100 equilibrated with sodium phosphate buffer, I 0.1, pH6.8. From the results for Dextran 500, it was shown that a linear relation exists between elution volume and the corresponding osmotic pressure calculated for the same concentration and incorporating the term containing the second virial coefficient. This relationship was used to predict the concentration dependence of elution volume for bovine serum albumin and myoglobin, proteins that partially penetrate Sephadex G-100. Possible consequences of osmotic effects are considered in relation to various types of column experiments, including differential chromatography.     

Sedimentation equilibrium in macromolecular solutions of arbitrary concentration. I. Self-associating proteins

Relations describing sedimentation equilibrium in solutions of self-associating macromolecules at arbitrary concentration are presented. These relations are obtained by using scaled-particle theory to calculate the thermodynamic activity of each species present at a given radial distance. The results are expected to be valid for solutions of globular proteins under conditions such that interactions between individual solute molecules may be approximated by a hard-particle potential. Sedimentation equilibria in solutions containing either a nonassociating solute or a solute that self-associates according to several different schemes are simulated using the derived relations. The results of these simulations are presented in terms of the dependence of apparent weight-average molecular weight upon solute concentration. Simple empirical relations are presented for estimating the true weight-average molecular weight from the apparent weight-average molecular weight, without reference to any particular self-association scheme. The weight-average molecular weight estimated in this fashion is within a few percent of the true weight-average molecular weight at all experimentally realizable solute concentrations ( < 400 g/L).     

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.     

Augmented water binding and low cellular water content in erythrocytes of camel and camelids.

We investigated a link between hemoglobin primary structure, hemoglobin hydrophobicity-hydrophilicity, and erythrocyte water content in various mammalian species. Some hemoglobin molecules, particularly those of the camel and camelids, contain more charged amino acid residues and are more hydrophilic than the hemoglobins of human and a number of other mammalian species. To test the in vivo significance of these alterations of hemoglobin primary structure, we determined the osmotically unresponsive erythrocyte water fractions in mannit solutions of various osmolarities at 4 degreesC. Among the species investigated, the size of the osmotically unresponsive erythrocyte water fraction relates in a positive linear way to hemoglobin hydrophilicity. The extreme low total erythrocyte water content of camel erythrocytes (1.1-1.3 g water/g dry mass) may be explained by a comparatively high osmotically unresponsive erythrocyte water fraction. It is proposed that alterations of hemoglobin sequences of camel and camelids may be the part of a natural selection process aimed at protecting these animals against osmotic dehydration in arid environments.     

Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions

Bacteria act to maintain their hydration when the osmotic pressure of their environment changes. When the external osmolality decreases (osmotic downshift), mechanosensitive channels are activated to release low molecular weight osmolytes (and hence water) from the cytoplasm. Upon osmotic upshift, osmoregulatory transporters are activated to import osmolytes (and hence water). Osmoregulatory channels and transporters sense and respond to osmotic stress via different mechanisms. Mechanosensitive channel MscL senses the increasing tension in the membrane and appears to gate when the lateral pressure in the acyl chain region of the lipids drops below a threshold value. Transporters OpuA, BetP and ProP are activated when increasing external osmolality causes threshold ionic concentrations in excess of about 0.05 M to be reached in the proteoliposome lumen. The threshold activation concentrations for the OpuA transporter are strongly dependent on the fraction of anionic lipids that surround the cytoplasmic face of the protein. The higher the fraction of anionic lipids, the higher the threshold ionic concentrations. A similar trend is observed for the BetP transporter. The lipid dependence of osmotic activation of OpuA and BetP suggests that osmotic signals are transmitted to the protein via interactions between charged osmosensor domains and the ionic headgroups of the lipids in the membrane. The charged, C-terminal domains of BetP and ProP are important for osmosensing. The C-terminal domain of ProP participates in homodimeric coiled-coil formation and it may interact with the membrane lipids and soluble protein ProQ. The activation of ProP by lumenal, macromolecular solutes at constant ionic strength indicates that its structure and activity may also respond to macromolecular crowding. This excluded volume effect may restrict the range over which the osmosensing domain can electrostatically interact. A simplified version of the dissociative double layer theory is used to explain the activation of the transporters by showing how changes in ion concentration could modulate interactions between charged osmosensor domains and charged lipid or protein surfaces. Importantly, the relatively high ionic concentrations at which osmosensors become activated at different surface charge densities compare well with the predicted dependence of 'critical' ion concentrations on surface charge density. The critical ion concentrations represent transitions in Maxwellian ionic distributions at which the surface potential reaches 25.7 mV for monovalent ions. The osmosensing mechanism is qualitatively described as an "ON/OFF switch" representing thermally relaxed and electrostatically locked protein conformations.     

Electrokinetic studies of the testosterone-aqueous d-glucose interface

Electro-osmosis and streaming-potential measurements were made across a testosterone-plug membrane, using water and aqueous solutions of d-glucose as permeants. The electrophoretic velocity of testosterone particles dispersed in these solutions was also measured, experiments being confined to the range where linear flux-force relationships hold. Phenomenological coefficients were evaluated by using these linear relations, and the results analyzed inthe light of the thermodynamics of irreversible processes. Saxen's relationship holds between electro-osmosis and streaming potential. Concentration dependence of the various phenomenological coefficients was also examined. Cross-phenomenological coefficients were found to decrease with increase in the concentration of d-glucose solutions. The results are explained on the basis of strong hydrogen-bonding between d-glucose and the surrounding water molecules. Such membrane parameters as pore size, average number of pores, and the membrane constant were evaluated. Electro-osmotic and electrophoretic data were used to estimate the zeta potential, in order to characterize the membrane-permeant interface. The dependence of the zeta potential on the concentration was also examined.     

Size as a parameter for solvent effects on Candida antarctica lipase B enantioselectivity

Changes in solvent type were shown to yield significant improvement of enzyme enantioselectivity. The resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B, CALB, was studied in eight liquid organic solvents and supercritical carbon dioxide, SCCO(2). Studies of the temperature dependence of the enantiomeric ratio allowed determination of the enthalpic (Delta(R-S)Delta H(++)) as well as the entropic (Delta(R-S)Delta S(++)) contribution to the overall enantioselectivity (Delta(R-S)Delta G(++)= -RTlnE). A correlation of the enantiomeric ratio, E, to the van der Waals volume of the solvent molecules was observed and suggested as one of the parameters that govern solvent effects on enzyme catalysis. An enthalpy-entropy compensation relationship was indicated between the studied liquid solvents. The enzymatic mechanism must be of a somewhat different nature in SCCO(2), as this reaction in this medium did not follow the enthalpy-entropy compensation relation.     

Volume regulation by flounder red blood cells in anisotonic media

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.     

Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

Osmotic flow caused by polyelectrolytes

Osmotic flow of water caused by high concentrations of anionic polyelectrolytes across semipermeable membranes, permeable only to solvent and simple electrolyte, has been measured in a newly designed flow cell. The flow cell features small solution and solvent compartments and an efficient stirring mechanism. We have demonstrated that, while the osmotic pressure of the anionic polyelectrolytes is determined primarily by micro-counterions, the osmotic flow is determined by solution-dependent properties as embodied in the hydrodynamic frictional coefficient which is determined by the polymer backbone segment of the polyelectrolyte. The variation of the osmotic permeability coefficient, L(p)(o), with concentration and osmotic pressure closely correlated with the concentration dependence of this frictional coefficient. These studies confirm previous work that the kinetics of osmotic flow across a membrane impermeable to the osmotically active solute is primarily determined by the diffusive mobility of the solute.     

Chain length-dependent interaction of free fatty acids with the erythrocyte membrane

Free fatty acids protect erythrocytes against hypotonic haemolysis in a certain low concentration range and become haemolytic at higher concentrations. The chain length dependence of this biphasic behaviour was investigated using human erythrocytes. The results can be summarized as follows: (i) A critical minimum chain length is required for both effects. Octanoic acid (C8) and fatty acids with a shorter chain length do not have any effect on the osmotic resistance of erythrocytes. (ii) Decanoic acid (C10) decreases the extent of hypo-osmotic haemolysis and does not become haemolytic at higher concentrations. (iii) Dodecanoic acid (C12) represents the minimum chain length for the typical concentration-dependent biphasic behaviour with protection against hypo-osmotic haemolysis at a certain low concentration range and subsequent haemolysis at higher concentrations. (iv) Tetradecanoic acid (C14) exhibits two concentration ranges of protection against hypo-osmotic haemolysis, each followed by haemolytic concentrations. (v) The observed effects are not correlated with the critical micellar concentrations of the investigated fatty acids.     

Na+ Recirculation and Isosmotic Transport

The Na(+) recirculation theory for solute-coupled fluid absorption is an expansion of the local osmosis concept introduced by Curran and analyzed by Diamond & Bossert. Based on studies on small intestine the theory assumes that the observed recirculation of Na(+) serves regulation of the osmolarity of the absorbate. Mathematical modeling reproducing bioelectric and hydrosmotic properties of small intestine and proximal tubule, respectively, predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1 (-/-) mice, and the inverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow. The model reproduces the volume responses of cells and lateral intercellular space (lis) following replacement of luminal NaCl by sucrose as well as the linear dependence of volume absorption on luminal NaCl concentration. Analysis of solvent drag on Na(+) in tight junctions provides explanation for the surprisingly high metabolic efficiency of Na(+) reabsorption. The model predicts and explains low metabolic efficiency in diluted external baths. Hyperosmolarity of lis is governed by the hydraulic permeability of the apical plasma membrane and tight junction with 6-7 mOsm in small intestine and < or = 1 mOsm in proximal tubule. Truly isosmotic transport demands a Na(+) recirculation of 50-70% in small intestine but might be barely measurable in proximal tubule. The model fails to reproduce a certain type of observations: The reduced volume absorption at transepithelial osmotic equilibrium in AQP1 knockout mice, and the stimulated water absorption by gallbladder in diluted external solutions. Thus, it indicates cellular regulation of apical Na(+) uptake, which is not included in the mathematical treatment.     

A model to explain the osmotic pressure behavior of hemoglobin and serum albumin

Previously published osmotic pressure data on hemoglobin and bovine serum albumin were used to determine the osmotically unresponsive solvent volume per unit dry mass of protein. A model is presented that accounts for the osmotic pressure of globular proteins based on a surface-associated osmotically unresponsive solvent volume. The model also accounts for changes in the osmotically unresponsive solvent volume owing to changes in pH, cosolute salt concentration, protein conformation, and protein aggregation.     

Diffusional solute flux during osmotic water flow across the human red cell membrane

The effect of solvent drag on the unidirectional efflux of labeled water, urea, and chloride from human red cells was studied by means of the continuous flow tube method under conditions of osmotic equilibrium and net volume flow. Solvent (water) flow out of cells was created by mixing cells equilibrated in 100 mM salt solution with a 200-mM or 250-mM salt solution, while flow of water into cells was obtained by equilibrating the cells in the higher concentration and mixing them with the 100-mM solution. Control experiments constitute measurements of efflux of [14C]ethanol in normal cells and 3H2O in cells treated with p-chloromercuribenzosulfonate under the conditions described above. In both instances, the solute is known to penetrate the membrane through nonporous pathways. As anticipated, the tracer flux of neither urea nor chloride showed any dependence on net solvent flow, regardless of the direction. If one assumes the recently reported reflection coefficient for urea of 0.7, the urea tracer flux should change by at least 24% under volume flow conditions. Since such changes would be easily detected with our method, we conclude that the pathways for water, for urea, and for chloride are functionally separated.     

Alcohol effects on rapid kinetics of water transport through lipid membranes and location of the main barrier

The effect of 1-alkanols (from 1-butanol up to 1-dodecanol) on the water permeability of dimyristoylphosphatidylcholine vesicle membranes was studied by measuring the osmotic swelling rate as functions of 1-alkanol concentrations and temperatures above the gel-to-liquid-crystalline phase transition. For 1-butanol and 1-hexanol, the activation energy for water permeation was invariant with the addition of alkanols, whereas for 1-octanol, 1-decanol and 1-dodecanol, the activation energy decreased depending on the alkanol concentration, and the extent of the decrease was larger for alkanol with a longer hydrocarbon chain. These results suggests that hydrocarbon moiety beyond seven or eight carbon atoms from the head group in phospholipid molecules constitutes the main barrier for water permeation through the dimyristoylphosphatidylcholine vesicle membrane. The relative volume change of the vesicle due to osmotic swelling increased with the addition of 1-alkanols. Presumably, the membrane structural strength is weakened by the presence of 1-alkanols in the membrane. Contrary to the dependence of the swelling rate upon the alkanol carbon-chain length, no significant difference in the effect on the relative volume changes was seen among the 1-alkanols. This result suggests that weakening of the membrane structure is caused by perturbation of the membrane/water interface induced by incorporation of 1-alkanols into the membrane.