Ion transport through dimethyl sulfoxide (DMSO) induced transient water pores in cell membranes

It is well known that dimethyl sulphoxide (DMSO) increases membrane permeability, which makes it widely used as a vehicle to facilitate drug delivery across biological membranes. However, the mechanism of how DMSO increases membrane permeability has not been well understood. Recently, molecular dynamics simulations have demonstrated that DMSO can induce water pores in biological membranes, but no direct experimental evidence is so far available to prove the simulation result. Using FluxOR Tl? influx assay and intracellular Ca2? imaging technique, we studied the effect of DMSO on Tl? and Ca2? permeation across cell membranes. Upon application of DMSO on CHO-K1 cell line, Tl? influx was transiently increased in a dose-dependent manner. The increase in Tl? permeability induced by DMSO was not changed in the presence of blockers for K? channel and Na?-K? ATPase, suggesting that Tl? permeates through transient water pores induced by DMSO to enter into the cell. In addition, Ca2? permeability was significantly increased upon application of DMSO, indicating that the transient water pores induced by DMSO were non-selective pores. Furthermore, similar results could be obtained from RAW264.7 macrophage cell line. Therefore, this study provided experimental evidence to support the prediction that DMSO can induce transient water pores in cell membranes, which in turn facilitates the transport of active substances across membranes.     

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 mu g/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd=3.34 +/- 0.36 nM without tagetone and Kd,t=5.86 +/- 0.86 nM with tagetone; p<0.05, two tailed Student's t-test) without affecting maximal binding (Bmax=488 +/- 24 fmoles/mg protein, and Bmax,t=500 +/- 25 fmoles/ mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 mu M with and without tagetone respectively). GABA was able to decrease merocyanine Delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 mu M. Tagetone, at a concentration of 50 mu g/ml and in the presence of GABA 30 mu M or 60 mu M, enhanced the ability of GABA alone on decreasing Delta A570-610. Tagetone alone did not change Delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine Delta A value are mainly exerted at the membrane potential level (Delta Psim). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding; on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.     

The mechanism of the stabilization of the hexagonal II (HII) phase in phosphatidylethanolamine membranes in the presence of low concentrations of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO), a water-miscible organic solvent, has been used as a cryoprotectant for cells. It is known that DMSO stabilizes the HII phase of phosphatidylethanolamine (PE) membranes rather than the Lalpha phase, while most other water-miscible organic solvents such as acetone and ethanol destabilize the HII phase. To elucidate the mechanism for this stabilizing effect of DMSO on the HII phase, we have investigated its effects on the structures and physical properties of PE membranes. X-ray diffraction data indicated that dipalmitoleoylphosphatidylethanolamine (DPOPE) membranes in H2O at 20 degrees C were in the Lalpha phase and that an Lalpha to HII phase transition occurred at X=0.060 (mole fraction of DMSO) in water/DMSO mixtures. As the DMSO concentration increased, the basis vector length of the dioleoylphosphatidylethanolamine (DOPE)/ 16 wt% tetradecane membrane and also of the DPOPE/ 16 wt% tetradecane membrane in the HII phase decreased, suggesting that the spontaneous curvature of these membranes increased. We have also investigated the effects of DMSO on the physical properties of the PE membranes, and compared them with those of acetone. As the DMSO concentration increased, the excimer to monomer fluorescence intensities of pyrene-phosphatidylcholine in the PE membranes decreased, indicating that the membrane fluidity decreased, and also the generalized polarization value of the Laurdan fluorescent probe in the DPOPE membrane increased, indicating that the polarity of the membrane interface decreased. On the other hand, acetone had the opposite effects to DMSO. The interaction free energy between the membrane surface segments and solvent increased with an increase in DMSO concentration. It decreased the amount of solvent in the membrane interface, inducing an increase in the spontaneous curvature. This can reasonably explain the effects of DMSO on the phase stability and the physical properties of the membranes.     

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes.

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 micrograms/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd = 3.34 +/- 0.36 nM without tagetone and Kd,t = 5.86 +/- 0.86 nM with tagetone; p < 0.05, two tailed Student's t-test) without affecting maximal binding (Bmax = 488 +/- 24 fmoles/mg protein, and Bmax,t = 500 +/- 25 fmoles/mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 microM with and without tagetone respectively). GABA was able to decrease merocyanine delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 microM. Tagetone, at a concentration of 50 micrograms/ml and in the presence of GABA 30 microM or 60 microM, enhanced the ability of GABA alone on decreasing delta A570-610. Tagetone alone did not change delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine delta A570-610 value are mainly exerted at the membrane potential level (delta psi m). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding: on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.     

Biological membranes as bilayer couples. III. Compensatory shape changes induced in membranes

We have previously proposed the hypothesis that asymmetric membranes behave like bilayer couples: the two layers of the bilayer membrane can respond differently to a particular perturbation. Such a perturbation, for example, can result in the expansion of one layer relative to the other, thereby producing a curvature of that membrane. In experiments with erythrocytes and lymphocytes, we now demonstrate that different membrane perturbations which have opposite effects on membrane curvature can compensate and neutralize one another, as expected from the bilayer couple hypothesis. This provides a rational basis, for example, for understanding the effects of amphipathic drugs on a variety of cellular phenomena which involve shape changes of membranes.     

Ion transport through dimethyl sulfoxide (DMSO) induced transient water pores in cell membranes

Abstract

It is well known that dimethyl sulphoxide (DMSO) increases membrane permeability, which makes it widely used as a vehicle to facilitate drug delivery across biological membranes. However, the mechanism of how DMSO increases membrane permeability has not been well understood. Recently, molecular dynamics simulations have demonstrated that DMSO can induce water pores in biological membranes, but no direct experimental evidence is so far available to prove the simulation result. Using FluxOR Tl⁺ influx assay and intracellular Ca²⁺ imaging technique, we studied the effect of DMSO on Tl⁺ and Ca²⁺ permeation across cell membranes. Upon application of DMSO on CHO-K1 cell line, Tl⁺ influx was transiently increased in a dose-dependent manner. The increase in Tl⁺ permeability induced by DMSO was not changed in the presence of blockers for K⁺ channel and Na⁺-K⁺ ATPase, suggesting that Tl⁺ permeates through transient water pores induced by DMSO to enter into the cell. In addition, Ca²⁺ permeability was significantly increased upon application of DMSO, indicating that the transient water pores induced by DMSO were non-selective pores. Furthermore, similar results could be obtained from RAW264.7 macrophage cell line. Therefore, this study provided experimental evidence to support the prediction that DMSO can induce transient water pores in cell membranes, which in turn facilitates the transport of active substances across membranes.     

Desferrioxamine protects human red blood cells from hemin-induced hemolysis

Hemin binding to red cell membranes, its effect on red cell hemolysis, and it interaction with desferrioxamine (DFO) in these processes were investigated. DFO interacted with hemin via the iron moiety. Blockage of the binding groups in DFO prevented interaction of DFO with hemin, implying the importance of the hydroxamic acid groups in DFO-hemin interactions. Since hemolysis is a result of hemin association with the membrane components, its binding in the presence and absence of DFO was studied. DFO strongly inhibited hemin-induced lysis in a concentration-dependent manner. With 50 microM hemin, 1 mM DFO completely inhibited lysis. Preincubation of ghost membranes with DFO (1 mM) inhibited binding of hemin (50 microM) to membranes by 42%. After ghost membranes were preincubated with hemin (50 microM), the addition of DFO (1 mM) removed 20% of the membrane-bound hemin. It is suggested that DFO may have an important role in alleviating the hemin-induced deleterious effects on the red cell membrane, especially in hemolytic anemias associated with unstable, autoxidized hemoglobins.     

Role of the interaction between puerarin and the erythrocyte membrane in puerarin-induced hemolysis

Adverse drug reactions (ADR), especially intravenous hemolysis, have largely limited the application of puerarin injections in clinics. This study investigated the underlying mechanisms of puerarin-induced hemolysis. Our results show that puerarin induced concentration-dependent and time-dependent hemolysis when human erythrocytes were incubated in saline solution with more than 2 mM puerarin for over 2 h. However, incubation in PBS or addition of 1 mM of lidocaine to the saline solution completely abolished the hemolysis. Providing materials that could start ATP synthesis did not reverse the hemolysis, and puerarin did not affect Na⁺–K⁺–ATPase activity. In addition, puerarin (0.1–2 mM) did not cause calcium influx or exhibited pro-oxidant activity in erythrocytes. Puerarin exhibited different influences on the membrane microviscosity of erythrocytes in saline and PBS. Moreover, 1 mM lidocaine inhibited 8 mM puerarin-induced reduction of membrane microviscosity in saline solution. SDS–PAGE analysis of membrane proteins revealed that 2 mM puerarin treatment induced the appearance of several new protein bands but attenuated the expression of protein bands 2.1, 3, 4.1, 4.2 and 5. These results suggest that high concentrations of puerarin-induced hemolysis were associated with the changes of membrane lipids and of the composition of erythrocytes membrane proteins but not with ATP depletion, pro-oxidation and calcium influx. These changes could be related to the intercalation of amphiphilic puerarin at high concentration into the erythrocyte membrane in certain media, resulting in membrane disorganization and, eventually, cytolysis. Hence, in clinics, determining the optimal dose of puerarin is critical to avoid overdosing and ADR.     

Solvation properties of raft-like model membranes

Dimethyl sulfoxide (DMSO) is a universal water-soluble solvent widely used in many biotechnological and medical applications, such as cells cryopreservation, and for the treatment of different human diseases (e.g. amyloidosis). Despite the great number of reported studies, the effects of DMSO on the physico-chemical properties of biological membranes are poorly understood. Often, these studies are limited to model membranes composed of phosphatidylcholines (PCs) and cholesterol (Chol). In this work, we explored the effect of DMSO on liposomes composed of the natural egg sphingomyelin (ESM) and Chol as raft-like model membranes.With a multi-technique approach we probe the structure and the thermal stability of ESM/Chol bilayer at different Chol mole fractions. In particular, we investigate the ESM-solvent interactions to clarify the role of DMSO in perturbing the solvating conditions of lipid vesicles and show that the addition of DMSO increases the thermal stability of vesicles. An increase of transition temperature, a decrease of both enthalpy and entropy as well as a decrease of the cooperativity of the gel to liquid phase transition are observed at 0.1 DMSO mole fraction. Fluorescence experiments with the probe Laurdan and FTIR spectra strongly indicate that DMSO exerts a dehydration effect on the membrane. Besides, FTIR measurements with tungsten hexacarbonyl, in combination with fluorescence data of the probe NBD-PE, indicate that DMSO promotes the formation of a highly packed membrane by reducing the thickness of the membrane.     

Piracetam inhibits the lipid-destabilising effect of the amyloid peptide Abeta C-terminal fragment

Amyloid peptide (Abeta) is a 40/42-residue proteolytic fragment of a precursor protein (APP), implicated in the pathogenesis of Alzheimer's disease. The hypothesis that interactions between Abeta aggregates and neuronal membranes play an important role in toxicity has gained some acceptance. Previously, we showed that the C-terminal domain (e.g. amino acids 29-42) of Abeta induces membrane permeabilisation and fusion, an effect which is related to the appearance of non-bilayer structures. Conformational studies showed that this peptide has properties similar to those of the fusion peptide of viral proteins i.e. a tilted penetration into membranes. Since piracetam interacts with lipids and has beneficial effects on several symptoms of Alzheimer's disease, we investigated in model membranes the ability of piracetam to hinder the destabilising effect of the Abeta 29-42 peptide. Using fluorescence studies and 31P and 2H NMR spectroscopy, we have shown that piracetam was able to significantly decrease the fusogenic and destabilising effect of Abeta 29-42, in a concentration-dependent manner. While the peptide induced lipid disorganisation and subsequent negative curvature at the membrane-water interface, the conformational analysis showed that piracetam, when preincubated with lipids, coats the phospholipid headgroups. Calculations suggest that this prevents appearance of the peptide-induced curvature. In addition, insertion of molecules with an inverted cone shape, like piracetam, into the outer membrane leaflet should make the formation of such structures energetically less favourable and therefore decrease the likelihood of membrane fusion.     

Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by alpha-tocopherol

The oxidation of rat red blood cells (RBC) by molecular oxygen was performed in an aqueous suspension with an azo compound as a free-radical initiator. The RBC were oxidized at a constant rate by a free-radical chain mechanism, resulting in hemolysis. The extent of hemolysis was proportional to the concentration of free radical. alpha-Tocopherol in RBC membranes suppressed the oxidation and hemolysis to produce an induction period. Tocopherol was constantly consumed during the induction period, and hemolysis developed when tocopherol concentrations fell below a critically low level. Among the membrane lipids, phosphatidylethanolamine, phosphatidylserine, and arachidonic acids were predominantly oxidized in the absence of tocopherol. In the presence of tocopherol, however, such lipid changes were suppressed during a 120-min incubation even when hemolysis started. Membrane proteins as well as lipids were oxidized. The formation of proteins with high molecular weight and concomitant decrease of the low-molecular-weight proteins were observed on gel electrophoresis with the onset of hemolysis. This study clearly showed the damage of RBC membranes caused by oxygen radical attack from outside of the membranes, and suggested that membrane tocopherol even below a critically low level could suppress lipid oxidation but that it could not prevent protein oxidation and hemolysis.     

Piracetam inhibits the lipid-destabilising effect of the amyloid peptide Aβ C-terminal fragment

Amyloid peptide (Aβ) is a 40/42-residue proteolytic fragment of a precursor protein (APP), implicated in the pathogenesis of Alzheimer's disease. The hypothesis that interactions between Aβ aggregates and neuronal membranes play an important role in toxicity has gained some acceptance. Previously, we showed that the C-terminal domain (e.g. amino acids 29-42) of Aβ induces membrane permeabilisation and fusion, an effect which is related to the appearance of non-bilayer structures. Conformational studies showed that this peptide has properties similar to those of the fusion peptide of viral proteins i.e. a tilted penetration into membranes. Since piracetam interacts with lipids and has beneficial effects on several symptoms of Alzheimer's disease, we investigated in model membranes the ability of piracetam to hinder the destabilising effect of the Aβ 29-42 peptide. Using fluorescence studies and ³¹P and ²H NMR spectroscopy, we have shown that piracetam was able to significantly decrease the fusogenic and destabilising effect of Aβ 29-42, in a concentration-dependent manner. While the peptide induced lipid disorganisation and subsequent negative curvature at the membrane-water interface, the conformational analysis showed that piracetam, when preincubated with lipids, coats the phospholipid headgroups. Calculations suggest that this prevents appearance of the peptide-induced curvature. In addition, insertion of molecules with an inverted cone shape, like piracetam, into the outer membrane leaflet should make the formation of such structures energetically less favourable and therefore decrease the likelihood of membrane fusion.     

Membrane solubility of ethanol in chronic alcoholism. The effect of ethanol feeding and its withdrawal on the protection by alcohol of rat red blood cells from hypotonic hemolysis

Membranes from ethanol-fed rats are resistant to the in vitro effects of ethanol on membrane structure and function. We have proposed that the resistance arises from adaptive changes in membrane composition which lower the solubility (partition coefficient) of ethanol in these membranes. The partition of ethanol (and other alcohols and anesthetics) into red blood cells protects the cells from hypotonic hemolysis. Here, we show that the protection by alcohols and anesthetics of red blood cells from ethanol-fed rats is greatly attenuated. This finding indicates that the membrane solubility of these agents is lowered in chronic alcoholism and thus explains the resistance to the acute effects of ethanol. The protection from hemolysis decreases over 2 weeks of ethanol-feeding and returns to normal values within 1 day after ethanol withdrawal. These changes are associated with a parallel increase in total and free serum cholesterol during ethanol feeding and a return to normal values within a day after withdrawal. However, we find only a slight increase in the cholesterol/phospholipid ratio of the red blood cell membranes during the development of ethanol tolerance. In rats fed a cholesterol and saturated fat diet, the increase in serum cholesterol is also associated with an attenuation of the protection from hypotonic hemolysis.     

Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.

We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes.     

The effects of 3-methylindole on hemolysis, transport of Na+, and ATPase activities of bovine erythrocytes.

Biochemical effects of 3MI on cellular membranes were investigated. This study was conducted to examine the effects of 3MI on the hemolysis of erythrocytes, the transport of 22Na+ in resealed erythrocyte ghosts, and on the ATPase activities of erythrocyte membranes. The percent of hemolysis as a function of 3MI incubation time was sigmoidal. Seventy-five percent of the hemoglobin was released with the second 2 hr of incubation during which the concentration of 3MI in the cells reached a plateau of 2500 mug/ml of packed RBC. The effect of 3MI at a subhemolytic concentration on passive and active 22Na+ transport were not significant. The total and Mg2+-dependent ATPase activities in the membranes were significantly increased after 1 hr of incubation with 3MI at concentrations of 100, 200, 300, 400 and 500 mug/ml (P less than or equal to 0/ml (P smaller than or equal to 0.02).     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Mechanisms of hepatic transport of cyclosporin A: an explanation for its cholestatic action?

The hepatic transport of the immunosuppressive Cyclosporin A (CyA) was studied using liposomal phospholipid membranes, freshly isolated rat hepatocytes and bile canalicular plasma membrane vesicles from rat liver. The Na(+)-dependent, saturable uptake of the bile acid 3H-taurocholate into isolated rat liver cells was apparently competitively inhibited by CyA. However, the uptake of CyA into the cells was neither saturable, nor temperature-dependent nor Na(+)-dependent, nor could it be inhibited by bile salts or CyA-derivatives, indicating passive diffusion. In steady state depolarization fluorescence studies, CyA caused a concentration-dependent decrease of anisotropy, indicating a membrane fluidizing effect. Ion flux experiments demonstrated that CyA dramatically increases the permeability of Na+ and Ca2+ across phospholipid membranes in a dose- and time-dependent manner, suggesting a iontophoretic activity that might have a direct impact on cellular ion homeostasis and regulation of bile acid uptake. Photoaffinity labeling with a [3H]-labeled photolabile CyA-derivative resulted in the predominant incorporation of radioactivity into a membrane polypeptide with an apparent molecular weight of 160,000 and a minor labeling of polypeptides with molecular weights of 85,000-90,000. In contrast, use of a photolabile bile acid resulted in the labeling of a membrane polypeptide with an apparent molecular weight of 110,000, representing the bile canalicular bile acid carrier. The photoaffinity labeling as well as CyA transport by canalicular membrane vesicles were inhibited by CyA and the p-glycoprotein substrates daunomycin and PSC-833, but not by taurocholate, indicating that CyA is excreted by p-glycoprotein. CyA uptake by bile canalicular membrane vesicles was ATP-dependent and could not be inhibited by taurocholate. CyA caused a decrease in the maximum amount of bile salt accumulated by the vesicles with time. However, initial rates of [3H]-taurocholate uptake within the first 2.5 min remained unchanged at increasing CyA concentrations. In summary, the data indicate that CyA does not directly interact with the hepatic bile acid transport systems. Its cholestatic action may rather be the result of alterations in membrane fluidity, intracellular effects and an interaction with p-glycoprotein.     

Theory of cation-phospholipid-induced shape changes in a lipid bilayer couple

A quantitative model of ion binding and molecular interactions in the lipid bilayer membrane is proposed and found to be useful in examining the factors underlying such membrane characteristics as shape, sidedness, stability and vesicle size at various cation concentrations. The lipid membrane behaves as a bilayer couple whose preferential radius of curvature depends on the expansion or contraction of one monolayer relative to the other. It is proposed that molecular packing may be altered by electrostatic repulsion of adjacent like-charged phospholipid headgroups, or by bringing two headgroups closer together by divalent cation crossbridging. The surface concentrations of each type of cation-phospholipid complex can be described by simple binding equilibria and the Gouy-Chapman-Stern formulation for the surface potential in a diffuse double layer. The asymmetric distribution of acidic phospholipids in most biological membranes can account for the differential effects of identical ionic environments on either side of the bilayer. The fraction of vesicle material which tends to have a right-side-out orientation may be approximated by a normal distribution about the mean curvature. The theory generates vesicle sidedness distributions that, when fitted to experimental results from human erythrocyte membranes, provide an alternative method of estimating intrinsic cationphospholipid dissociation constants and other molecular parameters of the bilayer. The results also corroborate earlier suggestions that the Gouy-Chapman theory tends to overestimate free counter-ion concentrations at the surface under large surface potentials.     

Effect of salicylate on the elasticity, bending stiffness, and strength of SOPC membranes

Salicylate is a small amphiphilic molecule which has diverse effects on membranes and membrane-mediated processes. We have utilized micropipette aspiration of giant unilamellar vesicles to determine salicylate's effects on lecithin membrane elasticity, bending rigidity, and strength. Salicylate effectively reduces the apparent area compressibility modulus and bending modulus of membranes in a dose-dependent manner at concentrations above 1 mM, but does not greatly alter the actual elastic compressibility modulus at the maximal tested concentration of 10 mM. The effect of salicylate on membrane strength was investigated using dynamic tension spectroscopy, which revealed that salicylate increases the frequency of spontaneous defect formation and lowers the energy barrier for unstable hole formation. The mechanical and dynamic tension experiments are consistent and support a picture in which salicylate disrupts membrane stability by decreasing membrane stiffness and membrane thickness. The tension-dependent partitioning of salicylate was utilized to calculate the molecular volume of salicylate in the membrane. The free energy of transfer for salicylate insertion into the membrane and the corresponding partition coefficient were also estimated, and indicated favorable salicylate-membrane interactions. The mechanical changes induced by salicylate may affect several biological processes, especially those associated with membrane curvature and permeability.     

The mechanism of the hemolytic activity of polyene antibiotics

The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca2+ or Ba2+ but not by Mg2+, Na+ or SO42-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO4(2-) to permeate.