Albumin enhances the diffusion of lipophilic drugs into the membrane bilayer

In earlier work, we reported on the manner with which lipophilic drug molecules interact with the cell membrane in order to (a) enter the bilayer and laterally diffuse to their respective protein sites of action, or (b) penetrate this biological barrier to reach the cell interior. A remaining uncertainty is how lipophilic molecules reach the hydrophobic membrane core after traversing the aqueous medium and membrane polar surface. Here we present preliminary data using deuterium NMR, demonstrating the role of bovine serum albumin in facilitating this process. Our observation allows us to postulate a mechanism by which the passive transport of lipophilic ligands across the membrane can be greatly enhanced through the assistance of carrier proteins.     

Neither lipophilicity nor membrane-perturbing potency of phenothiazine maleates correlate with the ability to inhibit P-glycoprotein transport activity

Although phenothiazines are known as multidrug resistance modifiers, the molecular mechanism of their activity remains unclear. Since phenothiazine molecules are amphiphilic, the interactions with membrane lipids may be related, at least partially, to their biological effects. Using the set of phenothiazine maleates differing in the type of phenothiazine ring substitution at position 2 and/or in the length of the alkyl bridge-connecting ring system and side chain group, we investigated if their ability to modulate the multidrug resistance of cancer cells correlated with model membrane perturbing potency. The influence exerted on lipid bilayers was determined by liposome/buffer partition coefficient measurements (using the absorption spectra second-derivative method), fluorescence spectroscopy and calorimetry. Biological effects were assessed by a flow cytometric functional test based on differential accumulation of fluorescent probe DiOC(2)(3) by parental and drug-resistant cells. We found that all phenothiazine maleates were incorporated into lipid bilayers and altered their biophysical properties. With only few exceptions, the extent of membrane perturbation induced by phenothiazine maleates correlated with their lipophilicity. Within the group of studied derivatives, the compounds substituted with CF(3)- at position 2 of phenothiazine ring were the most active membrane perturbants. No clear relation was found between effects exerted by phenothiazine maleates on model membranes and their ability to modulate P-glycoprotein transport activity.     

A Fickian diffusion transport process with features of transport catalysis. Doxorubicin transport in human red blood cells

The transport of the antineoplastic drug doxorubicin (Adriamycin) in human red blood cells was investigated by measuring the net efflux from loaded cells. Previous data indicated that doxorubicin transport was a Fickian diffusion transport process of the electrically neutral molecule through the lipid domain of the cell membrane (Dalmark, 1981 [In press]). However, doxorubicin transport showed saturation kinetics and a concentration-dependent temperature dependence with nonlinear Arrhenius plots. The two phenomena were related to the doxorubicin partition coefficient between 1-octanol and a water phase. This relationship indicated that the two phenomena were caused by changes in the physiochemical properties of doxorubicin in the aqueous phase and were not caused by interaction of doxorubicin with cell membrane components. The physicochemical properties of doxorubicin varied with concentration and temperature because of the ability of doxorubicin to form polymers by self-association in aqueous solution like other planar aromatic molecules through pi-electron orbital interaction. The hypothesis is proposed that doxorubicin transport across cell membranes takes place by simple Fickian diffusion.     

Cationomycin and monensin partition between serum proteins and erythrocyte membrane: consequences for Na+ and K+ transport and antimalarial activities

The ionophore properties of cationomycin and monensin were studied on human erythrocytes by measuring Na+ influx by 23Na NMR and concomitant K+ efflux by potentiometry in the presence of increasing amounts of serum. Both ion currents (Na+ or K+) decreased linearly with the reciprocal of serum amount. The serum effects on ion currents were stronger with cationomycin than with monensin. Assuming this decreased transport activity was due to drug binding to serum proteins, a partition coefficient between the protein and the membrane phase was determined for each ionophore by using a novel model. This partition coefficient is about 30 times higher for cationomycin than for monensin; the same result was obtained with purified human serum albumin, indicating that albumin may be the major ionophore binding protein of serum. In parallel, we also measured IC50 for 50% in vitro growth inhibition of Plasmodium falciparum, the agent of malaria. In the presence of increasing serum concentrations, the antimalarial activity was decreased for both ionophores. Serum effect was less severe for monensin than for cationomycin, in agreement with the weaker interaction of monensin with proteins as shown from the partition coefficient values. A correlation was established between the ion transport currents (sodium and potassium) and the IC50 measured on P. falciparum in the presence of the various concentrations of serum. The relative value of the ion transport currents (expressed as percentage of control in absence of serum) can be indicative of the ionophore unbound fraction in the medium.     

Assessing the effects of surfactants on the physical properties of liposome membranes

Knowledge of the partition process of environmentally significant molecules between biological membranes and their surroundings is of vital importance to explain their activity and toxicity, as well as phenomena like absorption, distribution and metabolism. In this research effort, we have studied membrane interactions of three surfactants: t-octylphenoxypolyethoxyethanol (Triton X-100), cetyltrimethylammonium chloride (CTAC) and dodecylbenzene sulphonate (SDBS). Unilamellar liposomes (LUVs) of egg yolk phosphatidylcholine (EPC) were used as membrane models. The partition coefficient, a fundamental parameter in assessing the behaviour of xenobiotic compounds, was determined for SDBS and Triton X-100 by derivative spectrophotometry and fluorescence quenching. The effect of these surfactants upon the physico-chemical characteristics (fluidity, diameter and surface charge) of the liposome membrane was also determined. Results show that all the three surfactants cause an increase in fluidity of the liposome membrane, although for low surfactant concentrations uncharacteristic membrane rigidity was observed, probably due to a change in lipid packing density.     

Membrane processes associated with the osmotic-pulse incorporation of inositol hexaphosphate

In previous studies (Biochem. Biophys. Res. Commun. 144, 779-786 (1987); Prog. Clin. Biol. Res. 292, 65-75 (1989)), we showed that inositol hexaphosphate (IHP), when added to erythrocyte membrane ghosts in the range 0.6-2.5 mM, caused a large disruption of skeletal protein-protein interactions as monitored by electron paramagnetic resonance techniques. IHP incorporated into intact cells by an osmotic-pulse method (J. Cell. Physiol. 129, 221-229 (1986)) leads to cells with markedly decreased oxygen affinity. Exposure of the red cells to higher levels of IHP during the osmotic pulse leads to less lysis and more normal cellular indices after healing of the transiently-disrupted membrane (J. Lab. Clin. Med. 113, 58-66 (1989)). In order to determine what effect higher levels of IHP had on skeletal proteins and bilayer lipids of membrane ghosts, spin labeling studies were performed. The main findings were: (a) There was a concentration-dependent alteration in skeletal protein interactions. At concentrations greater than 25 mM IHP, the effectiveness of IHP to disrupt skeletal protein interactions was diminished. (b) No apparent alteration of the motion or order of phospholipids or the lipid water interface of intact cells into which IHP was incorporated occurred, suggesting that higher levels of IHP do not alter the physical state of the lipid bilayer.     

Subfractionation of cell populations by partitioning in dextran-poly (ethylene glycol) aqueous phases

Partitioning of cells in dextran-poly(ethylene glycol) aqueous two-phase systems depends on the interaction between the surface properties of the cells and the physical properties of the phases. The latter can be manipulated to a considerable extent by selection of polymer concentrations and ionic composition and concentration. If salts (e.g., phopshate) are used that have an unequal affinity for the two phases, an electrostatic potential difference between the phases results and, at appropriately high polymer concentrations, the partition coefficient of cells is determined predominantly by membrane charge-associated properties. By “balancing” the magnitude of the electrostatic potential difference against that of the interfacial tension (primarily a function of polymer, but also phosphate, concentrations) one can obtain phase systems that give usable partition coefficients for most cell populations (1). In work under way in our laboratory on the effects of different chemical and enzymatic modifications on the relative surface properties of rat red blood cells of different ages, we have now found that certain phase compositions did not resolve such treated cell subpopulations while other phase compositions did. Thus not all charged phase systems in which cell populations as a whole have usable partition coefficients are equally capable of detecting or subfractionating cell subpopulations. It is therefore essential, before drawing conclusions on the nonseqarability of cell subpopulations, to test cell separability in charged phase systems of different compositions if the system initially chosen does not afford a subfractionation.     

Inositol tripyrophosphate: a new membrane permeant allosteric effector of haemoglobin

Nine inositol tripyrophosphate (ITPP) salts have been synthesized. Their ability to act as allosteric effectors of haemoglobin (Hb) has been measured in vitro with free Hb and whole blood. All the synthesized compounds bound to free Hb and were also able to cross, to a certain extent, the plasma membrane of the red blood cells (RBCs) in whole blood samples, lowering the affinity of Hb for oxygen. The oxy-haemoglobin dissociation curves were significantly shifted towards higher values of oxygen partial pressures, both for free Hb and for intracellular Hb in whole blood.     

Whole blood optical biosensor

The future of rapid point-of-care diagnostics depends on the development of cheap, noncomplex, and easily integrated systems to analyze biological samples directly from the patient (e.g. blood, urine, and saliva). A key concern in diagnostic biosensing is signal differentiation between non-specifically bound material and the specific capture of target molecules. This is a particular challenge for optical detection devices in analyzing complex biological samples. Here we demonstrate a porous silicon (PSi) label-free optical biosensor that has intrinsic size-exclusion filtering capabilities which enhances signal differentiation. We present the first demonstration of highly repeatable, specific detection of immunoglobulin G (IgG) in serum and whole blood samples over a typical physiological range using the PSi material as both a biosensor substrate and filter.     

Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface

The virulence of the malaria parasite Plasmodium falciparum is related to its ability to express a family of adhesive proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the infected red blood cell surface. The mechanism for the transport and delivery of these adhesins to the erythrocyte membrane is only poorly understood. In this work, we have used specific immune reagents in a flow cytometric assay to monitor the effects of serum components on the surface presentation of PfEMP1. We show that efficient presentation of the A4 and VAR2CSA variants of PfEMP1 is dependent on the presence of serum in the bathing medium during parasite maturation. Lipid-loaded albumin supports parasite growth but allows much less efficient presentation of PfEMP1 at the red blood cell surface. Analysis of the serum components reveals that lipoproteins, especially those of the low-density lipoprotein fraction, promote PfEMP1 presentation. Cytoadhesion of infected erythrocytes to the host cell receptors CD36 and ICAM-1 is also decreased in infected erythrocytes cultured in the absence of serum. The defect appears to be in the transfer of PfEMP1 from parasite-derived structures known as the Maurer's clefts to the erythrocyte membrane or in surface conformation rather than a down-regulation or switching of particular PfEMP1 variants.     

Transport of steroid hormones facilitated by serum proteins.

The affinities with steroid hormones (alpha-estradiol, ethynylestradiol, progesterone, androsterone, dehydroisoandrosterone and testosterone) were observed for Cohn's fraction IV-1 and V (albumin). It was estimated from the comparison with the binding coefficient K (protein-bound form/free form of hormone) in a 3.5% (w/v) bovine serum albumin (BSA) solution that 40-80% of bound hormone in bovine serum is the BSA-bound form. It becomes clear in a liquid membrane system consisting of a hexane source phase (I), a water phase and a hexane receiving phase (II) that the transport flux of hormone is governed primarily by the partition coefficients between the water/hexane phases. In the case of a hormone with a lower partition coefficient, the uptake process from the hexane phase (I) to the water phase is a rate-determining step in the transport system and the serum proteins accelerate the transport of hormones, while with an increase in the partition coefficient the rate-determining step changes from the uptake step to the release step from the water phase to the hexane phase (II) and the hormone transport is decelerated owing to the significant decrease of free hormone concentration in the aqueous phase by the associated with serum proteins for the system having the restricted amount of hormone in the hexane source phase.     

Cerebrocrast promotes the cotransport of H+ and Cl- in rat liver mitochondria

Considering that cerebrocrast stimulates oligomycin-inhibited state 3 respiration simultaneously with mitochondrial transmembrane potential (Deltapsi) dissipation, the mechanism underlying the uncoupler activity of cerebrocrast was assessed by its ability to permeabilize the mitochondrial inner membrane to H(+) or to K(+) or to cotransport anions with H(+). The partition coefficient of cerebrocrast in mitochondrial membrane and its ability to act as a membrane-active compound disturbing membrane lipid organization were also investigated. Cerebrocrast induced no permeabilization of mitochondrial inner membrane to H(+) or K(+), but it was able to transport H(+) in association with Cl(-). Cerebrocrast showed a strong incorporation into the mitochondrial membrane, with a partition coefficient (Kp(m/w)) of 2.7(+/-0.1)x10(5). Cerebrocrast also reduced, in a concentration dependent manner, the phase transition temperature, the cooperative unit size, and the enthalpy associated with the phase transition temperature of DMPC membrane bilayers. It was concluded that the uncoupler activity of cerebrocrast is due to its ability to promote the cotransport of H(+) with Cl(-) through the rat liver mitochondrial inner membrane, and that this cerebrocrast mechanism of action may be potentiated by alterations of membrane lipid organization and membrane lateral heterogeneity.     

A quantitative structure-activity approach to chemoreception: Importance of lipophilic properties

A multiparameter regression analysis utilizing physico-chemical parameters, and commonly known as the Hansch approach has been extended to a set of existing data on olfactive threshold and olfactive stimulatory effectiveness of organic molecules.The compatibility of various parameters has been studied with adequate statistical methods, and we conclude that the lipophilic character (expressed by the octanol/water partition coefficient) of the molecules is an essential parameter related to preliminary non-specific action and transport across the muceous/lipid interphase and the membrane of the olfactive organ.Such an approach allows the refocusing of the problem of the relationship between structure and olfactive properties.     

The transport of potassium through lipid bilayer membranes by the neutral carriers valinomycin and monactin

Summary Stationary conductance experiments on neutral and negatively charged bilayer membranes in the presence of valinomycin or monactin agree with a recently proposed carrier transport model, which is common to both carrier types. This model assumes an interface reaction between a cation from the aqueous solution and a carrier molecule from the membrane phase to establish charge transport across the interface. The transport across the membrane interior is described by some kind of Eyring model. The discussion of the current-voltage characteristic, the dependence of membrane conductance on the carrier and K⁺ concentrations, and the comparison with appropriate experiments allow correlation of the different rate constants of the transport model. The results show that the rate constants partly depend on the surface charge of the membranes. This dependency can be described by introducing the Gouy-Chapman theory for charged surfaces into the transport model.It was found that the carrier molecules could be added either to the aqueous phase or to the membrane-forming solution. The quantitative treatment of this phenomenon gives an evaluation of the partition coefficient of the carrier molecules between the membrane bulk phase and water.     

Lipid-mobilizing effect of reduced glutathione and its intensifying action on lecithin-cholesterol acyltransferase activity in blood serum of rats

Effect of reduced glutathione (50 mg/100 g) on lipid distribution between organs (liver and kidney) and lecithin-cholesterol acyltransferase (LCAT) activity in blood serum of rats was investigated. The accumulation of common lipids as a result of speeding up the absorbtion of blood serum unsaturated fatty acids and relative decrease of lipids unsaturation in the liver and lipid content dynamics in kidneys owing to the intensification of two processes in this organ: the transport of polyene fatty acids in composition of blood serum lipoprotein lipids to kidney cells and peroxidation of membrane phospholipids were found out. The activating effect of GSH (in vivo and in vitro) on LCAT activity of rat blood serum was shown. It was summarised that GSH-intensification of blood serum etherification ability may be a basic component of reduced glutathione lipid mobilization effect.     

Some Observations on the Partition of Na+ and K+ into a Lipid Phase

It is likely that sodium and potassium must traverse a lipid membrane surrounding cells and that this membrane has to do with intracellular cation selection. Electrolyte theory is inadequate to predict the partition of salts of these cations between water and a lipid phase. The data obtained here demonstrate the partition and the cation selection to be a function of the anion species (or ionized lipid), the solvent and the presence of the unionized form of the lipid. Specificity in lipid partition is not synonymous with the cation specificity in precipitation of ionic crystals.     

Amantadine partition and localization in phospholipid membrane: a solution NMR study

Quantification of membrane partition potential of drug compounds is of great pharmaceutical interest. Here, a novel approach combining liquid-state NMR diffusion measurements and fast-tumbling lipid/detergent bicelles is used to measure accurately the partition coefficient K(p) of amantadine in phospholipid bilayers. Amantadine is found to have a strong membrane partition potential, with K(p) of 27.6 in DMPC and 37.8 in POPC lipids. Electrostatic interaction also plays a major role in the drug's affinity towards biological membrane as introduction of negatively charged POPG dramatically increases its K(p). Saturation transfer difference experiments in small bicelles indicate that amantadine localizes near the negatively charged phosphate group and the hydrocarbon chain of bilayer lipid. The approach undertaken in this study is generally applicable for characterizing interactions between small molecules and phospholipid membranes.     

Isolation and purification of bacterial membrane proteins by the use of organic solvents: The lactose permease and the carbodiimide-reactive protein of the adenosinetriphosphatase complex of escherichia coli

Techniques for the solubilization and fractionation of integral membrane proteins have been developed in recent years. A small portion of membrane protein (about 2%, proteolipid fraction) will partition into chloroform or 1-butanol, and, in several cases, these proteins retain functional activity. A virtually complete solubilization can be achieved at neutral pH by use of aprotic solvents, like hexamethylphosphoric triamide or N-methylpyrrolidone. At relatively low concentrations (< 3 M) aprotic solvents inhibited β-D-galactoside transport by whole cells and the derivative membrane vesicles of Escherichia coli, but this inhibition could be largely reversed by a simple washing procedure. At higher concentrations of aprotic solvent (5–6 M), 50–80% of the total protein of lactose transport-positive membrane vesicles was solubilized. When these extracts were added to intact lactose transport-negative membrane vesicles, lactose transport was reconstituted, the required energy being provided by either respiration (e.g., addition of D-lactate) or by a K⁺ diffusion potential established with the aid of valinomycin. The dicyclohexylcarbodiimide (DCCD)-reactive subunit of the E. coli ATPase complex was found to partition into chloroform, and to be amenable to further purification in organic solvent. Ether precipitation and chromatography on DEAE-cellulose and hydroxypropyl-Sephadex G-50 yielded an homogeneous polypeptide of an apparent molecular weight of 9,000. The purified and unlabeled DCCD-reactive protein was incorporated into K⁺-loaded liposomes, and a membrane potential was generated by the addition of valinomycin. There are indications that the DCCD-reactive protein alone made the membrane specifically permeable for protons.     

Different Permeability of Potassium Salts across the Blood-Brain Barrier Follows the Hofmeister Series

The passage of ions across biological membranes is regulated by passive and active mechanisms. Passive ion diffusion into organs depends on the ion-pairing properties of salts present in the serum. Potassium ions could affect brain activity by crossing the blood-brain barrier (BBB) and its accumulation in the extracellular cerebral space could precipitate seizures. In the present study, we analyze passive diffusion of a series of potassium salts in the in vitro isolated guinea pig brain preparation. Different potassium counter-anions confer ion-pairing and lipophilicity properties that modulate membrane diffusion of the salt. Extracellular recordings in different cortical areas demonstrated the presence of epileptiform activities that strongly relate to anion identity, following the qualitative order of the Hofmeister series. Indeed, highly lipophilic salts that easily cross the BBB enhanced extracellular potassium concentration measured by ion-selective electrodes and were the most effective pro-epileptic species. This study constitutes a novel contribution for the understanding of the potential epileptogenicity of potassium salts and, more generally, of the role of counter-anions in the passive passage of salts through biological membranes.     

二甲基亚砜对生物膜的作用机理

二甲基亚砜被广泛应用于生物、化学和药学领域,这些应用大多与其增加生物膜的通透性、促进活性分子跨膜传输的作用密切相关。本文对二甲基亚砜增加生物膜通透性的理论及实验研究做简要综述,主要强调二甲基亚砜在生物膜中诱导水性孔道形成的分子动力学模拟及其相关的实验研究。