Changes in the lipid physical state in human erythrocyte membranes subjected lead exposure in vitro

The effect of lead acetate on the physical state of membrane lipids in human erythrocytes in vitro was studied using the lipophilic fluorescence probe 1,6-diphenyl-1,3,5-hexatriene and spin probes 16-doxyl-stearate and iminoxyl palmitic acid. It was shown that 2-10 microM lead acetate causes an increase in both intensity and polarization of fluorescence of 1,6-diphenyl-1,3,5-hexatriene, indicating changes in the microviscosity of the lipid bilayer of erythrocyte membranes. Judging from the parameters of EPR spectra of 16-doxyl stearate and iminoxyl palmitic acid incorporated into erythrocyte membranes, 2-10 microM lead acetate increases the heterogeneity of the lipid bilayer in surface and deep hydrophobic layers of the erythrocyte membrane.     

Effect of the external electrostatic field on the conformation of a macromolecule containing charged groups

A method for calculating the free energy of a macromolecule containing charged groups in electrostatic field in aqueous solution was proposed. The non-electrostatic component of free energy was calculated with consideration of van der Waals interactions between uncharged parts of the macromolecule. The electrostatic component of free energy was calculated with regard for the interactions of charged groups of the macromolecule with each other and with water molecules. It was found that, depending on the strength of external electric field, the free energy of the system passes through a minimum, whereas the internal energy passes through a maximum. By minimizing the free energy, relative changes in the mean radius 'r' and the distance between the termini of the macromolecule 'h' were calculated. It was found that, at some values of field strength, both 'r' and 'h' decrease. An increase in strength led to an increase in 'r' and 'h'. The regularities observed depend on the charge of the macromolecule and the spatial redistribution of macromolecules and counterions.     

The electrochemical stability of erythrocyte membranes

The temperature measurements of the parameters of the medium kinetic pH curves when realizing the electrical breakdown of the erythrocyte membranes at the expense of the diffusional potential difference have been carried out. The energy values of the activation processes in antiport Cl-/OH- and potassium ions going out of the cells have been calculated. The values of activation energy of the given processes are different in the temperature range lying lower and higher 30-37 degrees C interval what the authors connect with the ratio change of contributions of lipid and protein components into kinetics of the processes. The values of the activation energy processes when erythrocyte mass was previously incubated with pro-oxidants, as well exposed to ultraviolet radiation have been obtained.     

The polyphosphoinositide phosphodiesterase of erythrocyte membranes

1. A new assay procedure has been devised for measurement of the Ca(2+)-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [(32)P]P(i). They are incubated under appropriate conditions for activation of the phosphodiesterase and the released (32)P-labelled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. 2. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5mm-Ca(2+). For both esters, optimum rates of release were seen at pH6.8-6.9. Mg(2+) did not provoke release of either ester. 3. Ca(2+) provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca(2+). 4. In the presence of Mg(2+), the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. 5. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. 6. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca(2+) and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bisphosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg(2+)-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.     

The influence of steroid hormones on in vitro NOx production by porcine fetal membranes

The aim of the study was to examine: 1/ allantochorial concentrations of nitrate/nitrite (NOx) and 2/ plasma concentration of NOx in pigs on days 25, 35, 40 and 60 of pregnancy as well as 3/ the influence of estradiol-17beta (E(2)) and/or progesterone (P(4)) on NOx production by porcine fetal membranes on the studied days of pregnancy. Total NOx concentration was determined using a microplate assay method based on the Griess reaction. Fetal membrane NOx content gradually increased from day 25 to day 60 of gestation. Blood plasma NOx concentration decreased from day 25 to 40, and then plasma NOx concentration significantly increased on day 60. In addition, the stimulatory effect of E(2), P(4) and E(2)+P(4) on NO in vitro production by porcine fetal membranes was demonstrated. The stimulatory effect of steroid hormones on NOx release depended on steroid dose and day of pregnancy. It is possible that the observed differences in the strength of the stimulatory action of E(2), P(4) and E(2)+P(4) on fetal membrane NOx production are associated with an activation of different isoforms of nitric oxide synthase.     

The effects of erythrocyte membranes on the nucleation of sickle hemoglobin

Pathology in sickle cell disease begins with nucleation-dependent polymerization of deoxyhemoglobin S into stiff, rodlike fibers that deform and rigidify red cells. We have measured the effect of erythrocyte membranes on the rate of homogeneous nucleation in sickle hemoglobin, using preparations of open ghosts (OGs) with intact cytoskeletons from sickle (SS) and normal adult (AA) red cells. Nucleation rates were measured by inducing polymerization by laser photolysis of carboxy sickle hemoglobin and observing stochastic variation of replicate experiments of the time for the scattering signals to reach 10% of their respective maxima. By optical imaging of membrane fragments added to a hemoglobin solution we contrast the rate of nucleation immediately adjacent to membrane fragments with nucleation in a region of the same solution but devoid of membranes. From analysis of 29,272 kinetic curves obtained, we conclude that the effect of AA OGs is negligible (10% enhancement of nucleation rates +/-20%), whereas SS OGs caused 80% enhancement (+/-20%). In red cells, where more membrane surface is available to Hb, this implies enhancement of nucleation by a factor of 6. These experiments represent a 10-fold improvement in precision over previous approaches and are the first direct, quantitative measure of the impact of erythrocyte membranes on the homogeneous nucleation process that is responsible for polymer initiation in sickle cell disease.     

The influence of the molecular structure of lipid membranes on the electric field distribution and energy absorption

We consider the influence of the molecular structure of phospholipid membranes on their dielectric properties in the radio frequency range. Membranes have a stratified dielectric structure on the submolecular level, with the lipid chains forming a central hydrophobic layer enclosed by the polar headgroups (HGs) and bound water layers. In our numerical model, isotropic permittivities of 2.2 and 48.8 were assigned to the lipid chain and bound water layers, respectively. The HG region was assumed to possess an anisotropic static permittivity with 142.2 and 30.2 in the tangential and normal directions, respectively. The permittivities of the HG and bound water regions have been assumed to disperse at frequencies around 51 and 345 MHz to become 2.2 and 1.8, respectively, in both the normal and tangential directions. Electric field distribution and absorption were calculated for phospholipid vesicles with 75 nm radius as an example. Significant absorption has been obtained in the HG and bound water regions. Averaging the membrane absorption over the layers resulted in a decreased absorption below 1 GHz but a more than 10-fold increase above 1 GHz, compared to a model with a homogeneous membrane of averaged properties. We propose single particle dielectric spectroscopy by AC electrokinetics at low-bulk medium conductivities for an experimental verification of our model.     

In vitro effects of ozone on human erythrocyte membranes: an EPR study

The effects of ozone at different concentrations (10, 30, 45 g/m3) on fluidity and thermotropic properties of erythrocyte membranes were investigated by EPR using two spin probes: 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA). The effect of ozone on the erythrocyte membrane fluidity was a dose-dependent process. The ozone at concentration of 10 g/m3 caused rigidization of the membrane while at concentration of 45 g/m3 increased fluidity both on the surface and in the deeper hydrocarbon region of the membrane. Temperature transitions close to the polar heads region (monitored by 5-DSA) were not sensitive to an increase in ozone concentration. In the case of 16-DSA, low temperature thermotropic transition (around 20 degrees C) gradually decreased with the increase of ozone concentration. High temperature transition (around 40 degrees C) significantly differed at the ozone concentration of 10 g/m3 and 45 g/m3, being higher and lower, respectively, as compared to untreated cells. For the ozone concentration of 45 g/m3 the disappearance of the low temperature break and the appearance of two breaks at 37 degrees C and 16 degrees C were observed.     

Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

Treatment of microsomal membranes from cotyledons of Phaseolus vulgaris with ozone raises the liquid-crystalline to gel lipid phase transition temperature and results in the formation of distinct domains of gel phase lipid in the membranes. Liposomes prepared from the total lipid extracts of ozone-treated membranes undergo phase separations just a few degrees below the transition temperature for intact membranes, indicating that the formation of gel phase lipids is largely attributable to ozone-induced alterations in the membrane lipids. Levels of unsaturated fatty acids as well as the sterol to phospholipid ratio are markedly reduced in the ozone-treated membranes, and the neutral lipid fraction from treated membranes shows, an increased propensity to induce the formation of gel phase phospholipid when incorporated into liposomes of egg phosphatidylcholine. Since gel phase phospholipid also forms in naturally senescing plant membranes and appears to be attributable to changes in the neutral lipid fraction, the effects of natural senescence and ozone on membranes have been compared.     

Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models

Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada. Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 μM; (c) X-ray diffraction studies showed that PPA in the 0.1–0.5 mM range induced increasing structural perturbation to DMPC, but no effects on DMPE multibilayers were detected.     

The effect of isoflurane on erythrocyte membranes studied by ATR-FTIR

The effect of isoflurane on erythrocyte membranes has been investigated by means of attenuated total reflection infrared spectroscopy. Infrared spectra were measured on sonicated erythrocyte ghosts layered upon a ZnSe crystal covered with D(2)O saline solutions containing increasing amounts of isoflurane. At clinically relevant anesthetic concentrations and 37 degrees C, significant changes in the structural and dynamic properties of the membrane phospholipid bilayers are observed. Both the acyl chain methylene symmetric and asymmetric stretching modes and the carbonyl ester stretching band displayed frequency shifts interpreted as transitions toward disordered liquid-like structure accompanied by dehydration of the phospholipid polar heads. In turn, no secondary structure-linked changes are observed in the amide I region of membrane proteins. Higher anesthetic concentrations (500-900 microM), resulted in progressive detachment of the multilayers from the ATR crystal and irreversible formation of denatured protein. Polarization studies in correspondence of the acyl lipid methylene stretching bands indicated that isoflurane decreases the dichroic ratio thus inducing disorder in the orientation of the lipid acyl chains.     

NMR investigation of the influence of procaine and its metabolites on the water exchange through human erythrocyte membranes

The effect of procaine hydrochloride and its metabolites on the diffusional water exchange through erythrocyte membranes was investigated at 37 degrees C and at concentrations ranging between 5 X 10(-5) M and 5 X 10(-1) M by using the NMR manganese doping method. Procaine hydrochloride and 2-diethylaminoethanol have a moderate stimulating effect on the water exchange, of up to 20% at concentrations ranging between 10(-3) and 10(-2) M, while an increasing inhibitory effect was found at higher concentrations. The p-aminobenzoic acid has no effect on the water exchange up to 10(-2) M and, at higher concentrations, and apparent decreasing inhibition was noticed which is thought to be an artefact due to the uptake of Mn2+ by the cells. The temperature dependence studies suggest that procaine HCl enhances the uptake of Mn2+ by the cells. An opposite effect was found for rigid erythrocytes. The p-aminobenzoic acid and 2-diethylaminoethanol appeared to be more effective than procaine hydrochloride in increasing the uptake of Mn2+.