Interaction of pentoxifylline with human erythrocytes. I. Interaction of xanthine derivatives with human erythrocyte ghosts

The interaction of pentoxifylline and other xanthine derivatives with human erythrocyte ghosts was studied. By fluorescence spectroscopy it was found that xanthine derivatives have two modes of binding to erythrocyte ghosts. One is a high-capacity binding to erythrocyte membranes. It seems that the 5-oxohexyl side chain of pentoxifylline is important for this. The second type may be a binding to proteins on the membranes and is specific for pentoxifylline and caffeine. From the circular dichroism spectra, it was presumed that the second binding mode of pentoxifylline occurs at hydrophobic regions of beta-structure of the membrane proteins. The relative high specificity in the interaction of pentoxifylline with erythrocytes should be related to its unique physiological activity on erythrocytes.     

顺-二氯二氨合铂(Ⅱ)与红细胞膜蛋白的相互作用

顺铂可与红细胞膜蛋白结合,并使膜收缩蛋白交联聚合,巯基是顺铂的可能结合部位,顺铂造成膜蛋白构象或在膜内组装的改变,这些结果支持了我们提出的顺铂作用的多靶分子模型。     

PHOTOCHEMICAL BINDING OF PHENOTHIAZINES ON BIOLOGICAL MEMBRANE PROTEINS

Abstract— The photobinding of phenothiazine derivatives (chlorpromazine, fluphenazine, promazine and promethazine) was studied on four different types of biological membranes (microsomes, myelin and synaptosomes from rat brain as well as human erythrocytes). The photoreaction was performed by ultraviolet irradiation of the tritiated compounds in their long wavelength absorption band (313 nm) and bound photoproducts were analysed by autoradiography of the proteins separated by polyacrylamide gel electrophoresis. The specificity of binding is low, however, a 34000 dalton band is intensely labeled on synaptic membranes with chlorpromazine and fluphenazine. All the phenothiazines bind on erythrocyte membrane proteins and specially on band 4.2 and on a peptide located before actin on the electrophoresis gel. These results show the generality of the phenothiazine photobinding on membrane proteins. These photobinding properties can be used for the identification and localization of some of these proteins.     

ERYTHROPOIETIC PROTOPORPHYRIA: PHOTODYNAMIC TRANSFER OF PROTOPORPHYRIN FROM INTACT ERYTHROCYTES TO OTHER CELLS

Erythrocytes in patients with erythropoietic protoporphyria (EPP) contain large amounts of protoporphyrin and are regarded as the main source of protoporphyrin in this disease. Cells in the skin of EPP patients accumulate protoporphyrin released from the erythrocytes and upon sun exposure endothelial cells are photodamaged. In the present study a light-induced transfer of protoporphyrin directly from EPP erythrocytes to cultured cells is demonstrated. Erythrocytes were layered upon cultured cells and irradiated. The nearness of erythrocyte and cultured cell membranes potentiated the transfer of protoporphyrin between these cells. This transfer was rapid and preceded the release of protoporphyrin to proteins in the medium. Further irradiation of the protoporphyrin-enriched cultured cells, after removal of the erythrocytes, caused severe photodamage to the cells and survival was dependent on both the amount of protoporphyrin transferred and on the light fluence. Clinical observations and the results of this study indicate that light energy may be involved in two steps in the pathophysiology of EPP: (A) light-induced release of protoporphyrin from erythrocytes to endothelial cells and (B) photodynamic damage to protoporphyrin-enriched endothelial cells.     

Temperature-dependent changes in erythrocytes' cytosol state during natural and artificial hypobiosis

At present, the question of how the structural state of the erythrocyte cytosol is arranged to maintain essential permeabilities successfully both at normal temperature and during periods with a significant body temperature reduction during hypobiosis remains unanswered. In the present work, we performed comparative investigations of temperature-dependent changes in the cytosol state of erythrocytes from animals subjected to natural (winter hibernating ground squirrels) or artificial hypobiosis. The cytosol state was evaluated by the ESR method of spin probes (TEMPON) within the temperature range of 0-50 degrees C. Erythrocyte resistance to acid hemolysis, which is limited by the permeability of membranes for protons and the state of the anion channel, were determined using the method described by Terskov and Getelson [Biofizika 2 (1957) 259]. A change in cytosol microviscosity of erythrocytes was found as well as a temperature-dependent increase in acid resistance of erythrocytes. Our investigations allow us to conclude that physiological changes occurring in a mammalian organism during natural and artificial hypobiosis are accompanied by structural modifications of the erythrocyte cytosol. The temperature range where these modifications are observed (8, 15, 40 degrees C) suggests that the most probable modifying link is spectrin and/or the sites of its interaction with membrane. The interaction of cytoskeletal components with the cell membrane plays a key role in regulation of membrane permeability, suggesting an important role of this interaction in the adaptive reactions of erythrocytes.     

Erythrocyte membrane alterations in Huntington disease: effects of gamma-aminobutyric acid.

The interaction of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) with erythrocyte membranes from patients with Huntington disease and normal controls has been studied by electron spin resonance. GABA affects the physical state of erythrocyte membrane proteins in control and Huntington disease differently. In addition, after exposure of spin-labeled Huntington disease erythrocyte membranes to 0.1 mM GABA, the relevant electron spin resonance parameters reflecting the physical state of membrane proteins are indistinguishable from those of untreated control membranes. These findings support the concept that this disease is associated with a generalized membrane defect.     

Preparation of liposomes containing Ceramide 3 and their membrane characteristics

Liposomes composed of Ceramide 3, [2S,3S,4R-2-stearoylamide-1,3,4-octadecanetriol], and L-alpha-dipalmitoylphosphatidylcholine (DPPC) were prepared by varying the amount of Ceramide 3, and the effects of Ceramide 3 on the liposome formation, particle size, dispersibility, microviscosity and phase transition temperature were examined by means of a microscopy, a dynamic light scattering method, a fluorescence polarization method, a differential scanning calorimetry (DSC) and so on. All the DPPC was able to contribute to the formation of liposomes up to 0.130 mol fraction of Ceramide 3. The particle size of liposomes was almost unaffected by the addition of Ceramide 3. The dispersibility of liposomes containing Ceramide 3 was maintained for at least 15 days. The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel state was independent of the mole fraction of Ceramide 3. The phase transition temperature from gel to liquid crystalline states of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating a cooperative interaction between DPPC and Ceramide 3 molecules. However, a sharp DSC peak became broad and split at higher mole fractions of Ceramide 3, suggesting a phase separation in the mixed DPPC/Ceramide 3 liposomal bilayer membranes. These phenomena were suggested to be related to the previously observed fact for the mixed DPPC/Ceramide 3 monolayers that Ceramide 3 interacts with DPPC in the liquid-expanded phase with consequent phase separation accompanied with domain formation.     

Binding of sulfonamides to erythrocyte proteins and possible drug-drug interaction

The mode of binding of sulfonamides to erythrocyte proteins and possible drug-drug interaction between those compounds in erythrocytes resulting in changes in tissue levels were studied in rats using zonisamide (a novel antiepileptic agent possessing a sulfonamide group), several other sulfonamides and some antiepileptics without a sulfonamide group. In Michaelis-Menten plottings, the sulfonamide was found to be concentrated into erythrocytes in vitro and in vivo in a saturable high-affinity mode and in a linear low-affinity mode at ordinary therapeutic plasma levels through a simple diffusion process. Concentration in erythrocytes was affected by the presence of albumin in the extracellular medium. The cellular sulfonamide was readily replaced by extracellular sulfonamides in vitro. Even in vivo, erythrocyte levels of zonisamide were lowered by administration of other sulfonamides, although the plasma and tissue levels were not significantly changed since the plasma and tissue compartments of zonisamide were large relative to the erythrocyte compartment at ordinary therapeutic dose levels of zonisamide in animals and man. Therefore, disposition of zonisamide was not significantly influenced by other sulfonamides, but it is suggested that drug-drug interaction affecting the tissue levels may occur for a combination of sulfonamides with extremely different affinities for erythrocytes and low therapeutic plasma levels.     

ALTERATIONS IN ERYTHROCYTE BAND 3 ORGANIZATION INDUCED BY THE PHOTOSENSITIZER, HEMATOPORPHYRIN DERIVATIVE

Abstract— Photosensitization of erythrocytes in the presence of hematoporphyrin derivative causes cross-linking of membrane proteins. This cross-linking is associated with partial lysis of the cells and an increased susceptibility to heat-induced membrane fragmentation. The effect of photosensitization on the organization of erythrocyte band 3 was monitored using the technique of time-resolved phosphorescence anisotropy. Band 3 rotational diffusion was somewhat restricted upon photooxidation, indicating aggregation of this major integral membrane protein.     

PHOTOSENSITIZATION BY 2-CHLORO-3, 11-TRIDECADIENE-5, 7, 9-TRIYN-1-OL: DAMAGE TO ERYTHROCYTE MEMBRANES, Escherichia coli, AND DNA

The natural product 2-chloro-3,11-tridecadiene-5,7,9-triyn-1-ol (1) photosensitized the inactivation of Escherichia coli in the presence of near-ultraviolet light (320-400 nm; NUV) under both aerobic and anaerobic conditions. A series of E. coli strains differing in DNA repair capabilities and catalase proficiency exhibited indistinguishable inactivation kinetics following treatment with the chemical plus NUV. The presence of carotenoids did afford some protection to E. coli against inactivation under aerobic conditions, consistent with the involvement of singlet oxygen. The photosensitized hemolysis of human erythrocytes occurred more rapidly in the absence than in the presence of oxygen. Aerobically, the onset of hemolysis was partially inhibited by NaN3 and by 2,6-di-t-butyl-4-methylphenol (BHT) but not by superoxide dismutase (SOD). The aerobic lipid peroxidation observed in the membranes of erythrocyte ghosts was completely inhibited by BHT, and partially by NaN3, but not by SOD. These results suggest that either lipid peroxidation of the membrane is not the main cause of photohemolysis or that BHT has insufficient access to intact erythrocyte lipids to protect them. Aerobically, crosslinking of membrane proteins was also observed; it was not affected by SOD, but was partially inhibited by BHT and NaN3. The anaerobic photosensitized hemolysis of erythrocytes was more rapid; a radical mechanism was suggested since BHT inhibited the hemolysis to a greater extent than under aerobic conditions. Neither lipid peroxidation nor protein crosslinking was observed under conditions believed to be anaerobic. A light-dependent electron transfer to cytochrome c was obtained under argon but not under oxygen. Although induced mutations were not observed in the experiments with E. coli, 1 was capable of damaging both supercoiled pBR322 and Haemophilus influenzae transforming DNA in a manner that seemed to be equivalent under aerobic and anaerobic conditions. In conclusion, 1 can behave as typical photodynamic molecule under aerobic conditions but, in contrast to most photodynamic molecules, it is also phototoxic under anaerobic conditions. The extent to which the radical reactions detected under anaerobic reactions compete with the photodynamic processes when oxygen is present is not known.     

Affinity interactions on a liposome surface detected by ultrasound velocimetry.

In this work, we performed targeted immobilization of immunoglobulins by means of bacterial S-layer proteins from Bacillus coagulans E38-66/V1 recrystallized on liposomes, which were exploited as immobilization matrix for antibody (Ab)-human IgG. The study of interaction of rabbit or swine anti-human IgG as antigens (Ag) was performed by means of measuring changes of ultrasound velocity. We showed that at a temperature of 25 degrees C, the increment of ultrasound velocity [u] linearly decreased following an increase of concentration of Ag. The decrease of [u] was presumably due to changes of hydration of the membrane due to the binding process. Approximately 10 times lower changes of [u] were observed at 45 degrees C for Ag-Ab interaction as well as for nonspecific interaction of Ag with liposomes covered by S-layer without Ab. No substantial differences in the behaviour of [u] were observed for interactions of human IgG with rabbit or swine anti-human IgG.     

PHOTODYNAMIC ACTION OF MEROCYANINE 540 IN ARTIFICIAL BILAYERS AND NATURAL MEMBRANES: ACTION SPECTRA AND QUANTUM YIELDS

The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC540) in liposomes and isolated erythrocyte membranes were obtained using electron spin resonance techniques. Oxygen consumption was measured by spin label oximetry in the presence of histidine for fully-saturated dimyristoylphosphatidylcholine vesicles, mono-unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine vesicles and erythrocyte membranes. The quantum yield for the photogeneration of 1O2 by membrane-bound MC540 in aqueous buffer was determined to be 0.065 +/- 0.005, which is approx. 1/10 of the value determined for Rose Bengal under similar conditions. Using unilamellar liposomes and isolated erythrocyte membranes containing MC540 at different monomer/dimer ratios, we have observed that the action spectra of 1O2 generation closely overlap the absorption spectra of the monomeric dye in these systems. It is likely that factors which affect the monomer-dimer equilibrium of MC540 will influence the production of 1O2. These findings have important implications for the phototherapeutic efficacy of MC540.     

Dark interaction of 5-methoxypsoralen and 8-methoxypsoralen with erythrocyte ghosts: a fluorescence and circular dichroism study

The dark interaction of 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) with plasma membranes was studied using human erythrocyte ghosts as a model. In the presence of ghosts, modifications of the fluorescence characteristics of 5-MOP were observed, together with a quenching of the fluorescence of the tryptophan (Trp) residues of membrane proteins (up to 25%). Moreover, the appearance of an induced circular dichroism indicates that 5-MOP is located in a chiral environment. In contrast, only slight effects were observed in the case of 8-MOP. It is concluded that 5-MOP molecules are located partly within chiral protein sites of the membrane in such a way that a F?rster energy transfer can occur from the Trp residues to the psoralen molecules.     

REACTIVITY OF PHOTOCHEMICALLY-GENERATED LIPID HYDROPEROXIDES IN CELL MEMBRANES WITH GLUTATHIONE PEROXIDASE

The ability of glutathione peroxidase (Gpx) to catalyze the reductive inactivation of photochemically-generated lipid hydroperoxides (LOOHs) was investigated, using hematoporphyrin derivative (HPD) as a photosensitizing agent and erythrocyte ghosts as membrane targets. Glutathione peroxidase was reactive toward photoperoxidized membranes only after their exposure to phospholipase A2 (PLA2). Iodometrically-determined LOOH values were typically 30-40% greater than values measured by enzymatic assay using Gpx and glutathione reductase. A consistent result was obtained when photooxidized membranes were treated with PLA2 and GSH/Gpx followed by iodometric assay, viz. persistence of approximately 40% of the starting LOOH. Whereas photooxidized egg phosphatidylcholine liposomes underwent total LOOH loss when incubated with PLA2 and GSH/Gpx, no net loss was observed with photooxidized cholesterol/dimyristoyl-phosphatidylcholine liposomes. The results suggest that cholesterol hydroperoxides in ghost membranes account for the Gpx-resistant fraction of LOOHs.     

Effects of incorporation of various amphiphiles into recipient liposome membranes on inter-membrane protein transfer.

To obtain information about the factors governing spontaneous inter-membrane protein transfer, we examined the effects of incorporation of various amphiphilic compounds in dimyristoylphosphatidylcholine (DMPC) liposomes on protein transfer from influenza virus-infected cells to the liposomes, and analyzed the physical properties of these liposome membranes. The incorporation of amphiphilic compounds, negatively charged dicetylphosphate (DCP), dipalmitoylphosphatidylserine (DPPS) or positively charged dimethyldipalmitoylammonium (DMDPA), into DMPC liposomal membranes enhanced protein transfer. The liposomes containing DCP, DPPS or DMDPA were unaffected by osmotic shock caused by external addition of glucose, suggesting a decrease in lipid packing in the liposomal membranes. Furthermore, calorimetric study of these liposomes showed that a phase separation occurred partially in the liposomal membranes. Accordingly, the membranes of DMPC liposomes containing DCP, DPPS and DMDPA should be distorted due to the coexistence of two phases, gel and liquid crystalline, in the membranes. Consequently, the membrane distortion could be responsible for the enhancement effects of the amphiphiles on the inter-membrane protein transfer from influenza virus-infected cells to the liposomes.     

Explorations of ABO-Rh antigen expressions on erythrocyte dielectrophoresis: changes in cross-over frequency

A quadrupole dielectrophoretic microdevice was utilized to examine the ABO-Rh dependencies on erythrocyte polarizations. This important step toward medical microdevice technology would transform key clinical blood tests from the laboratory into the field. Previous work in dielectrophoretic microdevices demonstrated that the large number of ABO antigens on erythrocyte membranes impacts their dielectrophoretic signature at 1 MHz. This work explores the dielectrophoretic behavior of native human erythrocytes categorized by their ABO-Rh blood types and directly compares these responses to the same erythrocyte sample modified to remove the A and B antigens. A β(1-3)-galactosidase enzyme was utilized to cleave the ABO polysaccharide backbone at the galactosidase bonds. The enzymatic reaction was optimized by comparing agglutination of the native and modified blood cells in addition to UV-Vis and HPLC analysis of the reaction effluent for saccharide residues. Next, the dielectrophoretic behaviors of the native and modified erythrocytes were visually verified in a quadrupole electrode microdevice over a frequency range from 100?kHz to 80?MHz. The lower cross-over frequency (COF), which transitions from negative to positive dielectrophoresis, for ABO blood types tested (A+, A-, B+, B-, AB+, O+ and O-) differed over the range from 17 to 47?MHz. The COFs of the corresponding enzyme-modified erythrocytes were also determined and the range narrowed to 29-41 MHz. A second COF in the 70-80?MHz range was observed and was reduced in the presence of the transmembrane Rhesus factor. These results suggest that antigen expression on erythrocyte membrane surfaces influence cell polarizations in nonuniform AC fields.     

Phospholipid Bilayers as Molecular Models for Drug-Cell Membrana Interactions. The Case of the Antiinflamatory Drug Diclofenac

Phospholipids are amphipatic molecules with long hydrophobic acyl chains and zwitterionic polar heads which assemble into different types of molecular aggregates. The most relevant is the bilayer because of its relation with cell membranes, which are very complex entities. For this reason, simpler molecular models based on phospholipids bilayers are widely used. We have determined the bilayer structure of phospholipids located in the outer and inner monolayers of most cell membranes, and use them as molecular models to study the way different chemicals of biological interest interact with cell membranes. We present the results of our studies on the nonsteroidal anti-inflammatory drug diclofenac, from which little is known about its effects on human erythrocytes. This report presents the following evidence that diclofenac interacts with the human red cell membrane: a) X-ray diffraction and fluorescence spectroscopy of phospholipids bilayers show that diclofenac interacts with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy studies on human erythrocytes it was observed that the drug induced morphological changes different from their normal biconcave shape.     

Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry.

The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes.

The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF–CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes.     

DSC and Raman study on the effect of lysozyme and bovine serum albumin on phospholipids liposomes

In this paper, the effect of increasing amounts of lysozyme (Lyso) and bovine serum albumin (BSA) on the behaviour of lecithin (DMPC) and cephalin (DMPE) liposomes was investigated by means of Raman and DSC techniques. The results showed that both proteins affected, but in a different way, both lecithin and cephalin liposomes. In the samples with lower Lyso concentrations (up to 2 % w/w), a small decrease on the main transition temperature (T _m) was observed, whereas T _m increased by further addition of Lyso (up to 15.0 % w/w). At the same time, an increase of about 20 % in the ΔH of the transition was observed. Pre-transition was also affected in a greater extent by protein presence. When BSA interacted with liposomes, a smaller increase in the T _m values was observed with a contemporary increase of about 8 % in the associated ΔH. The data suggested that the BSA–liposomes interaction involves only the external surface of the bilayer, excluding thus any penetration into the liposomal hydrophobic core. On the contrary, a partial penetration into the bilayer is suggested when Lyso is added to liposomes. Both considered proteins strengthened the overall bilayer structure of DMPC liposomes, suggesting a decrease in the membrane permeability. Moreover, Lyso secondary structure changed by interaction with liposomes, as demonstrated by the Raman spectra behaviour, in particular in the case of DMPE.