Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.     

Modulation of membrane receptor endocytosis by chemical effectors of membrane fluidity

Several chemical effectors were used to induce changes in spleen B cell membrane fluidity. Membrane fluidity was monitored by fluorescence polarization analysis of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and cell viability was checked not to be affected by the treatments. Membrane immunoglobulin (Ig) endocytosis by the living B cells with modified or unmodified membranes was quantitatively measured by flow cytometry, using a previously described method (Métézeau et al., 1982, 1984). The kinetics of endocytosis of membrane Ig was not affected by chemical effectors increasing membrane fluidity. On the contrary, increasing membrane microviscosity resulted in the slowing down and eventually the blocking of membrane Ig endocytosis. It is suggested that a step depending on membrane microviscosity is involved in the process of endocytosis; this step may become rate limiting when membranes are artificially rendered or naturally become (i.e. for pathological or particularly differentiated cells) more viscous.     

Modulation of cardiac mitochondrial membrane fluidity by age and calorie intake

The aim of the present study was to determine the effects of dietary restriction (DR) on the age-related changes in membrane fluidity, fatty acid composition and free radical damage of mitochondrial membranes obtained from the rat left ventricle. Mitochondrial membrane preparations were obtained from the left ventricles of 6- and 24-month-old, male, Fischer 344 rats that were allowed to eat throughout their life either ad lib (Group A) or only 60% of the amount consumed by the ad lib fed group (Group B). Our results show that the membrane fluidity of the 24 month Group A hearts was less than that of the 6 month group A hearts. No differences in membrane fluidity were observed between the 6 and 24 month DR groups. The fatty acid composition of the mitochondrial membranes of the two ad lib fed groups differed: the long-chain polyunsaturated 22:4 fatty acid was higher in the older group, although linoleic acid (18:2) was lower. DR eliminated the differences. No statistically significant difference in the overall polyunsaturated fatty acid content was noted. However, the peroxidizability index was higher in the membranes of the 24 month Group A hearts but not in the 24 month Group B hearts. Finally, the degree of lipid damage, as assessed in vitro by the induced production of reactive oxygen species, was elevated in the 24 month Group A hearts. No difference was observed between the young and old DR groups. Considered together, these results suggest that DR maintains the integrity of the cardiac mitochondrial membrane fluidity by minimizing membrane damage through modulation of membrane fatty acid profile.     

Oxidation of liposomal membrane suppressed by flavonoids: quantitative structure-activity relationship

Antioxidant activity of the set of 12 flavonoids in heterogeneous membrane system of dioleoyl phosphatidylcholine liposomes stressed by peroxidative damage induced by 2,2'-azobis-(2-amidinopropane)hydrochloride azoiniciator and evaluated by the thiocyanate method was assessed. Antioxidant activity (pIC(50)) was correlated with 19 molecule parameters calculated for the minimum energy conformers of the compounds tested. The linear regression analysis revealed that the parameter of hydration energy E(HYDR) (R = -0.747) was the best predictor of antioxidant activity in a liposomal system.     

Modulation of serotonin binding in rat brain by membrane fluidity

Preincubation of rat brain homogenates with increasing concentrations of n-hexanol reduced specific serotonin (5-HT) binding and increased membrane fluidity as measured by fluorescence depolarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. At 5 mM ascorbate maximal reductions of both membrane fluidity and specific 5-HT binding were observed. Both effects were enhanced in the presence of ferrous sulphate and oxygen. In the presence of ascorbate (5.7 mM) only one 5-HT binding site was observed in contrast with high and low affinity binding sites (K_D1 = 0.08 ± 0.04 nM, K_D2 = 28.8 ± 1.3 nM) found in the absence of ascorbate. The ascorbate induced decrease of specific 5-HT binding may be explained by lipid peroxidation, which decreases membrane fluidity, and by ascorbate's reducing properties. Since different correlations were found between membrane fluidity and specific 5-HT binding depending upon the presence of ascorbate or n-hexanol, the results suggest that membrane fluidity is a critical factor, however, just one of several determinants in 5-HT binding studies.     

Modulation of sarcoplasmic reticulum Ca2+-pump activity by membrane fluidity

Intramolecular excimerization of 1,3-di-1-pyrenylpropane [Py(3)Py] was used to assess the fluidity of sarcoplasmic reticulum membranes (SR); on the basis of the spectral data, the probe incorporates completely inside the membrane probably somewhere close to the polar head groups of phospholipid molecules, however not in the very hydrophobic core. The excimerization rate is very sensitive to lipid phase transitions, as revealed by thermal profiles of dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers. Cholesterol abolishes pretransitions and broadens the thermal profiles of the main transitions which vanish completely at 50 mol % sterol. Excimer formation in liposomes of SR total lipid extracts does not show any sharp transitions, as in the case of DMPC and DPPC. However, the plots display discontinuities at about 20 degrees C which are broadened by cholesterol and not observed at 50 mol % sterol. Also cholesterol has been incorporated in native SR membranes by an exchange technique allowing progressive enrichment without changing the phospholipid/protein molar ratio. As in liposomes, discontinuities of excimer formation at 20 degrees C are broadened by cholesterol enrichment. The full activity of uncoupled Ca2+-ATPase is only affected by cholesterol above a molar ratio to phospholipid of 0.4. However, a significant decrease in activity (about 20%) is only noticed at a ratio of 0.6 (the highest technically achieved); at this ratio, about 28 lipid molecules per Ca2+-ATPase are expected to be relatively free from cholesterol interaction. The vesicle structure is still intact at this high ratio, as judged from the absence of basal activity (not Ca2+ stimulated).(ABSTRACT TRUNCATED AT 250 WORDS)     

Deuterated phytoestrogen flavonoids and isoflavonoids for quantitation

Isotopically and isomerically pure polydeuterated flavonoids and isoflavonoids have been prepared for quantitation of these compounds in biological matrices. Various deutero-labeling techniques are presented and methods for establishing the isotopical and isomerical purity of deuterated products are discussed.     

Modulation of membrane fluidity in living protoplasts of Nicotiana plumbaginifolia by catalytic hydrogenation

A homogeneous water-soluble Ru catalyst, has been incorporated into mesophyll protoplasts isolated from Nicotiana plumbaginifolia leaves. In the presence of hydrogen gas this complex causes an extensive loss of unsaturated fatty acid bonds and a concomitant increase in microviscosity of the cellular membranes. Although the gradual reduction of the level of unsaturation, per se, is accompanied by considerable cell damage, there is an optimum reaction time where approximately 50% of the protoplasts are still living and about 20% of the double bounds initially present in fatty acyl residues have undergone hydrogenation. The possible mechanism of the self regulatory process competing with the hydrogenation in the early stages of the reaction is also discussed.     

Modulation of the kinetic characteristics of the sarcoplasmic reticulum ATPase by membrane fluidity

(Ca²⁺ + Mg²⁺)-ATPase from sarcoplasmic reticulum has been reconstituted with dipalmitoylphosphatidylcholine, and the activating effect of ATP and Ca²⁺ on this enzyme has been studied at different temperatures. It has been found that two kinetic forms of the enzyme are interconverted at about 31°C, and this is possibly related to a phase change in the phospholipid which is more directly associated with the protein. Above 31°C the enzyme is less dependent on ATP activation at high ATP concentrations but shows positive cooperativity for Ca²⁺ activation. On the other hand, below 31°C, the reconstituted enzyme is more dependent on ATP for activation at high ATP concentrations than the purified ATPase and does not show cooperativity for Ca²⁺ activation.     

Modulation of membrane fusion by membrane fluidity: temperature dependence of divalent cation induced fusion of phosphatidylserine vesicles

We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90 degrees light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 A) or large unilamellar vesicles (LUV, diameter 1000 A) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 degrees C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 degree C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 degrees C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 degrees C and above), subsequent collapse of the vesicles' internal space causes complete release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity of isoflavonoids for lipid bilayers evaluated with liposomal systems

Three parameters, i.e., the proportion of the amount incorporated into the liposomes, the partition coefficient in a system of n-octanol/phosphate buffered saline, and the retention times by HPLC, were measured to determine the lipophilicity of isoflavonoids. The presence of a hydroxyl group at 5-position of the A-ring and a methoxyl group at 4'-position of the B-ring in the isoflavonoid structure increased the three parameters. The localization of isoflavonoids in lipid bilayers was investigated by a liposome system with fluorescent probes. The location of the isoflavonoid depended on its structure. The cytotoxicity of isoflavonoids was investigated by a colony-formation assay with Chinese hamster lung fibroblast V79 cells. The structure-activity relationship of the cytotoxic activity partly reflected those of the three parameters. This suggests that the biological activities of isoflavonoids in vitro could be attributable to their affinity for lipid components in the cases where the estrogen receptors have no role.     

Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles.

P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues. P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport [3H]daunomycin. In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of [3H]daunomycin and [3H]vinblastine. Accumulation of both cytotoxic agents was stimulated by ATP, exhibited temperature dependence and osmotic sensitivity, and followed Michaelis-Menten kinetics. Alterations in CMV lipid fluidity were induced by the known fluidizers, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) and benzyl alcohol, and were assessed by fluorescence polarization techniques using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). Both A2C (2.5-5.0 microM) and benzyl alcohol (10-20 mM) produced a dose-dependent increase in CMV lipid fluidity. Moreover, both fluidizers, at the above doses, significantly inhibited (p < 0.05) the ATP-dependent accumulation of [3H]daunomycin. [3H]Vinblastine accumulation was also inhibited by A2C (p < 0.05). Lower doses of A2C (0.6 microM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation. Kinetic analysis revealed that A2C (5.0 microM) noncompetitively inhibited [3H]daunomycin accumulation and uncompetitively inhibited [3H]vinblastine accumulation with apparent Ki values of approximately 1.5 and approximately 1.2 microM, respectively. Verapamil competitively inhibited P-gp-mediated accumulation of [3H]daunomycin but failed to alter the fluidity of CMV. Taken together, the present results demonstrate that while increases in membrane fluidity of CMV are not necessarily required to inhibit P-gp-mediated drug accumulation, they can inhibit these processes, at least in CMV. Alterations in the physical state of CMV, therefore, appear to be at least one important modulator of P-gp function.     

Modulation of isoniazid susceptibility by flavonoids in Mycobacterium

In the course of a project to identify plant natural products which modulate the susceptibility of different strains of fast-growing mycobacteria to the first-line antituberculotic isoniazid (INH), several flavonoids without significant antimycobacterial activities at the tested concentrations were screened for their ability to decrease the minimum inhibitory concentrations (MICs) of INH. Flavonoids with different substitution patterns, namely epicatechin, isorhamnetin, kaempferol, luteolin, myricetin, quercetin, rutin and taxifolin were tested to examine structure–activity relationships (SARs) of these compounds. Different mycobacterial strains, i.e. Mycobacterium smegmatis (ATCC 14468), M. smegmatis mc²155 (ATCC 700084), M. smegmatis mc²2700, M. phlei (ATCC 11758) and M. fortuitum (ATCC 6841) were used. The strongest synergistic effects were observed in M. smegmatis mc²155 followed by M. phlei, whereas the tendency of INH potentiation by certain flavonoids remained the same within each strain. Myricetin was the most efficient intensifier of INH susceptibility in all tested strains causing a decrease of the MIC of INH up to 64-fold at 16 μg/ml, followed by quercetin. Structure–activity relationships of flavonoids as intensifiers of INH susceptibility in mycobacteria indicate that they overlap with SARs for their radical-scavenging properties, however the potentiation of INH activity cannot only be explained by their radical-scavenging activity alone.     

Biophysical interpretation of membrane fluidity by catastrophe theory

We have regrouped the data of two examples where membrane fluidity was progressively modified by pharmacological and physiological agents. In our approach, each initial condition is determined by control parameters (depth of the membrane bilayer explored, concentration of agents). The fluidity is expressed as a state parameters followed on the control space. Then, according to Catastrophe Theory, the results are depicted as tridimensional patterns which can be recognized as bifurcation sets. Consequently, the fluidity is considered as resulting in a compromise phenomenon (normal factor) between two structurally attracting configurations (of hydrophilic and hydrophobic nature). The concepts of membrane activity and membrane function are then discussed on the basis of physiological functionality of biomembranes. The main application of this research interests the pharmacological domain. Indeed, a new classification of drugs could be proposed. According to the loss of membrane functionality, some drugs could imply a too high differentiation of attractors (splitting factor) and others could imply the destruction of the compromise. The first type is characterized by the physical destruction of the membrane. In the second type, the entity of the bilayer is preserved but the membrane is destructurated.     

Adenylate cyclase and membrane fluidity

Summary The relationships between membrane fluidity as induced by drug addition and the stimulation of adenylate cyclase by hormones (mainly catecholamines), GTP, Gpp(NH)p and NaF are reviewed. In particular, the data corresponding to pigeon erythrocyte membranes are reviewed and compared with other data published in the literature. A brief summary of the theories involved in fluidity measurements and their significance at the molecular level is also given for anisotropy of fluorescence and electron spin resonance.One of the conclusions is that the cationic drugs and neutral alcohols by perturbing preferentially the inner half-layer of the bilayer induced in pigeon erythrocyte membrane correlated multiphasic changes on fluidity and adenylate cyclase activity.This and other experimental data concerning the regulation of the adenylate cyclase are discussed in regard to a new interpretation of cyclase stimulation: the repressor hypothesis. In cell membrane the catalytic unit C is repressed by its association with a repressor complex made of the hormone receptor R and the regulatory protein N. The activation of cyclase activity is the dissociation of the catalytic unit C from the repressor complex R.N according to the equilibrium: R.N.C (inactive) R.N + C (active). Hormones, metal ions (magnesium), and nucleotides (GTP) are the allosteric ligands which shift this equilibrium towards the dissociation. state with the liberation of the active form, membrane-bound, C unit. Gpp(NH)p, fluoride and forskolin will also shift the equilibrium toward the right. GDP and free receptors favour the associated repressed state of the system.     

Modulation of rat distal colonic brush-border membrane Na+-H+ exchange by dexamethasone: role of lipid fluidity

Earlier studies by our laboratory have suggested a relationship between an amiloride-sensitive Na+-H+ exchange process and the physical state of the lipids of rat colonic brush-border membrane vesicles. To further assess this possible relationship, a series of experiments were performed to examine the effect of dexamethasone administration (100 micrograms/100 g body wt. per day) subcutaneously for 4 days on Na+-H+ exchange, lipid composition and lipid fluidity of rat distal colonic brush-border membrane vesicles. The results of these studies demonstrate that dexamethasone treatment significantly: (1) increased the Vmax of the Na+-H+ exchange without altering the Km for sodium of this exchange process, utilizing the fluorescent pH-sensitive dye, acridine orange. 22Na flux experiments also demonstrated an increase in amiloride-sensitive proton-stimulated sodium influx across dexamethasone-treated brush-border membrane vesicles; (2) increased the lipid fluidity of treated-membrane vesicles compared to their control counterparts, as assessed by steady-state fluorescence polarization techniques using three different lipid-soluble fluorophores; and (3) increased the phospholipid content of treated-membrane vesicles thereby, decreasing the cholesterol/phospholipid molar ratio of treated compared to control preparations. This data, therefore, demonstrates that dexamethasone administration can modulate amiloride-sensitive Na+-H+ exchange in rat colonic distal brush-border membrane vesicles. Moreover, it adds support to the contention that a direct relationship exists between Na+-H+ exchange activity and the physical state of the lipids of rat colonic apical plasma membranes.