Docosahexaenoic acid promotes micron scale liquid-ordered domains. A comparison study of docosahexaenoic versus oleic acid containing phosphatidylcholine in raft-like mixtures.

The understanding of the functional role of the lipid diversity in biological membranes is a major challenge. Lipid models have been developed to address this issue by using lipid mixtures generating liquid-ordered (Lo)/liquid-disordered (Ld) immiscibility. The present study examined mixtures comprising Egg sphingomyelin (SM), cholesterol (chol) and phosphatidylcholine (PC) either containing docosahexaenoic (PDPC) or oleic acid (POPC). The mixtures were examined in terms of their capability to induce phase separation at the micron- and nano-scales. Fluorescence microscopy, electron spin resonance (ESR), X-ray diffraction (XRD) and calorimetry methods were used to analyze the lateral organization of the mixtures. Fluorescence microscopy of giant vesicles could show that the temperature of the micron-scale Lo/Ld miscibility is higher for PDPC than for POPC ternary mixtures. At 37°C, no micron-scale Lo/Ld phase separation could be identified in the POPC containing mixtures while it was evident for PDPC. In contrast, a phase separation was distinguished for both PC mixtures by ESR and XRD, indicative that PDPC and POPC mixtures differed in micron vs nano domain organization. Compared to POPC, the higher line tension of the Lo domains observed in PDPC mixtures is assumed to result from the higher difference in Lo/Ld order parameter rather than hydrophobic mismatch.

Building up of the liquid-ordered phase formed by sphingomyelin and cholesterol.

The long-range and molecular orders and dynamics in codispersions of egg sphingomyelin-cholesterol have been investigated by synchrotron x-ray diffraction and electron spin resonance using phosphatidylcholine spin-labeled at several positions on the sn-2 chain. Mixtures containing 0, 17, 33, 41, 50 mol% cholesterol exhibited a single phase by x-ray diffraction methods. The temperature dependence of the d-spacing between 20 and 50 degrees C is attenuated with increasing proportions of cholesterol, becoming invariant for cholesterol contents of 41 and 50 mol% on completion of the liquid-ordered phase. Electron spin resonance revealed two sites for 17 and 33 mol% cholesterol. One site is highly ordered and the other is less ordered than the fluid phase of pure sphingomyelin as shown by the molecular and the intramolecular order parameters reflecting the segmental motions of the probe. The two-sites exchange rate indicates a mean lifetime of the sites of approximately 0.1 micros during which the lipid displacement is approximately 1 nm. The short lifetime of the sites probed by ESR and the single phase detected by x-ray diffraction support in this binary mixture, the building up of the Lo phase by a progressive accumulation of randomly distributed sphingomyelin-cholesterol condensed complexes rather than by diffusional exchange between extended domains.

Compared effects of cholesterol and 7-dehydrocholesterol on sphingomyelin-glycerophospholipid bilayers studied by ESR.

The ESR of 7- and 16-doxylstearic spin-labeled fatty acids (7NS and 16NS, respectively) reveal the distinct influence of cholesterol or cholesterol precursor analogue, delta7-dehydrocholesterol, on the molecular ordering and the fluidity of lipid mixtures containing sphingomyelin (SM). The phase-separation of sphingomyelin domains mixed within fluid glycerophospholipids (phosphatidylethanolamine and phosphatidylserine) can be followed by ESR as a function of the temperature and in the presence of sterols [cholesterol (CHOL) or 7-dehydrocholesterol (DHCHOL)]. The time scale of spin-label exchange among phases is appropriate to follow the occurrence of the specific sphingomyelin/sterol association forming liquid ordered (Lo) microdomains which separate from the fluid surrounding phase Lalpha. Sphingomyelin embedded within the fluid bilayer associates with both sterols below 36 degrees C to give a phase Lo traceable by ESR in the form of a highly anisotropic component. Above 36 degrees C, the contribution in the ESR spectrum, of the Lo phase formed by 7-dehydrocholesterol with sphingomyelin is reduced by contrast with cholesterol forming a temperature-stable liquid ordered phase up to 42 degrees C. The consequences of this destabilization of the SM/sterol microdomains are envisioned in the biosynthesis defect where the precursor 7-dehydrocholesterol substitutes, for a significant part, the embryonic cell cholesterol.

[Physical arrangement of membrane lipids susceptible to being used in the process of cell sorting of proteins]. / Arrangement physique des lipides membranaires susceptibles d'être utilisés par les processus d'adressage cellulaire des protéines.

Detection of immiscible lipid domains in biological membranes offers an alternative support to protein sorting. Liquid ordered domains ("rafts") comprising cholesterol and saturated sphingolipids incorporate saturated glycosyl-phosphatidylinositol (GPI)-anchored or acylated (palmitoyl- and myristoyl-) proteins or particular transmembrane protein sequences. These lipid domains can be isolated in the form of Detergent resistant membranes (DRM) from biological plasma membrane preparations. Caveolae appear to be a differentiated fraction of plasma membranes comprising such numerous cross-linked microdomains associated with caveolin in different cell types. While the biological relevance of such membrane domains is evidenced in vivo by co-patching of proteins sharing the identical affinity for sphingolipids and by the disruption of co-patching following cell cholesterol depletion, only a few physical studies confort the principle of membrane heterogeneity. Results are now presented where cholesterol addition in a tertiary lipid mixture forces outphase-separation, as a realistic model where the lipid segregation can promote protein sorting to the segregated Lo phase. A lipid mixture comprising phosphatidylserine, phosphatidylethanolamine and sphingomyelin of natural origin in the ratio (1/4/3: mole/mole) has been rendered neatly heterogeneous after the addition of cholesterol (27 mole%). Xray diffraction (Small angle Xray scattering) showed the splitting of two neatly resolved lamellar diffractions in the presence of cholesterol. Above 37 degrees C the heterogeneity was traceable by a broadened diffraction spot up to the complete get-to-liquid transition of sphingomyelin at temperatures > 40 degrees C where the spot became again symmetrical and narrow. The large temperature range where the immiscible lamellar phases are detected, the specific requirement for cholesterol association with sphingomyelin, the positive influence of calcium and the reversibility of domain formation support the occurrence for such domains at the inner side of the plasma membrane whereon lipids-bound proteins concentrate.

Cell-generated nitric oxide inactivates rat hepatocyte mitochondria in vitro but reacts with hemoglobin in vivo.

BACKGROUND & AIMS: Nitric oxide forms inactive iron-nitrosyl complexes within hepatic mitochondria in vitro. However, when formed in vivo, NO might react instead with hemoglobin. The aim of this study was to compare the effects of cell-derived NO on rat hepatocyte mitochondria in vitro and in vivo. METHODS: First, hepatocytes were cultured in vitro for 24 hours under a porous membrane supporting macrophages that were stimulated by endotoxin. Second, hepatic macrophage hyperplasia was induced in vivo by preadministration of killed Corynebacterium parvum; 7 days later, rats received endotoxin and were killed after 6 hours. Third, mitochondria were exposed to sodium nitroprusside in vitro, washed, mixed with blood, and recovered. RESULTS: Iron-nitrosyl complexes and hepatocyte mitochondrial dysfunction were observed in the in vitro model and prevented by an NO synthase inhibitor. In the in vivo model, however, despite a 130-fold increase in plasma nitrate levels and formation of hemoglobin-NO complexes in blood, no iron-nitrosyl complex was detected in hepatic mitochondria, and hepatic mitochondrial function was not impaired. In the third model, mitochondria lost preformed iron-nitrosyl complexes when exposed to blood. CONCLUSIONS: Although NO reacts with hepatocyte mitochondria in vitro, in vivo it reacts with sinusoidal hemoglobin without detectable impairment of hepatic mitochondrial function.

Metabolic activation of the nitroaromatic antiandrogen flutamide by rat and human cytochromes P-450, including forms belonging to the 3A and 1A subfamilies.

The in vitro metabolic activation of flutamide, a nitroaromatic antiandrogen which produces hepatitis in a few recipients, was first studied with male rat liver microsomes. There was no electron spin resonance evidence for the reduction of flutamide by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase into a nitro anion free radical. In contrast, flutamide was oxidatively transformed by cytochrome P-450 into reactive metabolite(s) that covalently bound to microsomal proteins. Covalent binding required oxygen and NADPH, and was decreased by the nucleophile glutathione and by the cytochrome P-450 inhibitors SKF 525-A, piperonyl butoxide and troleandomycin (an inhibitor of the cytochrome P-450 3A subfamily). Covalent binding was increased markedly by pretreatment with dexamethasone (an inducer of the cytochrome P-450 3A subfamily) and moderately by pretreatment with beta-naphthoflavone (an inducer of the 1A family). Covalent binding was immunoinhibited markedly by anticytochrome P-450 3A immunoglobulin G and moderately by anticytochrome P-450 1A immunoglobulin G. Covalent binding was much lower with liver microsomes from female rats (not expressing P-450 3A2). Covalent binding of flutamide also occurred with human liver microsomes (where it was inhibited by troleandomycin), and with yeast microsomes expressing human liver cytochromes P-450 1A1, 1A2 or 3A4. We concluded that flutamide was oxidatively transformed into chemically reactive metabolite(s) by rat and human cytochromes P-450, including forms belonging to the 3A and 1A subfamilies.

Generation of free radicals during the reductive metabolism of nilutamide by lung microsomes: possible role in the development of lung lesions in patients treated with this anti-androgen.

The pulmonary metabolism of nilutamide, a nitroaromatic anti-androgen drug leading to pulmonary lesions in a few recipients, has been investigated in rats. Incubation of nilutamide (1 mM) with rat lung microsomes and NADPH under anaerobic conditions led to the formation of the nitro anion free radical, as indicated by ESR spectroscopy. The steady state concentration of this radical was not decreased by CO or SKF 525-A (two inhibitors of cytochrome P450), but was decreased by NADP+ (10 mM) or p-chloromercuribenzoate (0.47 mM) (two inhibitors of NADPH-cytochrome P450 reductase activity). Anaerobic incubations of [3H]nilutamide (0.1 mM) with rat lung microsomes and a NADPH-generating system resulted in the in vivo covalent binding of [3H]nilutamide metabolites to microsomal proteins; covalent binding required NADPH; it was decreased in the presence of NADP+ (10 mM), or in the presence of the nucleophile glutathione (10 mM), but was unchanged in the presence of carbon monoxide. Under aerobic conditions, in contrast, the nitro anion free radical was reoxidized by oxygen, and its ESR signal was not detected. Covalent binding was essentially suppressed. Instead, there was consumption of NADPH and oxygen, and production of superoxide anion and hydogen peroxide. We conclude that nilutamide is reduced by rat lung microsomes NADPH-cytochrome P450 reductase into a nitro anion free radical. In anaerobiosis, the radical is reduced further to covalent binding species. In the presence of oxygen, in contrast, this nitro anion free radical undergoes redox cycling, with the generation of reactive oxygen species.

Generation of free radicals during the reductive metabolism of the nitroaromatic compound, nilutamide.

Incubation of the antiandrogen nilutamide with rat liver microsomes and NADPH, under anaerobic conditions, led to the formation of the nitro anion free radical, as indicated by electron spin resonance spectroscopy. The steady-state concentration of the nitro anion free radical was not decreased by SKF 525-A, carbon monoxide or metyrapone (3 inhibitors of cytochrome P-450) but was decreased by NADP+ or p-chloromercuribenzoate (2 inhibitors of NADPH-cytochrome P-450 reductase). Under aerobic conditions, the nitro anion free radical was reoxidized by oxygen, and the electron spin resonance signal was not detected. This redox cycle was associated with NADPH oxidation, consumption of oxygen, and formation of superoxide anion, hydrogen peroxide and glutathione disulfide. Under anaerobic conditions, nilutamide was further reduced to chemically reactive metabolites. Anaerobic incubation of [3H]nilutamide (0.1 mM) with rat liver microsomes and a NADPH-generating system resulted in the in vitro covalent binding of [3H]nilutamide metabolites to microsomal proteins; covalent binding required NADPH; it was decreased in the presence of NADPH-cytochrome P-450 reductase inhibitors (methylene blue, 2'-adenosine monophosphate) or in the presence of the nucleophile glutathione, but was unaffected by cytochrome P-450 inhibitors (SKF 525-A, CO). Covalent binding was decreased markedly under aerobic conditions. We conclude that nilutamide is reduced by microsomal NADPH-cytochrome P-450 reductase into a nitro anion free radical. In the presence of oxygen, this nitro anion free radical undergoes redox cycling with oxygen, and forms reactive oxygen species. In anaerobiosis, it is reduced further to covalent binding species.

Effects of platelet-activating factor (PAF), lyso-PAF and lysophosphatidylcholine on phosphatidylcholine bilayers, an ESR, 31P-NMR and X-ray diffraction study.

The effect of asymmetric phospholipids, such as platelet-activating factor (PAF), lyso-PAF and lysophosphatidylcholine, on phosphatidylcholine bilayers has been examined using ESR, 31P-NMR and X-ray diffraction methods. ESR and 31P-NMR experiments have been performed on oriented multibilayers. ESR measurements of 5-doxyl stearic acid, as a spin probe, show that PAF disorients phosphatidylcholine bilayers when present at molar ratios greater than 40%. This is manifest as a broadening of the local director orientation distribution, a parameter required to simulate the lineshape. Despite the marked change of the regular in-plane orientation of the films, there are only slight changes in the order parameter of the acyl chains. Cholesterol orients films containing asymmetric phospholipids, in a way consistent with the formation of 1/1 stoichiometric complexes between cholesterol and the asymmetric phospholipid. Such complexes can be detected with 3-doxyl cholestane, a spin-labelled sterol analogue interacting with lyso-PAF and PAF. Simulations of 31P-NMR resonance linewidth of oriented multibilayers of PAF/egg lecithin mixtures indicate a rippled structure which accounts for the perturbed distribution of the local director observed by ESR spin probe measurements. Micellisation of the film can be discounted on the basis of the 31P-NMR linewideth for the concentration range investigated. X-ray diffraction studies of liposomes of dimyristoyl- and dioleoyl-phosphatidylcholine containing PAF relate the disorientation of the film with the emergence of a lamellar interdigitated phase of reduced (4.2 nm) repeat distance. This interdigitated phase coexists with the lecithin lamellar phase (repeat spacing 6.0 nm) at temperatures below and above the gel-to-liquid crystal transition temperature of the lecithin. Cholesterol/PAF mixtures give X-ray diffraction patterns indexing a lamellar repeat distance of 6.7 nm. Cholesterol also prevents formation of interdigiatated lamellae with the various asymmetric phospholipids. A model is proposed where the asymmetric phospholipid segregates from lecithin to form 'blister-like' structures within the film consisting of thin interdigitated lamellae. Formation of complexes between cholesterol and the asymmetric lipid prevents the creation of these structures.

Short chain phospholipids in membrane protein crystallization: a 31P-NMR study of colloidal properties of dihexanoyl phosphatidylcholine.

The colloidal features of short chain phospholipids can be deduced from 31P-NMR analysis by comparison with available data on phospholipid aqueous dispersion. In this study with dihexanoyl phosphatidylcholine, detergent phase separation was obtained by temperature shift and by addition of the precipitating agent polyethylene glycol. The 31P-NMR spectra indicate that the detergent micelles fuse to enter the hexagonal HII and lamellar phases. Consequences for the crystallization of membrane proteins are discussed.

Binding of two spin-labelled derivatives of chlorpromazine to human erythrocytes.

The binding to human intact erythrocytes of two different spin-labelled derivatives of chlorpromazine has been studied. The influence of the positively charged side chain of the drug has been the focus of our attention. The positively charged amphiphilic compound (spin derivative I) is water-soluble up to 80 microM at pH values below 5.9. The apolar analogue (spin derivative II) aggregates in aqueous buffer from the lowest concentration tested. Both spin derivatives undergo a slow reduction inside the erythrocyte. The reduced nitroxides are readily reoxidized by adding a low, non-quenching, concentration of potassium ferricyanide to the intact erythrocytes. The fractions of spin label I and II bound to the erythrocyte membrane or to the erythrocyte-extracted lipids remain constant as a function of the temperature (3-42 degrees C) and as a function of the concentration of the spin label up to 150 microM. E.s.r. spectra of both spin labels show a two-component lineshape when they are bound to intact erythrocytes. Below 35 degrees C for the positively charged spin probe, and below 32 degrees C for the apolar spin probe, the simulation of the lineshape shows that more than 50% of the spectrum originates from a slow-motion component. This slow-motion component is also found in erythrocyte-extracted lipids probed by the positively charged spin label below 25 degrees C. In contrast, no slow-motion component is detected in the range 4-40 degrees C for the apolar spin label in erythrocyte-extracted lipids. In this environment the apolar probe experiences a single fast anisotropic motion with an exponential dependence on 1/temperature. Detailed lineshape simulations take into account the exchange frequency between binding sites where the probe experiences a fast motion and binding sites where it experiences a slow motion. The exchange frequency is strongly temperature-dependent. Characterization of the different motions experienced inside the different locations has been achieved and compared for whole erythrocytes and for the extracted lipids. The biochemical nature of the binding sites (membrane protein/acidic phospholipid) giving rise to the slow-motion component is discussed as a function of the polarity of the spin-labelled drug and as a function of the temperature controlling the fluidity of the lipid bulk and influencing the distribution of the drug inside the membrane.

Influence of PAF-acether on lecithin-oriented multibilayers monitored by ESR: interaction of PAF-acether with cholesterol.

The influence of PAF-acether on natural ovolecithin oriented multibilayers is detected by ESR of intercalated 5 doxyl stearic acid. Simulation of lineshapes demonstrates an enlarged orientational distribution of the local director of the phospholipid phase and a small increase of the order parameter. The amount of PAF-acether required to destabilize the ovolecithin lamellar phase depends on the degree of hydration. By contrast, cholesterol displays an organizing effect on the PAF-acether phase. Simulation establishes a sharp orientational distribution of the local director when cholesterol reaches stoichiometric ratio relative to PAF-acether. At the same time, cholesterol increases the order parameter.

Binding of spin-labeled clofibrate to lipoproteins.

The binding of spin-labeled clofibrate to native and partially delipidated lipoproteins is a rapid, linear and non-saturable process observed up to the critical micellar concentration of the drug. Low-density lipoproteins (LDL) display a lower affinity for the drug than very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) relative to their respective specific volume. Unlike various lipophilic drugs, uptake of spin-labeled clofibrate does not correlate with lipoprotein lipid volume. Spin-labeled clofibrate binding to LDL is enhanced when the temperature increases above 25 degrees C. The binding to HDL and VLDL is less temperature-sensitive. The simulation of the ESR spectra has shown that two types of motion should be superimposed for the spin-labeled clofibrate in HDL, in LDL or in partially delipidated LDL. From 40 down to 25 degrees C for HDL and LDL, a fast anisotropic motion is observed. From 25 degrees C down to 5 degrees C, a two-component motion takes place, including a slow isotropic motion of the probe tumbling in a highly hydrophobic environment. Interactions of spin-labeled clofibrate with the apolipoproteins in HDL and LDL are assumed from the emergence of this strongly immobilized component observed when the temperature decreases. In contrast, for spin-labeled clofibrate inserted in the apolar core of VLDL, ESR shows only one component in the whole temperature range (5-40 degrees C). The location of the spin-labeled drug inside the various lipoprotein particles is discussed as a function of temperature.

A nuclear magnetic resonance study of the interactions of antimalarial drugs with porphyrins.

Haematins (hydroxyferriprotoporphyrin IX) constitute a possible receptor for antimalarial drugs such as chloroquine or quinine. This paper reports the study of the interactions of these two molecules with two tetrapyrrole (haematin and uroporphyrin I) by 1H-NMR spectroscopy. This method provided us with the geometry of the interactions in aqueous medium. The interaction consists of a close stacking of the porphyrin ring and the quinoleine moiety of the drugs. Using a porphyrin ring current model it was possible to reach the spatial relationships of the interacting species. It was concluded that hydrophobic forces play a key role in the interaction. The porphyrin plane can accommodate wide structural variations of the interacting species, leading to a weak specificity. The consequences on the mode of action of antimalarial drugs are discussed.

Tryptamine-adenosine 5'-monophosphate interactions as studied by nuclear magnetic resonance and relaxation.

The conformation of tryptamine-adenosine 5'-monosphate and of their 1:1 complex in neutral aqueous solution at 297 K has been investigated by proton NMR and relaxation. The dependences of the proton chemical shift as a function of the tryptamine and AMP concentrations yield an association constant of 6.5 +/- 0.5 1 . mol-1. The reorientation correlation time of the complex tau R = (2.5 +/- 0.1) . 10(-10) s has been determined from the deuteron and ESR linewidth measurements on specifically labelled AMP. The proton longitudinal relaxation shows that the adenine and indole rings are head-to-head stacked 0.31 +/- 0.01 nm apart as confirmed by proton chemical shift measurements. In this complex, the AMP ribose ring takes the 3'-endo (N) conformation and the orientation of the adenine base is anti, whereas the tryptamine aminoethyl residue, in the gauche conformation, is most likely bound to the phosphate by coulombic interactions.

Proton NMR and spin lattice relaxation study of nucleoside di- and triphosphates in neutral aqueous solutions.

The average conformations of adenosine, inosine and guanosine di- and triphosphates in neutral aqueous solution have been investigated by 1H vicinal couplings, chemical shifts and T1 relaxation time measurements at 250 MHz. Comparison of chemical shifts with those of the corresponding nucleotide monophosphates suggests that the beta-phosphate group is in all cases oriented towards the base and close to H3'. The vicinal coupling constants indicate that the proportion of the S conformer of the ribose moiety is 55--60% and that the gauche-gauche rotamer of the CH2-OP exocyclic group is predominant. The preferential orientations of the base have been determined by minimization of the standard deviation about the mean of the molecular reorientation correlation times derived from the H8, H1', H2' and H3' relaxation times and computed interproton distances. The problem of the correlation between the syn-anti equilibrium and the N equilibrium S interconversion has been examined. Typical magnetization recovery curves after a 180 degree pulse have been simulated in the case of ATP, taking into account cross relaxation effects. It is shown that in most of the molecules under consideration the syn orientation of the base is predominant whereas for ATP the syn and anti are equivalent.

Conformations of purine ribosyl 5'-nucleotides bound to glycogen phosphorylase b. A proton T2 relaxation time investigation.

The conformation of 5'-nucleotides in the active site of glycogen phosphorylase b has been deduced from linewidth measurements of protons H-1', H-8 and H-2. It is shown by selective deuteration of the purine ring in position 8 that the orientation of the base is anti in the case of strong activators like AMP and syn in that of weak activators like IMP. The orientation correlation time of the nucleotides in the active site is nearly that of the enzyme, i.e. 160 ns at 21 degrees C.