Cholesterol Oxidase Treatment Research Articles

Effects of cholesterol oxidase on neurotransmission and acetylcholine levels at the mice neuromuscular junctions

Membrane cholesterol oxidation is a hallmark of redox and metabolic imbalance, and it may accompany neurodegenerative disorders. Using microelectrode recordings of postsynaptic responses as well as fluorescent dyes for monitoring synaptic vesicle cycling and membrane properties, the action of enzymatic cholesterol oxidation on neuromuscular transmission was studied in the mice diaphragms. Cholesterol oxidase (ChO) at low concentration disturbed lipid-ordering specifically in the synaptic membranes, but it did not change markedly spontaneous exocytosis and evoked release in response to single stimuli. At low external Ca2+ conditions, analysis of single exocytotic events revealed a decrease in minimal synaptic delay and the probability of exocytosis upon plasmalemmal cholesterol oxidation. At moderate- and high-frequency activity, ChO treatment enhanced both neurotransmitter and FM-dye release. Furthermore, it precluded a change in exocytotic mode from full-fusion to kiss-and-run during high-frequency stimulation. Accumulation of extracellular acetylcholine (without stimulation) dependent on vesamicol-sensitive transporters was suppressed by ChO. The effects of plasmalemmal cholesterol oxidation on both neurotransmitter/dye release at intense activity and external acetylcholine levels were reversed when synaptic vesicle membranes were also exposed to ChO (i.e., the enzyme treatment was combined with induction of exo-endocytotic cycling). Thus, we suggest that plasmalemmal cholesterol oxidation affects exocytotic machinery functioning, enhances synaptic vesicle recruitment to the exocytosis and decreases extracellular neurotransmitter levels at rest, whereas ChO acting on synaptic vesicle membranes suppresses the participation of the vesicles in the subsequent exocytosis and increases the neurotransmitter leakage. The mechanisms underlying ChO action can be related to the lipid raft disruption.

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells.

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

Abstract 563: Working Out the Kinks of SR-BI-mediated Cholesterol Transport

High density lipoproteins (HDL) facilitate reverse cholesterol transport (RCT), the process by which cholesterol effluxed from peripheral tissues is delivered via selective uptake to the liver for excretion. These processes are mediated by HDL's high-affinity receptor, scavenger receptor BI (SR-BI). SR-BI is an 82 kDa glycoprotein with two transmembrane domains (TMD), two short cytoplasmic N/C-terminal domains, and a large extracellular domain. Our laboratory solved the NMR structure of the C-terminal TMD and nearby extracellular domain. This peptide contains four conserved proline residues, an amino acid that confers structural flexibility. In our structure, P408 and P412 are in an extracellular alpha-helix, P438 lies at a helix-loop junction, and P459 introduces a kink into the TMD helix. We hypothesized that these proline residues support SR-BI-mediated cholesterol transport. To test this hypothesis, we mutated each proline to alanine and transiently expressed wild-type (WT), P408A-, P412A-, P438A-, or P459A-SR-BI in COS-7 cells. Total lysate and cell surface expression of SR-BI was examined by immunoblot analysis and flow cytometry, respectively. All mutants expressed on the cell surface, except P408A-SR-BI, which was absent from whole cell lysates. P412A- and P438A-SR-BI were significantly impaired in their ability to efflux cell-associated [ 3 H]cholesterol to HDL, as compared to WT-SR-BI. Likewise, cellular binding of [ 125 I]HDL and selective uptake of [ 3 H]cholesteryl oleyl ether were reduced with P412A- and P438A-SR-BI. We assessed SR-BI’s ability to modulate membrane cholesterol pools by cholesterol oxidase treatment. SR-BI enhances the accessibility of cholesterol to the exogenous enzyme, and a proportional increase in cholestenone is detected by thin layer chromatography. Cholestenone was increased with WT-SR-BI but not P412A- or P438A-SR-BI, suggesting defective membrane cholesterol distribution. Taken together, key proline residues in the near-C-TMD extracellular domain support efficient HDL binding, selective uptake, cholesterol efflux, and membrane cholesterol organization. These critical residues appear to support SR-BI in a conformation that allows for efficient RCT.

Regulation of Kv7.2/Kv7.3 channels by cholesterol: Relevance of an optimum plasma membrane cholesterol content

Kv7.2/Kv7.3 channels are the molecular correlate of the M-current, which stabilizes the membrane potential and controls neuronal excitability. Previous studies have shown the relevance of plasma membrane lipids on both M-currents and Kv7.2/Kv7.3 channels. Here, we report the sensitive modulation of Kv7.2/Kv7.3 channels by membrane cholesterol level. Kv7.2/Kv7.3 channels transiently expressed in HEK-293 cells were significantly inhibited by decreasing the cholesterol level in the plasma membrane by three different pharmacological strategies: methyl-β-cyclodextrin (MβCD), Filipin III, and cholesterol oxidase treatment. Surprisingly, Kv7.2/Kv7.3 channels were also inhibited by membrane cholesterol loading with the MβCD/cholesterol complex. Depletion or enrichment of plasma membrane cholesterol differentially affected the biophysical parameters of the macroscopic Kv7.2/Kv7.3 currents. These results indicate a complex mechanism of Kv7.2/Kv7.3 channels modulation by membrane cholesterol. We propose that inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol depletion involves a loss of a direct cholesterol-channel interaction. However, the inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol enrichment could include an additional direct cholesterol-channel interaction, or changes in the physical properties of the plasma membrane. In summary, our results indicate that an optimum cholesterol level in the plasma membrane is required for the proper functioning of Kv7.2/Kv7.3 channels.

Direct Imaging of Mobile Nanodomains in the Live Cell Plasma Membrane by using a Two-Color Photobleaching Approach

We recently developed a method termed TOCCSL (Moertelmaier,APL-2005) (‘Thinning out Clusters while Conserving Stoichiometry of Labeling’) which allowed for the first time the direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane (Brameshuber,JBC-2010). Our method sensed these platforms by their property to assemble the putative raft markers glycosylphosphatidylinositol-anchored monomeric GFP (mGFP-GPI) and GM1-Bodipy on a time-scale of seconds on the cell membrane of living CHO and Jurkat T-cells. In order to resolve platforms we used a special photobleaching protocol to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single-molecule brightness analysis. To further validate our method we extended TOCCSL by utilizing two-color co-localization in combination with stoichiometric photobleaching (Ruprecht,SoftMatter-2010). Glycosylphosphatidylinositol-anchored SNAP-tag (GPI-SNAP) was stably expressed by CHO cells and labeled with SNAP-AF488 and SNAP-AF647 at an equimolar ratio. For both colors we were able to observe a homogeneous surface staining with a density of 300-1000 molecules/μm². To reduce the surface density we applied TOCCSL parallel to both color channels. An appropriate recovery time yielded a stoichiometrically reduced surface density enabling the localization of single fluorescent GPI-SNAPs in both color channels with a resolution down to a few ten nanometers. After correction of chromatic aberration we used an automatic algorithm to detect pairs of co-localized GPI-SNAPs within a diffraction limited distance. The number of co-localized GPI-SNAPs and their stability was found to be comparable to our previous measured mGFP-GPI data: around 30% of GPI-SNAPs homo-associated with a lifetime of seconds and disappeared after cholesterol-oxidase treatment - thus supporting the lipid raft concept.

Palmitoylation is required for efficient Fas cell death signaling

Localization of the death receptor Fas to specialized membrane microdomains is crucial to Fas-mediated cell death signaling. Here, we report that the post-translational modification of Fas by palmitoylation at the membrane proximal cysteine residue in the cytoplasmic region is the targeting signal for Fas localization to lipid rafts, as demonstrated in both cell-free and living cell systems. Palmitoylation is required for the redistribution of Fas to actin cytoskeleton-linked rafts upon Fas stimulation and for the raft-dependent, ezrin-mediated cytoskeleton association, which is necessary for the efficient Fas receptor internalization, death-inducing signaling complex assembly and subsequent caspase cascade leading to cell death.

Fas ligand is localized to membrane rafts, where it displays increased cell death–inducing activity

AbstractFas ligand (FasL), a member of the TNF protein family, potently induces cell death by activating its matching receptor Fas. Fas-mediated killing plays a critical role in naturally and pathologically occurring cell death, including development and homeostasis of the immune system. In addition to its receptor-interacting and cell death–inducing extracellular domain, FasL has a well-conserved intracellular portion with a proline-rich SH3 domain–binding site probably involved in non-apoptotic functions. We report here that, as with the Fas receptor, a fraction of FasL is constitutively localized in rafts. These dynamic membrane microdomains, enriched in sphingolipids and cholesterol, are important for cell signaling and trafficking processes. We show that FasL is partially localized in rafts and that increased amounts of FasL are found in rafts after efficient FasL/Fas receptor interactions. Raft disorganization after cholesterol oxidase treatment and deletions within the intracellular FasL domain diminish raft partitioning and, most important, lead to decreased FasL killing. We conclude that FasL is recruited into lipid rafts for maximum Fas receptor contact and cell death–inducing potency. These findings raise the possibility that certain pathologic conditions may be treated by altering the cell death–inducing capability of FasL with drugs affecting its raft localization.

Increased cholesterol decreases uterine activity: functional effects of cholesterol alteration in pregnant rat myometrium

Uterine quiescence is essential for successful pregnancy. Cholesterol and triglycerides are markedly increased in pregnancy. Cholesterol is enriched in microdomains of the plasma membrane known as rafts and caveolae. Both lipid rafts and caveolae have been implicated in cellular signaling cascades. The purpose of this work was to investigate whether manipulation of cholesterol content alters uterine contractility. Late pregnancy (19-21 days) rats were humanely euthanized and strips of longitudinal myometrium were then dissected. Force and Ca(2+) measurements were simultaneously recorded and cholesterol increased by the addition of 5 mg/ml cholesterol or 0.25 mg/ml low-density lipoproteins (LDLs) or reduced by 2% methyl-beta-cyclodextrin (MCD) or 2 U/ml cholesterol oxidase addition to the perfusate. Both LDLs and cholesterol profoundly inhibited spontaneous uterine force production and associated Ca(2+) transients; frequency, amplitude, and duration of contraction were all significantly reduced compared with preceding control contractions. Force and Ca(2+) were also reduced by cholesterol when 1 nM oxytocin was used to stimulate the myometrium. Uterine activity was significantly increased by cholesterol extraction with MCD or cholesterol oxidase treatment. Electron microscopy confirmed the lipid raft disrupting effect of MCD, as formerly electron microscopy-visible caveolae in the myometrial cell membrane all but disappeared after MCD treatment. These data show that uterine smooth muscle cell cholesterol content is critically important for functional activity. A novel finding of our study is that cholesterol is inhibitory for force generation. It may be one of the mechanisms operating to maintain uterine quiescence throughout gestation and may also contribute to difficulties in labor suffered by obese women.

Cholesterol affects African swine fever virus infection

African swine fever virus (ASFV) enters cells by receptor mediated endocytosis and requires a fusion event between the viral envelope and the limiting membrane of the endosome at low pH. In order to investigate the role of cholesterol in the early stages of ASFV infection, we have studied the effect of the removal of cell and viral membrane cholesterol by cholesterol oxidase treatment of cells and virions, as well as the effect of some inhibitors of cholesterol synthesis on the infectious pathway. In addition, we have investigated viral infection in cholesterol-depleted Vero cells. Both cholesterol-depleted and cholesterol oxidase-treated Vero cells were unaltered in their ability to bind or internalize the virus, but were blocked in ASFV fusion and subsequent virus replication. Our results indicate that ASFV infection is affected by cholesterol in the target membrane.

Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum

The factors involved in shuttling cholesterol among cellular membranes have not been defined. Using amphotericin B selection, we previously isolated Chinese hamster ovary cell mutants expressing defects in intracellular cholesterol transport. Complementation analysis among seven mutants identified one cell line, mutant 3-6, with a unique defect. The present analysis revealed three key features of mutant 3-6. First, the movement of cholesterol both from the endoplasmic reticulum and through lysosomes to the plasma membrane was normal. However, when intact 3-6 cells were treated with sphingomyelinase, movement of plasma membrane cholesterol to acyl CoA:cholesterol acyltransferase in the endoplasmic reticulum was defective. Cellular cholesterol was mobilized to this enzyme upon activation by 25-hydroxycholesterol. Second, mutant 3-6 did not utilize endogenously synthesized sterol or low density lipoprotein-derived cholesterol for growth as effectively as parental Chinese hamster ovary cells. Finally, despite normal movement of cholesterol to the plasma membrane, mutant 3-6 was amphotericin B resistant. The plasma membrane cholesterol content was normal as assessed by cholesterol oxidase treatment and Semliki Forest virus fusion, which suggests that the 3-6 mutation alters the organization of cholesterol in the plasma membrane. Our characterization of this mutant cell line should facilitate the identification of gene(s) required for this transport pathway.

Olive oil normalizes the altered distribution of membrane cholesterol and Na +Li + countertransport activity in erythrocyte of hypertensive patients

The effects of olive oil (OO)- and high-oleic sunflower oil (HOSO)-enriched diets on erythrocyte membrane cholesterol distribution (by means of cholesterol oxidation after continuous cholesterol oxidase treatment) and Na +Li + countertransport activity in control subjects and patients with untreated essential hypertension (with or without concomitant hypercholesterolemia) have been examined. The participants were 12 normotensive and sixteen hypertensive women who consumed in randomized order the two monosaturated fatty acid (MUFA) diets over 4-week periods separated by a 4-week washout period. The half-times for cholesterol oxidation were significantly higher in hypertensive women, ranging from an increase of 38 to 57% in the normo-(20.6 ± 2.8 min; P < 0.001) and hyper-(23.4 ± 4.2 min; P < 0.001) cholesterolemic groups. There was a general decrease by 75% in the half-time for cholesterol oxidation after HOSO diet. Interestingly, the oxidation rates were almost normalized after OO diet. The activity of Na +Li + countertransport was found to be significantly higher in hypertensive women, ranging the increase from 22 to 57% in the normo-(0.314 ± 0.043 mmol × [h × liter cell] −1; P < 0.01) and hyper-(0.405 ± 0.086 mmol × [h × liter cell] −1; P < 0.01) cholesterolemic groups. This transport system was further activated after HOSO diet, while almost restored after OO diet. These findings suggest that dietary OO, but not HOSO, is helpful for normalizing the impaired distribution of membrane cholesterol and reducing elevated activity of Na +Li + countertransport in erythrocyte of hypertensive patients. This action of OO also indicates that alterations in membrane cholesterol distribution may be relevants for the pathogenesis of hypertension. The effects, however, cannot be exclusively attributed to the content of MUFAs (mainly oleic acid) in the diet, as HOSO was unable to induce favorable changes.

Oxidation of Nuclear Membrane Cholesterol Inhibits Nucleoside Triphosphatase Activity

Oxygen derived free radicals can oxidize membrane cholesterol. We have previously shown that cholesterol in the nuclear membrane can modulate nuclear nucleoside triphosphatase (NTPase) activity. Nucleocytoplasmic transport of peptides and mRNA via the nuclear pore complex may be regulated by the NTPase. The purpose of the present study was to determine if oxidation of nuclear cholesterol could alter NTPase activity. Nuclear membrane cholesterol was oxidized in situ with cholesterol oxidase (to selectively oxidize cholesterol) and NTPase activity measured. HPLC analysis confirmed the formation of cholesterol oxides. The activity of the NTPase was strikingly inhibited by cholesterol oxidase treatment. The Vmax of the NTPase was significantly decreased after cholesterol oxidase treatment but the Km value was unchanged. The sensitivity of NTPase activity to varying cholesterol oxidase concentrations also suggested that cholesterol located in the inner leaflet of the nuclear membrane appeared to be more important in the modulation of NTPase activity than that in the cytoplasmic leaflet. Our results indicate that oxidation of nuclear membrane cholesterol inhibits NTPase activity. These results have implications for peptide and mRNA flux across the nuclear membrane during conditions where lipid oxidation may be expected.

Analysis of hydroxy and keto cholesterols in oxidized brain synaptosomes.

A rapid method for the simultaneous determination of cholesterol and its oxidation products as well as alpha-to-copherol and tocopherolquinone in brain subcellular fractions is described. The samples are saponified and extracted with hexane. It is not necessary to remove cholesterol in the sample before analyzing for oxysterols. The hexane extract can be used for the assay of cholesterol compounds by capillary gas chromatography and tocopherol compounds by liquid chromatography using a procedure reported previously. Oxidation of synaptosomes by a mixture of Fe2+ plus ascorbate resulted in the production of 7-keto-, 7 alpha-hydroxy-, 7 beta-hydroxy-, and 5 alpha, 6 alpha-epoxycholesterols. The identities of these products were confirmed with gas chromatography/mass spectrometry. Cholesterol oxidase treatment did not result in the formation of any of the above compounds. Thus the types and amounts of the products of oxidation of cholesterol were dependent upon the oxidizing agent. Extraction of the oxysterols under milder conditions without saponification using sodium dodecyl sulfate cannot be used since such treatment results in low recovery of oxysterols. Oxidation of synaptosomes by low concentrations of ferrous iron and ascorbate resulted in (i) low levels of oxidation of cholesterol which could be followed by estimating the production of oxysterols and (ii) oxidation of a substantial percentage of alpha-tocopherol. The proposed procedure will be useful in monitoring the oxidation of small quantities of membrane cholesterol in vitro.

Distribución y movimiento del colesterol en la membrana de eritrocito de pacientes con hipertensión esencial

The participants were 12 healthy subjects, 12 normocholesterolaemic hypertensive and 12 hypercholesterolaemic hypertensive patients. Venous blood samples were collected in Trabajo ganador del VIII Premio Sociedad Andaluza de Medicina Interna. «Dr. Lopez Laguna» 0.38% sodium citrate. Erythrocyte membrane cholesterol distribution and transbilayer movement were measured according to the continuous cholesterol oxidase treatment. Erythrocyte Na + -Li + countertransport activity was determined by measurements of Li + efflux in the presence or absence of Na + .The statistical analysis was conducted for significance by using ANOVA for paired data and correlations were carried out by linear regression analysis using Pearson's correlation coefficient. Our findings are consistent with the presence of cholesterol-rich domains randomly distributed in the human erythrocyte. Cholesterol appeared to be similarly enriched in the cytofacial monolayer but the size of such structural cholesterol pool was greater in erythrocytes of hypertensive patients than in those of healthy subjects. There was also a decrease in the movement rate of erythrocyte membrane cholesterol, which contributed to keep impaired the asymmetric distribution of cholesterol in hypertensive patients. In addition, there was a strong correlation between the half-time for membrane cholesterol distribution or transbilayer movement and erythrocyte Na + -Li + countertransport activity, a well known marker of inherited predisposition to essential hypertension. Our data indicate that the steady-state distribution of membrane cholesterol and cell cholesterol exchange may be intimately linked to the etiology of essential hypertension, and to other diseases which are associated to alterations in the lipid metabolism.

Phase transitions of rat stratum corneum lipids by an electron paramagnetic resonance study and relationship of phase states to drug penetration

In order to relate barrier function to stratum corneum structure and the thermal transitions of corneum lipids, samples from hairless rat skin were investigated by using ESR and drug penetration techniques. The phase transition of stratum corneum lipids was estimated using a deeper probe (16-doxyl-stearic acid) inserted in the lipid bilayers and measuring the rotational correlation time, τ c. Results of ESR study showed that stratum corneum lipids underwent thermal transitions at 39.3 ± 1.6°C and 63.6 ± 2.6°C roughly similar to the data obtained by differential scanning calorimetry measurements. Cholesterol oxidase treatment decreased the fluidity of the lipids at lower temperatures. The treatment of straum corneum with laurocapram (1%) and isopropyl myristate (IPM, 2%) little changed both phase transition temperatures, although the treatment highly increased the molecular motion of the lipids. The flux ( J s) of lipophilic drugs (β-estradiol, indomethacin and betahistine) through the skin was enhanced with increasing temperatures, with an increase in the diffusion constant within skin and a decrease in the lag time. There was a good relationship between log J s or log permeability coefficient ( K p) and 1 τ c in the temperature range of 45 to 64°C. The calculated activation energy ( ΔE) for diffusion of these drugs cross skin was 17–40 kcal/mol. Judging from our data, stratum corneum lipids of rat probably exist as the gel, crystalline state below 39°C, the mesomorphic state between 39 and 64°C and the fluid, liquid-crystalline state at temperatures of 64°C or above. These results are in line with that the permeability of these lipophilic drugs through the intercellular lipids disordered is highly increased.

Erythrocyte membrane cholesterol distribution in patients with untreated essential hypertension

To determine whether the membrane cholesterol distribution is associated with the increased activity of sodium-lithium countertransport in the erythrocytes of normocholesterolaemic and hypercholesterolaemic patients with untreated essential hypertension. Erythrocytes were prepared from venous blood samples obtained from normotensive subjects and hypertensive (normocholesterolaemic and hypercholesterolaemic) patients. The membrane cholesterol distribution between the inner and outer monolayers was measured by means of cholesterol oxidation to cholestenone after continuous cholesterol oxidase treatment. The sodiumlithium countertransport activity was determined by measurements of external sodium (150 mmol/l)-stimulated lithium efflux. The statistical analysis was conducted by analysis of variance with Tukey's correction and correlations were performed by linear regression analysis using Pearson's correlation coefficient. The half-times for cholesterol oxidation were significantly higher in patients with untreated essential hypertension, either with (25.4 +/- 5.6 min) or without (21.7 +/- 2.9 min) concomitant hypercholesterolaemia, than in the controls (15.3 +/- 2.8 min). Sodium-lithium counter-transport activities were also higher in the hypertensive patients (0.410 +/- 0.094 and 0.304 +/- 0.037 mmol/h per litre cell for the hypercholesterolaemic and normocholesterolaemic groups, respectively) than in the controls (0.262 +/- 0.081 mmol/h per litre cell). In single-regression analysis, the half-time for membrane cholesterol oxidation was positively correlated to the erythrocyte cation flux mediated by the sodium-lithium countertransport. It is hypothesized that the sodium-lithium countertransport activity might be influenced by membrane structural cholesterol domains.

Transbilayer movement of erythrocyte membrane cholesterol in human essential hypertension.

To study whether the rate of transbilayer movement of membrane cholesterol is impaired in erythrocyte membrane of normo- and hypercholesterolaemic patients with untreated essential hypertension. An observational case-control study. Erythrocytes were prepared from venous blood samples obtained from normotensive subjects and hypertensive patients. The rate of transbilayer movement of membrane cholesterol was monitored in intact erythrocytes, using a radiolabelled cholesterol tracer. Erythrocytes were treated briefly or continuously with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the period of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected the transbilayer movement of radiolabelled cholesterol. There were no significant differences between the diffusion of cholesterol across the erythrocyte membrane of normo- and hypercholesterolaemic hypertensive patients, but the rates were significantly lower than that estimated in control subjects. The mean +/- SD half-times for the process were 55.1 +/- 8.8 and 11.3 +/- 2.1 min in controls, 63.1 +/- 9.2 and 15.8 +/- 2.3 min in normocholesterolaemic hypertensive patients, and 66.2 +/- 9.4 and 16.2 +/- 1.7 min in hypercholesterolaemic hypertensive patients, after a brief and after a continuous cholesterol oxidase treatment, respectively. There is a reduction in the transbilayer movement of membrane cholesterol in erythrocytes of patients with untreated essential hypertension.

Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation.

Caveolae are a membrane specialization used to internalize molecules by potocytosis. Caveolin, an integral membrane protein, is associated with the striated coat present on the cytoplasmic surface of the caveolae membrane. We now report that oxidation of caveolar cholesterol with cholesterol oxidase rapidly displaces the caveolin from the plasma membrane to intracellular vesicles that colocalize with Golgi apparatus markers. After the enzyme is removed from the medium, caveolin returns to caveolae. When untreated cells are gently homogenized, caveolin on the plasma membrane is accessible to both anti-caveolin IgG and trypsin. After cholesterol oxidase treatment, however, Golgi-associated caveolin is inaccessible to both of these molecules. Brefeldin A, which inhibits ER to Golgi trafficking, blocks the appearance of caveolin in the Golgi apparatus but does not prevent caveolin from leaving the plasma membrane. Indirect immunogold localization experiments show that in the presence of cholesterol oxidase caveolin leaves the plasma membrane and becomes associated with endoplasmic reticulum and Golgi compartments. Surprisingly, the loss of caveolin from the plasma membrane does not affect the number or morphology of the caveolae.

Distribution of erythrocyte membrane cholesterol in human essential hypertension

To determine whether the cholesterol distribution is impaired in erythrocyte membranes of normo- and hypercholesterolaemic patients with untreated essential hypertension. Observational case-control study. Erythrocytes were prepared from venous blood samples obtained from normotensive subjects and hypertensive patients. The membrane cholesterol distribution was measured by cholesterol oxidation to cholestenone after continuous cholesterol oxidase treatment. The membrane cholesterol content was determined. The ability of cells to be labelled with [3H]-cholesterol was also tested. The cholesterol distribution was asymmetric in erythrocyte membranes of the control subjects, and hypertensive patients. The oxidation rate was faster in normotensive subjects, and no differences were found between normo- and hypercholesterolaemic hypertensive patients. Total cholesterol mass was lower in erythrocyte membranes of hypertensive patients, as indicated by a higher incorporation of radioactive cholesterol. These data suggest that the membrane cholesterol distribution is impaired in patients with untreated essential hypertension, and support the presence of cholesterol-rich domains in the erythrocyte membrane inner monolayer.

Effect of cholesterol oxidase treatment on physical state of renal brush border membranes: evidence for a cholesterol pool interacting weakly with membrane lipids.

Treatment with cholesterol oxidases has shown that cholesterol is heterogeneously distributed in brush borders isolated from the apical membrane domain of the renal and intestinal epithelial cells [Bloj, B., & Zilversmit, D. B. (1982) J. Biol. Chem. 257, 7608-7614; El Yandouzi, E. H., & Le Grimellec, C. (1992) Biochemistry 31, 547-551]. To better understand the origin of cholesterol heterogeneity, the effects of cholesterol oxidation by Brevibacterium sp. cholesterol oxidase on the physical state of renal brush border membrane vesicles were determined using steady-state fluorescence polarization and differential phase fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH). Selective quenching by trinitrobenzenesulfonate indicated that DPH distributes equally between outer and inner membrane leaflets. Oxidation of 90% of the cholesterol decreased the steady-state anisotropy of DPH, determined at 37 degrees C, by 14%. This modification corresponded to a change in the lipid order, the rotational rate of the probe being unaffected. Oxidation of the cholesterol corresponding to the readily accessible pool (30% of the total cholesterol), on the other hand, had a very limited effect on the membrane physical state. This contrasted with the linear decrease in both anisotropy and fluorescence lifetime of DPH obtained when cholesterol was replaced by cholestenone in liposomes made of phosphatidylcholine/sterol (1/1 molar ratio). These results indicate that, in brush border membranes, the cholesterol readily accessible to cholesterol oxidase interacts only weakly with the other membrane lipids.