Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface. (1/160)

In addition to its effect on high density lipoprotein (HDL) cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI) was recently reported to stimulate free cholesterol (FC) flux from Chinese hamster ovary (CHO) cells stably expressing mouse SR-BI, a novel function of SR-BI that may play a role in cholesterol removal from the vessel wall where the receptor can be found. It is possible that SR-BI stimulates flux simply by tethering acceptor HDL particles in close apposition to the cell surface thereby facilitating the movement of cholesterol between the plasma membrane and HDL. To test this, we used transiently transfected cells and compared the closely related class B scavenger receptors mouse SR-BI and rat CD36 for their ability to stimulate cholesterol efflux as both receptors bind HDL with high affinity. The results showed that, although acceptor binding to SR-BI may contribute to efflux to a modest extent, the major stimulation of FC efflux occurs independently of acceptor binding to cell surface receptors. Instead our data indicate that SR-BI mediates alterations to membrane FC domains which provoke enhanced bidirectional FC flux between cells and extracellular acceptors.  (+info)

Two moles of O2 consumption and one mole of H2O2 formation during cholesterol peroxidation with cholesterol oxidase from Pseudomonas sp. strain ST-200. (2/160)

Cholesterol oxidase from Pseudomonas sp. strain ST-200 oxidized cholesterol and cholestanol to 6beta-hydroperoxycholest-4-en-3-one and 5alpha-cholestan-3-one respectively. The former was converted spontaneously to several oxysteroids such as 6-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, with the consumption of 2 mol of O(2) and the formation of 1 mol of H(2)O(2) for each mole of cholesterol oxidized. An oxidized form of the cholesterol oxidase dehydrogenates cholesterol, probably to the 5-en-3-one derivative. A reduced form of the enzyme, yielded from the cholesterol dehydrogenation reaction, dioxygenated cholest-5-en-3-one to 6beta-hydroperoxycholest-4-en-3-one.  (+info)

Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum. (3/160)

The kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated. BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s-1 and 105 s-1 for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s-1 for BCO and 232 s-1 for SCO (in the presence of 1% Thesit and 10% 2-propanol). With reduced SCO the rate of Delta5-6-->Delta4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s-1). With oxidized SCO and BCO the rate of isomerization is much faster ( approximately 300 s-1), thus it is not rate-limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates approximately 250 s-1 and 1.3 s-1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme-product complexes.  (+info)

Increased proximal tubular cholesterol content: implications for cell injury and "acquired cytoresistance". (4/160)

BACKGROUND: Acute renal failure (ARF) leads to secondary adaptive changes that serve to protect proximal tubules from subsequent ischemic or toxic damage [so-called "acquired cytoresistance" (CR)]. A characteristic of CR is increased plasma membrane resistance to attack. Therefore, this study sought to identify potential changes in plasma membrane lipid composition in CR tubules/renal cortex and, if present, to test whether they might mechanistically contribute to the CR state. METHODS: Renal cortices/isolated tubules were obtained from CR mouse kidneys (18-hr postinduction of ischemia reperfusion, myoglobinuria, or ureteral obstruction). Their plasma membrane phospholipid/cholesterol profiles were compared with those observed in either control tissues or tissues obtained one to two hours post-renal damage (that is, prior to emergence of CR). RESULTS: Either no changes or inconsistent changes in phospholipid profiles were observed in CR tissues. Conversely, CR (vs. control) tissues demonstrated a consistent 25 to 50% increase in membrane cholesterol content. To ascertain whether cholesterol impacts tubule susceptibility to injury, its levels were reduced in proximal tubule (HK-2) cells with either (a) mevastatin, (b) a cholesterol "stripping" agent, (c) cholesterol oxidase, or (d) cholesterol esterase. Then cell susceptibility to injury [adenosine 5'-triphosphate (ATP) depletion; Fe-mediated oxidant stress] was assessed. In each instance, cholesterol reductions dramatically sensitized to superimposed injury (for example, a 2 to 3 times increase in the % of lactate dehydrogenase release). When cholesterol levels were restored to normal in CR tubules (with a "stripping" agent), an increased tubule susceptibility to injury resulted. Because cholesterol decreases membrane fluidity, the impact of a membrane-fluidizing agent (A2C) on cell injury was assessed. A2C dramatically sensitized HK-2 cells to superimposed attack. CONCLUSIONS: ARF leads to an up-regulation of proximal tubule cholesterol content. The latter may then contribute to acquired CR, possibly by stabilizing the plasma membrane via its antifluidizing effect.  (+info)

Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells. (5/160)

We investigated the mechanism of endothelin receptor type A (ETA) internalization in Chinese hamster ovary cells using two assays; flow cytometric quantification of cell surface myc-ETA and in situ localization of Cy5-labeled ET-1. In both assays, agonist-dependent internalization of myc-ETA was inhibited by nystatin and filipin, both of which disrupt internalization via caveolae, whereas it was barely affected by chlorpromazine and hypertonic sucrose, both of which disrupt internalization via clathrin-coated pits. In addition to myc-ETA, ET-1 caused intracellular translocation of caveolin-1 and this translocation was also blocked by nystatin but not by chlorpromazine. These results strongly argue that ETA is internalized via caveolae but not clathrin-coated pits. Treatment of the cells with cholesterol oxidase reduced cellular cholesterol and caused intracellular translocation of caveolin-1 but did not affect cell surface localization of myc-ETA. In cholesterol oxidase-treated cells, however, both chlorpromazine and hypertonic sucrose effectively blocked ET-1-induced myc-ETA internalization and nystatin was less effective than in untreated cells. Accordingly, expression of a dominant negative form of beta-arrestin blocked myc-ETA internalization in cholesterol oxidase-treated cells but not in untreated cells. These results suggest that, in Chinese hamster ovary cells, 1) agonist-occupied ETA can be internalized either via caveolae or clathrin-coated pits; 2) of the two, the former is the default pathway; and 3) the oxidative state of cell surface cholesterol is one of the factors involved in the pathway selection.  (+info)

Comparison of a new method for the direct and simultaneous assessment of LDL- and HDL-cholesterol with ultracentrifugation and established methods. (6/160)

BACKGROUND: Automated electrophoresis combined with enzymatic cholesterol staining might improve routine assessment of LDL- and HDL-cholesterol (LDLC and HDLC), as an alternative to the Friedewald equation and precipitation. A new method (Hydrasys; SEBIA) that adapts the cholesterol esterase/cholesterol oxidase reaction within urea-free gels was evaluated. METHODS: Fresh sera from 725 subjects (512 dyslipidemics) were analyzed by electrophoresis, in parallel with sequential ultracentrifugation, beta-quantification, calculation, and precipitation. RESULTS: Electrophoresis was linear up to 4 g/L cholesterol, with a detection limit of 0.042 g/L cholesterol/band. Within-run, between-run, between-batch, and between-operator imprecision (CVs) were 1.6%, 2.0%, 1.5%, and 2.7% for LDLC, and 3.9%, 4.3%, 5.5%, and 4.9% for HDLC, and remained unchanged up to 6.3 g/L plasma triglycerides (TGs). Precision decreased with very low HDLC (<0.25 g/L). Serum storage for 3-7 days at +4 or -80 degrees C did not interfere significantly with the assay. Agreement with beta-quantification was stable for LDLC up to 5.07 g/L (r = 0.94), even at TG concentrations >4 g/L (r = 0.91). Bias (2.88% +/- 12%) and total error (7.84%) were unchanged at TG concentrations up to 18.5 g/L. Electrophoresis predicted National Cholesterol Education Program cut-points with <0.04 g/L error, exactly and appropriately classified 79% and 96% of the subjects, and divided by 2.4 (all subjects) and 5.8 (TGs >1.5 g/L) the percentage of subjects underestimated by calculation. One-half of the patients with TGs >4 g/L had LDLC >1.30 g/L. For HDLC, correlation was better with precipitation (r = 0.87) than ultracentrifugation (r = 0.76). Error (-0.10% +/- 26%) increased when HDLC decreased (<0.35 g/L). Direct assessment of the LDLC/HDLC ratio detected 45% more high-risk subjects than the calculation/precipitation combination. CONCLUSIONS: Electrophoresis provides reliable quantification of LDLC, improving precision, accuracy, and concordance over calculation, particularly with increasing plasma TGs. Implementation of methods to detect low cholesterol concentrations could extend the applications for HDLC assessment.  (+info)

Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack. (7/160)

BACKGROUND: Cholesterol is a major component of plasma membranes, forming membrane microdomains ("rafts" or "caveolae") via hydrophobic interactions with sphingolipids. We have recently demonstrated that tubule cholesterol levels rise by 18 hours following diverse forms of injury, and this change helps to protect kidneys from further damage (so-called acquired cytoresistance). The present study was undertaken to better define the effects of membrane cholesterol/microdomains on tubule homeostasis and cell susceptibility to superimposed attack. METHODS: Plasma membrane cholesterol was perturbed in normal mouse proximal tubular segments with either cholesterol esterase (CE) or cholesterol oxidase (CO). Alternatively, cholesterol-sphingomyelin complexes were altered by sphingomyelinase (SMase) treatment. Changes in cell energetics (ATP/ADP ratios + ouabain), viability [lactate dehydrogenase (LDH) release], phospholipid profiles, and susceptibility to injury (Fe-induced oxidant stress, PLA2, Ca2+ ionophore) were determined. The impacts of selected cytoprotectants were also assessed. RESULTS: Within 15 minutes, CE and CO each induced approximately 90% ATP/ADP ratio suppressions. These were seen prior to lethal cell injury (LDH release), and it was ouabain resistant (suggesting decreased ATP production, not increased consumption). SMase also depressed ATP without inducing cell death. After 45 minutes, CE and CO each caused marked cytotoxicity (up to 70% LDH release). However, different injury mechanisms were operative since (1) CE, but not CO, toxicity significantly altered cell phospholipid profiles, and (2) 2 mmol/L glycine completely blocked CE- but not CO-mediated cell death. Antioxidants also failed to attenuate CO cytotoxicity. Disturbing cholesterol/microdomains with a sublytic CE dose dramatically increased tubule susceptibility to Fe-mediated oxidative stress and Ca2+ overload, but not PLA2-mediated damage. CONCLUSION: Intact plasma membrane cholesterol/microdomains are critical for maintaining cell viability both under basal conditions and during superimposed attack. When perturbed, complex injury pathways can be impacted, with potential implications for both the induction of acute tubular damage and the emergence of the postinjury cytoresistance state.  (+info)

Presence of oxidized cholesterol in caveolae uncouples active platelet-derived growth factor receptors from tyrosine kinase substrates. (8/160)

Platelet-derived growth factor receptor beta (PDGFRbeta) in fibroblasts is concentrated in caveolae where it controls the tyrosine phosphorylation of multiple proteins. Caveolae are enriched in cholesterol and sphingolipids, but the role of these lipids in PDGFR signal transduction is unknown. We report that introduction of cholest-4-en-3-one into caveolae membranes uncouples PDGFR autophosphorylation from tyrosine phosphorylation of neighboring proteins. Cholest-4-en-3-one appears to interfere with the normal interaction between PDGFR and its partners. The results suggest that tightly packed caveolae lipids form a membrane platform that functions as a lipid scaffold for organizing the molecular interactions of multiple signaling pathways.  (+info)

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