betadex and mevastatin

Cholesterol is a prominent component of nerve terminals. To examine cholesterol's role in central neurotransmission, we treated hippocampal cultures with methyl-beta-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used hippocampal cultures from Niemann-Pick type C1-deficient mice defective in intracellular cholesterol trafficking. These conditions revealed an augmentation in spontaneous neurotransmission detected electrically and an increase in spontaneous vesicle endocytosis judged by horseradish peroxidase uptake after cholesterol depletion by methyl-beta-cyclodextrin. In contrast, responses evoked by action potentials and hypertonicity were severely impaired after the same treatments. The increase in spontaneous vesicle recycling and the decrease in evoked neurotransmission were reversible upon cholesterol addition. Cholesterol removal did not impact on the low level of evoked neurotransmission seen in the absence of synaptic vesicle SNARE protein synaptobrevin-2 whereas the increase in spontaneous fusion remained. These results suggest that synaptic cholesterol balances evoked and spontaneous neurotransmission by hindering spontaneous synaptic vesicle turnover and sustaining evoked exo-endocytosis.

Topics: Animals; beta-Cyclodextrins; Cells, Cultured; Cholesterol; Disease Models, Animal; Evoked Potentials; Hippocampus; Intracellular Signaling Peptides and Proteins; Lovastatin; Mice; Mice, Knockout; Neuroglia; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C; Proteins; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Synaptic Vesicles; Vesicle-Associated Membrane Protein 2

Lipids, including cholesterol, are critical components of the cell membrane where they are enriched in microdomains, lipid rafts, which organize and concentrate receptors and intracellular proteins involved in signal transduction. The present study examined the effects of cholesterol depletion on serotonin (5-HT) binding and signaling via 5-hydroxytryptamine(7) (5-HT(7)) receptors in stably transfected HeLa cells. Immunohistochemical, ligand-binding and biotinylation experiments demonstrated that the studied cells expressed high levels of 5-HT(7) receptors at their surface with a pharmacological profile resembling 5-HT(7) receptors in native tissue. Depletion of cholesterol, by combined treatment with mevastatin, fumonisin B(1) and mevalonate or methyl-beta-cyclodextrin (MbetaCD), caused highly significant reductions in the B(max) values of [(3)H]5-HT- and [(3)H]-(R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol ([(3)H]SB269970)-binding to 5-HT(7) receptors. Cholesterol depletion also reduced the total level of 5-HT(7) receptor protein determined by Western blot analysis. None of the examined treatments affected the affinity of [(3)H]5-HT- or [(3)H]SB269970-binding to 5-HT(7) receptors. Treatment with serotonin caused strong inductions in the phosphorylation states of Ser(63)-ATF-1 and Ser(133)-CREB. These effects of serotonin on signal transduction were significantly counteracted by pre-treatment with cholesterol synthesis inhibitors. Altogether, the present study demonstrates that cholesterol depletion decreases binding of both agonist and antagonist radioligands to 5-HT(7) receptors and counteract 5-HT(7) receptor-mediated intracellular signaling.

Topics: 5-Methoxytryptamine; Activating Transcription Factor 1; beta-Cyclodextrins; Binding, Competitive; Blotting, Western; Cholesterol; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Fumonisins; HeLa Cells; Humans; Lovastatin; Mevalonic Acid; Phenols; Phosphorylation; Radioligand Assay; Receptors, Serotonin; Serine; Serotonin; Signal Transduction; Sulfonamides; Transfection; Tritium

Lipid rafts are cholesterol-enriched microdomains in plasma membranes. The functional activity of many membrane proteins, including death and growth factor receptors, depends on their insertion in lipid rafts. We have previously demonstrated the presence of lipid rafts in keratinocytes and shown that lipid rafts are involved in the control of keratinocyte proliferation and metabolic activity. In this work, we investigated the effect of lipid-raft disruption on HaCaT keratinocyte survival. Lipid rafts could be disrupted or rearranged with cholesterol-targeting detergents: methyl-beta-cyclodextrin and filipin III. Moreover, cholesterol oxidation by a specific oxidase or blocking of cholesterol synthesis by mevastatin had a similar effect on lipid rafts. All cholesterol-modifying substances caused cell death in a concentration-dependent manner. More detailed studies on the effects of cyclodextrin revealed apoptotic cell death at concentrations >or=0.5% (w/v). The molecular mechanism of apoptosis precipitated by raft disruption remains unknown but does not seem to be dependent of either membrane permeabilization or cell-cycle arrest imposed by cholesterol-modifying compounds.

Topics: Apoptosis; beta-Cyclodextrins; Cell Cycle; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Cholesterol; Cholesterol Oxidase; Detergents; Enzyme-Linked Immunosorbent Assay; Filipin; Humans; In Situ Nick-End Labeling; Keratinocytes; Lipids; Lovastatin; Membrane Microdomains; Microscopy, Confocal; Microscopy, Electron; Microscopy, Fluorescence; Time Factors

Release of cellular cholesterol and phospholipid mediated by helical apolipoprotein and ATP-binding cassette transporter (ABC) A1 is a major source of plasma HDL. We investigated the effect of calcium channel blockers on this reaction.. Expression of ABCA1, apoA-I-mediated cellular lipid release, and HDL production were enhanced in cAMP analogue-treated RAW264 cells by verapamil, and similar effects were also observed with other calcium channel blockers. The verapamil treatment resulted in rapid increase in ABCA1 protein and its mRNA, but not the ABCG1 mRNA, another target gene product of the nuclear receptor liver X receptor (LXR). By using the cells transfected with a mouse ABCA1 promoter-luciferase construct (-1238 to +57bp), verapamil was shown to enhance the transcriptional activity. However, it did not increase transcription of LXR response element-driven luciferase vector.. The data demonstrated that verapamil increases ABCA1 expression through LXR-independent mechanism and thereby increases apoA-I-mediated cellular lipid release and production of HDL.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; Apolipoprotein A-I; ATP Binding Cassette Transporter 1; ATP Binding Cassette Transporter, Subfamily G, Member 1; ATP-Binding Cassette Transporters; beta-Cyclodextrins; Bucladesine; Calcium Channel Blockers; Cell Line; Cholesterol; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Humans; Hydroxycholesterols; Lipoproteins; Lipoproteins, HDL; Liver X Receptors; Lovastatin; Macrophages; Mice; Nicardipine; Nifedipine; Orphan Nuclear Receptors; Phospholipids; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Stereoisomerism; Transfection; Verapamil

Fas is a member of the tumour necrosis factor receptor superfamily. Fas-mediated apoptosis is an essential mechanism protecting against skin cancer. Activation of Fas by specific ligand or agonistic antibodies leads to the formation of a membrane associated death-inducing signalling complex comprising aggregates of Fas, the Fas-associated death domain protein (FADD), and caspase-8. It has recently been suggested that activity of Fas is not only regulated by its cognate ligand but also by the association of this receptor with cholesterol-enriched lipid domains in the plasma membrane (lipid rafts). We report here that disruption of lipid rafts by cholesterol-depleting compounds (methyl-beta-cyclodextrin, filipin III, cholesterol oxidase, and mevastatin) leads to a spontaneous clustering of Fas in the non-raft compartment of the plasma membrane, formation of Fas-FADD complexes, activation of caspase-8, and apoptosis. We propose that in some cell types exclusion of Fas from lipid rafts leads to the spontaneous, ligand-independent activation of this death receptor, a mechanism that can potentially be utilized in anticancer therapy.

Topics: Apoptosis; beta-Cyclodextrins; Cell Line; Cholesterol; Cholesterol Oxidase; Cyclodextrins; Dose-Response Relationship, Drug; fas Receptor; Filipin; Humans; Keratinocytes; Ligands; Lovastatin; Membrane Fluidity; Membrane Microdomains

Alpha-synuclein contributes to the pathogenesis of Parkinson's disease (PD), but its precise role in the disorder and its normal function remain poorly understood. Consistent with a presumed role in neurotransmitter release and its prominent deposition in the dystrophic neurites of PD, alpha-synuclein localizes almost exclusively to the nerve terminal. In brain extracts, however, alpha-synuclein behaves as a soluble, monomeric protein. Using a binding assay to characterize the association of alpha-synuclein with cell membranes, we find that alpha-synuclein binds saturably and with high affinity to characteristic intracellular structures that double label for components of lipid rafts. Biochemical analysis demonstrates the interaction of alpha-synuclein with detergent-resistant membranes and reveals a shift in electrophoretic mobility of the raft-associated protein. In addition, the A30P mutation associated with PD disrupts the interaction of alpha-synuclein with lipid rafts. Furthermore, we find that both the A30P mutation and raft disruption redistribute alpha-synuclein away from synapses, indicating an important role for raft association in the normal function of alpha-synuclein and its role in the pathogenesis of PD.

Topics: alpha-Synuclein; Amino Acid Substitution; Animals; beta-Cyclodextrins; Brain Chemistry; Cell Compartmentation; Cell Membrane Permeability; Cells, Cultured; Cholesterol; Detergents; Digitonin; Fumonisins; HeLa Cells; Hippocampus; Humans; Kidney; Lovastatin; Membrane Lipids; Membrane Microdomains; Mevalonic Acid; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Mutation, Missense; Nerve Tissue Proteins; Nystatin; Protein Binding; Rats; Recombinant Fusion Proteins; Sphingolipids; Synucleins; Transfection

Cell degeneration in Alzheimer's disease is mediated by a toxic mechanism that involves interaction of the AbetaP peptide with the plasma membrane of the target cell. We report here that PC12 cells become resistant to the cytotoxic action of AbetaP when incubated in a medium that enriches cholesterol levels of the surface membrane. On the other hand, making cholesterol-deficient membranes by either cholesterol extraction with cyclodextrin or by inhibiting de novo synthesis of cholesterol makes PC12 cells more vulnerable to the action of AbetaP. Increasing cholesterol content of PS liposomes also suppresses AbetaP-dependent liposome aggregation. We suggest that by modifying the fluidity of the neuronal membranes, cholesterol modulates the incorporation and pore formation of AbetaP into cell membranes. This idea is supported by our finding that the enhanced cytotoxicity generated by lowering the membrane cholesterol content can be reversed by AbetaP calcium channel blockers Zn2+ and tromethamine.

Topics: Amyloid beta-Peptides; Animals; Anticholesteremic Agents; beta-Cyclodextrins; Cell Membrane; Cell Survival; Cholesterol; Cyclodextrins; Dose-Response Relationship, Drug; Liposomes; Lovastatin; PC12 Cells; Peptide Fragments; Phosphatidylserines; Rats; Tromethamine; Zinc

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.. 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).. 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.. 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.

Topics: Acute Kidney Injury; Animals; beta-Cyclodextrins; Cholesterol; Cholesterol Oxidase; Cyclodextrins; Cytoprotection; Kidney Cortex; Kidney Tubules, Proximal; Lovastatin; Male; Membrane Fluidity; Mice; Phospholipids; Sterol Esterase