betadex and acetovanillone

Using electrophysiological and optical techniques, we studied the mechanisms by which cholesterol depletion stimulates spontaneous transmitter release by exocytosis at the frog neuromuscular junction. We found that methyl-β-cyclodextrin (MCD, 10 mM)-mediated exhaustion of cholesterol resulted in the enhancement of reactive oxygen species (ROS) production, which was prevented by the antioxidant N-acetyl cysteine (NAC) and the NADPH oxidase inhibitor apocynin. An increase in ROS levels occurred both extra- and intracellularly, and it was associated with lipid peroxidation in synaptic regions. Cholesterol depletion provoked a rise in the intracellular Ca(2+) concentration, which was diminished by NAC and transient receptor potential vanilloid (TRPV) channel blockers (ruthenium red and capsazepine). By contrast, the MCD-induced rise in [Ca(2+)]i remained unaffected if Ca(2+) release from endoplasmic stores was blocked by TMB8 (8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride). The effects of cholesterol depletion on spontaneous release and exocytosis were significantly reduced by the antioxidant, intracellular Ca(2+) chelation with BAPTA-AM and blockers of TRPV channels. Bath application of the calcineurin antagonist cyclosporine A blocked MCD-induced enhancement of spontaneous release/exocytosis, whereas okadaic acid, an inhibitor of phosphatases PP1 and PP2A, had no effect. Thus, our findings indicate that enhancement of spontaneous exocytosis induced by cholesterol depletion may depend on ROS generation, leading to an influx of Ca(2+) via TRPV channels and, subsequently, activation of calcineurin.

Topics: Acetophenones; Acetylcysteine; Animals; beta-Cyclodextrins; Calcium; Calcium Signaling; Capsaicin; Cholesterol; Cyclosporine; Exocytosis; Neuromuscular Junction; Okadaic Acid; Ranidae; Reactive Oxygen Species; Ruthenium Red; Synaptic Membranes; TRPV Cation Channels

The association constants for the interactions of 2-hydroxy-4-methoxyacetophenone, 2-hydroxy-5-methoxyacetophenone, 2-hydroxy-6-methoxyacetophenone, 3-hydroxy-4-methoxyacetophenone and 4-hydroxy-3-methoxyacetophenone with β-cyclodextrin in water were measured by (1)H NMR and by isothermal titration calorimetry. Very good agreement was obtained between the different methods. The errors associated with the NMR method for measuring mM binding affinities were estimated to be 10-30%, and by isothermal titration calorimetry, 10-20%. Rotating frame nuclear Overhauser effect spectroscopy studies show that the solution phase host-guest complexes formed by β-cyclodextrin with these hydroxymethoxyacetophenone derivatives are not structurally well defined but that the hydroxymethoxyacetophenone derivatives are mostly associated with the narrow primary hydroxyl rim.

Topics: Acetophenones; beta-Cyclodextrins; Calorimetry; Macromolecular Substances; Magnetic Resonance Spectroscopy; Molecular Structure

Recent studies have indicated the importance of cholesterol-rich membrane lipid rafts (LRs) in oxidative stress-induced signal transduction. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, the major sources of reactive oxygen species, are implicated in cardiovascular diseases, including hypertension. We tested the hypothesis that NADPH oxidase subunits and activity are regulated by LRs in human renal proximal tubule cells. We report that a high proportion of p22(phox) and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D(1)-like receptor agonist, fenoldopam (1 micromol/L per 20 minutes) dispersed Nox subunits within LRs and non-LRs and decreased oxidase activity (30.7+/-3.3%). In contrast, cholesterol depletion (2% methyl-beta-cyclodextrin [beta CD]) translocated NADPH oxidase subunits out of LRs and increased oxidase activity (154.0+/-10.5% versus control, 103.1+/-3.4%), which was reversed by cholesterol repletion (118.9+/-9.9%). Moreover, NADPH oxidase activation by beta CD (145.5+/-9.0%; control: 98.6+/-1.6%) was also abrogated by the NADPH oxidase inhibitors apocynin (100.4+/-3.2%) and diphenylene iodonium (9.5+/-3.3%). Furthermore, beta CD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0+/-5.1% versus beta CD: 162.0+/-2.0%) or Nox4 (108.0+/-10.8% versus beta CD: 152.0+/-9.8%). We have demonstrated for the first time that disruption of LRs results in NADPH oxidase activation that is abolished by antioxidants and silencing of Nox2 or Nox4. Therefore, in human renal proximal tubule cells, LRs maintain NADPH oxidase in an inactive state.

Topics: Acetophenones; beta-Cyclodextrins; Cell Membrane; Cholesterol; Dopamine Agonists; Enzyme Activation; Enzyme Inhibitors; Fenoldopam; Humans; Immunoblotting; Isoenzymes; Kidney Tubules, Proximal; Membrane Glycoproteins; Membrane Microdomains; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; Onium Compounds; Reactive Oxygen Species; Receptors, Dopamine D1; RNA, Messenger; RNA, Small Interfering

Thermodynamic parameters of inclusion complex of beta-cyclodextrin (beta-CD) with paeonol and two of its isomers in aqueous solution have been determined with nano-watt-order isothermal titration calorimetry (ITC) and the host-guest inclusion structure has been investigated by using 1H NMR spectra at 298.2 K. The analysis of thermodynamic data reveals that stoichiometry of beta-CD complex with paeonol (Pae) or acetovanillone (Ace) is 1:1 whereas the inclusion complex of beta-CD with 2-hydroxyl-5-methoxyacetophone (Hma) is in 1:1 coexistence with 2:1 stoichiometry. Further analysis indicates that formation of all the complexes is simultaneously driven by enthalpy and entropy, the inclusion complexation of Pae.beta-CD, Ace.beta-CD and Ham.beta-CD2 is predominantly driven by entropy while Ham.beta-CD by enthalpy. The 1H NMR spectra data provide clear evidence of the inclusion phenomena, which shows that the aromatic ring of the guest molecule insert itself into the torus from the narrow side of the cavity.

Topics: Acetophenones; beta-Cyclodextrins; Calorimetry; Drug Compounding; Isomerism; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Structure; Solubility; Solvents; Thermodynamics; Titrimetry; Water