methyl-beta-cyclodextrin
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The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.
- MeSH
- analgetika farmakologie terapeutické užití MeSH
- beta-cyklodextriny * farmakologie MeSH
- bolest chemicky indukované farmakoterapie metabolismus MeSH
- CHO buňky MeSH
- cholesterol metabolismus MeSH
- Cricetulus MeSH
- HEK293 buňky MeSH
- kationtové kanály TRPM * metabolismus genetika MeSH
- lidé MeSH
- membránové mikrodomény metabolismus účinky léků MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- pregnenolon farmakologie MeSH
- pyrimidinony farmakologie MeSH
- sfingomyelinfosfodiesterasa * metabolismus farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In the present investigation, eleven human adipose tissue samples, two seal blubber samples and two pelican muscles samples were analyzed with regard to their concentrations of PCB parent compounds as well as to the respective chiral methylsulfonyl metabolites 3-MeSO2-CB 91, 4-MeSO2-CB 91, 3-MeSO2-CB 95, 4-MeSO2-CB 95, 3-MeSO2-CB 149, 4-MeSO2-CB 149, 3-MeSO2-CB 132, 4-MeSO2-CB 132, 3-MeSO2-CB 174, and 4-MeSO2-CB 174 and the achiral metabolites 3-MeSO2-CB 49, 4-MeSO2-CB 49, 3-MeSO2-CB 101, 4-MeSO2-CB 101, 3-MeSO2-CB 110, 4-MeSO2-CB 110 and 3-MeSO2-DDE. In order to verify enantioselective transformation processes and to compare the different enzymatic transformation pathways in birds and mammals, the enantioselective excesses of the chiral PCB-metabolites were determined by enantioselective gas chromatography with electron capture and mass spectrometric detection using modified cyclodextrin phases, including heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl-)-beta-cyclodextrin/OV1701 (1:1) for the parent PCBs and heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl-)-beta-cyclodextrin/SE52 (1:4) for the metabolites, respectively.
- MeSH
- cyklodextriny metabolismus MeSH
- dichlordifenyldichlorethylen analýza metabolismus MeSH
- látky znečišťující životní prostředí analýza metabolismus MeSH
- lidé MeSH
- methansulfonáty analýza metabolismus MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- polychlorované bifenyly analýza metabolismus MeSH
- stereoizomerie MeSH
- svaly chemie MeSH
- tuková tkáň chemie MeSH
- tuleňovití MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
We developed a method that enables us to distinguish between the same or the opposite enantiomer migration order (EMO) of two enantiomers of a chiral compound with two different selectors. The method is applicable to racemic samples and thus a standard of the pure enantiomeric form(s) is not required. First, complexation constants and mobilities of complexes of the two enantiomers with the first and second selector are determined. However, for a racemic sample it is not possible to deduce whether the first migrating enantiomer with one selector is the same one as the first migrating enantiomer with the second selector. A specific mixture of the two selectors is designed to resolve this. In case the two enantiomers exhibit the same, respectively the opposite EMO in the two selectors, the mixture does, respectively does not separate the racemic sample. Thus two peaks are detected in the first case, while a single coalescent peak is recorded in the opposite case. We demonstrate the method on a racemic sample of amphetamine. Its relative EMO is determined with three cyclodextrins, heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin.
Airway smooth muscle (ASM) membrane depolarization through KCl opens L-type voltage dependent Ca2+ channels (Ca(v)1.2); its opening was considered the cause of KCl contraction. This substance is used to bypass intracellular second messenger pathways. It is now clear that KCl also activates RhoA/Rho kinase (ROCK) pathway. ROCK isoforms are characterized as ROCK1 and ROCK2. Because ROCK1 seems the most abundant isotype in lung, we studied its participation in KCl stimulated bovine ASM. With methyl-beta-cyclodextrin (MbetaCD) we disrupted caveolae, a membrane compartment considered as the RhoA/ROCK assembly site, and found that KCl contraction was reduced to the same extent (~26%) as Y-27632 (ROCK inhibitor) treated tissues. We confirmed that KCl induces ROCK activation and this effect was annulled by Y-27632 or MbetaCD. In isolated plasmalemma, ROCK1 was localized in non-caveolar membrane fractions in Western blots from control tissues, but it transferred to caveolae in samples from tissues stimulated with KCl. Ca(v)1.2 was found at the non-caveolar membrane fractions in control and MbetaCD treated tissues. In MbetaCD treated tissues stimulated with KCl, contraction was abolished by nifedipine; only the response to Ca(v)1.2 opening remained as the ROCK component disappeared. Our results show that, in ASM, the KCl contraction involves the translocation of ROCK1 from non-caveolar to caveolar regions and that the proper physiological response depends on this translocation.
- MeSH
- centrálně působící myorelaxancia farmakologie MeSH
- chlorid draselný farmakologie MeSH
- hladké svalstvo účinky léků metabolismus MeSH
- kaveoly účinky léků metabolismus MeSH
- kinázy asociované s rho metabolismus MeSH
- orgánové kultury - kultivační techniky MeSH
- skot MeSH
- svalová kontrakce účinky léků fyziologie MeSH
- trachea účinky léků metabolismus MeSH
- transport proteinů účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
Interleukin 6 (IL-6) is a pleiotropic cytokine that mediates a variety of functions, including induction of the acute-phase response in hepatocytes. IL-6 initiates its action by binding to its cell surface receptor, followed by activation of Janus kinases and tyrosine phosphorylation of the signal transducer and transcription factor (STAT) 3. Although it has been suggested that cholesterol- and sphingolipid-enriched membrane domains, called lipid rafts, and caveolin are involved in this process, their roles in the earliest stages of IL-6-mediated signaling are far from being understood. Here we show that pretreatment of HepG2 hepatoma cells with methyl-beta-cyclodextrin (MbetaCD), which removes cholesterol and destroys lipid rafts, inhibited tyrosine phosphorylation of STAT3 in IL-6-activated, but not PV-activated cells. Furthermore, when the cells were lysed under conditions preserving lipid rafts, no IL-6- or PV-induced phosphorylation of STAT3 was observed. Although most of the STAT3 was found in large MbetaCD-resistant assemblies in both non-activated and IL-6-activated cells, its association with lipid rafts was weak or undetectable. The extent of IL-6-induced tyrosine phosphorylation of STAT3 was comparable in cells expressing low or high levels of caveolin. Similar STAT3 transducer complexes were observed in freshly isolated rat hepatocytes. The combined data suggest that STAT3 tyrosine phosphorylation occurs in preformed transducer complexes that can be activated in the absence of intact lipid rafts or caveolin.
- MeSH
- aktiny metabolismus MeSH
- buněčná membrána metabolismus účinky léků MeSH
- cholesterol metabolismus MeSH
- cytoskelet metabolismus účinky léků MeSH
- detergenty MeSH
- financování organizované MeSH
- fosfotyrosin metabolismus MeSH
- hepatocyty metabolismus účinky léků MeSH
- interleukin-6 farmakologie MeSH
- kaveoliny metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- multiproteinové komplexy chemie metabolismus MeSH
- nádorové buněčné linie MeSH
- rozpustnost účinky léků MeSH
- separace buněk MeSH
- signální transdukce účinky léků MeSH
- subcelulární frakce metabolismus MeSH
- transkripční faktor STAT3 metabolismus MeSH
- tubulin metabolismus MeSH
- vanadáty farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
The exposure of phosphatidylserine (PS) on the cell surface is a general marker of apoptotic cells. Non-apoptotic PS externalization is induced by several activation stimuli, including engagement of immunoreceptors. Immune cells can also be activated by aggregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs). However, it is unknown whether cell triggering through these proteins, lacking transmembrane and cytoplasmic domains, also leads to PS externalization. Here we show that engagement of GPI-APs in rodent mast cells induces a rapid and reversible externalization of PS by a non-apoptotic mechanism. PS externalization triggered by GPI-AP-specific monoclonal antibodies was dependent on the activity of H(+)-ATP synthase and several other enzymes involved in mast cell signaling but was independent of cell degranulation, free cytoplasmic calcium up-regulation, and a decrease in lipid packing as determined by merocyanine 540 binding. Surprisingly, disruption of actin cytoskeleton by latrunculin B or plasma membrane integrity by methyl-beta-cyclodextrin had opposite effects on PS externalization triggered through GPI-AP or the high affinity IgE receptor. We further show that PS externalization mediated by GPI-APs was also observed in some other cells, and its extent varied with antibodies used. Interestingly, effects of different antibodies on PS externalization were additive, indicating that independent stimuli converge onto a signaling pathways leading to PS externalization. Our findings identify the cell surface PS exposure induced through GPI-AP as a distinct mechanism of cell signaling. Such a mechanism could contribute to "inside-out" signaling in response to pathogens and other external activators and/or to initiation of other functions associated with PS externalization.
- MeSH
- apoptóza genetika účinky záření MeSH
- beta-cyklodextriny farmakologie MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- biologický transport fyziologie účinky léků MeSH
- buňky NIH 3T3 MeSH
- degranulace buněk fyziologie účinky léků MeSH
- financování organizované MeSH
- fluorescenční barviva farmakologie MeSH
- fosfatidylseriny metabolismus MeSH
- glykosylfosfatidylinositoly metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mastocyty metabolismus MeSH
- mitochondriální protonové ATPasy metabolismus MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- potkani Wistar MeSH
- pyrimidinony farmakologie MeSH
- receptory IgE metabolismus MeSH
- signální transdukce fyziologie účinky léků MeSH
- thiazolidiny farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
Electron paramagnetic resonance (EPR) spin trapping spectroscopy is an important method used in free radical research; however, its application in biological systems is hindered by EPR silencing of spin adducts. Previous studies in superoxide-generating chemical systems have shown that spin adducts can be partially stabilized by cyclodextrins. In this work, for the first time, this proposed protective effect of cyclodextrins is investigated in a real biological sample-in isolated thylakoid membranes and photosystem II (PSII) particles with EMPO as a spin trap. It is shown that (i) randomly methylated beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin form inclusion complexes with EMPO-superoxide adducts (EMPO-OOH), (ii) both cyclodextrins increase the intensity of the EMPO-OOH EPR signal in PSII particles up to five times, (iii) higher EMPO-OOH EPR signal intensity is a result of increased stability of EMPO-OOH, and (iv) the extent of the protection of EMPO-OOH adduct provided by cyclodextrins is different in thylakoids and PSII particles. Along with the spin trapping data, the toxicity of cyclodextrins is also discussed with particular focus on photosynthetic preparations. The presented data show that both tested cyclodextrins can be used as valuable tools to improve the sensitivity of spin trapping in biological samples.
- MeSH
- beta-cyklodextriny farmakologie MeSH
- cyklodextriny farmakologie MeSH
- elektronová paramagnetická rezonance metody MeSH
- fotosystém II (proteinový komplex) chemie izolace a purifikace metabolismus MeSH
- pyrroly MeSH
- senzitivita a specificita MeSH
- spin trapping MeSH
- Spinacia oleracea MeSH
- superoxidy chemie metabolismus MeSH
- techniky in vitro MeSH
- tylakoidy chemie účinky léků metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid-NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-β-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-β-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs.
- MeSH
- anticholesteremika farmakologie MeSH
- beta-cyklodextriny farmakologie MeSH
- cholesterol oxidasa farmakologie MeSH
- cholesterol nedostatek fyziologie MeSH
- elektrofyziologické jevy fyziologie MeSH
- fluidita membrány účinky léků fyziologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- membránové lipidy fyziologie MeSH
- metoda terčíkového zámku MeSH
- mozeček cytologie účinky léků fyziologie MeSH
- nervové vedení fyziologie MeSH
- nervový přenos fyziologie MeSH
- potkani Wistar MeSH
- receptory N-methyl-D-aspartátu účinky léků fyziologie MeSH
- simvastatin farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
