Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties.

• MeSH
• fluidita membrány účinky léků   MeSH
• krysa rodu rattus MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• lithium farmakologie   MeSH
• membránové lipidy metabolismus   MeSH
• peroxidace lipidů účinky léků   MeSH
• přední mozek účinky léků   metabolismus   MeSH
• spánková deprivace metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The fluorescent molecule diphenylhexatriene (DPH) has been often used in combination with fluorescence anisotropy measurements, yet little is known regarding the non-linear optical properties. In the current work, we focus on them and extend the application to fluorescence, while paying attention to the conformational versatility of DPH when it is embedded in different membrane phases. Extensive hybrid quantum mechanics/molecular mechanics calculations were performed to investigate the influence of the phase- and temperature-dependent lipid environment on the probe. Already, the transition dipole moments and one-photon absorption spectra obtained in the liquid ordered mixture of sphingomyelin (SM)-cholesterol (Chol) (2:1) differ largely from the ones calculated in the liquid disordered DOPC and solid gel DPPC membranes. Throughout the work, the molecular conformation in SM:Chol is found to differ from the other environments. The two-photon absorption spectra and the ones obtained by hyper-Rayleigh scattering depend strongly on the environment. Finally, a stringent comparison of the fluorescence anisotropy decay and the fluorescence lifetime confirm the use of DPH to gain information upon the surrounding lipids and lipid phases. DPH might thus open the possibility to detect and analyze different biological environments based on its absorption and emission properties.

• MeSH
• cholesterol chemie   MeSH
• difenylhexatrien chemie   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční polarizace MeSH
• lipidové dvojvrstvy chemie   MeSH
• molekulární konformace MeSH
• sfingomyeliny chemie   MeSH
• simulace molekulární dynamiky MeSH
• tranzitní teplota MeSH
• vztahy mezi strukturou a aktivitou MeSH
• změna skupenství MeSH
• Publikační typ
• časopisecké články MeSH

1,6-Diphenyl-1,3,5-hexatriene (DPH) is one of the most commonly used fluorescent probes to study dynamical and structural properties of lipid bilayers and cellular membranes via measuring steady-state or time-resolved fluorescence anisotropy. In this study, we present a limitation in the use of DPH to predict the order of lipid acyl chains when the lipid bilayer is doped with itraconazole (ITZ), an antifungal drug. Our steady-state fluorescence anisotropy measurements showed a significant decrease in fluorescence anisotropy of DPH embedded in the ITZ-containing membrane, suggesting a substantial increase in membrane fluidity, which indirectly indicates a decrease in the order of the hydrocarbon chains. This result or its interpretation is in disagreement with the fluorescence recovery after photobleaching measurements and molecular dynamics (MD) simulation data. The results of these experiments and calculations indicate an increase in the hydrocarbon chain order. The MD simulations of the bilayer containing both ITZ and DPH provide explanations for these observations. Apparently, in the presence of the drug, the DPH molecules are pushed deeper into the hydrophobic membrane core below the lipid double bonds, and the probe predominately adopts the orientation of the ITZ molecules that is parallel to the membrane surface, instead of orienting parallel to the lipid acyl chains. For this reason, DPH anisotropy provides information related to the less ordered central region of the membrane rather than reporting the properties of the upper segments of the lipid acyl chains.

• MeSH
• antifungální látky chemie   MeSH
• difenylhexatrien chemie   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční polarizace MeSH
• fosfatidylcholiny chemie   MeSH
• hydrofobní a hydrofilní interakce MeSH
• itrakonazol chemie   MeSH
• lipidové dvojvrstvy chemie   MeSH
• povrchové vlastnosti MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Surfactin, an anionic lipopeptide produced by Bacillus subtilis, is an antimicrobial that targets the cytoplasmic membrane. Nowadays it appears increasingly apparent that the mechanism of resistance against these types of antibiotics consists of target site modification. This prompted us to investigate whether the surfactin non-producing strain B. subtilis 168 changes its membrane composition in response to a sublethal surfactin concentration. Here we show that the exposure of B. subtilis to surfactin at concentrations of 350 and 650 μg/ml (designated as SF350 and SF650, respectively) leads to a concentration-dependent growth arrest followed by regrowth with an altered growth rate. Analysis of the membrane lipid composition revealed modifications both in the polar head group and the fatty acid region. The presence of either surfactin concentration resulted in a reduction in the content of the major membrane phospholipid phosphatidylglycerol (PG) and increase in phosphatidylethanolamine (PE), which was accompanied by elevated levels of phosphatidic acid (PA) in SF350 cultures. The fatty acid analysis of SF350 cells showed a marked increase in non-branched high-melting fatty acids, which lowered the fluidity of the membrane interior measured as the steady-state fluorescence anisotropy of DPH. The liposome leakage of carboxyfluorescein-loaded vesicles resembling the phospholipid composition of surfactin-adapted cells showed that the susceptibility to surfactin-induced leakage is strongly reduced when the PG/PE ratio decreases and/or PA is included in the target bilayer. We concluded that the modifications of the phospholipid content of B. subtilis cells might provide a self-tolerance of the membrane active surfactin.

• MeSH
• Bacillus subtilis chemie   účinky léků   růst a vývoj   MeSH
• cyklické peptidy farmakologie   MeSH
• fluidita membrány účinky léků   MeSH
• fosfolipidy analýza   MeSH
• lipopeptidy farmakologie   MeSH
• mastné kyseliny analýza   MeSH
• membránové lipidy analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

UNLABELLED: Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. IN CONCLUSION: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling.

• MeSH
• algoritmy MeSH
• buněčná membrána chemie   metabolismus   MeSH
• cholesterol metabolismus   MeSH
• difenylhexatrien chemie   metabolismus   MeSH
• difuze MeSH
• fluorescenční polarizace MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus   MeSH
• receptory thyroliberinu chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• zelené fluorescenční proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PRINCIPAL FINDINGS: HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. SUMMARY: Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

• MeSH
• buněčná membrána chemie   účinky léků   metabolismus   MeSH
• cetomakrogol farmakologie   MeSH
• detergenty farmakologie   MeSH
• HEK293 buňky MeSH
• hydrofobní a hydrofilní interakce MeSH
• lidé MeSH
• pertusový toxin toxicita   MeSH
• receptory opiátové delta metabolismus   MeSH
• teplota MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We report sphingolipid-related reorganization of gel-like microdomains in the plasma membrane of living Saccharomyces cerevisiae using trans-Parinaric acid (t-PnA) and 1,6-diphenyl-1,3,5-hexatriene (DPH). Compared to control, the gel-like domains were significantly reduced in the membrane of a sphingolipid-deficient lcb1-100 mutant. The same reduction resulted from sphingolipid depletion by myriocin. The phenotype could be reverted when a myriocin-induced block in sphingolipid biosynthesis was bypassed by exogenous dihydrosphingosine. Lipid order of less-ordered membrane regions decreased with sphingolipid depletion as well, as documented by DPH fluorescence anisotropy. The data indicate that organization of lateral microdomains is an essential physiological role of these structural lipids.

• MeSH
• buněčná membrána chemie   metabolismus   MeSH
• difenylhexatrien chemie   MeSH
• fluorescenční polarizace MeSH
• fluorescenční spektrometrie MeSH
• membránové mikrodomény chemie   metabolismus   MeSH
• mutace MeSH
• nenasycené mastné kyseliny chemie   MeSH
• Saccharomyces cerevisiae MeSH
• sfingolipidy biosyntéza   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

AIMS: To find out membrane tolerance strategy to ethanol in Bacillus subtilis that possesses a powerful system of protection against environmental stresses. METHODS AND RESULTS: Cytoplasmic membranes of B. subtilis were severely affected by even short-term exposure to 3% (v/v) ethanol: the growth rate and membrane protein synthesis were markedly reduced, and no adaptive alterations in phospholipids were detected. Simultaneously, steady-state DPH fluorescence anisotropy (r(ss)) showed that the membrane rigidity increased substantially. Analysis of the membrane phosphoproteome using in vitro labelling with [γ-(32) P]ATP revealed the association of DnaK and GroEL chaperones with membrane, indicating a stress induction process. Upon a long-term 3% (v/v) ethanol stress, the cell growth accelerated slightly and the composition of polar head groups and fatty acids of membrane phospholipids underwent an extensive reconstruction. Correspondingly, membrane fluidity turned back to the original r(ss) values of the control cells. CONCLUSIONS: In B. subtilis, the adaptive response to short-term ethanol stress comprises the recruitment of molecular chaperones on the impaired membrane structure; consequently, the phospholipid synthesis is restored and membrane fluidity adapts properly to the continuing ethanol stress. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings underline the role of membrane lipids in establishing tolerance towards ethanol and also suggest the contribution of molecular chaperones to the membrane and cell recovery.

• MeSH
• Bacillus subtilis účinky léků   růst a vývoj   fyziologie   MeSH
• bakteriální proteiny metabolismus   MeSH
• buněčná membrána chemie   metabolismus   MeSH
• ethanol metabolismus   farmakologie   MeSH
• fluidita membrány účinky léků   MeSH
• fluorescenční polarizace MeSH
• fosfolipidy metabolismus   MeSH
• fyziologický stres MeSH
• mastné kyseliny analýza   MeSH
• membránové lipidy chemie   metabolismus   MeSH
• molekulární chaperony metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Biophysical studies of fluorescence anisotropy of DPH and Laurdan generalized polarization were performed in plasma membranes (PM) isolated from control and cholesterol-depleted HEK293 cells stably expressing pertussis toxin (PTX)-insensitive DOR-Gi1α (Cys351-Ile351) fusion protein. PM isolated from control, PTX-untreated, cells were compared with PM isolated from PTX-treated cells. Results from both types of PM indicated that i) hydrophobic membrane interior was made more accessible to water molecules and more chaotically organized in cholesterol-depleted samples, ii) cholesterol depletion resulted in an overall increase in surface area of membrane, membrane fluidity, and mobility of its constituents. Analysis of DOR-Gi1α coupling in PTX-treated and PTX-untreated cells indicated that cholesterol depletion did not alter the agonist binding site of DOR (Bmax and Kd) but the ability of DOR agonist DADLE to activate G proteins was markedly impaired. In PTX-untreated membranes, EC50 for DADLE-stimulated [35S]GTPγS binding was shifted by one order of magnitude to the right: from 4.3±1.2×10(-9) M to 2.2±1.3×10(-8) M in control and cholesterol-depleted membrane samples, respectively. In PTX-treated membranes, EC50 was shifted from 4.5±1.1×10(-9) M to 2.8±1.4×10(-8) M. In summary, the perturbation of optimum PM organization by cholesterol depletion deteriorates functional coupling of DOR to covalently bound Gi1α as well as endogenously expressed PTX-sensitive G proteins of Gi/Go family while receptor ligand binding site is unchanged. The biophysical state of hydrophobic plasma (cell) membrane interior should be regarded as regulatory factor of DOR-signaling cascade.

• MeSH
• buněčné linie MeSH
• cholesterol metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• lidé MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptory opiátové delta agonisté   metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The effect of non-ionic detergents on baclofen (GABAB-R agonist)-stimulated G-protein activity was measured as a [(35)S]GTPgammaS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic--a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABAB-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58>Triton X-100>Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (rDPH) incorporated into the hydrophobic PM interior. Decrease of rDPH, in the order of Brij58>Triton X-100>Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time phi paralleled by an increase of diffusion wobbling constant Dw (analysis by time-resolved fluorescence according to "wobble-in-cone" model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58>Triton X-100>Digitonin.

• MeSH
• 2-naftylamin analogy a deriváty   MeSH
• buněčná membrána metabolismus   účinky léků   MeSH
• cetomakrogol farmakologie   MeSH
• difenylhexatrien MeSH
• difuze MeSH
• financování organizované MeSH
• fluorescenční barviva MeSH
• fluorescenční spektrometrie MeSH
• krysa rodu rattus MeSH
• laurany MeSH
• mozek metabolismus   účinky léků   MeSH
• oktoxynol farmakologie   MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptory GABA-B metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH