Melittin Stimulates Incorporation and Degradation of Sphingomyelin in Synaptosomal Plasma Membranes

Abstract: Melittin enhanced sphingomyelin (SPM) degradation by the neutral membrane-bound sphingomyelinase from calf brain synaptosomal plasma membranes (SYM) up to 20-fold. Melittin in concentrations as high as 100 μM did not significantly alter membrane fluidity of SYM as measured by fluorescence depolarization and electron spin resonance (ESR) using diphenylhexatriene and a doxy1 derivative of SPM, respectively. In the concentration range 100-1000 μM. melittin was observed to rigidify SYM. The incorporation of SPM.erivatives into the lipid bilayer of SYM.as demonstrated by ESR measurements. Melittin enhanced the uptake of SPM-derivatives into SYM.     

Tolerance to NaCl induces changes in plasma membrane lipid composition,fluidity and H+-ATPase activity of tomato calli

Two tomato ( Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 m M NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H⁺-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 m M NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 m M NaCl, growth of calli tolerant to 100 m M NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 m M NaCl. Growth in the presence of 100 m M NaCl provoked an increase of Na⁺ and Cl⁻ content, but no significant changes in K⁺ and Ca²⁺. As compared with NaCl-sensitive and 50 m M NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 m M NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 m M NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 m M NaCl showed lower ATP hydrolysis and ATP-dependent H⁺-pumping activities, as well as a lower passive permeability to H⁺ than plasma membrane from NaCl-sensitive and 50 m M NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H⁺-ATPase activity in salt tolerance of tomato calli is discussed.     

Change in Membrane Fluidity of Sand Dollar Egg Cortices Caused by Ca2+-Induced Exocytosis: Microscopic Analysis with Fluorescence Anisotropy

The surface membrane fluidity of sand dollar eggs after exocytosis was investigated by using a specially designed video-microscope system to measure the fluorescence depolarization of isolated cortices stained with hexadecanoylaminofluorescein. When unfertilized eggs were stained before isolation, the plasma membranes became labeled with fluorescent dye, but cortical vesicles did not. The fluorescence anisotropy of the isolated cortices increased from 0.256 to 0.285 during exocytosis induced by Ca²⁺. The increased anisotropy was not changed by lowering the Ca²⁺concentration after exocytosis. Dislodging of cortical vesicles by shearing with a stream of solution had no affect on the anisotropy. These results suggest that the fluidity of the plasma membrane decreases after exocytosis. When cortices were stained after isolation, both plasma membranes and cortical membrane organelles became labeled. These cortices possessed an anisotropy of 0.215. After dislogding the cortical organelles the anisotropy increased to 0.232. These results indicate that the fluidity of the cytoplasmic membrane leaflets of cortical organelles is higher than that of the plasma membrane. Therefore, it was suggested that only the outer leaflet of the plasma membrane becomes less fluid after exocytosis.     

Pitfalls in measuring fluorescence polarization in mitogen-stimulated T-lymphocytes

Fluorescence polarization measurements with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to detect changes in the fluidity of plasma membranes from T-lymphocytes stimulated with mitogens. When the cells were incubated with succinyl-concanavalin A an increase in fluorescence polarization was observed. This, however, could be shown to be due to the interaction of the mitogen with the label DPH and did not reflect changes in the plasma membrane. In purified plasma membranes a decrease rather than an increase of fluorescence polarization was observed.     

Fluidity characteristics of bovine thyroid plasma membranes

Highly purified plasma membranes of bovine thyroid were obtained by differential pelleting followed by discontinuous gradient centrifugation in a swing-out rotor. Subfractions of plasma membranes were prepared by affinity chromatography on Con A-Sepharose. The final membrane fractions were enriched 25-30-fold over homogenate in 5'-nucleotidase and alkaline phosphatase and displayed a protein to phospholipid ratio of 1.67 and a cholesterol to phospholipid molar ratio of 0.55. The phospholipid composition did not deviate appreciably from that of whole tissue except for the higher sphingomyelin level (22.5 vs. 14.0%). The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. The physical state of the membrane was studied by (i) calculation of the lipid structural order parameter SDPH from steady-state fluorescence anisotropy determinations of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH); (ii) estimation of the lateral diffusion coefficient of pyrene following excimer formation. These parameters were determined in native thyroid plasma membranes and in reconstituted vesicles, obtained by detergent dialysis from octylglucoside solubilized membrane components. The presence of membrane protein or neutral lipids induced more restraint on the movements of the fluorophores. The lipid order parameter, SDPH was mainly determined by the neutral lipids. Subfractions of plasma membrane enriched in luminal membranes have a slightly lower fluidity (higher SDPH and lower Ddiff values) than subfractions enriched in basolateral membranes. This difference appears to be due to both differences in lipid as well as protein composition. Under physiological conditions, no significant alterations in probe dynamics could be observed upon addition of thyrotropin or cholera toxin, even at micromolar concentrations.     

Membrane fluidity of Toxoplasma gondii: a fluorescence polarization study

Toxoplasma gondii membrane fluidity was investigated by fluorescence polarization. We used 1,6-diphenyl 1,3,5-hexatriene (DPH) as a fluorescent hydrophobic probe. Fluorescence anisotropy (r) and degree of order (s) showed high fluidity properties. Chemical analysis was performed on this parasite. We found a low cholesterol/phospholipid ratio, many unsaturated fatty acids chains, and high phosphatidylcholine and low sphingomyelin amounts. These results were in good agreement with the observed high fluidity. This may be related to the great adaptability of Toxoplasma gondii in infesting a wide variety of host cells.     

Possible Mechanism of Dantrolene Stabilization of Cultured Neuroblastoma Cell Plasma Membranes

Abstract: Some reports have suggested that dantrolene interacts directly with the membrane bilayer. We investigated effects of dantrolene on changes in membrane properties induced by compound 48/80 (C48/80), a membrane stimulator. The addition of C48/80 for 1 min elicited a rapid, dose-dependent Ca²⁺ influx, which was reduced to 14% by the absence of external Ca²⁺. Dantrolene inhibited the C48/80-induced increase in Ca²⁺ permeability of plasma membranes in a concentration-dependent manner (0.33–10 µ M , IC₅₀ value was 5 µ M ). We next examined C48/80-induced changes in structural and dynamic membrane properties by electron spin resonance (ESR). The ratio h ₀/ h ₋₁ was determined to evaluate membrane fluidity. C48/80 increased the membrane fluidity in a concentration-dependent manner (0.1–0.56 mg/ml). Dantrolene (10 µ M ) itself did not change the membrane fluidity, but it significantly reduced the C48/80-induced increase in membrane fluidity (0.56 mg/ml). Moreover, the C48/80-induced increase in fluidity was dependent on extracellular Ca²⁺. We conclude that dantrolene protects neuroblastoma cell plasma membrane from C48/80-induced membrane perturbation, which causes Ca²⁺ influx and an increase in membrane fluidity. These findings strongly suggest that dantrolene directly stabilizes the neuronal plasma membrane.     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦（TriticumsativumL．）根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯（DPH）荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H －AT－Pase活力降低,暗示膜蛋白的构象和功能发生了改变。结果表明,质膜膜脂物理状态改变可能是植物感受渗透胁迫的原初响应。     

Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A_(549) cells to cisplatin

Alterations of membrane lipid biophysical properties of sensitive A549 and resistant A549/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Mero-cyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A549 cell and 0.194 for the resistant A549/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A549/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that alterations of the membrane fluidity of both     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦(Triticum sativum L.)根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯(DPH)荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H     

DPH标记细胞膜的动力学与膜脂流动性的荧光偏振校正测量

用稳态荧光技术测得经过校正的荧光成分,由此算出用DPH标记的细胞膜的偏振度。方法是作荧光偏振值在随时间变化的曲线,将其外推至零标记时间求出该时间的荧光偏振值。用此法测定了艾氏腹水癌细胞的膜流动性。结果表明流动性比用整个细胞测得之值小,说明膜脂的有序程度和包装密度比胞浆中的脂大。实验结果和用三房空模型分析所得的理论值符合较好,提示荧光探剂的标记过程主要受分子扩散所控制。     

Alterations in erythrocyte membrane lipid composition and fluidity in primary lipoprotein lipase deficiency.

Lipid composition of plasma lipoproteins and erythrocyte ghost membranes has been studied in 16 healthy normolipidaemic subjects and in 16 patients affected by primary lipoprotein lipase deficiency, resulting in severe chylomicronaemia and in cholesterol-depleted low-density lipoproteins and high-density lipoproteins. A significant decrease in membrane cholesterol/phospholipid ratio was observed in lipoprotein lipase deficient patients compared to controls (3.27 +/- 0.33 vs. 3.95 +/- 0.50, mean +/- S.D.; P less than 0.0001). There was also an increase in the erythrocyte membrane phosphatidylcholine/sphingomyelin ratio in lipoprotein lipase deficient patients compared to controls (1.53 +/- 0.10 vs. 1.05 +/- 0.13; P less than 0.0001) due to a concurrent increase in phosphatidylcholine and decrease in sphingomyelin relative concentrations in these patients. Erythrocyte ghost membrane fluidity was determined by fluorescence anisotropy and found to be higher in membranes from lipoprotein lipase deficient patients. This increase in membrane fluidity can be attributed in part to changes in membrane cholesterol and phospholipid concentrations in response to abnormal plasma lipoprotein composition.     

Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition

Administration of high-dose ethinylestradiol to rats decreases bile flow, Na,K-ATPase specific activity, and liver plasma membrane fluidity. By use of highly purified sinusoidal and bile canalicular membrane fractions, the effect of ethinylestradiol administration on the protein and lipid composition and fluidity of plasma membrane fractions was examined. In sinusoidal fractions, ethinylestradiol (EE) administration decreased Na,K-ATPase activity (32%) and increased activities of alkaline phosphatase (254%), Mg2+-ATPase (155%), and a 160-kDa polypeptide (10-fold). Steady-state and dynamic fluorescence polarization was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH) was significantly higher in canalicular compared to sinusoidal membrane fractions. Ethinylestradiol (5 mg/kg per day for 5 days) selectively increased sinusoidal polarization values. Similar changes were demonstrated with the probes 2- and 12-anthroyloxystearate. Time-resolved fluorescence polarization measurements indicated that EE administration for 5 days did not change DPH lifetime but increased the order component (r infinity) and decreased the rotation rate (R). However, 1 and 3 days after EE administration and with low doses (10-100 micrograms/kg per day for 5 days) the Na,K-ATPase, bile flow, and order component were altered, but the rotation rate was unchanged. Vesicles prepared from total sinusoidal membrane lipids of EE-treated rats, as well as phospholipid vesicles, demonstrated increased DPH polarization, as did intact plasma membrane fractions. Liver plasma membrane fractions showed no change in free cholesterol or cholesterol/phospholipid molar ratio, while esterified cholesterol content was increased with high-dose but not low-dose ethinylestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of amyloid β-peptide on the fluidity of phosphatidylcholine membranes: Uses and limitations of diphenylhexatriene fluorescence anisotropy

There is accumulating evidence that peptide-induced perturbations in the order and dynamics of cellular membranes may play a role in the neurotoxicity of amyloid β-peptide (Aβ). Several studies have reported that Aβ decreases fluidity of membranes based on an Aβ-induced increase in the fluorescence anisotropy of diphenylhexatriene (DPH). However, the effect of Aβ on the membrane fluidity is still a subject of controversy, because other studies that employed pyrene as a fluorescent probe have shown that Aβ has the opposite effect. To reveal the reason for this discrepancy, we have examined the effect of Aβ on the fluidity of phosphatidylcholine membranes using spectroscopic methods. The fluorescence anisotropy of DPH is dramatically increased on addition of Aβ to DPH-containing phosphatidylcholine membranes. However, Aβ does not affect the Raman spectrum of the membrane, which is sensitive to the packing order of the hydrocarbon chains of lipids. We have also found that circular dichroism (CD) bands of DPH appear during incubation of DPH-containing membranes with Aβ, whereas DPH is an achiral molecule. The observed CD bands of DPH are induced by a chiral environment of Aβ but not by that of the lipids, because positive CD bands appear regardless of the d/l-chirality of phosphatidylcholine. The findings obtained from CD measurements provide evidence that DPH molecules translocate from the membrane to Aβ. The peptide-mediated extraction of DPH from the membrane may cause changes in the fluorescence anisotropy of DPH, even though Aβ does not affect the fluidity of membranes.     

Use of a FACS III for fluorescence depolarization with DPH

We have previously demonstrated age-related differences in human lymphocyte membrane fluidity, by use of steady-state polarization measurements on bulk cell suspensions with the fluorescence probe DPH. However, for exact analysis of the possible functional importance of these changes, single-cell measurements were deemed of interest. We have now used an analog division device to measure fluorescence depolarization "p" of DPH in real time with a FACS III flow cytometer. The measurements are reliable, as we have been able to confirm the differences in DPH "p" between monocytes and lymphocytes previously shown in bulk suspension and to demonstrate the expected differences in fluidity of lipid-modulated cells. We also found significant differences in DPH "p" between lymphocytes of young and elderly blood donors. Lymphocyte subsets did not differ in polarization values but did differ in fluorescence intensity with Th less than Ts less than B = NK cells.     

Membrane fluidity measured by fluorescence polarization using an EPICS V cell sorter

We have characterized the measurement of fluorescence polarization on single cells using an EPICS V cell sorter. A critical analysis is made of the balancing and calibration of the system. The system is highly linear for polarization measurements. Cellular membranes were labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) to measure membrane fluidity. Fluorescence polarization histograms had coefficients of variation as low as 7%. Cells labeled with DPH after 24 hr incubation in medium lacking serum showed a significantly higher fluorescence polarization than cells in medium containing serum. The fluorescence polarization measured at 15 degrees C was 0.311 compared to 0.270 at 25 degrees C for cells labeled with DPH, verifying that temperature affects the membrane fluidity as measured by flow cytometry.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.     

Isolation and partial characterization of basolateral membranes from rat proximal colonic epithelial cells

The isolation of basolateral membranes from rat proximal colonic epithelial cells is described. Cells were harvested using a technique combining chelation of divalent cations with mechanical dissociation. After homogenization, differential centrifugation yielded a 'crude' membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 10-14-fold over homogenate in ouabain-sensitive sodium-potassium dependent adenosine triphosphatase and ouabain-sensitive potassium-dependent phosphatase specific activities. SDS-polyacrylamide gel electrophoresis of this membrane revealed at least 18 protein bands with molecular weights of 14600-200000. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, free cholesterol and fatty acids were the major lipid components of this membrane. The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. Membranes and their liposomes were studied, using the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH), by steady-state fluorescence polarization. The fluorescence anisotropy was greater in the intact membranes compared to their liposomes, indicating greater fluidity in the liposomes. Compositional studies suggested that the high fluidity of this membrane was due to its low ratios of protein/lipid (w/w), cholesterol/phospholipid (mol/mol), and sphingomyelin/phosphatidylcholine (mol/mol).     

Changes in plasma membrane fluidity of corn (Zea mays L.) roots after Brij 58 treatment

Summary The detergent Brij 58 has been introduced to reverse plasma membrane (PM) vesicles from the right-side-out to the inside-out form. The aim of the present work was to investigate the effect of Brij 58 on the formation of an ATP-dependent proton gradient and on the fluidity of the lipid phase of PM vesicles. PMs of corn (Zea mays L.) roots were isolated by phase-partitioning. The fluidity of PMs was estimated by measurement of fluorescence polarization with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The PMs of corn roots were relatively rigid. The hydrophobic part of the lipid bilayer was more fluid than the hydrophilic part. After intercalation of Brij 58 into the lipid bilayer the membrane fluidity changed in a concentration-dependent manner. Treatment with the detergent Brij 58 increased the degree of fluorescence polarization for TMA-DPH, while it decreased it for DPH. This effect was saturated at a detergent-to-protein ratio of 1 4 for both fluorescence probes. Although the biophysical characteristics of the membrane were changed after Brij 58 treatment, the formation of ATP-dependent proton gradients could still be measured with those vesicles. The generation of an ATP-dependent proton gradient with Brij 58-treated PM vesicles suggests that the detergent treatment indeed turned the originally right-side-out vesicles to sealed inside-out vesicles. The limits of the effect caused by Brij 58 in the context of PM enzyme activities are discussed.Abbreviations Brij 58 polyoxyethylene 20 cetyl ether - DPH 1,6-diphenyl-1,3,5-hexatriene - HCF III hexacyanoferrate (III) - ISO inside-out - PM plasma membrane - RSO right-side-out - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene     

Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)