Effects of alcohols on ADP-induced aggregation and membrane fluidity of gel-filtered bovine blood platelets

Summary The effects of four alcohols—n-propyl,n-butyl,n-amyl andn-hexyl alcohol—on the ADP-induced aggregation of gel-filtered bovine platelets were examined. All four alcohols inhibited the aggregation, the order of their effects beingn-propyln-amyl<n-hexyl. Comparison of the inhibitory effects of the alcohols with their physico-chemical properties showed that their degrees of inhibition depended on their hydrophobicities. Moreover, it was suggested that their interaction with the lipid layer of the membrane was important for the inhibition. Studies on the effects of alcohols on the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene-labeled platelets showed that the membrane fluidity of the platelets increased in the same concentration range in which aggregation inhibition was observed. Since the alcohols inhibited aggregation without affecting Ca²⁺ mobilization in the platelets, as revealed in this study, it was concluded that inhibition of platelet aggregation was due to perturbation of membrane lipids by the alcohols. This hypothesis is supported by several recent studies on the effects of cholesterol and cations, which suggest that a relatively rigid membrane favors platelet aggregation.     

Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change

The effects of long-chain cis-unsaturated fatty acids with different alkyl chain lengths and different numbers of double bonds on aggregation of bovine platelets and membrane fluidity were investigated. All the cis-unsaturated fatty acids tested inhibited aggregation and at the same time increased membrane fluidity in accordance with their inhibitory effects. The saturated fatty acids and trans-unsaturated fatty acid tested for comparison had much lower or no effects on aggregation and membrane fluidity. The inhibitory effects of mono cis-unsaturated fatty acids increased with increase of their alkyl chain length. cis-Unsaturated fatty acids with two or more double bonds had more inhibitory effects than mono-unsaturated fatty acids. The position of the double bonds had less influence than the number of double bonds. We also examined the effects of cis-unsaturated fatty acids on membrane fluidity with diphenylhexatriene and anthroyloxy derivatives of fatty acids as probes and observed increased fluidity to be considerable in the membrane. The alcohol analogs of cis-unsaturated fatty acids also inhibited aggregation and increased membrane perturbation. These results suggest that the inhibition of platelet aggregation by cis-unsaturated compounds is due to perturbation of the lipid layer.     

Effects of alcohols on fluorescence anisotropies of diphenylhexatriene and its derivatives in bovine blood platelets: relationships of the depth-dependent change in membrane fluidity by alcohols with their effects on platelet aggregation and adenylate cyclase activity.

The effects of three short-chain alkyl alcohols and benzyl alcohol on the membrane fluidity of bovine blood platelets were investigated by studies on the fluorescence anisotropies of diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and its anionic propionic acid derivative (DPH-PA). These alcohols decreased the fluorescence anisotropy of DPH, which is thought to be located within the hydrophobic core of the membrane, in concentration ranges that inhibited platelet aggregation. On the other hand, they had little or no effects on the fluorescence anisotropy of DPH-PA which is thought to be located in the interfacial region of the lipid bilayer. Likewise, they had little or no effects on the fluorescence anisotropy of TMA-DPH, which is also thought to be located in the interfacial region of the lipid bilayer, either when the probe was located in the outer layer of the plasma membrane or when the probe was located in the inner membrane compartment. These results suggest that alcohols mainly increase the fluidity in the central region of the lipid bilayer. Consistent with their effects on the fluorescence anisotropy of DPH, these alcohols increased the intracellular cyclic AMP concentration. Thus alcohols may inhibit platelet function due to stimulation of adenylate cyclase, which is mediated by perturbation of the central region of the membrane lipid bilayer.     

Inhibitory effects of long-chain alkyltrimethylammonium ions on aggregation of bovine platelets and the relation of their effects to Ca2+ mobilization

The inhibitory efefcts of alkyltrimethylammonium ions on ADP-and thrombin-induced aggregation of bovine platelets were investigated. The ammonium cations inhibited the two aggregation reactions to similar extents. The relationship between their inhibitory efects on ADP-induced aggregation and their alkyl chain lengths from C₈ to C₁₈ was investigated. Results showed that the inhibitory effects of ammonium cations increased with increase of their alkyl chain lengths up to C₁₆, and that the increase was linear with chain lengths of up to C₁₄. This linear relation and slope of the linear regression line suggested that the inhibitory effects of the ammonium cations depended on their partitioning into the membrane. However, unlike long-chain unsaturated fatty acids, they did not affect the membrane fluidity of the platelets, Fluorescence analysis of fura-2 loaded platelets revealed that, in the concentration range where the alkyltrimethylammonium ions inhibited aggregation, they inhibited agonist-induced increase in cytosolic Ca²⁺ both in the presence and absence of extracellular Ca²⁺. These results suggest that inhibition of platelet aggregation by alkyltrimethylammonium ionsis mainly due to their inhibition of increase in cytoplasmic Ca²⁺ by inhibition of both intracellular Ca⁺ mobilization and Ca²⁺ uptake.     

Lanthanum and some other cation-induced changes in fluidity of synaptosomal membrane studied with nitroxide stearate spin labels.

Using nitroxide fatty acid spin labels, the effects of some cations such as La3+, Cd2+ and Hg2+ on synaptosomal membranes were studied by observing changes in their ESR spectra. The labels were incorporated almost instantaneously into synaptosomes isolated from rat brain cortex. ESR spectra of the spin-labeled synaptosomes were significantly braodened immediately upon adding La3+, Ce3+, Cd2+ or Hg2+ but hardly affected by Ca2+, Sr2+ and Ba2+. The magnitude of the change in the separation of the outer two peaks in ESR spectra (2T') depends on the number (n) of methylene units between the polar head group and the spin-label (nitroxide) group; that is, it increases with decreasing n. Among these ions, the effect of La3+ was the greatest and appeared to be in parallel with the amount of La3+ bound with the synaptosomes. On the other hand, K+, Rb+ or Li+ causes hardly any significant changes.     

Relationship of the effects of nigericin on the aggregation and cytoplasmic pH of bovine platelets in the presence of different cations

The effect of nigericin on aggregation of bovine platelets was investigated in media containing the chloride salts of various alkali metal cations of quaternary ammonium cations. In medium with K+, which has the highest permeability with the ionophore among the cations tested, nigericin slightly enhanced both ADP- and thrombin-induced aggregation. In medium with Na+, nigericin scarcely affected ADP-induced aggregation, and slightly inhibited thrombin-induced aggregation. In media with Cs+, choline and tetramethylammonium, it inhibited the aggregations induced by both ADP and thrombin. Measurement of the cytoplasmic pH with the fluorescent probe 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein showed that nigericin increased the intracellular pH in K+ medium and caused its stable decrease (of about 0.6) in Cs+, choline and tetramethylammonium media, but caused only a small transient decrease in medium with Na+. These results suggest that the effects of nigericin on platelet aggregation are mainly due to its effects on the cytoplasmic pH. This conclusion is supported by the findings that the effects on platelet aggregation of other types of ionophore tested were also proportional to their effects on the cytoplasmic pH.     

Spin-labeling of adenosine triphosphatase in sarcoplasmic reticulum membrane and change in the state of the spin labels induced by deoxycholate.

Fragmented sarcoplasmic reticulum (SR) was reacted with a thiol-directed spin label, N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)maleimide, under various conditions. It was found that ATP inhibited the binding of the label to SR protein in the initial phase of the reaction, but as the incubation time was extended up to 18 h, the amount of label bound to SR protein in the control and ATP-containing samples became almost identical. The Ca2+-dependent ATPase control and ATP-containing samples became almost identical The Ca2+-dependent ATPase (ATP phosphohydrolase [EC 3.6.1.3]) of SR was protected by the presence of ATP during incubation with relatively low concentrations of spin label, irrespective of the total amount of label bound, although with increasing concentration of bound label the ATPase activity decreased. Deoxycholate slightly reduced the rotational freedom of the label bound to SR protein and decreased the initial rate of quenching of protein-bound nitroxide by ascorbate. From an analysis of these results, it was concluded that the binding of deoxycholate to protein decreases the accessibility of ascorbate to the protein-bound label.     

Relationship between inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase by cholesterol feeding and short-term changes in membrane fluidity during neonatal development

Both 5% cholesterol feeding and fasting produced a decrease in the hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity, although certain diurnal variations remained during the second day of treatment. Supplementation of 5% cholesterol to the diet produced a significant increase in cholesterol content of hepatic microsomes, whereas no significant variations were observed after fasting. The phospholipid content of hepatic microsomes did not change by fasting. However, cholesterol feeding produced a clear decrease in microsomal phospholipids. After 7 hr of cholesterol feeding, an increase of nearly 3-fold in the cholesterol/lipidic phosphorus molar ratio was found. Fasting had no effect on this molar ratio. The changes observed by cholesterol feeding agree with a mechanism of regulation of hepatic reductase by alteration in membrane fluidity, a mechanism that would be already operative during the neonatal period.     

胁迫条件对一土生青霉菌总酚积累及其清除自由基能力的影响

旨在阐明胁迫条件对一土生青霉菌总酚积累及其抗氧化活性的影响。采用固体培养基培养青霉菌,以紫外线辐射、添加H2O2水溶液和降低培养基营养物质含量作为胁迫手段,检测受到胁迫后,菌体总酚的积累及酚类清除自由基的能力。菌丝体总酚含量以Folin-Ciocalteu法测定,自由基清除率以分光光度法测定。结果表明,在胁迫条件下,各实验组菌丝体的总酚含量较对照组均有显著提高,添加H2O2组的总酚含量最高达63.86mg/g,紫外线辐射组的总酚含量最高达58.4mg/g,营养胁迫组的总酚含量最高达43.19mg/g。各实验组酚类提取物对羟自由基的清除率最高,其中添加1mmol/LH2O2组为35.28%,紫外线辐射40s组为69.97%,75%营养胁迫组为50.83%。因此,胁迫条件可增加该青霉合成酚类化合物及提高其抵抗胁迫的能力。     

Distribution of phenolic compounds and antioxidant activity between young and old leaves of Carthamus tinctorius L. and their induction by salt stress

In the present work, we investigated the effect of salt stress on the distribution of safflower (Carthamus tinctorius L.) antioxidant system in relation to leaf age. The study was carried out under growth chamber conditions using seedlings of three cultivars which were subjected to 0 and 50 mM NaCl for 3 weeks. Leaf growth, water content, lipid peroxidation, and phenolic compound (total polyphenols, total flavonoids, and proanthocyanidins) concentration were measured at two leaf stages (young and old leaves). Leaf growth was affected by salinity only in Kairouan cultivar that also showed a significant decrease in old leaf water content. By contrast, Gabes and Tazarka cultivars maintained their old leaf water content constant and showed a reduction in that of young leaves. This could be attributed to a higher aptitude of the latter two cultivars to use absorbed sodium and chloride for osmotic adjustment in old leaves, keeping potassium for specific functions. Salt-induced lipid peroxidation was observed only in old leaves, whereas the accumulation of the major phenolic compounds under saline conditions was higher in young leaves, except in Gabes cultivar where no significant difference was found between the two leaf stages. A significant variability was also found between the three cultivars. The better behavior of salt-challenged leaves of Gabes and Tazarka cultivars compared to that of Kairouan cultivar may be related to their higher water content and the accumulation of polyphenols, in particular flavonoids that were shown to be efficiently involved in the restriction of salt-induced oxidative damages.     

ESR spectral analysis of the molecular motion of spin labels in lipid bilayers and membranes based on a model in terms of two angular motional parameters and rotational correlation times.

Electron spin resonance (ESR) spectral line shapes are calculated for a nitroxide spin-labeled molecule undergoing rapid restricted rotations (twisting) about its long molecular axis while simultaneously tumbling within a cone. Explicit expressions are derived for the hyperfine splittings and g-values, as well as for the secular contributions to the motionally modulated linewidths. The present model is useful for analyzing the restricted twisting and tumbling motions, and rotational correlation times, of spin-labeled molecules in bilayers. Simulated spectra compare well with experimental spectra of lecithin bilayers marked with cholestane spin label, over a wide temperature range.     

ESR characterization and metallokinetic analysis of Cr(V) in the blood of rats given carcinogen chromate(VI) compounds.

It has been shown that bio-trace metal elements are related to many diseases and the aging process. For many years, carcinogen hexavalent chromium (VI) has been known to be toxic to animals, but its dynamic toxicological mechanism is not sufficiently elucidated. Bioinorganic chemistry in terms of metallokinetic analysis of beneficial or toxic metal ions and their complexes is an important investigation for understanding their biochemical and physiological roles. We have tried to examine the real-time behavior of paramagnetic metal ions and complexes in animals, in which electron spin resonance (ESR) was capable of measuring paramagnetic species in chemical and biological systems. On the basis of our previous results on stable nitroxide spin probes, we have developed the in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) method to analyze time-dependent ESR signal changes due to paramagnetic metal ions and their complexes in real time. When K2Cr2O7 or Na2Cr2O7 in saline was intravenously administered to rats, two ESR signals due to pentavalent chromium(V) were detectable in the circulating blood of rats. Cr(V) detected in the blood was indicated to be in the CrO(O4) and CrO(S2O2) coordination modes after the study on model complexes. From the changes of ESR signal intensities due to Cr(V) in the blood, the metallokinetic parameters were obtained using the pharmacokinetic analysis and the curve-fitting methods. The obtained results are important for understanding carcinogen chromate in terms of the formation of Cr(V) in animals. In addition, we propose the BCM-ESR method, which is useful to analyze the disposition of paramagnetic metal species in the blood of living animals.     

Stabilizing effects of cholesterol on changes in membrane permeability and potential induced in red blood cells by lysolecithin.

Effects of cholesterol on permeability of K+ ion and on change in membrane potential induced by lysolecithin were studied. Cholesterol inhibited K+ release from rabbit red blood cells treated with lysolecithin (1.25 micrograms/ml), 3.3 X 10(-6) M of cholesterol being the optimum concentration for blocking K+ release. Changes in membrane potential, monitored by changes in intensity of fluorescence of cyanine dye, were induced by lysolecithin and inhibited by cholesterol. The inhibitory action on both K+ permeability and membrane potential varied with the cholesterol concentration. The observed effects are thought to be due to membrane-stabilizing activities such as decreasing membrane fluidity and hardening the membrane at the fluid-phase transition temperature. These properties of cholesterol may have significance in relation to transformed cells (tumor cells, lymphomed cells).     

Transbilayer incorporation of 1-pyrenebutyltrimethylammonium by blood platelets and its application for analyzing changes in physico-chemical properties of the membrane lipid bilayer induced by platelet activation.

The binding of cationic butyltrimethylammonium derivative of pyrene to bovine platelets was initially rapid and then increased gradually, unlike the bindings of other anionic and neutral derivatives of pyrene tested. The rate of increase in binding of the cationic probe depended on temperature and was due to its incorporation into the cytoplasmic side of the platelet membranes, as shown quantitatively by monitoring decrease in its extractability with albumin. The penetration into the inner membrane compartment did not reach equilibrium even after 4 h at 37 degrees C. Slow penetration of a fluorescent probe such as this is useful in studies on the physico-chemical properties of the outer layer and cytoplasmic side of the platelet membranes and their changes. Initial rapid binding of the cationic probe to platelets, representing the binding of the probe to the outer layer of the plasma membrane, was increased by ionomycin-induced platelet activation. Fluorescence spectra in the presence of a relatively high concentration of the cationic probe showed increase of the excimer of the cationic probe accompanied with the incorporation of the probe to the cytoplasmic side. On ionomycin-induced activation, the excimer-to-monomer intensity ratio of the probe in the cytoplasmic side of the platelet membranes decreased, possibly due to decrease in fluidity of the lipid layer near the probe or change in distribution of the probe.     

Relationship between cell adherence and proteoglycan synthesis in cultures of human peripheral blood mononuclear cells: effects of concanavalin A.

Human, blood-derived mononuclear cells (MC), stimulated with Concanavalin A (Con A), synthesized a chondroitin sulfate (CS) proteoglycan (PG), which was elaborated largely by T-cells. Following Con A stimulation, the majority of MC adhered to the culture dish by day 2; but as incubation progressed to day 10 the proportion of non-adherent (NAd) MC increased in a fashion which approximately paralleled the accumulation of PG in the medium. Cell kinetic studies suggest that, following Con A stimulation, there was an inverse relationship between the amount of newly synthesized cellular PG and adherence, which appears to be related to a reciprocal effect on PG synthesis of the declining adherent (Ad) cell density with time of culture. In the stimulated cultures, NAd cells contained much more newly synthesized CS/cell than Ad cells up to day 6 of incubation. Cell type analysis, using monoclonal antibodies against specific cell surface markers, suggested that the higher PG synthesis in the NAd population may, at least in part, be due to a greater proportion of T-helper cells.