Shuji Kitagawa

ASYMMETRY OF MEMBRANE FLUIDITY IN THE LIPID BILAYER OF BLOOD PLATELETS : FLUORESCENCE STUDY WITH DIPHENYLHEXATRIENE AND ANALOGS

SummaryMembrane fluidity of bovine platelets was examined with diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and anionic propionic acid derivative (DPH-PA). After addition of these probes to platelet suspensions at 37°C, the fluorescence intensity of DPH-PA reached equilibrium within 2 min, whereas those of DPH and TMA-DPH increased gradually. With increase in the fluorescence intensity of TMA-DPH, its fluorescence anisotropy decreased significantly, but the fluorescence anisotropies of DPH-PA and DPH did not change during incubation. The gradual increase of fluorescence intensity of TMA-DPH was due to its penetration into the cytoplasmic side of the platelet membrane, as shown quantitatively by monitoring decrease in its extractability with albumin. Transbilayer movement of TMA-DPH was markedly temperature-dependent, and was scarcely observed at 15°C. The fluorescence intensity of TMA-DPH was much higher in platelet membranes and vesicles of extracted membrane lipids than the initial intensity in intact platelets. Moreover, the fluorescence anisotropy of TMA-DPH was much lower in the former preparations than the initial value in intact platelets. These results suggest that binding sites for TMA-DPH in the cytoplasmic side of the platelet membrane are more fluid than those in the outer leaflet of the plasma membrane. Platelet activation by ionomycin induced specific change in the fluorescence properties of TMA-DPH without causing transbilayer incorporation of the probe.

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.

EPR study on the interaction of hexavalent chromium with glutathione or cysteine: Production of pentavalent chromium and its stability

Abstract The reduction of hexavalent chromium (Cr(VI)) by glutathione was studied by EPR spectrometry and comparing it with that by cysteine. The characteristics of production of the pentavalent chromium (Cr(V)) species were studied. Two Cr(V) species, which were characterized by g values of 1.995–1.996 and 1.985–1.986, were detected at pH values above 5.0, whereas at pH 3.0 and 4.0 a single species of Cr(V) with a g value of 1.989–1.990 was found. The Cr(V) species were relatively long-lived and most stable at pH 7.0, where signal of two Cr(V) species were observed for more than 30 min. The intensities of the Cr(V) signals were pH-dependent, increasing with an increase in pH from 3.0 to 8.0. At neutral pH, the signal corresponding to the species of the larger g value increased markedly with an increase in glutathione concentration. Stable production of Cr(V) by glutathione was also confirmed by EPR measurements at 77 K. On the other hand, the characteristics of Cr(V) generation by cysteine were quite different. Production of the Cr(V) species was confirmed by a sharp single signal with a g value of 1.984–1.987. The signal intensity corresponding to Cr(V) generation did not change much with a change in pH from 3.0 to 6.0; at pH 7.0 only a small signal was observed, and at pH 8.0 no signal was observed. Moreover, the life-time of the Cr(V) signal was shorter than that observed for the reduction with glutathione. These results suggest that Cr(V) may be stabilized in glutathione solution by its suitable redox potential and ligand structure.

Inhibitory mechanism of cis-polyunsaturated fatty acids on platelet aggregation: the relation with their effects on Ca2+ mobilization, cyclic AMP levels and membrane fluidity

The in vitro inhibitory effects of cis-polyunsaturated fatty acids, linolenic (18:2 delta 9,12), alpha-linoleic (18:3 delta 9,12,15) and eicosatrienoic (20:3 delta 11,14,17) acid, on bovine platelet aggregation and their inhibitory mechanism were investigated. These fatty acids inhibited platelet aggregation induced by ADP and thrombin to similar extent. Fluorescence analyses with fura-2-loaded platelets showed that, in the concentration ranges that inhibited aggregation, they also inhibited agonist-induced increase in cytoplasmic Ca2+. According to radioimmunoassay study, addition of these fatty acids increased cyclic AMP contents in the presence of theophylline corresponded with their inhibitory effects on aggregation. These fatty acids induced a 1.6-1.8-fold increase over basal concentration of cyclic AMP in the concentration ranges that fully inhibited aggregation. On the other hand, saturated fatty acid, stearic acid, affected neither aggregation nor cyclic AMP levels. As reported previously [1985) Biochim. Biophys. Acta 818, 391), these unsaturated fatty acids induced increase in membrane fluidity in the same concentration range. These results suggest that inhibition of platelet aggregation by cis-polyunsaturated fatty acids is due to the increase in cyclic AMP levels. This increase seems to be due to stimulation of adenylate cyclase which is mediated by membrane perturbation.

ESR analysis with long-chain alkyl spin labels in bovine blood platelets. Relationship between the increase in membrane fluidity by alcohols and phenolic compounds and their inhibitory effects on aggregation

Four spin-labeled probes (5-doxylstearic acid (5-NS), its methyl ester (5-NMS), 16-doxylmethylstearate (16-NMS) and 4-(N,N-dimethyl-N-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-ox yl (CAT-15)) were used to monitor membrane fluidity change in bovine platelets induced by three alkyl alcohols, benzyl alcohol and two phenolic compounds. The relationship between the increase in membrane fluidity induced by these compounds and their inhibitory effects on platelet aggregation was observed. Experiments with the four probes showed that n-hexyl alcohol induced decreases in the order parameter of 5-NS and apparent rotational correlation times of the other probes at the same minimal alcohol concentration. The decreases were observed in the concentration range that inhibited aggregation. n-Amyl alcohol and n-butyl alcohol decreased the values of the parameters of the above mentioned only at higher concentrations that were dependent on their hydrophobicities. Like alkyl alcohols, benzyl alcohol and phenolic compounds decreased the values of the parameters in the concentration ranges in which these compounds inhibited platelet aggregation. The concentration of these compounds causing 50% inhibition of platelet aggregation, the IC50 values, and data on 5-NS-labeled platelets indicated that they inhibited aggregation and decreased the value of the order parameter at lower concentrations relative to their Poct values in comparison to the effective concentrations of alcohols. Phenolic compounds also decreased the values of the apparent rotational correlation times of 5-NMS and 16-NMS. These results indicate that the inhibition of platelet aggregation by alcohols and phenolic compounds is due to membrane perturbation in wide range in depths within the lipid bilayer.

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

SummaryThe 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-propyl<n-butyl<n-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 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.

Inhibition of ADP-induced aggregation of bovine platelets by saturated fatty acids and its relation with the change of membrane surface charge

The inhibitory effects of saturated fatty acids with 4 to 18 carbon atoms on ADP-induced aggregation of bovine platelets were investigated. The inhibitory effects of the acids increased with increase of their alkyl chain length up to C14. On the other hand, from C16 the inhibitory effects tended to decrease with increase of chain length, and stearic acid (C18) was not inhibitory. There was a linear relationship between the inhibitory effects and alkyl chain lengths up to C12. This linear relation and the slope of the linear regression line suggested that the inhibitory effects of the acids depended on their partition into the membrane. The fatty acids decreased the fluorescence of the surface charge probe 2-p-toluidinylnaphthalene-6-sulfonate, indicating that they increased the negative charge on the membrane surface. The relative effects of the acids on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that the inhibition of platelet aggregation by saturated fatty acids is due to a change in the membrane surface charge of the platelet plasma membrane.

Effects of four types of reagents on ADP-induced aggregation and Ca2+ mobilization of bovine blood platelets.

The effects of four types of reagents--a stimulant analog (ATP), reagents increasing cAMP (theophylline and (-)-isoproterenol), Ca2+ blockers (chlorpromazine, procaine, dibucaine and tetracaine) and nonspecific membrane-reactive reagents (n-butanol, n-hexanol and linoleate) - on ADP-induced Ca2+ mobilization and aggregation of platelets were investigated. All the reagents tested inhibited the aggregation. Of these reagents, those increasing cAMP and the stimulant analog inhibited the aggregation at least partly by inhibiting Ca2+ mobilization, whereas Ca2+ blockers and nonspecific membrane-reactive reagents must have inhibited the aggregation by different mechanisms, because they had: (1) no effect on ADP-induced Ca2+ mobilization, (2) accelerated it, or (3) themselves stimulated Ca2+ mobilization. The results showed that the inhibitory effects of Ca2+ blockers were at least partly due to competition with Ca2+ for binding sites on the outside of the membrane, whereas the effects of the nonspecific membrane-reactive reagents tested were due to membrane perturbation.

Effects of anions on ADP-stimulated aggregation of bovine blood platelets

Abstract ADP-stimulated aggregation of bovine blood platelets was observed in media containing isotonic potassium salts of various monovalent anions. The aggregation depended on the anion in the medium, the order of aggregation being Cl − , Br − >I − >SCN − , ClO 4 − . After 30-min incubation, the extent of aggregation of platelets in Cl − or Br − medium was little changed, whereas, that in SCN − or ClO 4 − medium was remarkably decreased. This anion dependency of aggregation may be due to change in the membrane potential.