Specific receptor sites for 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) on rabbit platelet and guinea pig smooth muscle membranes

By using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF), we have directly identified its specific binding sites on rabbit platelet plasma membranes. The equilibrium dissociation constant for 3H-PAF is 1.36 (+/- 0.05) X 10(-9) M at 0 degrees C. The number of binding sites is 1.61 (+/- 0.34) X 10(12)/mg of membrane, which corresponds to approximately 150-300 receptors/platelet (depending on membrane vesicle orientation). Binding of 3H-PAF to rabbit platelet plasma membrane is rapid (t1/2 less than 5 min at 0 degrees C) and reversible. For a series of PAF analogues, their affinity for the receptor sites parallels with their relative potency to induce platelet aggregation. PAF can cause contraction of smooth muscle of heart, parenchymal strip, trachea, and ileum. Specific PAF receptor binding was demonstrated with purified plasma membrane from several smooth muscles and from polymorphonuclear leukocytes but not from presumably PAF nonresponsive cells such as erythrocytes and alveolar macrophages. It is likely that the interaction of PAF with these binding sites initiates the specific responses of platelets, polymorphonuclear leukocytes, and smooth muscles.     

Oxidative response and membrane modification of diabetic platelets challenged with PAF

Alterations in the functional activities of platelets (PLT) in type I diabetes have been widely observed. These changes play a key role in the development of cardiovascular complications in diabetes. Various functional activities of PLT are the result of the interaction of numerous stimuli with PLT plasma membrane. This study was designed to evaluate the oxidative response and membrane modifications of diabetic PLT stimulated by platelet activating factor (PAF). The oxidative response was assessed by employing luminol- and lucigenin-amplified chemiluminescence. Luminol-amplified chemiluminescence is sensitive to the release of hydrogen peroxide whereas lucigenin-amplified chemiluminescence is sensitive to the production of superoxide anion. Membrane fluidity and polarity were studied using fluorescence spectroscopy. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). The diabetic group consisted of 20 type I diabetic children with good metabolic control. Our results show a significant decrease in the luminol- and lucigenin-amplified chemiluminescence of PAF stimulated PLT in the diabetic group with respect to controls. These data indicate a decrement in the release of reactive oxygen species by diabetic PLT. We observed a significant increase in steady-state fluorescence anisotropy of diabetic PLT membrane that reflects a decrease in membrane fluidity. Laurdan showed a blue shift of the fluorescence emission and excitation spectra in diabetic PLT with respect to the control group, indicating a decrease in membrane polarity. The addition of PAF to PLT induced a red shift of Laurdan spectra in both groups, indicating an increase in membrane polarity. Our study [table: see text] demonstrates an altered oxidative response to PAF stimulation of diabetic PLT, probably due to altered generation or handling of reactive oxygen species, and alterations in the physico-chemical properties of the plasma membrane which could influence various functional activities of PLT.     

Effect of PAF on polyrnorphonuclear leucocyte plasma membrane polarity: a fluorescence study

The effect of PAF on the plasma membrane polarity of polymorphonuclear leukocytes (PMNs) was investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-1auroyl) naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surrounding. Laurdan shows a marked steady-state emission blue-shift in non-polar solvents, with respect to polar solvents. Our results demonstrate that PAF (10(-7) M) induces a blue shift of the fluorescence emission spectra of Laurdan. These changes are blocked in the presence of the PAF antagonist, L-659,989. Our data indicate that the interaction between PAF and PMNs is accompanied by a decrease in polarity in the hydrophobic-hydrophilic interface of the plasma membrane.     

Platelet-activating factor binding to human platelet membranes

Previously reported methods for quantifying platelet-activating factor (PAF) binding to rabbit platelet membranes were modified for studies of PAF binding to human platelet membranes. The membranes were prepared by the "glycerol lysis" method and PAF binding was quantified by using polyethylene glycol precipitation to recover membrane-bound PAF. Optimal PAF binding required buffers containing 3 to 10 mm KCl and either 5 to 10 mM MgCl2 or 5 to 10 mM CaCl2. NaCl was not as effective as KCl and concentrations of NaCl greater than 3 mM strongly inhibited PAF binding. Maximal binding occurred after incubation for 60 min at 0 degree C and was reversed by the addition of excess unlabeled PAF. PAF binding was saturable. Scatchard analysis of PAF binding to 50 micrograms of membrane protein revealed 10.3 +/- 1.7 x 10(11) receptors per milligram of membrane protein and the receptors had a Kd of 7.6 +/- 1.9 nM. The calculated receptor number, binding affinity, and specificity of binding are similar to those previously calculated for PAF binding to intact human platelets, suggesting that the membrane binding site for PAF is the PAF receptor.     

Pharmacologic characterization of the rabbit neutrophil receptor for platelet-activating factor

The characteristics of receptors for platelet-activating factor (PAF) on rabbit neutrophils are investigated in this report. The presence of PAF-specific binding to rabbit neutrophils was confirmed using radiolabeled ligand binding assays and a rabbit peritoneal neutrophil membrane preparation. Binding of PAF to the neutrophil membranes was reversible and reached equilibrium within 30 min. Scatchard analysis of PAF-specific binding to the rabbit neutrophil membranes revealed a dissociation constant (Kd) for PAF of 0.41 +/- 0.045 nM and a Bmax of 0.32 +/- 0.11 pmol of PAF receptor/mg of protein. The order of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF to rabbit peritoneal neutrophil membranes was determined. For the competition assays, 100 micrograms of neutrophil or platelet membrane protein, 0.18 nM 3H-PAF, and varying amounts of PAF antagonist were incubated at room temperature for 1 hr. PAF receptor antagonists tested were ONO-6240, brotizolam, kadsurenone, WEB-2086, L-652-731, BN-52021, CV-3988, triazolam, alprazolam, and verapamil. The orders of potencies of these PAF receptor antagonists were similar for inhibition of 3H-PAF binding to rabbit peritoneal neutrophil and platelet membranes (correlation coefficient, r = 0.97). PAF had a significantly higher affinity for rabbit neutrophil membranes (Kd = 0.41 +/- 0.045 nM), as compared with its affinity for rabbit platelet membranes (Kd = 0.87 +/- 0.092 nM). In addition, sodium was found to inhibit 3H-PAF specific binding to rabbit platelet membranes and not to affect 3H-PAF binding to neutrophil membranes. These data indicate that, although PAF receptors on rabbit platelets and neutrophils exhibit similar orders of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF, the disparity in Kd of PAF for the receptors and the effect of NaCl on the binding of 3H-PAF reveal subtle differences between the cell types.     

Effects of platelet activating factor and related lipids on phase transition of dipalmitoylphosphatidylcholine

Recent evidence localizing the inflammatory mediator, platelet activating factor, (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to the membranes of stimulated neutrophils raises the possibility that PAF may, in addition to its activities as a mediator, alter the physical properties of membranes. Accordingly, the effects of PAF and related alkyl ether and acyl analogs on phase transition thermodynamics of dipalmitoylphosphatidylcholine (DPPC) were studied using fluorescence polarization of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). PAF, its ester analog (1-palmitoyl-2-acetylphosphatidylcholine) and both the corresponding alkyl and acyl lysophospholipid analogs (each at a concentration of 10 mol%) significantly decreased the phase transition temperature and broadened the phase transition of DPPC (P less than 0.05). The relative potency of the lipids in causing this effect was ester-PAF greater than or equal to PAF greater than or equal to lyso-PAF greater than lyso-PC suggesting that the fluidization of the synthetic membranes was attributable to both the 2-position acetyl group and the 1-position alkyl linkage. Furthermore, using various related compounds, increases in chain length and degree of unsaturation in the 2-position were shown to enhance the depression in transition temperature and broadening of the phase transition. Phase transition thermodynamics were also assessed using differential scanning calorimetry. Similar depression in the phase transition temperature was measured for PAF and both the alkyl and acyl lysophospholipids. Broadening of the phase transition for DPPC by the various analogs was assessed by calculation of transition peak width and cooperative unit. Data from fluorescence polarization and differential scanning calorimetry provide similar though not identical results and support the hypothesis that the unique features of PAF may alter membrane physical properties and could ultimately explain some of its biologic actions.     

Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4 degrees C.

Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.     

Monoclonal anti-idiotypic antibodies to platelet activating factor (PAF) and their interaction with PAF receptors.

Monoclonal anti-idiotypic antibodies (3C3F3E4 and 10D3F8H7) that interact with platelet activating factor (PAF) receptors were generated using an auto-anti-idiotypic approach by immunizing mice with an aldehydic analog of PAF coupled to bovine thyroglobulin. The resulting hybridomas were screened for anti-idiotypic antibody (anti-anti-PAF) with F(ab')2 fragments of affinity-purified polyclonal rabbit anti-PAF antibody. These antibodies displayed internal image properties of PAF and were considered as Ab2 beta according to the following criteria: (a) they bound to F(ab')2 fragments of the affinity-purified rabbit polyclonal anti-PAF antibody that had high affinity for PAF; (b) they inhibited [3H]PAF binding to rabbit polyclonal anti-PAF antibody and its F(ab')2 fragment in a concentration-dependent manner; (c) they displaced [3H]PAF from the anti-PAF antibody/[3H]PAF complex specifically; (d) they inhibited [3H]PAF binding to PAF receptors on rabbit platelet membranes dose dependently; (e) they displaced [3H]PAF from the [3H]PAF/PAF receptor complex specifically; and (f) they stimulated rabbit platelets to aggregate, and this aggregation could be inhibited or totally blocked by specific PAF receptor antagonists WEB 2086 and SRI 63-441. All of the above are consistent with the first successful production of monoclonal antibodies that mimic PAF and interact specifically with the PAF binding domain of PAF receptors on rabbit platelet membranes.     

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

Aging and the biophysical properties of cell membranes

Fluorescence spectroscopy was used to examine the biophysical characteristics of human platelet membranes as a function of subject age. The structural order of membrane lipid domains was determined with the use of 1,6-diphenyl-1,3,5-hexatriene (DPH), a fluorescent probe that preferentially localizes in the hydrocarbon core of synthetic and biological membranes. Over the age range of subjects examined (17 to 86 years) the structural order of platelet membranes, as reflected by the steady-state fluorescence polarization of DPH, increased substantially. The magnitude of the observed increase in membrane structural order is sufficient to affect membrane-related cell functions including platelet aggregation. A major contributor to the increase in structural order of platelet membranes may have been an increase in the concentration of cholesterol in serum and tissue with age. The changes observed here in platelet membranes may be a general phenomenon of aging, as changes of similar type and magnitude have been observed in lymphocyte membranes and brain with age in other studies.     

Changes in membrane properties of erythrocytes and polymorphonuclear cells in psoriasis

Using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and its cationic derivative, 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene, we evaluated membrane fluidity in living polymorphonuclear leukocytes and in erythrocytes of psoriatic patients. Our results have shown that erythrocyte membranes of psoriatic patients exhibit a decrease of fluidity. These changes were not associated with any relevant modifications of the cholesterol to phospholipid molar ratio. Moreover, we observed a decrease in polymorphonuclear leukocytes membrane fluidity associated with changes in chemotactic migration. Our results indicate changes of membrane fluidity involving membranes different from the epidermal cells and suggest the hypothesis of a defective membrane-cytoskeleton interaction in psoriasis.     

Platelet and erythrocyte membrane changes in Alzheimer's disease

Previous reports have suggested that the physical properties of cell membranes and calcium homeostasis in both the central and peripheral nervous system are changed in Alzheimer's disease (AD). This study has examined the biophysical properties of erythrocyte and platelet membranes by measuring the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and possible related changes in lipid peroxidation. In addition, we have studied calcium homeostasis by measuring thrombin-stimulated changes in intraplatelet free calcium and Ca2(+)-ATPase activity in AD and healthy age and sex-matched controls. Our results show that there was no significant difference in the fluorescence anisotropy of DPH in erythrocyte membranes isolated from the three groups. There was also no significant difference in lipid peroxidation levels in erythrocytes and plasma of AD patients compared to controls. However, there was a significant reduction in the fluorescence anisotropy of DPH in platelet membranes from AD patients, compared with healthy controls. Recent evident suggests that the increase in platelet membrane fluidity results from alterations in internal membranes. We measured the specific activities of enzyme markers associated with intracellular and plasma membranes in platelets from AD patients and healthy controls. There was a significant reduction in the specific activity of antimycin A-insensitive NADH-cytochrome-c reductase (a specific marker for smooth endoplasmic reticulum (SER)), in AD patients compared to controls, but no change in the specific activity of bis(p-nitrophenyl)phosphate phosphodiesterase (a specific marker for plasma membrane). We have also shown that SER mediated [Ca2+] homeostasis is possibly impaired in AD platelets, i.e., the percentage of thrombin-stimulated increase in intraplatelet [Ca2+] above basal levels was significantly higher in AD compared to matched controls and there were significant reductions in the specific activities of Ca2+/Mg2(+)-ATPase and Ca2(+)-ATPase (but not Mg2(+)-ATPase) in AD platelets. Finally electron microscopic analysis of platelets showed that there was a significant increase in the incidence of abnormal membranes in AD patients compared to controls. The ultrastructural abnormalities seem to consist of proliferation of a system of trabeculated cisternae bounded by SER. These results suggest that both SER structure and function might be defected in AD platelets, which could explain the fluidity changes observed here.     

Binding of bovine factor Va to phosphatidylcholine membranes.

The interaction of bovine factor Va with phosphatidylcholine membranes was examined using four different fluorescence techniques: 1) changes in the fluorescence anisotropy of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to monitor the interaction of factor Va with 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), 2) changes in the fluorescence anisotropy of N-(lissamine rhodamine B sulfonyl) diacyl phosphati-dylethanolamine (Rh-PE) incorporated into SUVs prepared from 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), 3) changes in the fluorescence anisotropy of fluorescein-labeled factor Va (labeled in the heavy chain) upon interaction with POPC SUVs, 4) fluorescence energy transfer from fluorescein-labeled factor Va to rhodamine-labeled POPC SUVs. In the first two sets of experiments, labeled lipid vesicles were titrated with unlabeled protein, whereas, in the latter two types of experiments, labeled factor Va was titrated with vesicles. For the weak binding observed here, it was impossible from any one binding experiment to obtain precise estimates of the three parameters involved in modeling the lipid-protein interaction, namely, the dissociation constant Kd, the stoichiometry of binding i, and the saturation value of the observable Rmax from any one experiment. However, a global analysis of the four data sets involving POPC SUVs yielded a stable estimate of the binding parameters (Kd of approximately 3.0 microM and a stoichiometry of approximately 200 lipids per bound factor Va). Binding to DMPC SUVs may be of slightly higher affinity. These observations support the contention that association of factor Va with a membrane involves a significant acidic-lipid-independent interaction along with the more commonly accepted acidic-lipid-dependent component of the total binding free energy.     

脊髓损伤后脊髓神经细胞膜PAF受体特性的变化

采用３ＨＰＡＦ放射配体结合试验方法测定脊髓神经细胞膜上ＰＡＦ受体的特异性位点,观察脊髓损伤后２、６ｈ、１、３周脊髓神经细胞膜ＰＡＦ受体结合特性变化。结果显示,脊髓神经细胞膜上存在ＰＡＦ高、低亲和力结合位点,脊髓损伤后２、６ｈ、１周组ＰＡＦ受体高、低亲和力位点Ｋｄ值和Ｂｍａｘ均有不同程度下降,与对照组比较,有显著性差异（Ｐ＜０．０５）。表明在伤后早期ＰＡＦ受体亲和力增加,结合位点减少。提示ＰＡＦ受体在脊髓损伤后继发性损害病理生理过程中起一定作用。     

Fluorescence studies on the interaction of the tumor promoter phorbol myristate acetate and related compounds with rat liver plasma membranes.

The interaction of the potent tumor-promoting agent phorbol myristate acetate (PMA) with purified rat liver plasma membranes suspended in phosphate-buffered saline (PBS), pH 7.4, was studied by fluorescence spectrophotometry. Exposure of membranes to PMA caused up to 21% decrease of the native membrane emission, i.e. the fluorescence of both tryptophan and tyrosine, compared to non-treated membranes. The decrease in the membrane emission varied with both the PMA and the membrane concentration. Treatment of rat liver plasma membranes with biologically less active analogs of PMA, phorbolol myristate acetate (PHMA) and 4a alpha-phorbol didecanoate (4a alpha-PDD), resulted in a 5-10% decrease of the native membrane emission. These studies suggest that PMA causes alterations in membrane structure which are due, at least in part, to conformational changes in the membrane proteins.     

A study of the Interaction Between Cetirizine and Plasma Membrane of Eosinophils, Neutrophils, Platelets and Lymphocytes using A fluorescence Technique

The effect of cetirizine on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils, platelets and lymphocytes was investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4- trimethylammonium-phenyl)-6-phenyl-1, 3, 5-hexatriene (TMA-DPH) incorporated in the membrane. The results demonstrate that cetirizine (1 mug/ml) induced a significant increase in the Hpid order in the exterior part of the membrane and a decrease in membrane heterogeneity in eosinophils, neutrophils and platelets. Moreover, cetirizine blocked the PAF induced changes in membrane fluidity in these cells. Cetirizine did not influence significantly the plasma membrane of lymphocytes. These data may partially explain the effect ofcetirizine on inflammatory cell activities.     

Binding of platelet activating factor by isolated membranes from U937 cells

Platelet activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC) has been known to have biological effect on cells. The mechanisms of the effect of the potent phospholipid on cells has not been established. We have used 1-O-[3H]alkyl-2-acetyl-GPC [( 3H]PAF) to study the interaction on the isolated membranes of U937 cells. The binding process was time, protein concentration, temperature dependent and reversible. The binding of [3H]PAF to the U937 cell membranes was slightly inhibited by the addition of PAF analogue, 3-O-Hexadecyl-2-acetyl-sn-glycerol-1-phosphorylcholine. U937 cell membranes showed high affinity binding sites for PAF with equilibrium dissociation constant (Kd) of 5 x 10(-9) M. The displacement of bound [3H]PAF with 500-fold excess of nonlabeled PAF was not altered suggesting that the bound [3H]PAF was not degraded during the binding. Binding of [3H]PAF on U937 cell membranes was inhibited by PAF antagonist, 59227RP. The kinetic of the inhibition by PAF antagonist is competitive suggesting that PAF and PAF antagonist bind at the same site.