The platelet-activating factor receptor antagonist L-659,989 inhibits phospholipase D activity.

The platelet-activating factor (PAF) receptor antagonist L-659,989 [(+/-)-trans-2-(3-methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran)] has been reported to be a specific inhibitor of the PAF receptor and as such, it is widely used for assessment of PAF receptor mediated biological effects. We report here that L-659,989 may not be as specific as previously reported because it is also a potent inhibitor of phospholipase D activity. At concentrations of 30 micrograms/ml, L-659,989 inhibited basal and agonist-stimulated phospholipase D activity by about 55% and 70-100% respectively, through a mechanism that may involve the generation of intracellular ceramides. Another PAF receptor antagonist, WEB-2086, did not affect phospholipase D activity at concentrations up to 50 micrograms/ml. Either of these inhibitors when present at 20 micrograms/ml are reported to fully block the effects of PAF. Furthermore, L-659,989 directly inhibited the activity of bacterial PLD in vitro. These results indicate that caution is required in the interpretation of results derived from the use of L-659,989.     

Bacterial lipopolysaccharide up-regulates platelet-activating factor-stimulated Ca2+ mobilization and eicosanoid release in human Mono Mac 6 cells.

When human monocytic Mono Mac 6 cells were treated with bacterial LPS (10 ng/ml, 72 h), they showed an increase in phagocytic activity, superoxide anion production, and expression of monocyte/macrophage-associated cell surface Ag. In these more mature (LPS-treated) cells but not in untreated cells, platelet-activating factor (PAF) (100 nM) produced a three- to fourfold increase in cytosolic free Ca2+ concentration. The cytosolic free Ca2+ concentration increase was inhibited by the PAF receptor antagonist L-659,989 (10 microM) and by EGTA (2 mM), indicating receptor-dependent Ca2+ influx. Furthermore, L-659,989 (10 microM), as well as PAF (1 microM), inhibited specific [3H]PAF binding in LPS-treated but not in untreated cells. Consistent with these results, PAF (100 nM) stimulated release of arachidonic acid and thromboxane B2 only in LPS-treated cells, and this could be inhibited by L-659,989 (10 microM) and EGTA (2 mM). Our data indicate that LPS up-regulates PAF-induced Ca2+ influx, resulting in arachidonic acid and eicosanoid release in Mono Mac 6 cells.     

Specific binding of platelet-activating factor (PAF) by human peripheral blood mononuclear leukocytes

Binding of platelet-activating factor (PAF) to human peripheral blood mononuclear leukocytes was time-dependent, reversible, and saturable. [3H]PAF binding to the cells was inhibited dose-dependently by unlabeled PAF and PAF receptor antagonists: L-659,989, triazolam, and alprazolam. Scatchard analysis of saturation binding data indicated one class of receptors for PAF with KD = 5.7 nM and Bmax = 18 fmol/10(6) cells (11,100 receptors/cell). PAF (10 nM) increased intracellular free calcium concentration in human lymphocytes and this effect was inhibited by L-659,989 dose-dependently. Our data suggest that human peripheral blood mononuclear leukocytes have specific receptors for PAF.     

Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids.     

Prolongation of parturition in the pregnant rat following treatment with a platelet activating factor receptor antagonist

The presence of platelet activating factor (PAF) in amniotic fluid of women only in labor is indicative of a role for PAF in parturition. In addition, stimulated amnion membrane produces both PAF and prostaglandin E2, each of which is capable of inducing myometrial contraction. To evaluate the involvement of PAF in the process of parturition, we administered the PAF receptor antagonist, L-659,989, to 17-day timed pregnant rats and followed the events of labor and delivery. Administration by mouth with L-659,989 of three concentrations (1.6, 16, and 48 mg/kg/day) did not alter the gestational period; however, the duration of parturition was increased from 2-fold to 5-fold by such treatment. No toxicity of the analog was apparent; treated dams showed no signs of morbidity, and fetal mortality was not significantly altered by treatment with the antagonist. Based on these experiments, it is suggested that the PAF receptor antagonist interferes with the normal progression of events of parturition and that PAF is an integral mediator in initiating the myometrial contraction necessary for expulsion of the fetus.     

(+/-)-trans-2-(3-Methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4,5- trimethoxyphenyl)tetrahydrofuran (L-659,989), a novel, potent PAF receptor antagonist

The title compound, L-659,989, is a highly potent, competitive, and selective antagonist of the binding of [3H]PAF to its receptors in platelet membranes from rabbits and humans. It exhibits equilibrium inhibition constants for PAF binding of 1.1 nM (rabbit) to 9.0 nM (human), values that are at least 1-2 orders of magnitude lower than those of other PAF antagonists tested. L-659,989 potently inhibits PAF-induced aggregation of rabbit platelets and degranulation of rat (ED50 4.5 nM) and human (ED50 10 nM) neutrophils. L-659,989 inhibits PAF-induced extravasation and lysosomal enzyme release in rats, and is active orally in female rats (ED50 0.2 mg/kg) with an extraordinary oral duration of action of 12 to 16 hours at 1.0 mg/kg p.o.     

Platelet activating factor-induced neuronal apoptosis is initiated independently of its G-protein coupled PAF receptor and is inhibited by the benzoate orsellinic acid

The bioactive lipid mediator platelet activating factor (PAF) is recognized as a key effecter of neuronal apoptosis, yet it is not clear whether its G-protein coupled receptor (PAFR) initiates or prevents PAF neurotoxicity. Using PAFR-/- and congenic wild-type mice, we show that PAF triggers caspase-3/7 activity and neuronal death in PAFR-/- but not PAFR+/+ cerebellar granule neurons. Restoring receptor expression by recombinant adenoviral infection protected cells from PAF challenge. Neuronal death was not mediated by nitric oxide or N-methyl-d-aspartate receptor signaling given that N-nitro-l-arginine methyl ester and MK-801 did not inhibit PAF-induced neuronal loss in PAFR-/- neurons. To intervene in PAFR-independent neurotoxicity, the anti-apoptotic actions of three structurally distinct PAF antagonists were compared to a panel of plant and fungal benzoic acid derivatives. We found that the PAF antagonist BN 52021 but not FR 49175 or CV 3988 inhibited PAFR-independent neurotoxicity. Orsellinic acid, a fungal-derived benzoic acid, blocked PAF-mediated neuronal apoptosis without affecting PAFR-mediated neuroprotection. These findings demonstrate that PAF can transduce apoptotic death in primary neurons independently of its G-protein coupled receptor, that PAFR activation is neuroprotective, and that orsellinic acid effectively attenuates PAFR-independent neuronal apoptosis.     

Platelet-activating factor in human endometrium

Platelet-activating factor (PAF) is a phospholipid actively produced by human endometrium and deeply involved in the processes of ovoimplantation and labor. We recently found that PAF represents a new autocrine growth factor for a human adenocarcinoma cell line, HEC-1A. Indeed, biologically active PAF is synthesized by HEC-1A cells, under progesterone control. In HEC-1A cells, PAF regulates intracellular calcium concentration ([Ca²⁺]), DNA synthesis and expression of early oncogenes. All these effects are blocked by the receptor antagonist L659,989. However, while nanomolar concentrations of PAF mobilize [Ca²⁺], only micromolar concentrations affect cell growth, suggesting heterogeneity of PAF receptors or signaling. Two distinct populations of PAF receptors are present in HEC-1A cells, which bind PAF in nanomolar and micromolar concentrations, respectively. Since HEC-1A cells are producing elevated concentrations of PAF and micromolar concentrations of the PAF antagonist L659,989 inhibit cell proliferation, an autocrine role for PAF is suggested in HEC-1A cells.     

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

A product of activated human granulocytes stimulates prostaglandin E2 synthesis in human amnion cells

Cell-free supernatant from formylmethionyl-leucyl-phenylalanine (fMLP)-activated granulocytes causes a time- and concentration-dependent stimulation of prostaglandin E2 (PGE2) production in amnion cells. PGE2 concentration in the culture medium after 36 h treatment with granulocyte supernatant (from 40 x 10(6) granulocytes/ml of amnion cell medium), 1.49 +/- 0.71 pg/ng DNA (n = 13), was significantly higher (p = 0.0015) than in control cells (0.33 +/- 0.23 pg/ng DNA, n = 13). Indomethacin abolished this stimulation. Granulocyte supernatant and human epidermal growth factor (hEGF) had an additive effect on amnion cell PGE2 production. Catalase, superoxide dismutase (SOD), protease inhibitors or the platelet-activating factor (PAF) antagonist L-659,989 had no effect. Actinomycin D, cycloheximide and mepacrine reduced the PGE2 production. The phospholipase A2 activity present in granulocyte supernatants was resistant to heating, whereas heating decreased their PGE2-stimulating activity by 92%. Exogenous phospholipase A2 had no effect on PGE2 synthesis. The granulocyte product could be precipitated with ammonium sulphate. On gel filtration of supernatant, two peaks of PGE2-synthesis stimulating activity were obtained (molecular weights 12,000 and 60,000). This data serve to explain the association of chorioamnionitis with preterm labor: activated granulocytes release a protein(s) that induces prostaglandin production in amnion cells, and thus promote labor.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Action of two juvenile hormone antagonists on lepidopteran epidermis

The juvenile hormone antagonist ETB (ethyl-4-2(t-butylcarbonyloxy)-butoxybenzoate) caused formation of precocious larval-pupal intermediates after the 4th (penultimate)-larval instar of the tobacco hornworm, Manduca sexta, when 50 μg were applied to any 3rd stage larvae or to 4th stage larvae within 12 hr after ecdysis. This dose was most effective within 12 hr after ecdysis to the 3rd stage. In the black mutant larval assay for juvenile hormone, ETB had activity, 0.75 μg per larva giving half-maximal score. In vitro ETB acted as a juvenile hormone to prevent the ecdysteroid-induced change in commitment at concentrations above 0.1 μg/ml with an ED₅₀ at 2.8 μg/ml and as a partial juvenile hormone antagonist to 0.1 μg/ml juvenile hormone I at concentrations between 10^?3 and 10^?2 μg/ml. By contrast, EMD (ethyl-E-3-methyl-2-dodecenoate) had little juvenile hormone-like activity in vitro up to its limits of solubility (100 μg/ml) and exhibited sporadic partial juvenile hormone antagonistic activity in vitro at concentrations between 1 and 100 μg/ml. Since these concentrations were 10–1000 times that of juvenile hormone I in the medium, EMD apparently is not an efficient competitor.     

Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation

The internalization of Aspergillus fumigatus into lung epithelial cells is critical for the infection process in the host. Gliotoxin is the most potent toxin produced by A. fumigatus. However, its role in A. fumigatus internalization into the lung epithelial cells is still largely unknown. In the present study, the deletion of the gliP gene regulating the production of gliotoxin in A. fumigatus suppressed the internalization of conidia into the A549 lung epithelial cells, and this suppression could be rescued by the exogenous addition of gliotoxin. At lower concentrations, gliotoxin enhanced the internalization of the conidia of A. fumigatus into A549 cells; in contrast, it inhibited the phagocytosis of J774 macrophages in a dose-dependent manner. Under a concentration of 100 ng/ml, gliotoxin had no effect on A549 cell viability but attenuated ROS production in a dose-dependent manner. Gliotoxin significantly stimulated the phospholipase D activity in the A549 cells at a concentration of 50 ng/ml. This stimulation was blocked by the pretreatment of host cells with PLD1- but not PLD2-specific inhibitor. Morphological cell changes induced by gliotoxin were observed in the A549 cells accompanying with obvious actin cytoskeleton rearrangement and a moderate alteration of phospholipase D distribution. Our data indicated that gliotoxin might be responsible for modulating the A. fumigatus internalization into epithelial cells through phospholipase D1 activation and actin cytoskeleton rearrangement.     

Platelet activating factor-induced apoptosis is inhibited by ectopic expression of the platelet activating factor G-protein coupled receptor

The pro-inflammatory lipid mediator platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) accumulates in ischemia, epilepsy, and human immunodeficiency virus-1-associated dementia and is implicated in neuronal loss. The present study was undertaken to establish a role for its G-protein coupled receptor in regulating neurotoxicity. PC12 cells do not express PAF receptor mRNA as demonstrated by northern analysis and RT-PCR. In the absence of the G-protein coupled receptor, PAF (0.1-1 micro m) triggered chromatin condensation, DNA strand breaks, oligonucleosomal fragmentation, and nuclear disintegration characteristic of apoptosis. Lyso-PAF (0.001-1 micro m), the immediate metabolite of PAF, did not elicit apoptotic death. Concentrations of PAF or lyso-PAF that exceeded critical micelle concentration had physicochemical effects on plasma membrane resulting in necrosis. Apoptosis but not necrosis was inhibited by the PAF antagonist BN52021 (1-100 micro m) but not CV3988 (0.2-20 micro m). Ectopic PAF receptor expression protected PC12 transfectants from ligand-induced apoptosis. PAF receptor-mediated protection was inhibited by CV3988 (1 micro m). These data provide empirical evidence that: (i) PAF can initiate apoptosis independently of its G-protein coupled receptor; (ii) PAF signaling initiated by its G-protein coupled receptor is cytoprotective to PC12 cells; (iii) the pro- and anti-apoptotic effects of PAF on PC12 cells can be pharmacologically distinguished using two different PAF antagonists.     

Pseudomonas aeruginosa toxin ExoU induces a PAF-dependent impairment of alveolar fibrin turnover secondary to enhanced activation of coagulation and increased expression of plasminogen activator inhibitor-1 in the course of mice pneumosepsis

Background

ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A₂ activity, was shown to induce vascular hyperpermeability and thrombus formation in a murine model of pneumosepsis. In this study, we investigated the toxin ability to induce alterations in pulmonary fibrinolysis and the contribution of the platelet activating factor (PAF) in the ExoU-induced overexpression of plasminogen activator inhibitor-1 (PAI-1).

Methods

Mice were intratracheally instilled with the ExoU producing PA103 P. aeruginosa or its mutant with deletion of the exoU gene. After 24 h, animal bronchoalveolar lavage fluids (BALF) were analyzed and lung sections were submitted to fibrin and PAI-1 immunohistochemical localization. Supernatants from A549 airway epithelial cells and THP-1 macrophage cultures infected with both bacterial strains were also analyzed at 24 h post-infection.

Results

In PA103-infected mice, but not in control animals or in mice infected with the bacterial mutant, extensive fibrin deposition was detected in lung parenchyma and microvasculature whereas mice BALF exhibited elevated tissue factor-dependent procoagulant activity and PAI-1 concentration. ExoU-triggered PAI-1 overexpression was confirmed by immunohistochemistry. In in vitro assays, PA103-infected A549 cells exhibited overexpression of PAI-1 mRNA. Increased concentration of PAI-1 protein was detected in both A549 and THP-1 culture supernatants. Mice treatment with a PAF antagonist prior to PA103 infection reduced significantly PAI-1 concentrations in mice BALF. Similarly, A549 cell treatment with an antibody against PAF receptor significantly reduced PAI-1 mRNA expression and PAI-1 concentrations in cell supernatants, respectively.

Conclusion

ExoU was shown to induce disturbed fibrin turnover, secondary to enhanced procoagulant and antifibrinolytic activity during P. aeruginosa pneumosepsis, by a PAF-dependent mechanism. Besides its possible pathophysiological relevance, in vitro detection of exoU gene in bacterial clinical isolates warrants investigation as a predictor of outcome of patients with P. aeruginosa pneumonia/sepsis and as a marker to guide treatment strategies.     

钙超载和血小板激活因子对肺组织释放降钙素基因相关肽的影响

在肠缺血／再灌注（Ｉ／Ｒ）损伤的大鼠模型中,用放射免疫法分析血管和离体肺灌洗液中降钙素基因相关肽（ＣＧＲＰ）。结果发现Ｉ／Ｒ损伤后血浆ＣＧＲＰ水平较自身伤前增高１６１．３％（Ｐ〈０．０１）,伤前预先用血小板激活因子受阻断剂（ＳＲ２７４１Ａ）,血浆ＣＧＲＰ仍然高于自身伤前１１７．８％（Ｐ〈０．０１）。而假手术组没有明显变化。另外,Ｉ／Ｒ损伤组大鼠离体肺灌洗液同正常对照相比,ＣＧＲＰ还下降了１７．４％     

Platelet-activating factor (PAF) mediation of rat anaphylactic responses to soluble immune complexes. Studies with PAF receptor antagonist L-652,731

A new synthetic compound, L-652,731 (trans-2,5-(3,4,5-trimethoxyphenyl) tetrahydrofuran), which has been demonstrated by Hwang et al. to be a potent and specific platelet-activating factor (PAF) receptor antagonist causes 100% inhibition of 1 microM PAF-induced neutrophil degranulation at 50 microM, but has no effect on neutrophil degranulation induced by precipitating immune complexes (323 micrograms/ml), fMet-Leu-Phe (10(-7) M), or the calcium ionophore A23187 (10(-5) M). Intravenous infusion of 1 mumol L-652,731 results in almost 100% inhibition of hypotension induced by PAF but not that induced by isoproterenol, histamine, bradykinin, or acetylcholine. With the use of this novel PAF receptor antagonist, the in vivo mediator role of PAF in the soluble immune complex-induced hypotension, extravasation, vascular lysosomal hydrolase secretion, and neutropenia in rats was determined. The hypotension, extravasation, and lysosomal hydrolase release induced by immune complex infusion take 2 to 10 min longer to occur than the same responses elicited by PAF infusion. The neutropenia response is immediate with both stimuli. L-652,731 when orally administered to rats (20 mg/kg, 1.5 hr before PAF infusion) inhibited PAF-induced hypotension (69%), extravasation (76%), vascular lysosomal hydrolase release (79%), and neutropenia (73%). The same L-652,731-dosing regimen inhibited immune complex-stimulated hypotension (87%), extravasation (77%), and vascular lysosomal hydrolase release (31%). The initial and complete neutropenia induced by immune complex infusion was not inhibited in L-652,731-pretreated rats, but the rate of return of neutrophils to the blood was faster in the latter rats. Rats with blocked circulation to the liver still exhibited extensive extravasation and vascular lysosomal hydrolase release in response to PAF, but there was no extravasation and greatly reduced hydrolase release in response to immune complexes. Thus PAF is indicated to be a major mediator of soluble immune complex-induced hypotension and vascular permeability and a minor mediator of immune complex-induced lysosomal hydrolase release in rats. PAF probably does not mediate the initial and complete neutropenia stimulated by immune complexes. The liver is probably the major site for PAF production in response to circulating immune complexes.     

Development of specific functionally active receptors for platelet-activating factor in HL-60 cells following granulocytic differentiation

A human promyelocytic leukemia cell line (undifferentiated HL-60 cells) as well as a granulocyte form of HL-60 cells induced in vitro by exposure to dimethyl sulfoxide were examined for binding, metabolism, and biological responses to platelet-activating factor (PAF). Undifferentiated and differentiated HL-60 cells each exhibit a high capacity to incorporate and metabolize [3H]PAF at 37 degrees C; however, the amount of [3H]PAF that is assimilated by both cell populations is greatly reduced and its metabolism abolished at less than or equal to 4 degrees C. At 0 degrees C HL-60 granulocytes bind more [3H]PAF than their undifferentiated counterparts. Binding to differentiated cells reaches equilibrium within 80 min and is saturable, reversible and specific; PAF receptor antagonists WEB 2086, L-659,989, BN 52021, and kadsurenone abolish this specific [3H]PAF binding. In contrast, [3H]PAF uptake by undifferentiated HL-60 cells is neither saturable nor sensitive to specific receptor antagonists. Scatchard analyses reveal 5850 +/- 850 binding sites per differentiated HL-60 cell with a dissociation constant of 0.66 +/- 0.15 nM. In the presence of cytochalasin B, PAF (200 nM) induces degranulation only in differentiated cells and this response also is blocked by PAF receptor antagonists. Our results demonstrate that HL-60 cells develop specific and functionally active PAF receptors only after chemically induced differentiation into granulocytes.