Platelet-activating Factor Binding Research Articles

Specific high affinity binding of platelet activating factor to intact human blood neutrophils and eosinophils.

Neutrophils and eosinophils are involved in various inflammatory reactions such as leukocyte migration, adherence and phagocytosis. A regulation of platelet-activating factor (PAF) receptors in intact human blood neutrophils and eosinophils is clinically important. Intact human blood neutrophils and eosinophils prepared under sterile conditions specifically bound [3H]PAF in the presence of fatty acid-free serum albumin (0.25% BSA). Excess unlabeled PAF (500 nM) or the specific PAF receptor antagonist WEB 2086 (1 microM) inhibited the [3H]PAF binding. PAF receptors on the surface of intact blood neutrophils and eosinophils had high affinity Kd values of 0.55 and 2.3 nM at 4 degrees C. The Bmax values were 200 fmol/2.5 x 10(6) neutrophils and 26 fmol/2.5 x 10(5) eosinophils. PAF receptors on the outer plasma membranes were functionally relevant as high dose PAF displaced WEB 2086 after 3 min preincubation mediating maximal cytosolic [Ca2+]i flux. High doses of PAF or phorbol myristate acetate (PMA) downregulated neutrophils and a low dose PAF decreased the specific [3H]PAF binding to eosinophils determined with WEB 2086 at 20 degrees C. Only neutrophils were significantly upregulated by low dose PAF (5 nM), lyso PAF or low dose PMA (1 nM). Up- and downregulation by PAF itself of neutrophil and eosinophil PAF receptors might explain their desensitization and some clinical controversy concerning the role of PAF in inflammatory and allergic diseases. The latter hypothesis would lead to a novel combination of antagonists against PAF receptors and PAF production.

Identification of Functional Platelet-Activating Factor Receptors on Human Keratinocytes

Platelet-activating factor (PAF) is a potent inflammatory mediator that has been shown to be produced by human keratinocytes and is thought to play a role in cutaneous inflammation. Immunofluorescence and radioligand binding studies were used to characterize PAF receptors (PAF-R) on human keratinocytes and the human epidermoid cell lines A-431 and HaCaT. Indirect immunofluorescence studies demonstrated anti-PAF-R staining of primary cultures of human keratinocytes, A-431 cells, and HaCaT cells. Primary cultures of human fibroblasts and the melanoma cell line SK-30 failed to show immunostaining above that seen with control antiserum. With indirect immunofluorescence studies of sections of normal human skin, a granular anti-PAF-R staining pattern was noted on the keratinocyte cell membranes. A-431 cells readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Binding studies on crude membrane preparations of A-431 cells conducted at 4 degrees C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single class of high-affinity (KD = 6.3 +/- 0.3 nM) PAF binding sites. The immunofluorescence and radioligand binding sites were shown to be functional PAF-Rs, as 10 pM to 1 microM PAF increased intracellular calcium in primary cultures of human keratinocytes, A-431 cells, and HaCaT cells, whereas PAF treatment of primary cultures of human fibroblasts or the melanoma cell line SK-30 did not result in changes in the intracellular calcium concentration. The structurally dissimilar PAF-R antagonists CV-6209, Ro19-3704, and alprazolam all inhibited the PAF-induced calcium changes in A-431 cells. The CV-6209 inhibition was seen at doses that competed with the PAF binding to these cells. These studies provide the first evidence for the presence of a functional PAF-R expressed on human keratinocytes, suggesting that this lipid mediator may play an important role in normal keratinocytes or in inflammatory dermatology.

Human umbilical vein endothelial cells: Specific binding of platelet-activating factor and cytosolic calcium flux

An interaction of the platelet-activating factor (Paf) with endothelial cells was investigated using human umbilical vein endothelial cells. Confluent endothelial cells bound [ 3H]Paf in the presence of 0.25% fatty acid-free serum albumin after culture in media containing either heat-inactivated foetal calf serum or serum substitute. The Scatchard analysis of the saturated specific [ 3H]Paf binding showed a B max of 2.5 fmol indicating 2800 binding sites per endothelial cell. [ 3H]Paf binding was partially reversible at 20 ° and 4 ° and endothelial cells partially metabolized [ 3H]Paf at 20 ° but not at 4 °. [ 3H]Paf binding and Paf-mediated increase of cytosolic free calcium were inhibited by specific Paf receptor antagonists which do not interfere with Paf metabolism. Immortalized umbilical vein endothelial cells bound [ 3H]Paf specifically after culture in the presence of insulin (20 hr, 0.4 U/mL) with non-specific binding in the absence of insulin. The results show that specific Paf binding mediated calcium flux in human endothelial cells.

Inhibition of PAF-induced human platelet aggregation by antithrombotic nipecotamides

Nipecotamides (piperidine-3-carboxamides) are potent inhibitors of platelet aggregation induced by a variety of agonists in vitro and in vivo. The inhibitory effects of six structural types of nipecotamides on human platelet aggregation induced by platelet-activating factor (PAF) in vitro, are studied. Evaluation of 15 racemates and stereoisomers of two nipecotamides showed that bis-nipecotoyl alkanes were more active than their mono congeners. Mono- and bis-nipecotoyl decanes were more potent than the corresponding hexanes. Lipophilicity was found to play a significant role in the antiplatelet activity of these compounds. The stereoselectivity in the PAF-antagonist potential of nipecotamides was less pronounced than that resulting from their action on ADP- or collagen-induced aggregation. Oxidation of the two benzylic carbon atoms of α,α′-bis[3-( N,N-diethylcarbamoyl)piperidino]- p-xylene·2HBr (A-1) to form 1,4-bis[3- N,N-diethylcarbamoyl)piperidino]benzenedicarboxamide (A-40K), which has a second set of carbonyl oxygens but lacks basic N atoms, resulted in a remarkable loss of ADP-antagonist potency while retaining PAF-antagonist activity. It is suggested that in addition to their membrane effects, nipecotamides act at other sites, including the PAF receptor. Double reciprocal plots of PAF binding to gel-filtered platelets (GFP) in the presence and absence of a typical nipecotamide (A-1C) were indicative of competitive inhibition ( K i = 19.28 μM). Scatchard analysis of 3H-PAF binding to GFP suggested the presence of high, intermediate ( I) and low affinity binding sites, of which the I site gave a K D app of 0.332 nM with an estimated 564 sites/platelet. Key interactions of nipecotamides with the PAF receptor appear to be the following (i) electrostatic interactions of the two amide oxygens with a primary set of electropositive areas spaced at 5–7 Å, (ii) in the case of appropriate compounds, electrostatic interactions of the two amide oxygens spaced at 10–12 Å with corresponding secondary receptor sites carrying positive electrostatic potential, (iii) a hydrophobic moiety fitting into a hydrophobic pocket in the receptor, and (iv) the cationic piperidine N + (at pH 7.4) interacting with a counterion, probably aspartic acid.

Presence of specific platelet-activating factor binding-sites in neuroblastoma N1E-115 cells

In this study we reported evidence for the existence of specific binding sites for platelet-activating factor (PAF) in neuroblastoma N1E-115 cells. The specific [ 3H]PAF binding reached a steady state level within 60 min at 25°C. Scatchard analysis of the specific [ 3H]PAF binding revealed the presence of two apparent populations of binding sites. The high-affinity binding site possessed a K d1 of 2.5 ± 0.6 pM and B max1 = 57.3 ± 20.0 fmol/mg protein. The low-affinity binding site possessed a K d2 = 3.2 ± 1.0 nM and B max2 = 4.4 ± 2.1 pmol/mg protein. Furthermore, the total [ 3H]PAF binding was partially displaced by unlabelled PAF, PAF antagonists BN 52021 and BN 50730 in a dose-dependent manner. This study confirms the presence of specific PAF receptors in neuronal cells.

Distinct biochemical responses of hepatic macrophages and endothelial cells to platelet-activating factor during endotoxemia

Acute endotoxemia is associated with activation of hepatic macrophages and endothelial cells. These cells release a variety of inflammatory mediators that have been implicated in tissue injury. In the present studies, we analyzed the biochemical responses of these cells to platelet-activating factor (PAF), a lipid autacoid released during hepatic inflammatory responses. To induce acute endotoxemia, rats were injected intravenously with lipopolysaccharide (LPS). Using the calcium sensitive fluorescent indicator dye Indo-1, we found that PAF induced a rapid and transient increase in intracellular calcium in both hepatic macrophages and endothelial cells. Induction of acute endotoxemia resulted in an increase in the amount of calcium mobilized by both cell types. Although endothelial cells from control rats were less responsive to PAF than macrophages, these cells were more sensitive to in vivo endotoxin. PAF was also found to cause a rapid decrease in intracellular pH in hepatic macrophages that was quantified by fluorescence image analysis using the pH sensitive dye SNAFL-calcein. This decrease occurred more rapidly in macrophages from endotoxemic rats. In cells from both control and endotoxemic rats, the effects of PAF on intracellular pH were inhibited by the specific PAF antagonist triazolam. In contrast to hepatic macrophages, PAF had no effect on intracellular pH in endothelial cells from either control or endotoxemic rats. Ligand binding studies demonstrated that both hepatic macrophages and endothelial cells possess high affinity binding sites for PAF. Macrophages expressed 6- to 7-fold more binding sites/cell than endothelial cells and exhibited a higher Kd. Whereas treatment of rats with LPS had no effect on the Kd for PAF binding to macrophages or on the number of binding sites, a significant increase in both of these receptor characteristics was observed in endothelial cells. Taken together, the present data suggest that the biochemical responses of endothelial cells and macrophages to PAF are distinct. Furthermore, cellular activation induced by PAF in endothelial cells appears to be independent of changes in intracellular pH.

Activation of the Phospholipase/Cyclooxygenase Cascade in the Rabbit Cornea by Platelet-Activating Factor is Challenged by PAF Receptor Antagonists

Platelet-activating factor (PAF) is a potent lipid inflammatory mediator which is generated in the cornea after injury. Its activity is regulated by interaction with specific receptors. The binding of PAF to its receptors initiates biochemical sequences that cluminate in the release of additional lipid mediators. An arachidonoyl-dependent phospholipase A2 is activated to release arachidonic acid from membrane phospholipids, especially phosphatidylcholine and ethanolamine. Arachidonic acid is then predominantly metabolized by the cyclooxygenase pathway to prostaglandins F2 alpha, E2 and D2, whereas the lipoxygenase pathway is not influenced by PAF. The release of arachidonic acid and prostaglandins stimulated by PAF is challenged by the PAF receptor antagonists BN 50727 and BN 50730. PAF acting intracellularly may also induce the synthesis of cyclooxygenase, presumably the 'inducible' isoform PGHS2, which has been implicated in the inflammatory response. Thus, the therapeutic use of PAF receptor angatonists could be potentially beneficial in the management of ocular inflammatory disease.

Characterization of platelet-activating factor binding to human airway epithelial cells: modulation by fatty acids and ion-channel blockers.

Radioligand-binding studies were performed in primary cultured human airway epithelial cells with [3H]PAF to determine whether these cells express platelet-activating factor (PAF) receptors. Scatchard analysis of PAF binding data revealed a single class of PAF binding sites with Kd 1.8 +/- 0.2 nM and Bmax. 21.0 +/- 2.1 fmol/10(6) cells (13,000 receptors/cell). PAF binding increased the intracellular free Ca2+ concentration ([Ca2+]i), indicating functional PAF receptors. Palmitate (C16:0), linoleic acid (C18:2 omega 6) or eicosapentaenoic acid (C20:5 omega 3) was incubated with the cells to test the effect on PAF binding. Incorporation of each fatty acid into cellular phospholipid occurred. [3H]PAF (1 nM) binding decreased in cells supplemented with C20:5 omega 3, but increased in the cells supplemented with C16:0. Scatchard analysis revealed that the inhibition of PAF binding by supplementation with C20:5 omega 3 was due to a decrease in both affinity and number of PAF receptors. PAF-stimulated increase in [Ca2+]i was also decreased by 60% in cells supplemented with C20:5 omega 3. Verapamil, a Ca(2+)-channel blocker, and amiloride, a Na(+)-channel blocker, inhibited specific binding of [3H]PAF to the cells, with IC50 4-5 microM and 0.2 mM respectively. Diphenylamine-2-carboxylate (DPC), a Cl(-)-channel blocker, dramatically increased PAF binding to the cell in a dose-dependent manner. Scatchard analysis revealed that verapamil and amiloride decreased both binding affinity and number of PAF receptors, whereas DPC increased PAF binding sites without affecting binding affinity. These results demonstrate that human airway epithelial cells have a functional receptor for PAF and that PAF receptor binding can be modulated by exogenous fatty acids and by ion-channel blockers.

Changes in [ 3H]PAF binding and PAF concentrations in gerbil brain after bilateral common carotid artery occlusion: a quantitative autoradiographic study

This study investigated the distribution of platelet activating factor (PAF) binding sites in the brain and their involvement in global ischemia in a model of bilateral common carotid occlusion in the gerbil. In sagittal sections of gerbil brain, labeling with [ 3H]PAF was mainly located in the cortex, hippocampus and cerebellum. The corpus striatum, the superior and inferior colliculi showed lower binding, while the thalamus was only weakly labeled. Scatchard analysis of the data obtained from displacement curves with unlabeled PAF revealed the presence of one or two populations of binding sites with different affinity constant values depending on the brain structures. When the gerbils were submitted to 10 min ischemia, similar autoradiography with [ 3H]PAF demonstrated a dramatic reduction of labeling in all brain structures, particularly in the hippocampus. Immunoreactive endogenous PAF concentrations in brain tissues showed a marked increase in ischemic animals: ( 8977.3±1113 pg/g wet weight) as compared to sham-operated control: ( 997.7±77 pg/g wet weight). Endogenous PAF levels returned to basal values following 30 min reperfusion. These results indicate that PAF may be involved in the early stages of brain ischemia in the gerbil and suggest that endogenous PAF produced during ischemia may contribute to the down-regulation of [ 3H]PAF binding sites in the brain.

Binding and Metabolism of Platelet-Activating Factor (PAF) by Isolated Rat Type II Pneumonocytes

The specific binding of platelet-activating factor (PAF) to isolated type II pneumonocytes from rat lung has been investigated employing an intact cell preparation. The dissociation constant (Kd) for the autacoid has been determined to be 0.46 × 10−9 M and approximately 3000 receptor sites per cell are present. In studies conducted on the metabolism of PAF in these cells, it was demonstrated that PAF is rapidly converted into 1-alkyl-2-acylglycerophosphocholine (alkyl-acyl GPC). After longer time intervals there was a substantial conversion of alkyl-acyl GPC into alkyl-acylglycerophosphoethanolamine (allkyl-acyl GPE). Both the alkyl-acyl GPC and alkyl-acyl GPE fractions were devoid of plasmalogens. The alkyl-acyl GPC fraction was further characterized and a distinct double peak could be visualized following thin-layer chromatography and the same lyso-compound was produced from both peaks following mild alkaline hydrolysis. By using appropriate standards it was concluded that the dual alkyl-acyl GPC peaks represent differences in the fatty acid present in the sn-2 position. One peak corresponds to the presence of saturated fatty acid in the sn-2 position and is probably due to the characteristic high capacity of the type II cells to produce disaturated glycerophospholipids.

Characterization of platelet-activating factor receptors on canine T lymphocytes.

The ability of purified canine T lymphocytes to selectively bind platelet activating factor (PAF) was characterized. Authentic radiolabeled PAF rapidly and selectively bound to T lymphocytes and reached saturation within 1 min. This binding was reversible and highly selective for (R) PAF because (S) PAF, lyso-PAF, and diacyl PAF did not displace the bound (R) PAF probe. Only increasing quantities of chemically pure (R) PAF displaced the radiolabeled (R) PAF probe. The binding maximum of PAF was determined to be 35 pM per 2 x 10(6) lymphocytes. Competitive radioligand binding studies and Scatchard analysis indicated a single class of high affinity receptors with a dissociation constant of 0.077 nM and a receptor density of 6419 receptors per cell. The ability of purified canine T lymphocytes to hydrolyze PAF to the biologically inactive metabolite lyso-PAF was also studied. Over a 30-min incubation period, about 5% of PAF was metabolized to lyso PAF. This rate of PAF hydrolysis was the same as the rate observed with the media without cells, suggesting a small degree of nonenzymatic hydrolysis. The effects of varying concentrations of authentic PAF on intracellular free Ca2+ release in purified T lymphocytes was evaluated using the fluorescent probe Fura-2 and excitation-emission spectrofluorometry. PAF below the concentration of 1.0 nM did not significantly increase intracellular Ca2+ in T lymphocytes. More than 1 nM PAF, intracellular-free Ca2+ modestly, but significantly, increased in T lymphocytes. In other experiments, canine PBMC proliferated in response to Con A and in the one way MLR. These proliferative responses were abolished when the selective PAF receptor antagonist SC-47014A was added to the culture medium. In the MLR, this inhibitory effect was dependent on the length of time that the antagonist was in the culture. Specifically, inhibition of proliferation was incrementally reversed when the PAF antagonist was introduced progressively later into the 7-day MLR stimulation period, suggesting that PAF receptor blockade prevents an MLR response from occurring, but is unable to suppress an existing MLR response. Although the Con A-induced mononuclear cell proliferation was abolished with PAF receptor antagonists, the addition of authentic biologically active PAF or PAF analogs did not alter the proliferative response to Con A. In conclusion, canine T lymphocytes possess high affinity receptors for PAF. These binding sites are highly selective and reversible. PAF binding slightly increases intracellular free Ca2+ in T lymphocytes and appears to be involved in lymphocyte proliferation in response to soluble plant mitogen and alloantigen.

Effect of epinephrine on the regulation of Na +/H + exchange in human platelets

In the present study, we investigated whether stimulation of platelets by epinephrine affects the Na +/H + exchanger, an antiport that regulates the cytosolic pH (pH i). Epinephrine alone failed to modulate Na +/H + exchange, as reflected by a constant fluorescence of the pH i indicator BCECF. In contrast, epinephrine accelerated Na +/H + exchange upon stimulation with a threshold concentration of platelet activating factor (PAF). The extra Na +/H + exchange was not caused by a better binding of PAF to platelets and occurred also in the presence of indomethacin, excluding a role for cyclooxygenase products. Epinephrine failed to mobilize Ca i 2+ (measured by fura-2 fluorescence) and did not activate protein kinase C ([ 32P]pleckstrin) or phospholipase C ([ 32P]phosphatidic acid). In combination with PAF, epinephrine left the PAF-induced mobilization of Ca i 2+ and accumulation of [ 32P]phosphatidic acid unchanged, but induced a 1.3-fold increase in the phosphorylation of pleckstrin. These data indicate that epinephrine enhances Na +/H + exchange via a direct effect of α 2a-adrenergic receptors on protein kinase C.

Inhibition of receptor-mediated platelet activation by nedocromil sodium

Background: Platelet activation by platelet activating factor (PAF) seems to be involved in the inflammatory process in asthma and may serve as a possible target for the antiinflammatory drug nedocromil sodium, which is known to inhibit cell activation by different stimuli. Methods: We investigated the effect and the mode of action of nedocromil sodium on platelet activation by PAF. In a set of healthy volunteers ( n = 45) we investigated seven different parameters of platelet activation by PAF, thrombin, and Ca 2+-ionophore. Results: Nedocromil sodium inhibited: (1) PAF-induced “shape change” reaction up to 78% (50% inhibitory concentration [IC 50]: 3 × 10 −9 mol/L), thrombin-mediated “shape change” up to 80% (IC 50: 2 × 10 −8 mol/L), but not the Ca 2+-ionophore-dependent reaction, (2) platelet aggregation by PAF up to 85% (IC 50: 2 × 10 −9 mol/L); (3) release of thromboxane B 2 up to 82% (IC 50: 5 × 10 −9 mol/L); (4) formation of inositol 1,4,5-triphosphate by PAF (IC 50 3 × 10 −7 mol/L), by thrombin (IC 50: 1 × 10 −9 mol/L), but not by Ca 2+ ionophore; (5) increase of intracellular free calcium (IC 50: 4 × 10 −7 mol/L); (6) formation of diacylglycerol (IC 50: 9 × 10 −9 mol/L), and (7) translocation of protein kinase C (IC 50: 1 × 10 −9 mol/L). Conclusions: In the concentration range of the IC 50 values found in these experiments, nedocromil sodium reduced PAF binding to platelets by only 10% to 20%, such that this interference cannot explain the observed effects of the compound. Inhibition of receptor-mediated platelet activation at an early stage in the signal transduction pathway, and without effect on Ca 2+-ionophore-—induced platelet activation, suggests an action of nedocromil sodium at the level of the cell membrane.

Isolation of a Phospholipid Inhibitor of Platelet Activating Factor-Induced Activity from Perfused Rat Liver: Identification as Phosphatidylglycerol

An endogenous inhibitor of platelet activating factor action was isolated from perfused rat liver. It was purified by thin-layer chromatography and high-performance liquid chromatography and subjected to chemical modifications in order to identify its structure. On the basis of its fast atom bombardment-mass spectrum it was characterized as phosphatidylglycerol composed mainly of 16:0/18:1 and 16:0/20:2 fatty acyl chains ([M + H] + at m/ z 749 and 775, respectively) and very minor levels of 18:0/18:1 and 18:0/20:2. The purified compound exhibited inhibition on rabbit platelet aggregation induced by 5 × 10 −10 M platelet activating factor (PAF) at an EC 50 value near 2.5 × 10 −6 M and on the serotonin secretion at an EC 50 7 × 10 −6 M. Other phospholipids isolated from the liver preparations, such as phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin (diphosphatidylglycerol), and phosphatidic acid, exhibited no inhibitory activity in the concentration range from 1 × 10 −4 to 1 × 10 −7 M nor did they induce any aggregation, or lysis, of the platelets. Of importance, phosphatidylglycerol could inhibit thrombin- and ADP-induced aggregation of rabbit platelets. These results suggested a possible site of inhibition common to the signal transduction pathway of these agonists. Preliminary binding experiments showed a noncompetitive type of inhibition on PAF binding to intact rabbit platelets.

Regulation of the receptor for platelet-activating factor on human platelets.

Human platelets possess about 300 receptors for platelet-activating factor (PAF) per cell with a Kd of about 0.2 nM. In the present study we investigated whether these receptors are subject to intracellular control mechanisms. Preincubation with the protein kinase C inhibitor staurosporine had no effect, and also agents that increase cyclic AMP failed to change the binding of [3H]PAF. The Ca(2+)-calmodulin inhibitors W-7 and sphingosine decreased PAF binding by 50-80%. Inhibition of energy metabolism induced a fall in adenylate energy charge [AEC = ([ATP] + 1/2[ADP])([ATP + ADP + AMP])] and an almost parallel decrease in specific [3H]PAF binding without changing the Kd. Restoration of the AEC restored the [3H]PAF binding. Abrupt arrest of energy metabolism during binding of [3H]PAF left the binding unchanged until the metabolic ATP level had decreased by about 90%. These data indicate that PAF receptors on human platelets are under close intracellular control, possibly via a Ca(2+)-calmodulin-dependent phosphorylation/dephosphorylation process.

Tumor necrosis factor induces enhanced responses to platelet‐activating factor and differentiation in human monocytic Mono Mac 6 cells

Human Mono Mac 6 cells exhibit characteristics of mature blood monocytes. Treatment of these cells with human recombinant human tumor necrosis factor (TNF) resulted in an increase in phagocytosis and phorbol myristate acetate-stimulated superoxide anion production at 12 h and growth retardation occurring at 24 h. Moreover, TNF induced a moderate increase of CD14 surface antigen expression, used as a phenotypic marker of monocyte/macrophage differentiation. Platelet-activating factor (PAF) stimulated a rapid rise in cytosolic free Ca++ ([Ca++]i) of 308 +/- 93 nM in TNF-treated cells compared to untreated cells (33 +/- 8 nM, n = 4). The effect of TNF was dose and time dependent, evident after 12 h and maximal at 48 h. The enhanced PAF-induced [Ca++]i rise was inhibited by the PAF receptor antagonist L-659,989 and EGTA, indicating receptor-dependent Ca++ influx. Furthermore, L-659,989 and PAF inhibited specific 3H-labeled PAF binding in TNF-treated, but not in untreated cells. Consistently, PAF stimulated arachidonic acid release only in TNF-treated cells. Preincubation of cells with anti-TNF monoclonal antibodies abolished TNF-induced effects, but failed to block lipopolysaccharide (LPS) effects. Distinct mechanisms of action by LPS were reflected by the different ability to induce surface antigen expression. In conclusion, the enhancement of PAF responses by TNF, associated with functional characteristics of differentiation in Mono Mac 6 cells, may represent a specific mechanism of cooperative interaction between PAF and TNF in inflammation, sepsis, immunoregulation and atherogenesis.

Forskolin inhibits platelet-activating factor binding to platelet receptors independently of adenylyl cyclase activation

The effect of forskolin on platelet-activating factor (PAF) receptor was investigated. Rabbit platelets treated with forskolin showed approximately a 9-fold increase in cAMP levels over the control. After treatment of platelets with forskolin prior to PAF binding, a 30–40% (P < 0.005) decrease in PAF binding was observed. The decrease in PAF binding caused by forskolin was concomitant with a decrease in the physiological responses of platelets induced by PAF. However, this forskolin-induced decrease in PAF binding was not a consequence of cAMP formation as the addition of a cAMP analog could not mimic the action of forskolin. Additionally, the inactive analog of forskolin, dideoxyforskolin, which does not activate adenylyl cyclase, also reduced PAF binding to its receptor. Reduction of PAF binding by forskolin and dideoxyforskolin was also observed with isolated platelet membranes. To understand the mechanism of forskolin induced changes in PAF binding, the involvement of a G-protein in this process was investigated. Cells treated with GTPγS showed approximately a 25% reduction in PAF binding. Addition of forskolin to the GTPγS treated cells resulted in a further reduction in PAF binding, suggesting the action of forskolin was independent of G-protein activation. The data suggests that the action of forskolin was independent of adenylyl cyclase or G-protein involvement. It is speculated that the action of forskolin on PAF binding was due to a direct effect of this molecule and its analog on the PAF receptor itself or to components of the post-receptor signalling for PAF.

Endotoxin transduces Ca 2+ signaling via platelet-activating factor receptor

Lipopolysaccharide (LPS) is a pathogenic substance causing severe multiple organ failures and high mortality, Although several LPS binding proteins have been identified, the molecular mechanism underlying the LPS signaling pathway still remains obscure. We have found that the LPS-induced Ca 2+ increase in platelets and platelet aggregation is blocked by selective platelet-activating factor (PAF) receptor antagonists, thus suggesting a cross-talk between LPS and the PAF receptor. Next, we confirmed this hypothesis using the cloned PAF receptors [(1991) Nature 349, 342–346; (1991) J. Biol. Chem. 266, 20400–20405] expressed in Xenopus occytes and Chinese hamster ovary (CHO) cells. In both systems, cells responded to LPS only when PAF receptors were expressed, and specific PAF binding was successfully displaced and reversibly dissociated by LPS. PAF receptor activation by LPS may represent a novel important pathway in the pathogenesis of circulatory collapse and systemic thrombosis caused by endotoxin.

Regulation of platelet-activating-factor receptors and the desensitization response in polymorphonuclear neutrophils.

Platelet-activating factor (PAF) desensitizes as well as stimulates its various target cells, We find that human polymorphonuclear neutrophils (PMN) exposed to PAF became maximally unresponsive to a second PAF challenge within 15-90 s in assays of Ca2+ mobilization and degranulation. The cells regained full PAF-sensitivity over the ensuing 20-40 min. These effects correlated with changes in PAF receptor availability. PMN treated with PAF, washed in regular buffer and assayed for PAF binding exhibited falls (maximal in 15 s), followed by rises (reaching control levels by 60 min), in the number of high-affinity PAF receptors. However, tracking studies showed that [3H]PAF accumulated on the cell surface for approximately 2 min before being internalized. Regular-buffer washes did not remove this superficial PAF, whereas a washing regimen using excess albumin to adsorb PAF removed 99% of the surface compound. PMN washed by the latter regimen after PAF exposure lost PAF receptors relatively slowly (maximal at approximately 5 min), but the ultimate extent of this loss and the rate at which receptor expression normalized were similar to those of cells washed in regular buffer. Neither cycloheximide nor actinomycin D influenced the course of the receptor changes, but two protein kinase C (PKC) blockers, staurosporine and 1-(5-isoquinolinesulphonyl)piperazine, inhibited the receptor-receptor-depleting actions of PAF. Indeed, a phorbol diester activator of PKC also caused PMN to decrease high-affinity PAF receptor numbers, and the two PKC blockers antagonized this action at concentrations that inhibited PAF-induced PAF receptor losses. We conclude that: (a) PAF induces PMN to down-regulate and then to re-express PAF receptors independently of protein synthesis; (b) these changes are likely to underlie the later stages and reversal of desensitization; (c) the onset (t < or = 2 min) of desensitization, however, precedes receptor down-regulation and must be due to receptor uncoupling from transductional elements; and (d) down-regulation of receptors for PAF appears to be mediated by PKC and/or elements inhibited by PKC blockers.

Involvement of tyrosine kinase and protein kinase C in platelet-activating-factor-induced c-fos gene expression in A-431 cells.

In A-431 cells, platelet-activating factor (PAF) induces the expression of c-fos and TIS-1 genes in both the absence and the presence of cycloheximide in a structurally specific and receptor-coupled manner. We have now investigated the molecular mechanisms of this response, particularly in relation to the role of protein kinases. Pretreatment of cells with genistein or methyl-2,5-dihydroxycinnamate (tyrosine kinase inhibitors) or staurosporine (a protein kinase C inhibitor) for 20 min abolished the c-fos expression induced by PAF. Interestingly, when genistein was added 90 s after addition of PAF, no inhibition was observed. Similarly, staurosporine did not inhibit c-fos expression when added 8 min after PAF addition to the cells. These inhibitions were dose-dependent (IC50 for staurosporine was 180 nM, and for genistein 50 microM). Simultaneous addition of PAF and phorbol 12-myristate 13-acetate (PMA) did not give a synergistic effect on c-fos expression. Pretreatment of cells with PMA had no effect on [3H]PAF binding, but abolished the PAF-induced gene expression. PAF-stimulated gene expression was desensitized if cells were pretreated with PAF. Interestingly, epidermal growth factor was able to stimulate c-fos expression in PAF-desensitized cells, and thus indicated involvement of distinct mechanisms for the two stimuli. Forskolin, an activator of adenylate cyclase, did not induce c-fos expression and had no effect on the PAF response. Exposure of cells to PAF for as little as 1 min, followed by its removal, was sufficient to activate the gene expression and demonstrated the rapidity and the exquisite nature of the signalling involved in this process. It is concluded that activation of PAF receptor (a proposed G-protein-coupled receptor) causes rapid production of signals which induce the expression of c-fos gene and that this is mediated via tyrosine kinase and protein kinase C.