Release Of PAF-acether Research Articles

Production of PAF-acether by synovial fluid neutrophils in rheumatoid arthritis.

PAF-acether (PAF) is a pro-inflammatory phospholipid molecule potentially involved in the pathogenesis of arthritis. PAF and related metabolites have been isolated in the synovial fluid from patients with arthritis. The aim of this study was to determine fluid and blood in patients with rheumatoid arthritis. Blood neutrophils from normal donors were also studied for their capacity to form PAF. Neutrophils were stimulated with the calcium ionophore A23187 (2 microM) for 1 to 60 min. PAF released in the medium and PAF associated to cells were measured. In synovial fluid neutrophils. PAF production began as soon as 1 min of stimulation (16.1 +/- 6.3 pmol per 1 x 10(6) cells) and reached a maximum at 20 min: 29.2 +/- 2.8 pmol per 1 x 10(6) cells (mean +/- SEM, n = 5). The amount of PAF released in the supernatant increased with the length of stimulation, similar amounts of PAF were produced by blood neutrophils isolated from the joint had a lower capacity to produce PAF than blood neutrophils from the same patients. The present results demonstrate the synthesis and release of PAF by synovial fluid neutrophils. They suggest that neutrophils may be source of PAF locally present in the joint. Newly synthesized PAF could participate in the amplification of the local inflammatory reaction.

Variations of blood PAF-acether levels during coronary artery surgery

Extracorporeal circulation (ECC) is associated with thrombocytopenia and transient leukopenia. After ECC and coronary artery bypass graft (CABG) surgery, some patients can develop pulmonary and cardiac dysfunction, which might be related to the release of various mediators such as thromboxane A 2, C 5a, and C 3a anaphylatoxins. The involvement of PAF-acether (PAF), a potent vasoactive thrombocytopenic and leukoneutropenic agent, has not been determined. Therefore, 10 patients were studied during and after CABG. The release of PAF, lipo PAF (PAF bound to blood lipoproteins), and lypo PAF (PAF precursor and metabolite) was measured in blood from the left atrium, radial artery, and pulmonary artery before and after CABG. PAF, lipo PAF, and lyso PAF were also determined during ECC at the entry and exit points of the oxygenator. Hemodynamic parameters, platelet, and leukocyte counts in the pulmonary artery were measured simultaneously. PAF did not increase significantly during ECC; it showed a transitory six-fold increase immediately after CABG in the radial artery (0.18 ± 0.13 v 1.09 ±0.36 ng/ mL, P < 0.05), but not in the pulmonary artery (0.10 ± 0.03 v 0.56 ± 0.21 ng/mL, P > 0.05). Blood PAF amounts in the radial artery were significantly higher than in the left atrium following ECC (1.09 ± 0.36 v 0.06 ± 0.04, P < 0.05), probably indicating PAF production in the heart. No variation of blood lipo PAF and lyso PAF was observed. No correlation was seen between PAF amounts and blood cell count. The positive correlation observed between PAF levels in the left atrium and the pulmonary arterial pressure (r = 0.80, P < 0.001) strengthens the putative physiologic role of this agent in pulmonary artery hypertension. The clinical use of PAF antagonists may help to determine PAF involvement in human physiology and pathology.

PAF acether release on antigenic challenge. A method for the investigation of drug allergic reactions.

This study describes an in vitro method to evaluate a PAF acether release test (PART) from white blood cells after antigenic challenge. PAF acether activity of the supernatant was tested by platelet aggregation. The aggregating power was abolished by using SRI 63-441 (Sandoz), a PAF acether inhibitor. This method was applied to 57 patients with allergic or pseudo-allergic reactions to drugs by using different drug protein conjugates. The results of PART were evaluated in relation to the clinical history (score of imputability) and to other tests (skin tests, lymphocyte transformation tests (LTT), IgE-RAST). A good correlation was found between the release of PAF acether and a high predictability score: sensitivity 75%, specificity 83.8%. PART also correlated with skin tests (75% agreement, n = 60), with LTT (67.7% agreement, n = 74) and in 65.6% of cases with positive penicillin IgE-RAST (n = 32). This method brings a new possibility for the investigation of drug-allergic and pseudo-allergic reactions.

Release of PAF-acether and eicosanoids from guinea-pig alveolar macrophages by FMLP: effect of cyclo-oxygenase and lipoxygenase inhibition

Adherent guinea-pig alveolar macrophages stimulated in vitro with FMLP (1 μM) for 5–60 min released PAF-acether, TXB 2 and LTB 4, with maximal levels being achieved after a 5 min incubation. The levels of eicosanoids were maintained during the 60 min incubation period, whereas the levels of PAF-acether decreased and were no longer detectable after 60 min incubation. Indomethacin (1 μM) completely suppressed the release of TXB 2 stimulated by FMLP (1 μM for 5 min) whilst potentiating the release of LTB 4. The selective 5-lipoxygenase inhibitor, BW A137C (1 μM), abolished the stimulated release of LTB 4 without affecting the release of TXB 2. BW755C (1–20 μM) caused a dose-related inhibition of TXB 2 release but did not affect LTB 4 release, while none of the drugs studied affected the release of PAF-acether. These results indicate that the FMLP-induced release of PAF-acether from guinea-pig alveolar macrophages is not susceptible to modulation by selective inhibition of lipoxygenase or cyclo-oxygenase and is therefore not secondary to the synthesis of LTB 4 or TXB 2.

Beneficial effects of prostaglandin E 2 in endotoxic shock are unrelated to effects on PAF-acether synthesis

The effects of endotoxic shock on the synthesis of PAF-acether by the stomach, duodenum and lung were examined in the rat. Furthermore, the effect of pretreatment with prostaglandin E 2 on endotoxin induced PAF-acether synthesis and changes in vascular permeability were examined. Administration of endotoxin resulted in significant increases in PAF-acether synthesis in all tissues studied. Such increases were apparent within 5–15 minutes of the administration of endotoxin, corresponding to the time when significant hypotension, hemoconcentration and increases in gastrointestinal vascular permeability were first observed. Pretreatment with prostaglandin E 2 resulted in a significant reduction of endotoxin-induced hypotension, hemoconcentration and changes in vascular permeability in the gastrointestinal tract. However, prostaglandin pretreatment did not significantly alter endotoxin-induced PAF-acether release from the gastrointestinal tissues studied. These results demonstrate that prostaglandin E 2 can significantly attenuate several of the systemic and gastrointestinal manifestations of endotoxic shock. The mechanism responsible for these beneficial actions appears to be unrelated to effects of prostaglandin E 2 on PAF-acether synthesis.

Circulatory effects of PAF-acether in newborn piglets

Platelet-activating factor (PAF-acether) is a lipid mediator that can exhibit potent vasoconstrictor influence in the pulmonary vessels. Therefore, the release of PAF-acether during inflammatory conditions in newborns might cause deleterious increases in pulmonary vascular tone. Thirty-four anesthetized open-chest newborn piglets were given 0.01-1 nmol PAF-acether iv. In separate experiments, animals were untreated or treated with either indomethacin (a cyclooxygenase inhibitor), SQ 29548 (a thromboxane receptor blocker), or LY 171883 (a leukotriene receptor blocker). The primary hemodynamic change was a 67 to 1,537% increase in the pulmonary vascular resistance index (PVRI) (P less than 0.01): mean pulmonary artery pressure (PAP) rose significantly at all doses tested, whereas only the largest dose consistently decreased cardiac index. Treatment with indomethacin or SQ 29548 prevented the decrease in cardiac index and attenuated the PAF-acether-induced rises in PAP and PVRI. Vehicle and LY 171883 had no effect. The inhibitory influence of indomethacin and SQ 29548 suggests that an important component of PAF-acether's pulmonary vasoconstrictor action is mediated (at least in the newborn piglet) by cyclooxygenase products, most likely thromboxane.

Biosynthesis of paf-acether factor-acether by human skin fibroblasts in vitro.

The synthesis and release of paf-acether by fibroblasts from normal human skin was investigated in vitro. When fibroblasts in suspension (1 X 10(6) cells) were stimulated with 2 microM Ca1+ ionophore A23187 (Io), they synthesized a material that aggregated aspirin-treated washed rabbit platelets and was identified as paf because 1) the platelet aggregation it induced was inhibited by BN 52021, an antagonist of paf putative receptors; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse phase HPLC elution. Paf production by fibroblasts occurred as soon as the first min of Io stimulation (287 +/- 92 pg/1 X 10(6) cells), reached a maximum at 5 min (369 +/- 85 pg/1 X 10(6) cells) and decreased thereafter. Half of the fibroblast-produced paf was recovered in supernatants. Addition of exogenous 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-paf) at 0.1 microM and/or acetyl-coenzyme A at 0.1 mM to fibroblasts during Io stimulation enhanced paf production by two- and three-fold, respectively. The paf precursors, i.e., 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC) and lyso-paf, were detected in fibroblasts either stimulated with Io or not. These precursors exhibited 80% hexadecyl and 20% octadecyl chains at the sn-1 position of the molecules, as determined by reverse phase HPLC and gas chromatography analysis. The present results are the first to demonstrate the synthesis and release of paf by fibroblasts from normal human skin. Such production within the dermis might account for the development of cutaneous inflammation and for the pathogenesis of many skin disorders.

Inhibitory effect of oral cetirizine on in vivo antigen-induced histamine and PAF-acether release and eosinophil recruitment in human skin

The use of a noninvasive skin chamber technique in vivo in pollen-sensitive patients allowed us to quantify the time-course release of histamine and the recruitment of inflammatory cells (i.e., neutrophils, monocytes, and eosinophils) in skin sites challenged with pollen, histamine, and compound 48 80 . The new H I-receptor antagonist, cetirizine 2 HCl, orally administered with 10 mg once a day to pollen-sensitive patients in a double-blind, crossover study versus placebo, induced a significant decrease in the wheal-and-flare cutaneous reaction induced by intradermal injection of pollen, histamine, and compound 48 80 . It also significantly inhibited the immediate histamine release occurring in skin chambers after pollen introduction, whereas it did not significantly inhibit the late release. In patients receiving placebo, we detected platelet-activating factor—acether in media collected at the sixth hour from chambers filled with pollen. With cetirizine 2 HCl treatment, platelet-activating factor—acether was not detected in chamber media. Interestingly, cetirizine 2 HCl significantly reduced the eosinophil recruitment observed on the superficial dermis 24 hours after pollen challenge.

Role of PAF-acether in the mediation of pathophysiological responses to aggregated immunoglobulins. Studies with the platelet-activating factor receptor antagonist BN 52021

Sprague-Dawley rats were challenged with an intravenous (i.v.) infusion of soluble aggregates of immunoglobulin G. Animals receiving a dose of aggregates of 40 mg/kg showed a significantly reduced time of lysis of diluted blood clot, which paralleled the appearance in plasma of tissue-type plasminogen activator. These changes occurred about 5–10 min after the challenge, which is a more protracted time-course than that observed in response to paf-acether. A significant increase in serum levels of N-acetylglucosaminidase was also observed in the animals several minutes after challenge. Blood neutrophil count showed a 50% reduction that reached its maximum at 10 min and was followed by an overshoot after 30 min. In experiments in rats previously depleted of circulating PMN by treatment with vinblastine, no significant differences were observed in N-acetylglucosaminidase release as compared to non-treated animals. Since prior evidence indicated that endogenously generated paf-acether could be a mediator responsible for these changes, at least to some extent, the compound BN 52021, a specific antagonist of the paf-acether receptor was given to these animals prior to the challenge with the complexes. All the above mentioned responses were significantly reduced by BN 52021, which is in keeping with the hypothesis involving endogenous paf-acether release in the mediation of these changes. By contrast, BN 52021 did not interfere with the clearance of the aggregates from the circulation, which seems to be a beneficial mechanism to reduce immune-mediated tissue injury. These data extend the number of paf-acether mediated pathophysiological changes that can be observed in response to immune aggregates.

The effects of N-3 polyunsaturated fatty acids on the generation of platelet-activating factor-acether by human monocytes.

Human leukocytes generate platelet-activating factor (PAF-acether), a lipid mediator of inflammation, from membrane alkyl phospholipids through the release of arachidonic acid or other fatty acids at the 2-position and subsequent acetylation. Because it was previously demonstrated that fish oil fatty acids suppress human leukocyte arachidonic acid release and metabolism, separate experiments were conducted to investigate the effects of dietary fish oil supplementation and in vitro incubation with fish oil fatty acids on calcium ionophore-stimulated PAF-acether generation in human monocytes. In subjects on their regular diets, a 4-hr incubation of monocyte monolayers with an optimally effective concentration of arachidonic acid of 1 micrograms/ml resulted in a 64% increase of calcium ionophore-induced net PAF-acether generation from 7.75 +/- 0.78 ng/10(6) cells for untreated monolayers to 12.70 +/- 1.21 ng/10(6) cells (mean +/- SEM). Treatment of monolayers with eicosapentaenoic acid (EPA) at the optimal concentration of 1 micrograms/ml decreased net PAF-acether generation by 28%. However, treatment of monocyte monolayers with docosahexaenoic acid did not appreciably affect net PAF-acether generation. The changes in PAF-acether release with each fatty acid added in vitro paralleled those in total PAF-acether generation; the percentage PAF-acether release remained unaffected. Three weeks of dietary supplementation with 18 g MaxEPA daily, providing 3.2 g EPA did not affect the PAF-acether generation of calcium ionophore-stimulated human monocyte monolayers. However, 6 weeks of dietary supplementation resulted in a 47% decrease of net total PAF-acether generation and a concomitant 59% decline in net PAF-acether release; the percentage release of PAF-acether was not affected. Thus, whether added to the diet or introduced in vitro, fish oil-derived fatty acids suppress PAF-acether generation by human monocyte monolayers.

Endogenic PAF-acether production by Guinea pig endothelial cells in experimental arterial thrombosis

Superfusion of PAF-acether over a branch of the mesenteric artery in the guinea pig invariably results in local endothelial injury and thrombus formation within 3–10 minutes. The thrombotic phenomena do not disappear when PAF-acether superfusion is discontinued, and even when forced embolization is induced. Within a very short interval renewal of thrombosis occurs at the same site. Several data point to a mechanism involving generation and release of endogenic PAF-acether. Recents findings on PAF-acether release by cultured endothelial cells indicate that in the in vivo situation this phenomenon could well be responsible for maintaining the thrombotic status as demonstrated by ultrastructural analysis. In a later stadium polymorphonuclear leukocytes are also involved in total thromboformation.

Decrease in IgE Fc receptor expression on mouse bone marrow-derived mast cells and inhibition of paf-acether formation and of beta-hexosaminidase release by dexamethasone.

The effect of dexamethasone (DM) on the immunologic and nonimmunologic release of paf-acether and of the granule marker beta-hexosaminidase (BHEX) from mouse bone marrow-derived mast cells (BMMC) was studied. BMMC (1 X 10(6] in a modified Tyrode's solution containing 0.25% bovine serum albumin (BSA) were sensitized with an optimal dose of dinitrophenyl (DNP)-specific monoclonal IgE, and were washed before challenge with 40 ng/ml of DNP coupled to BSA. Preincubation of BMMC for 24 hr with 1 nM to 1 microM DM inhibited in a dose-dependent fashion the immunologic release of paf-acether and of BHEX as compared with control cells, with a half-maximal effect at 20 nM and 4 nM respectively. By contrast, the ionophore A23187 (1 microM)-induced release of paf-acether and of BHEX was unaffected by DM pretreatment. Finally, the antigen-induced increase in acetyltransferase activity, used as an index of cellular activation, was inhibited by 37 +/- 16% in 1 microM DM-treated BMMC as compared with untreated cells. Preincubation of BMMC with DM for 24 hr caused a dose-dependent inhibition of 125I-IgE binding to the cells, with a half-maximal effect at 14 nM. As determined by Scatchard analysis, the number of IgE Fc receptors was decreased by 55% in 1 microM DM-treated BMMC as compared with untreated cells, although the dissociation constants were comparable (control: 12.6 +/- 4.1 nM; DM-treated cells: 14.1 +/- 6.7 nM; mean +/- 1 SD; n = 3). Cytofluorometer analysis of BMMC sensitized with a saturating amount of purified monoclonal IgE, followed by addition of a fluoresceinated anti-mouse IgG (heavy and light chains), revealed a single cellular population for both DM-treated and untreated BMMC. This demonstrates that the DM-induced decrease in IgE Fc receptor expression was exhibited by every BMMC. The possible link between the decreased sensitization of the cells consequent to the reduction in IgE Fc receptor expression and the alteration of the secretory response and acetyltransferase activity was investigated. BMMC were incubated with IgE under experimental conditions giving half-sensitization of the cells. Upon antigen challenge, a 10.5 +/- 3.7% decrease in acetyltransferase activity and a 29.2 +/- 3.5% decrease in paf-acether release were observed with half-sensitized cells as compared with cells sensitized with a saturating amount of IgE.(ABSTRACT TRUNCATED AT 400 WORDS)

A role for PAF-acether (platelet-activating factor) in platelet-dependent vascular diseases?

Platelets-isolated or in conjunction with leukocytes-interact with vessel walls in many experimental and human diseases. Several mediators are held responsible for platelet activation and interaction with leukocytes, among which PAF-acether (platelet-activating factor) is a prime candidate. This phospholipid mediator is released by most inflammatory cells, including neutrophils, by isolated organs such as kidney and heart, is a potent platelet and neutrophil agonist, and exerts major vasoactive properties. Its biosynthesis involves a two-step enzymatic process yielding the active molecule from the membrane alkyl-ether choline-containing phospholipids. The first step implicates a phospholipase A2 that hydrolyzes a long-chain fatty acid (which can be arachidonic acid) from membrane phospholipids, leaving the intermediate compound lyso PAF-acether, a PAF-acether precursor that is acetylated by an acetyltransferase in a second step. It can also result from deacetylation of PAF-acether by an acetylhydrolase. PAF-acether release might explain the intervention of platelets in diseases such as glomerulonephritis and allergic vasculitis, in which the involvement of neutrophils and platelets is frequently noted. The end result of these complex sets of cell-to-cell interactions is the release of most known inflammatory mediators, influencing vascular permeability, cell infiltration, and smooth muscle contraction. Nevertheless, direct evidence for the implication of these rather well-defined cellular and molecular interactions in human pathologic states remains to be obtained.

Platelet release reaction and aggregation induced by canatoxin, a convulsant protein: evidence for the involvement of the platelet lipoxygenase pathway

Canatoxin is a toxic protein isolated from Canavalia ensiformis seeds. It induces death preceded by convulsions of spinal cord origin and also produces in vitro aggregation of platelets in rabbit, human and guinea-pig plasma. The aggregating effect is dose-dependent at nanomolar concentrations. Rabbit platelets pretreated with canatoxin became refractory to a second exposure to this protein or to collagen, but were still responsive to ADP, Paf-acether or arachidonic acid. [14C]-5-hydroxytryptamine was released from pre-labelled platelets on stimulation with canatoxin. Washed rabbit platelets, but not thrombin-degranulated ones, aggregated on stimulation with canatoxin provided that fibrinogen was added before the toxin. Canatoxin's pro-aggregating activity was inhibited by mepacrine, EDTA, caffeine, prostacyclin, adenosine monophosphate and also by the ADP scavenger system, creatine phosphokinase/creatine phosphate. Furthermore, 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW 755C), eicosatetraynoic acid (ETYA) and nordihydroguaiaretic acid (NDGA) were potent inhibitors of canatoxin-induced aggregation. In contrast, no inhibition was seen with indomethacin. The data indicate that canatoxin is mainly a release-reaction-promoting agent, being devoid of any direct aggregating activity. Thus the aggregation is totally dependent on the release of ADP. Furthermore, canatoxin-induced platelet activation is probably dependent on platelet phospholipase A2 and lipoxygenase activity but is not dependent on cyclo-oxygenase products or the release of Paf-acether.

Release of platelet-activating factor, slow-reacting substance, and vasoactive amines from isolated rat kidneys

AbstractRelease of platelet-activating factor, slow-reacting substance, and vasoactive amines from isolated rat kidneys. The present work brings the first evidence for the simultaneous release of Paf-acether (platelet-activating factor), slow-reacting substance (SRS), histamine, and serotonin from isolated rat kidneys stimulated with ionophore A 23187. However, with compound 48/80 we detected only SRS, histamine, and serotonin. Upon addition of antigen, kidneys from sensitized rats released Paf-acether and SRS of anaphylaxis. Paf-acether released in the perfusate was identified by its ability to aggregate aspirin-treated washed rabbit platelets in the presence of an ADP scavenger complex. It was also characterized by its inactivation by phospholipase A2, and it was eluted from high pressure liquid chromatography (HPLC) after 16 to 18 min, a retention identical to that of synthetic Paf-acether, that is, between sphingomyelin and lysophosphatidylcholine. The biological activity of SRS was detected after several purification steps including Amberlite XAD-2 and reverse phase HPLC (RP-HPLC). Kidney SRS exhibited a typical spasmogenic activity in isolated guinea-pig ileum preparation that was reversed by FPL 55712. When kidneys were incubated with [3H]arachidonic acid, radioactivity and biological activity comigrated in RP-HPLC with leukotrienes C and D. These results indicate that the kidney is capable of actively released inflammatory mediators.Libération du platelet-active facteur, de la lent-réagit substance, et d'aminés vasoactives, à partir de reins isolés de rats. Nous avons mis en évidence la libération de Paf-acéther (platelet-active facteur), lent-réagit substance (SRS), histamine, et sérotonine par le rein de rat isolé et stimulé par du ionophore A 23187. Une stimulation au 48/80 induit seulement la libération de la SRS, histamine, et sérotonine. En présence de l'antigène, les reins de rats sensibilisés libèrent du Paf-acéther et de la SRS. Le Paf-acéther rénal a été caractérisé par sa capacité à agréger les plaquettes lavées de lapin en présence d'aspirine et du complexe creatine phosphate/créatine phosphokinase, son inactivation par la phospholipase A2 de pancréas, et son temps de rétention en chromatographic liquide à haute pression (HPLC) entre la sphingomyéline et la lysophosphatidylcholine, de 16 à 18 min, identique à celui du Paf-acéther synthétique. La SRS a été détectée après purification sur colonne d'Amberlite XAD-2 et par HPLC en phase réverse (RP-HPLC). Elle a été caractérisée par sa capacité à contracter l'iléon de cobaye en présence d'atropine et d'antihistaminique, contraction qui est relaxée spécifiquement par le FPL 55712, par son incorporation d'acide arachidonique tritié et l'élution en RP-HPLC de la radioactivité à des temps de rétention identiques à ceux des leukotriènes C et D synthétiques. Ces résultats démontrent que le rein est capable de libérer des médiateurs de l'inflammation.

Cooperative effects of 1-O-alkyl-2-O-acetyl- [formula omitted]-glycero-3-phosphocholine (PAF-acether) and exogenous arachidonic acid in stimulation of human blood platelets

Cooperative effects of PAF-acether and arachidonic acid in blood platelet activation were studied in human platelet-rich plasma. Using a combination of low concentrations of PAP-acether and subthreshold amounts of exogenous arachidonic acid an enhancement of aggregation and an increased formation of malondialdehyde are obtained. Both effects are completely suppressed either by acetylsalicylic acid or by use of PAF-acether desensitized platelets, indicating that cyclooxygenase products and intact receptors for PAP-acether are of primary importance in the observed synergism. Stimulating activity of PAF-acether on the formation of cyclooxygenase products is also seen with use of higher concentrations of arachidonic acid during mild stimulation of human platelets. This effect of PAP-acether is dependent on the concentration used and is obviously due to changes in the platelet membrane as derived from experiments with lysed platelets. Enhanced capacity of platelets to synthesize endoperoxides and thromboxane in consequence of an in vivo release of PAF-acether into the circulation might be of pathobiochemical relevance in some circumstances.

Dansylcadaverine and rimantadine inhibition of phagocytosis, PAF-acether release, and phosphatidylcholine synthesis in human polymorphonuclear leukocytes

The effect of dansylcadaverine, amantadine, and rimantadine on phagocytosis, release of PAF-acether, phospholipid methylation, and phosphatidylcholine formation by the cholinephosphotransferase pathway was assessed in human peripheral polymorphonuclear leukocytes. All of the drugs induced a dose-dependent and reversible inhibition in the uptake of complement-coated zymosan particles as well as a reduction in the release of PAF-acether. Simultaneously, a marked reduction in the formation of phosphatidylcholine was observed. This was also dose-dependent and reversible, and showed this order of potency: dansylcadaverine > rimantadine > amantadine. Dansylcadaverine reduced phospholipid methylation to a lesser extent than the cholinephosphotransferase pathway. These data show that drugs that block receptor-mediated endocytosis inhibit the response of the human polymorphonuclear cell and suggest that this action may be mediated by their actions on phospholipid metabolism.

Antigen-initiated release of platelet-activating factor (PAF-acether) from mouse bone marrow-derived mast cells sensitized with monoclonal IgE.

Mouse bone marrow mast cells sensitized with monoclonal IgE and activated with specific antigen released 2.8 +/- 0.5 ng of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (PAF-acether)/ 10(6) cells. The PAF-acether was identified by its ability to aggregate fully aspirin-treated washed rabbit platelets in the presence of an adenosine diphosphate (ADP)-scavenger complex, by its co-chromatography with [3H]-labeled semi-synthetic PAF-acether and synthetic 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, and by its inactivation by phospholipases A2, C, and D and not by lipase A1. The antigen-initiated release of PAF-acether, leukotriene C4 (LTC4), and leukotriene B4 (LTB4), and the secretion of the granule marker beta-hexosaminidase were not diminished by washing the cells before challenge, indicating that they were due to the interaction of antigen with the IgE fixed on the cell membrane and not to phagocytosis of immune complexes formed in the fluid phase. The parallel antigen-induced dose-response relationship, along with the superimposable time-course of the extracellular appearance, of beta-hexosaminidase, PAF-acether, and both leukotrienes indicated that the origin of these diverse mediators was from a common cell type with IgE-Fc receptors. Ethanol extraction of antigen-stimulated bone marrow-derived mast cells revealed the early transient appearance of a cell-associated platelet-aggregating activity, the action of which on platelets, like PAF-acether, was independent of ADP and arachidonic acid metabolism. The cell-associated activity contained a novel product that eluted at 13 min during high performance liquid chromatography (HPLC) (solvent hexane:n-propanol:water, 46:46:8), permitting resolution from PAF-acether and lyso-PAF-acether (1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which eluted at 29 min and 30 min, respectively. The cell-associated material, which differs from lyso-PAF-acether, the putative precursor of PAF-acether, in being active in the bioassay on platelets may represent a newly recognized intermediate in the generation of PAF-acether. As the transiently present cell-associated intermediate has not been previously recognized, its detection may depend upon the relatively unique properties of the bone marrow-derived mast cell system in which IgE-dependent activation leading to product generation is complete within 5 min.(ABSTRACT TRUNCATED AT 400 WORDS)

Release of platelet-activating factor (PAF-acether) and 2-lyso PAF-acether from three cell types.

The release and formation of PAF-acether and of its deacetylated precursor (2-lyso PAF-acether) have been determined on mouse macrophages (M phi), human polymorphonuclear neutrophils (PMN) and rabbit platelets using specific secretagogue stimuli in the same experimental conditions. It was found that as opposed to M phi, PMN and platelets were good releasers of PAF-acether. However, the total amount of PAF-acether formed by M phi and PMN was larger than that formed by platelets. The total amount of 2-lyso PAF-acether varied also for the three cell types, with platelets being by far the best producer. Calculation of the amount of PAF-acether formed from 2-lyso PAF-acether indicated that M phi and PMN possess a higher acetylating ability than platelets.

Biosynthesis of platelet-activating factor (PAF-acether) II. Involvement of phospholipase A 2 in the formation of PAF-acether and lyso-PAF-acether from rabbit platelets

The role of phospholipase A 2 (PLA 2) in the formation of platelet-activating factor (PAF-acether) by rabbit platelets is supported by several pieces of evidence. First, the release of PAF-acether was accompanied by that of its deacetylated analog, lyso-PAF-acether. Second, EDTA, EGTA, db-AMPc, p′-bromophenacylbromide and 874 CB, which, in spite of their structural diversity, are all PLA 2 blockers, inhibited the release of both PAF-acether and of the lyso-compound. Third, addition of hog pancreas PLA 2 to platelets as well as platelet lysis resulted in the release of lyso-PAF-acether, thus mimicking the metabolic events initiating formation of PAF-acether. These results indicate that PLA 2 activation triggers both the second and the third pathway of platelet activation.