Unsaturated 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines (unsaturated platelet-activating factor): aggregation of human platelets after incubation with indomethacin, creatinephosphate/creatinephosphokinase, xylocain and hirudine, and serotonin release after incubation with indomethacin

Unsaturated platelet-activating factor (PAF) aggregates thrombocytes of healthy female volunteers and releases within 1 min up to 30.95% of the platelet serotonin. Indomethacin does not inhibit the aggregation but reduces the release of serotonin induced by unsaturated PAF in citrated platelet-rich plasma (PRP). Creatinephosphate combined with creatinephosphokinase (CP/CPK) inhibits the second phase, whereas xylocain inhibits the first and second phase of aggregation induced by unsaturated PAF. Hirudine shows no influence on the aggregation induced by unsaturated PAF.     

Bovine fibrinogen-induced 14C-serotonin and other compounds release from human blood platelets.

Bovine fibrinogen can aggregate human blood platelets by a direct mechanism (first wave) and also indirectly by releasing endogenous ADP (second wave). A primarily non-altered surface structure of the thrombocytes is important for the induction of the first wave. Under certain conditions a second aggregation wave, concomitant with the release and the availability reaction is elicited. Besides the cell's contact, the presence of ADP and calcium ions are necessary for the induction of the release reaction in human platelet rich plasma. The changes induced by bovine fibrinogen thus follows the same pattern as the irreversible phase of aggregation, linked to release as induced by ADP.     

Priming effect of lipopolysaccharide on acetyl-coenzyme A:lyso-platelet-activating factor acetyltransferase is MyD88 and TRIF independent

LPS has a priming effect on various stimuli. For instance, LPS priming enhances the production of platelet-activating factor (PAF), a proinflammatory lipid mediator that is induced by PAF itself. Among various enzymes responsible for PAF biosynthesis, acetyl-coenzyme A:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase is one of the enzymes activated by PAF receptor stimulation. In this study we investigated the priming effect of LPS on the acetyltransferase activation by PAF in TLR4-knockout (KO) mice, MyD88-KO mice, and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF)-KO mice. This enzyme was biphasically activated by LPS. Although the first peak occurred within 30 min in wild-type (WT), but not TLR4-KO or MyD88-KO, macrophages, the second phase reached a maximum within hours in WT, MyD88-KO, and TRIF-KO, but not in TLR4-KO, macrophages. Only in the second phase was the increase in acetyltransferase activity upon PAF receptor activation remarkably enhanced in WT, MyD88-KO, and TRIF-KO cells, but not in TLR4-KO cells. These data demonstrated that LPS exerted a priming effect on PAF receptor-mediated acetyltransferase activation through the TLR4-dependent, but MyD88- and TRIF-independent, pathway.     

Platelet-activating factor (PAF) acts as an intercellular messenger in the changes of cytosolic free Ca2+ in human neutrophils induced by opsonized particles

Addition of opsonized particles to human neutrophils in suspension leads to a biphasic elevation in the cytosolic free Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i during the second phase (greater than 3 min) is pronounced (about 400 nM), in contrast to the rise during the first phase, which is relatively small (less than 100 nM). The second and large rise in [Ca2+]i is brought about by messenger(s) released from the cell after addition of opsonized particles. This second rise in [Ca2+]i is not observed in the presence of the platelet-activating factor (PAF) antagonist WEB 2086, indicating that PAF can act as an intercellular messenger affecting Ca2+ homeostasis in human neutrophils.     

Platelet-activating factor activates two distinct effector pathways in human eosinophils

In granulocytes, platelet-activating factor (PAF) shares many of its biological effects with other chemotactic factors, such as FMLP, complement fragments, and lipid mediators. Two unique effects are that PAF is relatively resistant to pertussis toxin (PTX) and that PAF activates the inflammatory functions of eosinophils more strongly than it activates those of neutrophils. To investigate the molecular mechanisms of the responses of eosinophils to PAF, we analyzed superoxide anion production by a chemiluminescence method that provides real-time kinetic data for the cellular responses. We found that PAF induced bimodal superoxide anion production in human eosinophils, consisting of an intense, but transient, first phase and a larger and sustained second phase. In contrast, PAF induced essentially a transient unimodal response in human neutrophils. The two phases of eosinophil response were mediated by distinct cellular mechanisms: the second phase was highly dependent on cellular adhesion and beta(2) integrins, but the first phase was independent of both adhesion and beta(2) integrins. The upstream signaling mechanisms were also different: the second phase was mediated by PTX-resistant G-protein(s) and through activation of phosphatidylinositol 3-kinase, while the first phase was mediated by PTX-sensitive G-protein(s). Furthermore, the second-phase response was approximately 100-fold more resistant to inhibition by a competitive PAF receptor antagonist than the first phase. Thus, eosinophils and neutrophils react differently to PAF, and PAF activates two separate and distinct effector pathways in human eosinophils. These two activation pathways may explain the eosinophils' strong and diverse biological responses to PAF.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Biphasic increase in intracellular calcium induced by platelet-activating factor in macrophages

In single mouse macrophages stimulated by platelet-activating factor (PAF), the intracellular calcium concentration (Cai) monitored with fura-2 at room temperature presents a biphasic increase, including a transient and a more sustained component. After pulse administration of PAF, the first phase lasts for a few seconds and reaches a peak value of 0.5-1 microM Ca2+ at high PAF concentration. The amplitude of this peak is independent of extracellular Ca2+ concentration, suggesting that the initial Ca2+ transient is due to the release of Ca2+ from intracellular stores. The second phase of the response lasts for several minutes; its maximum amplitude is reached 1-2 min after the brief initial PAF stimulation. This phase, suppressed in zero external Ca2+ and increased in 10 mM Ca2+, is probably due to influx of Ca2+ through the plasma membrane. This secondary Ca2+ increase is blocked by 10-50 microM lanthanum. At low PAF concentration, the initial Ca2+ transient is not followed by a second phase, showing that the initial rises of Ca2+ and of its activator (presumably inositol trisphosphate) are not sufficient to trigger the second phase of Ca2+ increase.     

Activation of washed chicken thrombocytes by 1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (platelet-activating factor)

Washed, [3H]serotonin-labeled chicken thrombocytes aggregated and secreted [3H]serotonin when stimulated in vitro with platelet-activating factor (PAF), collagen and calcium ionophore A23187. The effective dose causing a 25% secretion of [3H]serotonin (ED25) from washed chicken thrombocytes was 10(-8) M for PAF, 5 X 10(-8) M for collagen and 3 X 10(-7) M for A23187. Chicken thrombocyte activation by PAF required Ca2+ and appeared to be mediated through a specific receptor for PAF.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Cardiac and coronary consequences of intracoronary platelet activating factor infusion in the domestic pig

In previous studies we have shown that platelet-activating factor (PAF) is a potent vasoactive substance with deleterious effects on coronary blood flow (CBF) and myocardial performance. The present study further investigates the effects of PAF during its sustained intracoronary infusion in the blood-perfused domestic pig (n = 16). PAF infusion (1-9 nmol/min) produced triphasic changes in CBF (n = 7): an initial brief phase of coronary dilation (14 +/- 2% above baseline), followed by severe reduction in CBF due to increase in coronary vascular resistance and a third phase of escape that was characterized by return of CBF towards baseline in spite of continuing PAF infusion. In 9 remaining pigs PAF infusion had a biphasic response: the first phase of coronary dilation rapidly turned into severe coronary constriction accompanied by severe systemic hypotension and death within a few min. PAF infusion caused a profound rise in systemic arterial and coronary venous thromboxane B2 levels, while 6-keto-PGF1 alpha and leukotriene C4-immunoreactivity levels were not changed. Indomethacin completely blocked the rise in thromboxane level during PAF infusion and abolished the constrictor effect of PAF on the coronary vessels. These data suggest that PAF might play a detrimental role on the coronary circulation and cardiac function, primarily through thromboxane A2 mediated mechanism.     

Cardiac and coronary consequences of intracoronary platelet activating factor infusion in the domestic pig

In previous studies we have shown that platelet-activating factor (PAF) is a potent vasoactive substance with deleterious effects on coronary blood flow (CBF) and myocardial performance. The present student further investigates the effects of PAF during its sustained intracoronary infusion in the blood-perfused domestic pig (n=16). PAF infusion (1–9nmol/min) produced triphasic changes in CBF (n=7): an initial brief phase of coronary dilation (14 ± 2%) above baseline), followed by severe reduction in CBF due to increase in coronary vascular resistance and a third phase of escape that was characterized by return of CBF towards baseline in spite of continuing PAF infusion. In 9 remaining pigs PAF infusion had a biphasic response: the first phase of coronary dilation rapidly turned into severe coronary constriction accompanied by severe systemic hypotension and death within a few min. PAF infusion caused a profound rise in systemic arterial and coronary venous thromboxane B₂ levels, while 6-keto-PGF_1α and leukotriene C₄-immunoreactivity levels were not changed. Indomethacin completely blocked the rise in thromboxane level during PAF infusion and abolished the constrictor effect of PAF on the coronary vessels. These data suggest that PAF might play a detrimental role on the coronary circulation and cardiac function, primarily through thromboxane A₂ mediated mechanism.     

Genetic evidence for distinct roles of COX-1 and COX-2 in the immediate and delayed phases of prostaglandin synthesis in mast cells.

Activation of mast cells by aggregation of their high-affinity IgE receptors stimulates prostaglandin (PG) D(2) synthesis and secretion. An immediate phase of PGD(2) synthesis, complete within 30 min, is followed by a delayed, second phase of PGD(2) production that reaches a maximum 4 to 8 h after activation. Activation of mast cells from COX-2 (-/-) mice stimulates the release of PGD(2) during the first 30 min, whereas activation of mast cells from COX-1 (-/-) mice does not generate any PGD(2) in the first 2 h. On the other hand, COX-2 (-/-) cells do not participate in delayed phase of PGD(2) synthesis, while COX-1 (-/-) cells secrete low levels of PGD(2) between 2 and 4 h after activation. These data demonstrate that (i) the first phase of PG synthesis is COX-1 dependent and (ii) the second, delayed phase of PG synthesis is dependent on activation-induced synthesis and activity of COX-2.     

Creatine kinase and creatine kinase isoenzyme responses to heat stress

During this investigation the effects of heat acclimation and exercise on creatine kinase and creatine kinase BB isoenzyme responses in various tissues and serum of male Sprague-Dawley rats were ascertained. Forty rats were randomly divided into two groups of 20 rats each. One group was housed at 22+/-1 degrees C and the other at 33+/-1 degrees C. Each of the two groups were subdivided into two subgroups of ten rats each. One subgroup of each group was subjected to a programme of treadmill running of progressive intensity over a period of 6 weeks at the temperature at which it was housed while the other served as a resting control. At the end of the acclimation programme the rats were running at 23 m/min for 80 min. On the day of sacrifice all four subgroups were subjected to a discontinuous exercise protocol (10 min running alternated by a 2-min rest period; repeated three times) at 30+/-1 degrees C on a rodent treadmill at 23 m/min. The tissues investigated were kidney, heart and muscle. The rats were anaesthetized with pentobarbital sodium (6 mg/100 g body mass) injected intraperitoneally. The tissues were freeze-clamped and stored in liquid air until analysed. The body temperature of the four subgroups at the end of the experimental protocol were not significantly different. Acclimation at 33+/-1 degrees C resulted in significantly lower creatine kinase activity levels. Exercise at 30+/-1 degrees C also resulted in decreased creatine kinase activity levels in both acclimated groups. A similar trend was observed regarding creatine kinase BB isoenzyme activity levels, especially in kidney.     

Activation of platelet-activating factor receptor-coupled G alpha q leads to stimulation of Src and focal adhesion kinase via two separate pathways in human umbilical vein endothelial cells

Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.     

Bioactive amide of prostaglandin E1 and ethanolamine plasmalogen analog of platelet-activating factor inhibits several pathways of human platelet aggregation

The influence of an amide of prostaglandin E1 and ethanolamine plasmalogen platelet-activating factor analog 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-11alpha, 15alpha-dioxy-9-keto-13-prostenoyl)ethanolamine (PGE1-PPAF) on platelet-activating factor (PAF)-, ADP-, and thrombin-induced human platelet aggregation has been studied. It was found that PGE1-PPAF inhibits the PAF-, ADP-, and thrombin-induced platelet aggregation in platelet-rich plasma. 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine inhibited PAF-induced aggregation up to 50% but had no influence on platelet aggregation induced by ADP or thrombin. The ethanolamine plasmalogen analog of PAF 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolami ne, having a palmitoyl residue instead of PGE1, did not inhibit platelet aggregation induced by PAF, ADP, or thrombin. We propose that inhibition of human platelet aggregation by PGE1-PPAF is mediated by its action on platelet PAF-receptors and the adenylate cyclase system.     

Triazolobenzodiazepines competitively inhibit the binding of platelet activating factor (PAF) to human platelets

PAF causes dose dependent platelet aggregation of human platelet rich plasma or gel filtered platelets (GFP). The benzodiazepines alprazolam and triazolam, but not diazepam (1-10 microM), inhibit PAF induced aggregation but have no effect on aggregation induced by other platelet agonists such as ADP, epinephrine and collagen. The IC50 for aggregation by PAF (4 nM) in GFP is 1 microM for both alprazolam and triazolam. The mechanism for this inhibition was explored by studying the binding of 3H-PAF(0.08 nM) to GFP in Tyrodes buffer containing albumin (0.35%), Mg++ (1mM) and Ca++ (0.5mM). GFP was incubated with different doses of the drug for 5 min prior to addition of 3H-PAF. Incubation was then carried out for 60 min at 25 degrees C to achieve binding equilibrium, as previously established. Alprazolam and triazolam, but not diazepam, caused competitive displacement of 3H-PAF from specific binding sites of GFP. The IC50 of alprazolam was 3.8 microM while that of triazolam was 0.82 microM. Lineweaver-Burk plots of 3H-PAF binding in the presence of inhibitor were also consistent with competitive inhibition. These results are consistent with the interpretation that the specific inhibition of PAF induced platelet aggregation by alprazolam and triazolam, respectively, is due to competitive inhibition of binding of PAF to its receptor.     

Cathepsin G-induced malignant progression of MCF-7 cells involves suppression of PAF signaling through induced expression of PAFAH1B2

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.     

Aggregation and folding of recombinant human creatine kinase

The processes of aggregation and refolding of recombinant human creatine kinase (rHCK) were studied. Most of the rHCK expressed in E. coli was present in the insoluble traction and it could be solubilized in 6 M urea solution. Unfolding of rHCK in 6 M urea showed biphasic kinetic courses (kappa1 = 6.5 x 10(-3) s(-1); kappa2 = 0.54 x 10(-3) s(-1)) as observed by maximum fluorescence wavelength change. During refolding of the rHCK dissolved in urea, significant aggregation was noticed following first-order kinetics. Aggregation rate constants were influenced by the concentration of NaCl, which increased the difference in transition-free energy (deltadeltaG), showing that stabilization of folding intermediates by NaCl could efficiently reduce the formation of insoluble aggregates. Formations of aggregate were also reduced by adjusting temperature, pH, and concentration of rHCK. Refolding of rHCK under the optimized condition which prevented the aggregation also showed multi-kinetic phases (kappa1 = 3.0 x 10(-3) s(-1); kappa2 = 0.64 x 10(-3) s(-1)). Under optimized conditions applied in this study, rHCK could correctly refold retrieving the high specific enzymatic activity.     

Selective activation of human monocytes by the platelet-activating factor analog 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine

The capacity of platelet-activating factor (PAF) and its 2-O-methyl analog (methoxy-PAF) to activate human monocytes, neutrophils and platelets were compared. Both PAF and methoxy-PAF increased monocyte cytotoxicity toward WEHI 164 cells with a maximal increase in cell killing at 100 pM to 1 nM. Methoxy-PAF was slightly, but significantly, more potent than PAF for increasing cytotoxicity. PAF and methoxy-PAF increased monocyte release of TNF two- to three-fold above control release with no difference in their potency. Methoxy-PAF increased cell-associated TNF maximally after 2 to 3 h of incubation and increased TNF release maximally after 5 to 18 h of incubation. PAF induced release of the neutrophil granule enzyme beta-glucuronidase with maximal net release of 15 to 20% at 100 nM PAF whereas methoxy-PAF did not induce release of beta-glucuronidase. Similarly, 10 nM PAF induced 30% platelet aggregation whereas methoxy-PAF induced aggregation only at 1000-fold higher concentrations. Analysis of PAF and methoxy-PAF metabolism by monocyte and serum acylhydrolases indicates that methoxy-PAF is substantially more resistant than PAF to degradation by these enzymes. These observations indicate that methoxy-PAF activates monocytes selectively and suggest that this phospholipid or a related compound could be used for in vivo immunotherapy.     

Platelet-activating factor stimulates phospholipase C activity in human endometrium.

Human preimplantation embryos secrete platelet-activating factor (PAF), which stimulates prostaglandin E2 synthesis from secretory endometrium. This study investigated the action of PAF on phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-specific phospholipase C activity in human endometrium. Slices of normal endometrium were incubated with 5 microCi/ml myo-[2-3H] inositol for 3 h at 37 degrees C in 95% O2 and 5% CO2 to label tissue phosphoinositides. Inositol phosphates were extracted using trichloroacetic acid precipitation and diethylether neutralization and production was measured using Dowex 1-X8 anion-exchange column chromatography. PAF induced rapid and concentration-dependent accumulation of inositol phosphates (IP) from secretory endometrium, but had no effect on endometrium removed in the proliferative phase of the menstrual cycle. The IP3 fraction was significantly elevated from a median value of 14.0 c.p.m. mg-1 dry wt [range: 8-41 c.p.m. mg-1 dry wt] to 28.0 c.p.m. mg-1 dry wt [range: 11-87 c.p.m. mg-1 dry wt, P less than 0.002] following 1 min exposure of secretory endometrium to PAF-acether, in the presence of 10 mM LiCl. PAF-induced hydrolysis of PtdIns(4,5)P2 was inhibited by the specific PAF receptor antagonist WEB 2086, in a dose-dependent manner (P less than 0.02), indicating that in human endometrium PtdIns(4,5)P2 hydrolysis is mediated via a PAF receptor. These results indicate that PAF receptor coupling activates endometrial PtdIns(4,5)P2-specific phospholipase C only in the secretory phase of the menstrual cycle, suggesting that the PAF response may be under ovarian steroid regulation. It is proposed that the ability of the endometrium to respond to PAF appears to be a feature of the preparation of this tissue for implantation and that the second messengers generated may play a role in cellular processes involved in the maternal recognition of very early human pregnancy.