Exogenous Platelet-activating Factor Research Articles

Enhanced Fc Receptor Expression In Sea Urchin Spermatozoa By Platelet-Activating Factor

Platelet-activating factor (PAF) is a signaling phospholipid that has a variety of reproductive roles, e.g. sperm capacitation and the acrosome reaction (an exocytotic process). PAF’s mechanism action involves G-protein receptor activation induction of intracellular calcium, which enhances sperm motility. In nonreproductive cells, the Fc receptor (FcR) functions in ligand-triggered transmission signals across the membrane which alters secretion, exocytosis, and increased intracellular calcium. This study looked at the impact of PAF on FcR expression in sea urchin spermatozoa. Sea urchin sperm were exposed to exogenous PAF (10-7M; 15 minutes) and evaluated for FcR activity via flow cytometry. A significant (P=0.01) difference (64.30%) in FcR wave activity between the PAF (mean 3.286) and control (2.0) groups was observed. Within waves, there were more (83.31%) high points (crests) in the PAF (mean 1.571) group than the control (mean 0.857) group. Similarly, there were more (49.96%) low (troughs) points in the PAF (mean 1.714) group than the control (mean 1.143) group. This is the first report to demonstrate a relative positive change in spermatozoa FcR expression when exposed to PAF. The FcR functions in ligand-triggered transmission of PAF via a G-protein coupled receptor mediated pathway leading to physiological alterations. Provided the known mechanism of action of PAF on spermatozoa physiology, the data suggests that the interaction of PAF and FcR play a pivotal role in sperm activity and fertilization potential.

Platelet-Activating Factor Deteriorates Lysophosphatidylcholine-Induced Demyelination Via Its Receptor-Dependent and -Independent Effects.

Accumulating evidence suggests that platelet-activating factor (PAF) increases the inflammatory response in demyelinating diseases such as multiple sclerosis. However, PAF receptor (PAFR) antagonists do not show therapeutic efficacy for MS, and its underlying mechanisms remain poorly understood. In the present study, we investigated the effects of PAF on an ex vivo demyelination cerebellar model following lysophosphatidylcholine (LPC, 0.5mg/mL) application using wild-type and PAFR conventional knockout (PAFR-KO) mice. Demyelination was induced in cerebellar slices that were cultured with LPC for 18h. Exogenous PAF (1μM) acting on cerebellar slices alone did not cause demyelination but increased the severity of LPC-induced demyelination in both wild-type and PAFR-KO mice. LPC inhibited the expression of PAF-AH, MBP, TNF-α, and TGF-β1 but facilitated the expression of IL-1β and IL-6 in wild-type preparations. Of note, exogenous PAF stimulated microglial activation in both wild-type and PAFR-KO mice. The subsequent inflammatory cytokines TNFα, IL-1β, and IL-6 as well as the anti-inflammatory cytokine TGF-β1 demonstrated a diverse transcriptional profile with or without LPC treatment. PAF promoted TNF-α expression and suppressed TGF-β1 expression indiscriminately in wild-type and knockout slices; however, transcription of IL-1β and IL-6 was not significantly affected in both slices. The syntheses of IL-1β and IL-6 were significantly increased in LPC-induced demyelination preparations without PAF but showed a redundancy in PAF-treated wild-type and knockout slices. These data suggest that PAF can play a detrimental role in LPC-induced demyelination probably due to a redundant response of PAFR-dependent and PAFR-independent effects on inflammatory cytokines.

Platelet-activating factor increases reactive oxygen species-mediated microbicidal activity of human macrophages infected with Leishmania (Viannia) braziliensis

Platelet-activating factor (PAF) is produced by macrophages during inflammation and infections. We evaluated whether PAF is able to modulate the infection of human macrophages by Leishmania braziliensis, the main Leishmania sp. in Brazil. Monocyte-derived macrophages were incubated with promastigote forms in absence or presence of exogenous PAF. We observed that the treatment of macrophages with low concentrations of PAF prior to infection increased the phagocytosis of L. braziliensis. More importantly, exogenous PAF reduced the parasitism when it was added before, during or after infection. In addition, treatment with a PAF antagonist (PCA 4248) resulted in a significant increase of macrophage infection in a concentration-dependent manner, suggesting that endogenous PAF is important to control L. braziliensis infection. Mechanistically, while exogenous PAF increased production of reactive oxygen species (ROS) treatment with PCA 4248 reduced oxidative burst during L. braziliensis infection. The microbicidal effects of exogenous PAF were abolished when macrophages were treated with apocynin, an NADPH oxidase inhibitor. The data show that PAF promotes the production of ROS induced by L. braziliensis, suggesting that this lipid mediator may be relevant to control L. braziliensis infection in human macrophages.

Potential Therapeutic Strategies for Severe Anaphylaxis Targeting Platelet-Activating Factor and PAF Acetylhydrolase

Characterization of mediators and mechanisms of anaphylaxis will allow for more specific and effective treatment with fewer side effects. Recent studies have shown that platelet-activating factor (PAF) is a pivotal mediator of the life-threatening manifestations of anaphylaxis. The putative role of PAF in human anaphylaxis is based on a large body of evidence in experimental and human anaphylaxis. In animal models of anaphylaxis, intravenous administration of PAF reproduces the severe physiologic derangements of anaphylaxis. PAF receptor knock-out mice are resistant to experimental anaphylaxis. Data from human anaphylaxis show that levels of PAF increase proportionately with the severity of anaphylaxis, whereas a deficiency in the enzyme that inactivates PAF predisposes to severe or fatal anaphylaxis. Many of the biologic effects of PAF appear to be transduced by nitric oxide production. PAF receptor antagonists protect against the lethal effects of exogenous PAF, but, importantly, also protect against experimental anaphylaxis following allergen challenge. Mice treated with an enzyme that inactivates PAF are similarly resistant to anaphylaxis. Some clinically available medications for anaphylaxis act at different points of the PAF pathway. Epinephrine, the first-line treatment for anaphylaxis, appears to act in part by phosphorylation and inactivation of the PAF receptor, whereas methylene blue, which reduces the actions of nitric oxide, can reverse severe anaphylaxis that is refractory to conventional treatment. Taken together, these studies have shown that therapies targeting the PAF pathway might hold the potential for more specific and effective treatments for this potentially life-threatening condition.

Platelet‐activating Factor in Iberian Pig Spermatozoa: Receptor Expression and Role as Enhancer of the Calcium‐Induced Acrosome Reaction

Platelet-activating factor (PAF) is a phospholipid involved in reproductive physiology. PAF receptor is expressed in some mammalian spermatozoa species where it plays a role in these germ-cell-specific processes. The aim of this study is to identify PAF receptor in Iberian pig spermatozoa and to evaluate PAF's effects on motility, viability and acrosome reaction. Semen samples from Iberian boars were used. PAF receptor identification was performed by Western blotting. Spermatozoa motility was analysed by computer-assisted sperm analysis system, whereas spermatozoa viability and acrosome reaction were evaluated by flow cytometry. Different PAF concentrations added to non-capacitating medium during 60 min have no effect on any spermatozoa motility parameter measured. Acrosome reaction was rapid and potently induced by 1 μm calcium ionophore A23187 showing an effect at 60 min and maximum at 240 min. PAF added to a capacitating medium is not able to induce spermatozoa acrosome reaction at any time studied. However, PAF, in the presence of A23187, significantly accelerates and enhances the calcium-induced acrosome reaction in a concentration-dependent manner in Iberian boar spermatozoa. Exogenous PAF does not affect at all spermatozoa viability, whereas slightly exacerbated the A23187-induced loss in viability. This work demonstrates that PAF receptor is expressed in Iberian pig spermatozoa and that its stimulation by PAF regulates the calcium-induced acrosome reaction. This work contributes to further elucidate the physiological regulation of the most relevant spermatozoa functions for successful fertilization: acrosome reaction.

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa

The aim of this study was to investigate the effect of platelet activating factor (PAF) on the quality characteristics of cryopreserved canine spermatozoa. Cryopreserved semen of 5 mixed-breed dogs was treated with different concentrations of exogenous PAF (1 x 10(-3) M, 1 x 10(-4) M, 1 x 10(-5) M and 1 x 10(-6) M) and examined at different time intervals (0, 30, 60 and 120 min). Cryopreserved semen treated without PAF was used as the control. Sperm quality was evaluated for motility (computer-assisted semen analysis, CASA), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (SYBR-14/PI assay and Hoechst 33258). Also, ATP content of spermatozoa was determined using a bioluminescence assay. Treatment of cryopreserved semen with 1 x 10(-3) M PAF at 120 min of incubation resulted in significantly higher total sperm motility compared with the control. It was observed that PAF-improved total sperm motility was concurrent with enhanced sperm motility patterns after treatment of cryopreserved semen. Treatment of cryopreserved semen with PAF did not improve either sperm mitochondrial function or plasma membrane integrity, as monitored by different fluorescent membrane markers. Furthermore, ATP content of cryopreserved spermatozoa was significantly higher when PAF was used at a concentration of 1 x 10(-3) M compared with the control and other PAF treatments, regardless of the incubation time. The findings of this study indicated that treatment with 1 x 10(-3) M PAF at 120 min of incubation rendered better quality of cryopreserved canine semen, which was associated with improved sperm motility parameters and ATP content. It can be suggested that exogenous PAF addition is beneficial as a supplement for canine semen extender used for cryopreservation.

Exogenous platelet-activating factor improves the motility of human spermatozoa evaluated with C.A.S.A.: Optimal concentration and incubation time

The objective of this study is to determine the optimal conditions for human semen incubation treated with exogenous platelet activating factor (ePAF) for intra-uterine insemination (IUI). This prospective study was carried out on 32 infertile men and each semen sample was processed with the ISolate Sperm Separation Medium, washed with sperm washing medium (SWM) and resuspended either in SWM alone (control samples), or with ePAF 0.1, 0.5, and 1.0 μM. Each concentration was subsequently incubated and evaluated at 5, 15, 30, and 60 min. The motility parameters were evaluated by the computer-aided sperm analysis (C.A.S.A.) system. Curvilinear velocity, straight line velocity, average path velocity, rapid and progressive motility significantly increased compared to control samples at an ePAF concentration of 0.1 μM (with at least 15 min of incubation). The best results were obtained with ePAF concentrations of: 0.1 μM (60 min of incubation) and 0.5 μM (30-60 min of incubation). In conclusion, results are enhanced when ePAF is added to standard semen preparation for IUI. An ePAF concentration of 0.1 μM, with an incubation time of 15 min, can be used for semen samples with normal motility. Whilst, for semen samples with poor motility, the ePAF concentration is best increased to 0.5 μM and/or the incubation time prolonged to 60 min.

Leading Process Branch Instability in Lis1+/− Nonradially Migrating Interneurons

Mammalian forebrain development requires extensive migration, yet the mechanisms through which migrating neurons sense and respond to guidance cues are not well understood. Similar to the axon growth cone, the leading process and branches of neurons may guide migration, but the cytoskeletal events that regulate branching are unknown. We have previously shown that loss of microtubule-associated protein Lis1 reduces branching during migration compared with wild-type neurons. Using time-lapse imaging of Lis1(+/-) and Lis1(+/+) cells migrating from medial ganglionic eminence explant cultures, we show that the branching defect is not due to a failure to initiate branches but a defect in the stabilization of new branches. The leading processes of Lis1(+/-) neurons have reduced expression of stabilized, acetylated microtubules compared with Lis1(+/+) neurons. To determine whether Lis1 modulates branch stability through its role as the noncatalytic beta regulatory subunit of platelet-activating factor (PAF) acetylhydrolase 1b, exogenous PAF was applied to wild-type cells. Excess PAF added to wild-type neurons phenocopies the branch instability observed in Lis1(+/-) neurons, and a PAF antagonist rescues leading process branching in Lis1(+/-) neurons. These data highlight a role for Lis1, acting through the PAF pathway, in leading process branching and microtubule stabilization.

Treatment of Sperm With Platelet-activating Factor Does Not Improve Intrauterine Insemination Outcome in Unselected Cases of Mild Male Factor Infertility: A Prospective Double-blind Randomized Crossover Study

To evaluate the effect of sperm treatment with exogenous platelet-activating factor (PAF) on intrauterine insemination (IUI) clinical pregnancy rate in cases of mild male factor infertility. PAF is a phospholipid mediator, which is present in human sperm. The study was performed in the Assisted Reproduction Unit of the 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital, Athens, Greece, and included 92 couples who presented with mild male factor infertility-all candidates for IUI. A maximum of 4 IUI cycles per couple with or without exogenous PAF treatment were performed and the main outcome measure was the clinical pregnancy rate (pregnancies confirmed by ultrasonography per 100 cycles). The overall clinical pregnancy rate after a maximum of 4 IUI cycles was comparable in cases with and without sperm treatment with PAF (12.24% vs 11.11%). Addition or exclusion of PAF sperm treatment in the same patients did not significantly alter the outcome. The generalized use of exogenous PAF for the preparation of sperm in unselected cases of mild male infertility does not improve the clinical outcome of IUI.

The emerging roles of PAF acetylhydrolase

Platelet-activating factor (PAF), a phospholipid autacoid with potent effects throughout the innate immune system, is selectively degraded by two small families of PAF acetylhydrolases (PAF-AHs). These Ca2+-independent phospholipases A2 display remarkable specificity for the length of the sn-2 residue, but this selectivity is lost as the residue gains oxygen functions. Two of the PAF-AHs therefore are specific oxidized phospholipid phospholipases that reduce inflammation, but also remove oxidatively truncated phospholipids that induce apoptosis. The roles of these enzymes are manifold, and their separate and combined functions are now being addressed in model systems and clinical studies.

Physalin B inhibits Rhodnius prolixus hemocyte phagocytosis and microaggregation by the activation of endogenous PAF-acetyl hydrolase activities

The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A 2 (PLA 2) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1 μg/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10 μg/insect) or PAF (1 μg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA 2 activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.

Role of platelet-activating factor in pneumolysin-induced acute lung injury

Acute respiratory failure is a major complication of severe pneumococcal pneumonia, characterized by impairment of pulmonary microvascular barrier function and pulmonary hypertension. Both features can be evoked by pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae. We hypothesized that platelet-activating factor (PAF) and associated downstream signaling pathways play a role in the PLY-induced development of acute lung injury. Controlled, ex vivo laboratory study. Female Balb/C mice, 8-12 wks old. Ventilated and blood-free-perfused lungs of wild-type and PAF receptor-deficient mice were challenged with recombinant PLY. Intravascular PLY, but not the pneumolysoid Pd-B (PLY with a Trp-Phe substitution at position 433), caused an impressive dose-dependent increase in pulmonary vascular resistance and increased PAF in lung homogenates, as detected by reversed-phase high-performance liquid chromatography coupled to tandem mass spectrometry. The pressor response was reduced in lungs of PAF receptor-deficient mice and after PAF receptor blockade by BN 50730. PLY and exogenous PAF increased thromboxane B2 in lung effluate, and thromboxane receptor inhibition by BM 13505 diminished the pressor response to PLY. Differential inhibition of intracellular signaling steps suggested significant contribution of phosphatidylcholine-specific phospholipase C and protein kinase C and of the Rho/Rho-kinase pathway to PLY-induced pulmonary vasoconstriction. Unrelated to the pulmonary arterial pressor response, microvascular leakage of PLY was diminished in lungs of PAF receptor-deficient mice as well. PAF significantly contributed to PLY-induced acute injury in murine lungs. The PAF-mediated pressor response to PLY depends on thromboxane and on the downstream effectors phosphatidylcholine-specific phospholipase C, protein kinase C, and Rho-kinase.

Leptin modulates the detrimental effect of Porphyromonas gingivalis lipopolysaccharide-induced cytosolic phospholipase A2 activation on salivary mucin synthesis via ERK-signal transduction

Activation of cytosolic phospholipase A2 (cPLA2) by bacterial LPS is considered a key step in the generation of proinflammatory lipid messengers, including platelet-activating factor (PAF), recognized as the most proximal mediator of inflammatory events triggered by bacterial infection. In this study, we report on the role of leptin in modulation of the detrimental consequences of cPLA2 activation in salivary gland acinar cells by the LPS of a periodontopathic bacterium, P. gingivalis. Employing mucous cells of rat sublingual gland, we show that the LPS-induced cPLA2 activation is associated with up-regulation in PAF generation and the impairment in mucin synthesis, and was subject to suppression by leptin. A potentiation in the countering capacity of leptin on the LPS-induced arachidonic acid release and PAF generation was attained in the presence of ERK inhibitor, PD98059, while the PI3K inhibitor, wortmannin had no effect. However, the prevention by leptin of the LPS detrimental effect on mucin synthesis was subject to suppression by the inhibitors of both PI3K and ERK. Moreover, amplification in the effect of leptin on the LPS-induced decrease in mucin synthesis was attained with cPLA2 inhibitor, MAFP as well as PAF receptor antagonist, BN52020, while the reversal of the leptin effect occurred in the presence of exogenous PAF. These findings demonstrate the involvement of leptin in countering the pathological consequences of cPLA2 activation by P. gingivalis LPS on salivary mucin synthesis through the involvement in MAPK/ERK and PI3K signaling events.

Acid-Induced Release of Platelet-Activating Factor by Human Esophageal Mucosa Induces Inflammatory Mediators in Circular Smooth Muscle

In a human in vitro model of esophagitis, we investigated the genesis of esophagitis-associated dysmotility by examining HCl-induced production of inflammatory mediators in the mucosa and investigating their effect on esophageal circular muscle. Muscularis propria was removed from organ donors' esophagi, leaving the mucosal tube intact. The tube was tied at both ends, forming a sac, and filled with HCl at pH 4. After 3 h of incubation, the supernatant surrounding the sac was analyzed or applied to circular muscle strips. HCl alone did not affect circular muscle contraction in response to electrical field stimulation (EFS), but supernatant of HCl-treated mucosa abolished contraction. The inhibition was reversed by the platelet-activating factor (PAF) antagonist CV3988 [(+/-)-3-(N-octadecylcarbamoyl)-2-methoxy) propyl-(2-thiazolioethyl) phosphate], whereas the PAF analog 2-O-methyl platelet-activating factor C-16 (PAF-16) inhibited EFS-induced contraction and acetylcholine (ACh) release in circular muscle strips. The hydrogen peroxide scavenger catalase reversed the inhibition in contraction, to the same extent as CV3988. We therefore measured PAF and hydrogen peroxide (H(2)O(2)) in mucosa, mucosa supernatant, and circular muscle. HCl increased PAF and interleukin (IL)-1beta (but not IL-6, prostaglandin E(2), or H(2)O(2)) in mucosa, and only PAF was released into the supernatant, presumably to affect circular muscle. In circular muscle, exogenous PAF induced sequential formation of IL-6, H(2)O(2), IL-1beta, and PAF. Release of PAF by the mucosa inhibits ACh release from circular muscle layer neurons and initiates sequential formation of inflammatory mediators in muscle, resulting in production of PAF by the muscle itself, possibly initiating in a self-sustaining cycle.

Platelet-Activating Factor Synthesis and Response on Pancreatic Islet Endothelial Cells: Relevance for Islet Transplantation

Recent data suggest that donor intraislet endothelial cells may survive islet transplantation and participate to the events that influence islet engraftment. However, the mechanisms that regulate islet endothelial behavior in this setting are poorly known. We obtained immortalized human (hIECs) and mouse (mIECs) islet endothelial cells by transfection with SV40-T-large antigen and studied the synthesis and response to Platelet-activating factor (PAF), a multipotent phospholipid that acts as endothelial mediator of both inflammation and angiogenesis. HIECs showed typical endothelial markers such as expression of vWF, CD31, and CD105, uptake of acetylated-LDL and binding to ULE-A lectin. Moreover, they expressed nestin, the PAF-receptor and possess surface fenestrations and in vitro angiogenic ability of forming tubular structures on Matrigel. Likewise, mIECs showed expression of vWF, CD31, nestin, PAF-receptor and CD105, and uptake of acetylated-LDL. HIECs and mIECs rapidly produced PAF under stimulation with thrombin in a dose-dependent way. Exogenous PAF or thrombin-induced PAF synthesis increased leukocyte adhesion to hIECS and mIECs and cell motility of both endothelial cell lines. Moreover, PAF or thrombin-induced PAF synthesis accelerated in vitro formation of vessel-like tubular structures when hIECs are seeded on Matrigel. Notably, gene-microarray analysis detected up-regulation of beta3 integrin gene on hIECs stimulated with PAF, that was confirmed at the protein level. Based on the novel development of immortalized islet endothelium, these results suggest that PAF may have a dual role that links inflammation to angiogenesis in the early events of islet transplantation.

Effect of sperm treatment with exogenous platelet-activating factor on the outcome of intrauterine insemination

To evaluate the effect of sperm treatment with exogenous platelet-activating factor (PAF) on IUI clinical pregnancy rate. Prospective randomized study. Assisted Reproduction Unit, 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital, Athens, Greece. Fifty-two couples with unexplained infertility, candidates for IUI. Sperm treatment with an exogenous mixture of PAF (final concentration, 10(-7) mol/L) in sperm-washing medium, direct swim-up technique of sperm preparation, a maximum of six IUI cycles per couple with or without PAF treatment. Clinical pregnancy rate (pregnancies confirmed by ultrasonography per hundred cycles). The overall clinical pregnancy rate after a maximum of six IUI cycles was significantly higher when sperm was treated with PAF compared with the rate after the direct swim-up technique (23.07% vs. 7.92%). Treatment of sperm with exogenous PAF might improve the clinical outcome of IUI in cases of unexplained infertility.

Cytosolic Phospholipase A2-α Is Necessary for Platelet-activating Factor Biosynthesis, Efficient Neutrophil-mediated Bacterial Killing, and the Innate Immune Response to Pulmonary Infection: cPLA2-α DOES NOT REGULATE NEUTROPHIL NADPH OXIDASE ACTIVITY

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.

Oxygen-dependent PAF receptor binding and intracellular signaling in ovine fetal pulmonary vascular smooth muscle

Circulating levels of platelet-activating factor (PAF) are high in the fetus, and PAF is active in maintaining high PVR in fetal hypoxia (Ibe BO, Hibler S, Raj J. J Appl Physiol 85: 1079-1085, 1998). PAF synthesis by fetal pulmonary vascular smooth muscle cells (PVSMC) is high in hypoxia, but how oxygen tension affects PAF receptor (PAF-r) binding in PVSMC is not known. We studied the effect of oxygen tension on PAF-r binding and signaling in fetal PVSMC. PAF binding was saturable. PAF-r density (B(max): fmol/10(6) cells; means +/- SE, n = 6), 25.2 +/- 0.77 during hypoxia (Po(2) <40 Torr), was higher than 13.9 +/- 0.44 during normoxia (Po(2) approximately 100 Torr). K(d) was twofold lower in hypoxia than normoxia. PAF-r protein expression, 35-40% greater in hypoxia, was inhibited by cycloheximide, a protein synthesis inhibitor, suggesting translational regulation. IP(3) release, an index of PAF-r-mediated cell signaling, was greater in hypoxia (EC(50): hypoxia, 2.94 +/- 0.61; normoxia, 5.85 +/- 0.51 nM). Exogenous PAF induced 50-90% greater intracellular calcium flux in cells during hypoxia, indicating hypoxia augments PAF-r-mediated cell signaling. PAF-r phosphorylation, with or without 5 nM PAF, was 40% greater in hypoxia. These data show 1) hypoxia upregulates PAF-r binding, PAF-r phosphorylation, and PAF-r-mediated intracellular signaling, evidenced by augmented IP(3) production and intracellular Ca(2+) flux; and 2) hypoxia-induced PAF-r phosphorylation results in activation of PAF-r-mediated signal transduction. The data suggest the fetal hypoxic environment facilitates PAF-r binding and signaling, thereby promoting PAF-mediated pulmonary vasoconstriction and maintenance of high PVR in utero.

The significance of platelet‐activating factor and fertility in the male primate: a review

Since its discovery nearly 30 years ago platelet-activating factor (PAF) has emerged as one of the more important lipid mediators known. PAF (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine) exists endogenously as a mixture of molecular species with structural variants of the alkyl moiety. PAF is a novel potent signaling phospholipid that has unique pleiotropic biological properties in addition to platelet activation. PAF also plays a significant role in reproduction and is present in the sperm of a number of primate species. PAF content in squirrel monkey sperm is significantly higher during the breeding season than the non-breeding season. PAF content in rhesus sperm has a significant relationship with sperm motility. PAF content in human sperm has a positive correlation with seminal parameters and pregnancy outcomes. The enzymes (lyso-PAF-acetyltransferase and PAF-acetylhydrolase) necessary for PAF activation and deactivation are present in primate sperm. PAF-acetylhydrolase may act as a "decapacitation factor". Removal of this enzyme during capacitation promotes PAF synthesis increasing primate motility and fertilization. PAF also plays a significant role in the fertilization process, enhancing the fertilization rates of oocytes. Enhanced embryo development has also been reported in oocytes fertilized with PAF-treated sperm. Exogenous PAF will also significantly improve primate artificial insemination pregnancy outcomes. PAF antagonists inhibit sperm motility, acrosome reaction, and fertilization thus suggesting the presence of receptors for PAF. The PAF-receptor is present on primate sperm, with altered transcript levels and distribution patterns on abnormal cells. Whereas, the exact mechanism of PAF in sperm function and reproduction is uncertain, its importance in normal primate fertility is substantial.

Delineation of the role of platelet-activating factor in the immunoglobulin G2 antibody response.

Localized aggressive periodontitis (LAgP) is a chronic inflammatory disease characterized by severe destruction of periodontal tissues surrounding the first molars and incisors. LAgP subjects produce large amounts of immunoglobulin G2 (IgG2) antibody against oral pathogens, and this response is inversely correlated with the severity of disease. We previously demonstrated that platelet-activating factor (PAF) is required for optimal IgG2 responses. The present investigation was designed to determine the mechanism of IgG2 induction by PAF. Exogenous PAF acetylhydrolase suppressed approximately 80% of pokeweed mitogen-stimulated IgG2 production, confirming that PAF is essential for optimal responses. PAF-activated leukocytes produced gamma interferon (IFN-gamma), a Th1 cytokine that has been associated with IgG2 responses in previous studies. The monocyte-derived cytokines interleukin-12 (IL-12) and IL-18 are upstream of IFN-gamma production, and IgG2 production was suppressed by neutralizing antibodies against these proteins. In addition, PAF induced monocyte-derived dendritic cells (DC) but not macrophages (MPhi) to secrete IL-12 and IL-18. This observation was interesting because monocyte differentiation in LAgP subjects is skewed to the DC phenotype. Although other investigators have implicated IFN-gamma in IgG2 production, its precise role in this response is controversial. Our studies suggest that IFN-gamma induces isotype switching to IgG2 but only in concert with the Th2 cytokine IL-4. Thus, it appears that the unique PAF metabolism of LAgP monocytes or DC promotes Th1 responses that are essential for optimal IgG2 antibody production. As IgG2 antibodies opsonize oral bacteria and promote their clearance and destruction, these alterations in PAF metabolism may be essential for limiting disease severity in LAgP patients.