Endogenous PAF Research Articles

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses.

BackgroundSmall, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses.ResultsThe APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays.ConclusionThis study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure–function analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0586-4) contains supplementary material, which is available to authorized users.

Oxidized phosphatidylcholine induces migration of bone marrow-derived mesenchymal stem cells through Krüppel-like factor 4-dependent mechanism

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) and PAF-like oxidized phospholipids including 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) are generated upon LDL oxidation. The aim of this study was to evaluate the question of whether POVPC can regulate migration of human bone marrow-derived stem cells (hBMSCs) and to characterize signaling mechanisms involved in the POVPC-induced cell migration. POVPC treatment resulted in dose- and time-dependent increase of hBMSCs migration. Treatment of cells with BN52021, a specific antagonist of PAF receptor, completely blocked cell migration induced by not only PAF but also POVPC. Silencing of endogenous PAF receptor expression using PAF receptor-specific small interfering RNA resulted in significant attenuation of cell migration induced by PAF or POVPC. Both PAF and POVPC induced expression of Krüppel-like factor 4 (KLF4) in hBMSCs. POVPC- or PAF-induced cell migration was abrogated by small interfering RNA-mediated depletion of endogenous KLF4. These results suggest that PAF receptor plays a pivotal role in POVPC-induced migration of human BMSCs through PAF receptor-mediated expression of KLF4.

Seminal Platelet-Activating Factor

Platelet-activating factor (PAF; 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine) is a potent signaling phospholipid with pleiotropic biologic activities. Since its discovery more than 30 years ago, numerous investigators have documented its presence in a variety of tissues. PAF and its receptors are present both in somatic and reproductive cells. PAF appears to be critical for many of the events surrounding fertilization, early preimplantation embryo development, implantation, and embryo development. PAF is present in the sperm of many mammalian species including humans. It is synthesized, metabolized, and used by sperm. PAF may in fact be one biomarker for sperm capacitation. Endogenous PAF concentrations are directly correlated to sperm motility, forward progression, fertilization, and implantation and pregnancy rates in humans and other mammalian species. Exogenous PAF improves pregnancy rates in human intrauterine inseminations. Although the exact mechanism for PAF as it relates to sperm function is unclear, its importance is substantial.

Human spermatozoan platelet-activating factor content is related to motility and morphology

Objective: Platelet-activating factor (PAF; 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine) is a ubiquitous phospholipid present in human sperm that is implicated in the mediation of a wide variety of reproductive processes. Angle et al. (1991) reported an inverse relationship between human sperm PAF content and motility. However, animal models demonstrate a positive relationship between PAF content and motility. Recent human data also suggest that pregnancy rates following intrauterine insemination are greater for sperm exposed to exogenous PAF. Further, endogenous sperm PAF content is greater in those specimens resulting in a pregnancy during in vitro fertilization than those not resulting in pregnancy. The objective of this study was to determine the relationship between PAF content in sperm and seminal parameters.

Impact of blood serum insulin-like growth factor I on platelet-activating factor in bull spermatozoa

The objective of this study was to examine differences in platelet-activating factor [1- O-alkyl-2-acetyl- sn-glycero-3-phosphorylcholine; PAF] in spermatozoa between two lines of Angus beef cattle divergently selected for blood serum insulin-like growth factor I (IGF-I) concentration. Endogenous lipids were extracted from the spermatozoa and endogenous PAF content was determined by radioimmunoassay. The amount of PAF detected in spermatozoa obtained from high IGF-I bulls ( n = 8) ranged from 0.145 to 3.571 pM/10 6 cells. The level of PAF extracted from spermatozoa obtained from low IGF-I- bulls ( n = 5) ranged from 0.001 to 1.024 pM/10 6 cells. Polynomial regression analysis revealed a significant cubic relationship (R 2 = 0.374; F = 6.292; P < 0.05) between spermatozoa PAF content and blood serum IGF-I concentration. Spermatozoa-derived PAF levels (mean ± SEM) were significantly higher (P < 0.05) in the high IGF-I group (1.90 ± 0.39 pM/10 6 cells) than in the low IGF-I group (0.59 ± 0.20 pM/10 6 cells). High IGF-I bulls have a greater than three-fold higher PAF content in their spermatozoa than low IGF-I bulls. The data demonstrate that not only is PAF present in bull spermatozoa but that levels are significantly higher in individuals with high serum IGF-I concentrations.

Lps Activates NF-κB P50-P65 Heterodimer in Rat Small Intestine • 573

NF-κB is a transcription factor which regulates many genes involved in the expression of pro-inflammatory cytokines and adhesion molecules. We have previously shown that: (1) PAF (1.5 μ/kg, IV) activated NF-κB, mainly as p50 homodimer, in rat intestine; (2) LPS induced intestinal PAF production and injury, which was blocked by PAF antagonists. Here we examined whether LPS activates NF-κB in rat small intestine and whether it was via endogenous PAF production. Young Sprague-Dawley rats were injected IV with PAF (1.5 μ/kg), LPS (8mg/kg) or vehicle (saline). At 30 min, 1 hr, 90 min and 2 hr, ileal nuclear extracts were assessed for NF-κB DNA-binding activity by electrophoretic mobility shift assay using a [32P]-labeled oligonucleotide probe specific for NF-κB. Supershift experiments were done to identify the NF-κB subunits. We found that LPS (8mg/kg) activated NF-κB in the intestine, predominantly as p50-p65 heterodimer. This effect was maximum 2 hr after injection. In contrast, PAF activates NF-κB, predominantly as p50-p50 homodimer, and the effect peaked at 30 min. Further, LPS-induced NF-κB activation was partially inhibited by WEB 2086 (PAF antagonist). Thus, LPS induces NF-κB activation via both PAF-dependent and PAF-independent pathways.

Bioactive lipids in excitatory neurotransmission and neuronal plasticity

Long-term potentiation (LTP), a model of activity-dependent synaptic plasticity and of certain forms of memory, comprises the persistent enhancement of excitatory neurotransmission that results from high-frequency activation. A presynaptic component of LTP is thought to be modulated by a retrograde messenger generated by the postsynaptic neuron. Arachidonic acid, nitrix oxide, carbon monoxide and PAF have each been proposed as retrograde messengers in LTP, but arachidonic acid, unlike PAF, requires NMDA receptor activation. A PAF antagonist (BN 52021) that provides neuroprotection in ischemia-reperfusion displaces [ 3H] PAF bound to presynaptic membranes, blocks PAF-induced glutamate exocytosis and inhibits LTP. An antagonist selective for the intracellular PAF binding site (BN 50730) did not affect LTP, nor did BN 52021 modify NMDA currents. LTP was induced with weak synaptic stimulation coupled with postsynaptically administered enzyme resistant mcPAF. Theta-burst stimulation (10 min) after bath applications of mcPAF (1 μM) induced APV-independent LTP that was blocked by 5 μM BN 52021. When this antagonist was infused into the hippocampus before or immediately after training, it impaired memory of inhibitory avoidance training in the rat. Memory was not altered if the antagonist is infused 30 or 60 min after training. Moreover, mcPAF enhances memory on retention test performance of step-down inhibitory avoidance habituation and learning in rats. Also, memory was studied using a caudate nucleus-dependent cued water maze task. Rats received an 8 trial (30 s intertrial interval) training session in which a visible cued escape platform was located in a different quadrant of the maze of each trial. Following trial 8, the rats received a unilateral post-training intra-caudate injection of mcPAF (1 μg/0.5 μl), BN 52021 (0.5 μg/0.5 μl) or vehicle. On a retention test session 24 h later, latency to mount the escape platform was used as a measure of memory. The retention test escape latencies of rats given mcPAF were significantly lower than those of the vehicle-injected controls, indicating a memory enhancing effect of mcPAF. Injection of mcPAF did not affect retention when administered 2 h post-training indicating a time-dependent effect of mcPAF on memory. The latencies for animals injected with BN 52021 were significantly higher than those of the controls, indicating that antagonism of endogenous PAF impairs memory. The findings show that PAF plays a role in memory formation in a caudate-mediated cued discrimination task. Administration of BN 52021 2 h post-training had no effect on retention, indicating a time-dependent effect of endogenous PAF on memory formation. PAF, the most potent bioactive lipid known, modulates excitatory synaptic transmission, neuronal plasticity and memory. When PAF production is overstimulated as in seizures or ischemia, it becomes neurotoxic.

Effects of the platelet-activating factor receptor antagonist WEB 2086 on whole blood coagulation and fibrinolysis in a thromboelastography assay.

The effects of WEB 2086 on human blood coagulation and fibrinolysis were studied in vitro using computerized thromboelastography and native whole blood. WEB 2086 (3.0, 30, 150, and 300 microM) had no apparent effect on the split point (SP), reaction time (R) and lysis of the clot. The biKoatugulierung time (K) was prolonged, the angle (alpha) and maximum amplitude (MA) were diminished, and the overall coagulation index (TEG index) was smaller. SP and R indicate onset of coagulation with the formation of fibrin strands, suggesting that WEB 2086 had no effect on the plasma factors. Since the Lysis parameter reflects fibrinolysis, WEB 2086 had no demonstrable fibrinolytic activity. K, alpha and MA involve platelet function and the integrity of fibrin; the fact that these factors were affected to the exclusion of SP and R suggests an effect largely on platelets. There was lack of a dose-response relationship at this concentration range (3.0-300 microM), probably due to saturation of PAF receptors even at the lowest effective concentration. These findings are consistent with the known effects of WEB 2086 on platelet function and suggest that thromboelastography may be a useful tool in the study of the activity of PAF receptor antagonists on coagulation profiles. Furthermore, since WEB 2086 is a highly specific PAF receptor antagonist devoid of intrinsic activity, and the samples used were not treated with exogenous PAF, these results suggest that endogenous PAF may play a significant role in the normal clotting mechanism.

Endotoxin-resistant mice are protected from PAF-induced bowel injury and death role of TNF, complement activation, and endogenous PAF production

C3H/HeJ (endotoxin-resistant) mice have been used widely in biological research. However, the mechanism of endotoxin (LPS) resistance is only partially understood. In this study, we first investigated the differences in response to PAF, a mediator of endotoxin shock, between normal and C3H/HeJ mice. We found that in control mice, PAF (2.5 micrograms/kg) caused shock, hemoconcentration, complement activation, intestinal hypoperfusion, and necrosis with 75% mortality, whereas all C3H/HeJ mice survived, without complement activation or intestinal injury, and manifesting only mild hypotension and hemoconcentration. PAF also caused elevated serum TNF-alpha in some control mice but not in C3H/HeJ mice. We also observed that PAF induces endogenous PAF production and intestinal phospholipase A2 activation in normal mice, whereas PAF production and phospholipase A2 are suppressed in C3H/HeJ mice. The low endogenous PAF production may account, at least in part, for the resistance to LPS and PAF of C3H/HeJ mice.

Comparative study of endotoxin‐induced hypotension in kininogen‐deficient rats with that in normal rats

1. The aim of this study was to clarify the role of endogenous bradykinin (BK) in the hypotensive response induced by lipopolysaccharide (LPS) by comparing the degree of hypotension caused by LPS in a strain of specific pathogen-free (SPF) Brown Norway (B/N), kininogen-deficient mutant Katholiek rats with that of B/N normal Kitasato rats. 2. The dose-dependent hypotensive responses caused by intravenous injection of BK (1-100 nmol kg-1) or platelet-activating factor (PAF, 0.003-1 microgram kg-1), were not different in the two strains of rats used. However, there was a strong difference in the hypotensive response induced by LPS in kininogen-deficient and normal rats; in normal rats the hypotensive response was composed of two phases (15 min and 70-80 min after LPS injection), but in kininogen-deficient rats LPS caused a delayed (second phase), but not an acute (first phase) hypotension. 3. We demonstrate that Hoe 140 (1 mg kg-1, i.v.) is a potent, selective, and long-lasting antagonist of the hypotensive effects of BK. Hoe 140 diminished the hypotension caused by LPS in normal rats to the level observed in kininogen-deficient rats, but had no effect on the hypotension caused by LPS in kininogen-deficient rats. 4. TCV309 (0.1 mg kg-1, i.v.) selectively inhibited the hypotension caused by repetitive injection of PAF for up to 180 min. Pretreatment with TCV309 caused a near complete inhibition of the LPS-induced hypotension in kininogen-deficient and normal B/N rats. 5. In the normal rats, dexamethasone (0.5 mg kg-1, i.p.) inhibited the second phase of the hypotension induced by LPS, but not the first phase of the hypotension. 6. A small amount of BK (0.1 nmol kg-1) potentiated the hypotensive action of PAF (0.01 microg kg-1),when they were injected simultaneously.7. In conclusion, we demonstrate that formation of endogenous BK contributes primarily to the acute,but not to the delayed hypotension afforded by endotoxin in the rat. In contrast, formation of endogenous PAF contributes to both the acute and the delayed hypotension afforded by endotoxin in vivo.

Mechanisms of the inflammatory response induced by extracts of Schistosoma mansoni larvae in guinea pig skin.

Penetration of skin by cercariae of Schistosoma mansoni is associated with a local inflammatory response characterised by leukocyte accumulation and tissue swelling. The significance of the inflammation and its relevance to infection of the host is unknown. In this study, we have investigated the mechanisms of the local inflammatory response induced by injecting extracts of Schistosoma mansoni cercariae into guinea pig skin. Intradermal injection of cercarial homogenate or cercarial transformation fluid induced a dose-dependent increase in local edema formation and accumulation of intravenously injected 111In-labeled eosinophils and neutrophils. The responses were rapid in onset and independent of new protein synthesis. The capacity of extracts to induce edema formation and leukocyte accumulation correlated significantly with their proteolytic activity. In addition, extract-induced inflammation was reduced by co-injection with Trasylol, soybean trypsin inhibitor, PMSF, or heparin. A combination of PMSF and heparin virtually abolished extract-induced inflammation. The known inhibitory effect of Trasylol on kallikrein prompted an investigation into the role of kinins in inducing local edema formation. The bradykinin antagonist HOE 140 substantially reduced extract-induced edema whereas bradykinin itself was weak at inducing leukocyte accumulation. The cyclooxygenase inhibitor ibuprofen, the platelet-activating factor antagonist WEB 2086, and the 5-lipoxygenase inhibitor PF5901 had no effect on edema formation. In contrast, extract-induced eosinophil accumulation was reduced by WEB 2086 and PF5901, suggesting that endogenous PAF and leukotriene B4 were involved in recruiting these cells to inflammatory sites. Thus, cercarial proteases induce a local inflammatory response in guinea pig skin that can be attributed, in part, to known mediators of inflammation. It remains to be established whether the response is protective for the host or whether it is beneficial for the parasite. Further understanding of the underlying mechanisms may provide a target for the pharmacologic control of infection in schistosomiasis.

Lipoxin A4 induces hyperadhesiveness in human endothelial cells for neutrophils

Lipoxin A4 (LXA4), but not lipoxin B4, induced in vitro a dose- dependent, slowly emerging hyperadhesiveness in human umbilical vein endothelial cells (HUVEC), leading to a 1.9-fold increase in the binding of neutrophils (polymorphonuclear neutrophil granulocytes [PMN]). The maximal response to LXA4 occurred at 1 nmol/L and after 30 minutes of treatment of HUVEC. These response kinetics were intermediate in comparison with those of fast-acting inducers of HUVEC adhesivity (eg, thrombin, leukotriene B4 [LTB4] or platelet activating factor [PAF]), needing 5 to 15 minutes, or to the slow inducer interleukin-1 (IL-1 beta), which requires hours. The maximal LXA4 effect was slightly lower than that of LTB4 (100 nmol/L) and thrombin (1 U/mL), and less than that of PAF (100 nmol/L) or IL-1 beta (2.5 U/mL) (2.2-, 2.0-, 2.4-, or 13.6-fold increases, respectively). The LXA4 effect was inhibited by the PAF receptor antagonist WEB-2086; however, it could not be blocked by pertussis toxin. LXA4 conferred a slow, sustained increase in HUVEC cytosolic calcium ion concentrations, whereas thrombin did so rapidly and transiently. LXA4 also caused PMN to become hyperadhesive. Thus, this novel effect of LXA4 on HUVEC appears to be associated with endogenous PAF expression and slow increases of cytosolic calcium concentrations but not pertussis- sensitive G proteins.

Lipoxin A4 Induces Hyperadhesiveness in Human Endothelial Cells for Neutrophils

Lipoxin A4 (LXA4), but not lipoxin B4, induced in vitro a dose-dependent, slowly emerging hyperadhesiveness in human umbilical vein endothelial cells (HUVEC), leading to a 1.9-fold increase in the binding of neutrophils (polymorphonuclear neutrophil granulocytes [PMN]). The maximal response to LXA4 occurred at 1 nmol/L and after 30 minutes of treatment of HUVEC. These response kinetics were intermediate in comparison with those of fast-acting inducers of HUVEC adhesivity (eg, thrombin, leukotriene B4 [LTB4] or platelet activating factor [PAF]), needing 5 to 15 minutes, or to the slow inducer interleukin-1 (IL-1 beta), which requires hours. The maximal LXA4 effect was slightly lower than that of LTB4 (100 nmol/L) and thrombin (1 U/mL), and less than that of PAF (100 nmol/L) or IL-1 beta (2.5 U/mL) (2.2-, 2.0-, 2.4-, or 13.6-fold increases, respectively). The LXA4 effect was inhibited by the PAF receptor antagonist WEB-2086; however, it could not be blocked by pertussis toxin. LXA4 conferred a slow, sustained increase in HUVEC cytosolic calcium ion concentrations, whereas thrombin did so rapidly and transiently. LXA4 also caused PMN to become hyperadhesive. Thus, this novel effect of LXA4 on HUVEC appears to be associated with endogenous PAF expression and slow increases of cytosolic calcium concentrations but not pertussis-sensitive G proteins.

PAF-induced death in NMRI mice — a suitable shock model for testing new PAF receptor antagonists. Correlation with eicosanoid related substances

The effects of PAF antagonists, of substances which influence the arachidonic acid metabolism, and of dexamethasone and ketotifen were evaluated in an acute PAF-induced mortality model in female NMRI mice. We established a dependence of sensitivity to PAF on strain (AB mice showed no dose dependence) and on sex of the animals as well as on the PAF charges used in our experiments. PAF produced resistance in surviving animals against the PAF-induced death on repeated application. The PAF antagonists, WEB 2170 and WEB 2086, provided the best dose-dependent protection against PAF toxicity, followed by dexamethasone, by the COX/LOX synthetase inhibitor X 86 (a BW 755 C-analogue) and by the PAF receptor antagonist BN 52021. Particularly remarkable was the excellent prevention by aspirin. Aspirin may not only inhibit the cyclooxygenase pathway but also endogenous PAF synthesis. Other drugs, i.e. indomethacin, the thromboxane receptor antagonist, BM 13177, the thromboxane synthetase inhibitor, HOE 944, as well as the lipoxygenase inhibitors (NDGA, esculetin, SHAM and phenidone) exerted a dose-dependent protection only at high doses.

Platelet activating factor (PAF) enhances mitosis in preimplantation mouse embryos

Preimplantation mouse embryos were used to determine whether the reported significant increase in embryo metabolism and viability achieved through supplementation of the culture medium with the ether phospholipid 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocoline (platelet activating factor, PAF) is attributable to an enhanced rate of mitosis. Blastocyst-stage embryos cultured in the presence of 0.186 to 18.6 microM exogenous PAF had a significantly (P < 0.01) higher mitotic index (the proportion of cells arrested in metaphase following incubation in colchicine) than those cultured without PAF. At the 8-cell stage, 29% more blastomeres were in metaphase in the PAF-treated group (P < 0.01) 8 h after the addition of colchicine, but by 16 h there was no difference between groups; thus, PAF increased the rate at which cells entered metaphase but did not increase the total number. The mitotic index showed a negative correlation with the number of cells within blastocysts. PAF had a significantly (P < 0.01) greater impact on the mitotic index of blastocysts with fewer cells. The action of PAF was specific, being completely blocked by the PAF-receptor antagonist WEB 2086 (33 microM). In the absence of exogenous PAF the mitotic index was lower with WEB 2086 than without, suggesting inhibition of the action of endogenous embryo-derived PAF. These results show that PAF stimulates the rates at which cells within the preimplantation mouse embryo enter metaphase in vitro and suggest that it would decrease their doubling time, perhaps accounting for the embryotrophic actions of PAF.

Effect of PAF antagonists in retinal diseases

Long-term potentiation (LTP), a model of activity-dependent synaptic plasticity and of certain forms of memory, comprises the persistent enhancement of excitatory neurotransmission that results from high-frequency activation. A presynaptic component of LTP is thought to be modulated by a retrograde messenger generated by the postsynaptic neuron. Arachidonic acid, nitrix oxide, carbon monoxide and PAF have each been proposed as retrograde messengers in LTP, but arachidonic acid, unlike PAF, requires NMDA receptor activation. A PAF antagonist (BN 52021) that provides neuroprotection in ischemia-reperfusion displaces [3H] PAF bound to presynaptic membranes, blocks PAF-induced glutamate exocytosis and inhibits LTP. An antagonist selective for the intracellular PAF binding site (BN 50730) did not affect LTP, nor did BN 52021 modify NMDA currents. LTP was induced with weak synaptic stimulation coupled with postsynaptically administered enzyme resistant mcPAF. Theta-burst stimulation (10 min) after bath applications of mcPAF (1 μM) induced APV-independent LTP that was blocked by 5 μM BN 52021. When this antagonist was infused into the hippocampus before or immediately after training, it impaired memory of inhibitory avoidance training in the rat. Memory was not altered if the antagonist is infused 30 or 60 min after training. Moreover, mcPAF enhances memory on retention test performance of step-down inhibitory avoidance habituation and learning in rats. Also, memory was studied using a caudate nucleus-dependent cued water maze task. Rats received an 8 trial (30 s intertrial interval) training session in which a visible cued escape platform was located in a different quadrant of the maze of each trial. Following trial 8, the rats received a unilateral post-training intra-caudate injection of mcPAF (1 μg/0.5 μl), BN 52021 (0.5 μg/0.5 μl) or vehicle. On a retention test session 24 h later, latency to mount the escape platform was used as a measure of memory. The retention test escape latencies of rats given mcPAF were significantly lower than those of the vehicle-injected controls, indicating a memory enhancing effect of mcPAF. Injection of mcPAF did not affect retention when administered 2 h post-training indicating a time-dependent effect of mcPAF on memory. The latencies for animals injected with BN 52021 were significantly higher than those of the controls, indicating that antagonism of endogenous PAF impairs memory. The findings show that PAF plays a role in memory formation in a caudate-mediated cued discrimination task. Administration of BN 52021 2 h post-training had no effect on retention, indicating a time-dependent effect of endogenous PAF on memory formation. PAF, the most potent bioactive lipid known, modulates excitatory synaptic transmission, neuronal plasticity and memory. When PAF production is overstimulated as in seizures or ischemia, it becomes neurotoxic.

Immunoregulatory functions of paf-acether. IV. Enhancement of IL-1 production by muramyl dipeptide-stimulated monocytes.

paf-Acether (paf) is a phospholipid mediator of inflammation released from monocytes along with IL-1. In this study, we have examined the role of paf on IL-1 production by human monocytes. When paf from 1 nM to 5 microM, but not its precursor lyso paf, was added to monocytes in the presence of muramyl dipeptide (MDP) or LPS, a marked increase in IL-1 activity over the value with MDP alone was observed. In contrast, paf alone had minimal activity over the same dose range. Antibodies against rHu IL-1 alpha and rHu IL-1 beta neutralized the increased IL-1 activity. Interestingly, MDP that prompts monocytes to synthesize IL-1, induced the synthesis of paf, as well. Most of the paf produced remained cell-associated and always preceded IL-1 synthesis. When the paf receptor antagonist, L-652,731 was added to monocytes, it prevented the enhancement of IL-1 activity induced by exogenous paf. In contrast, L-652,731 had little effect on MDP-induced IL-1 synthesis in the absence of exogenous paf. This may indicate that there are alternative mechanisms involved in the sequences of events leading to IL-1 production. It is also conceivable that the paf receptor antagonist is not able to compete or inhibit endogenous paf as well as it does for exogenous paf. Nevertheless, exogenous paf in association with a second signal, modulates IL-1 production from human monocytes in a positive manner. This may constitute another means through which paf can modulate inflammatory and immune reactions.

CV 6209 IS A NON‐COMPETITIVE ANTAGONIST OF PLATELET‐ACTIVATING FACTOR RECEPTORS ON GUINEA‐PIG RESIDENT PERITONEAL MACROPHAGES

1. The characteristics of antagonism of platelet-activating factor (Paf) receptors by the phospholipid Paf analogue, CV 6209, were studied in rabbit platelets and polymorphonuclear leucocytes (PMN) and in guinea-pig macrophages. 2. Paf-induced aggregation of PMN or platelets was antagonized in a competitive and specific manner by CV 6209 with no detectable difference between the pA2 values (approximately 9.5). 3. The specificity of CV 6209 (1-100 nmol/L) for Paf receptors in platelets and PMN was indicated by a lack of effect on A23187 (10 mumols/L) or fMLP (1 mumol/L) induced aggregation, respectively. 4. CV 6209 (1-100 nmol/L) was also a potent antagonist of Paf-induced prostacyclin (PGI2) generation by guinea-pig peritoneal macrophages. However, CV 6209 caused significant depression of the maximum response to Paf and a non-parallel shift in the concentration-response curve indicating a non-competitive type antagonism. 5. PGI2 generation induced by the ionophore A23187 was unaffected by CV 6209 (up to 100 nmol/L) whereas basal PGI2 production by macrophages was reduced by lower concentrations (10-100 nmol/L). These observations are not consistent with a direct effect of CV 6209 on the enzymes involved in PGI2 synthesis but do suggest that endogenous Paf regulates basal PGI2 generation. 6. The non-competitive antagonism of guinea-pig macrophage Paf receptors gives further support to the contention that these receptors are distinct from those mediating aggregation of platelets and PMN.

Induction of Tissue Injury and Altered Cardiovascular Performance by Platelet-Activating Factor: Relevance to Multiple Systems Organ Failure

PAF is a phospholipid formed from the action of phospholipase A2 upon cellular membranes in response to immunologic or hypoxic stimuli. PAF does not exist in its active form as a storage product within cells, but is synthesized rapidly after phospholipase A2 activation. A potent lipid released by multiple cell types in mammalian systems, the emerging perspective is that PAF is a major endogenous mediator influencing the pathogenesis and outcome of ischemia and conditions of circulatory shock. These effects appear to be especially relevant to the syndrome of MSOF during critical illness. All of the major criteria for validation of a shock factor have been fulfilled for PAF. First, PAF has been measured in biological fluid of animals during shock states, although this is not an easy task since PAF is formed in minute amounts and is rapidly metabolized. Nevertheless, combinations of high pressure liquid chromatography (HPLC) and bioassay methods employing washed rabbit platelets have been successfully utilized in this regard. Second, synthetic PAF has been injected into cell suspensions, isolated tissues, and live animals, where it produces most of the effects attributed to endogenous PAF released by immunologic or hypoxic stimuli. These studies have shown that PAF exerts a variety of pathophysiologic actions, including (1) cardiodepression (that is, a negative inotropic effect), (2) reductions in systemic blood pressure, (3) leakage of fluid from the microvasculature, (4) bronchoconstriction, and (5) platelet aggregation. All of these actions of PAF can initiate or exacerbate shock and ischemic injury in multiple organ systems. Third, specific PAF receptor antagonists have been found to markedly attenuate the severity of endotoxic, anaphylactic, hemorrhagic, and traumatic shock, as well as acute myocardial ischemia. In all these conditions, a variety of PAF receptor antagonists (including PAF analogues and structurally dissimilar substances) have improved survival and have retarded pathophysiologic processes believed to be important in causing tissue injury. These processes include lysosomal membrane damage and proteolysis. Moreover PAF receptor antagonists attenuate the release of secondary toxic factors in shock, such as myocardial depressant factor. Thus, administration of specific PAF receptor antagonists early in the course of circulatory shock and organ ischemia may prove to be useful therapeutic agents in a variety of life-threatening disorders. In addition to having direct actions, PAF appears to function as a pivotal agent in a chain of mediators producing tissue injury. Recent evidence suggests that tumor necrosis factors (i.e., cachectin) stim

ラット実験潰瘍とPAF(platelet-activating factor,血小板活性化因子)

Platelet-activating factor (PAF,1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a ether-linked phospholipid that exhibits a wide range of biological activities, that include stimulation of platelet and neutrophile, vasodilation, increase in vascular permeability, bronchoconstriction and pulmonary hypertension. Such actions have implicated PAF as a potent chemical mediator in anaphylactic or inflammatory processes. Furthermore, in recent studies, it has been elucidated that PAF was not only produced in response to pathological stimulation but also generated in normal animal tissues. But, it remains to be unclear what is the biological role of the endogenous PAF in normal tissues.On the other hand, there have also been many reports on gastric mucosal damage induced by exogenous PAF or protective effect of PAF antagonist in experimental ulcer model. However, these observations suggested that stomach was the target organ for PAF which contributed to pathological state (e. g. inflammation).This study focussed the relation between endogenou s PAF in stomach and the alteration of gastric microenvironment and suggested the following matters.1) It was elucidated that the endogenous PAF was indeed present in stomach of normal rat. The significantly high level of PAF was detected in the antrum.2) The endogenous PAF level dynamically altered in quick resp onse to stimuli which affected the gastric microenvironment (e. g. water-immersion stress).3) It was suggested that PAF might play some role in the formation or deterioration of gastric mucosal lesion induced by water-immersion stress and ethanol, because the specific PAF antagonists prevented such experimental ulcer.4) It was also suggested that the endogen ous PAF level in stomach was changed in association with physiological alteration, aging.Stomach maintains its homeostasis by many kinds of factors. PAF may play some biological role as one of such factors. Hence, the alteration of endogenous PAF level may cause the imbalance of gastric microenvironment which contributed to the etiopathogenesis in gastric mucosal lesion (especially in the early phase of ulceration), or may be expression of senescence.