Platelet-activating Factor Analogs Research Articles

Endotoxins Stimulate Neutrophil Adhesion Followed by Synthesis and Release of Platelet-activating Factor in Microparticles

Lipopolysaccharides and triacyl-cysteine-modified proteins of Gram-negative and positive organisms are potent endotoxins. Animal models show that the receptor for platelet-activating factor (PAF) is responsible for many of the deleterious effects of endotoxin, where regulated, localized PAF production localizes the inflammatory response. In contrast, biologically active analogs of PAF (PAF-like lipids) are generated by oxidative attack on phospholipids by chemical reactions that are unregulated and unlocalized. The identity and distribution of the PAF receptor ligand in endotoxemia is unknown. We found human polymorphonuclear leukocytes (PMNs) were a significant source of PAF receptor agonists after stimulation by either class of endotoxin. Production of PAF receptor agonists required that the PMN adhere to a surface, and adhesion (and therefore accumulation of PAF-like bioactivity) in response to endotoxic stimulation was delayed for several minutes. PAF-like oxidized phospholipids were found by mass spectroscopy, but biosynthetic PAF accounted for most of the phospholipid agonists arising from endotoxic stimulation. A significant portion of the PAF made by PMNs was secreted, in contrast to its near complete retention by other inflammatory cells. Endotoxic stimulation induced a respiratory burst with the production of superoxide and the formation and shedding of microparticles. Free and microparticle-bound PAF appeared in the media, and blocking microvesiculation with calpeptin blocked PAF release. The released material activated platelets, and platelets co-aggregated with endotoxin-stimulated PMNs. Adherent PMNs therefore behave differently than suspended cells and are a significant source of free PAF after endotoxin exposure. Leukocytes can couple endotoxic challenge to the widespread circulatory and inflammatory effects of endotoxin.

Hydroxyl-platelet-activating factor exists in blood of healthy volunteers and periodontal patients.

Periodontal diseases are localized chronic inflammatory conditions of the gingival and underlying bone and connective tissue. Platelet-activating factor (PAF), a potent inflammatory phospholipid mediator that has been previously detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid and in saliva, is implicated in periodontal disease. Our results from previous studies showed that the biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl-PAF analogue. In this study, hydroxyl-PAF analogue was detected for the first time in human blood derived from patients with chronic periodontitis as well as from periodontally healthy volunteers. The hydroxyl-PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray analysis. In addition, the quantitative determination of PAF and hydroxyl-PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of hydroxyl-PAF analogue levels to PAF levels in periodontal patients, suggesting that this bioactive lipid may play a role in oral inflammation.

Platelet-Activating Factor, a Pleiotrophic Mediator of Physiological and Pathological Processes

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid with diverse pathological and physiological effects. This bioactive phospholipid mediates processes as diverse as wound healing, physiological inflammation, apoptosis, angiogenesis, reproduction and long-term potentiation. Recent progress has demonstrated the participation of MAP kinase signaling pathways as modulators of the two critical enzymes, phospholipase A2 and acetyltransferase, involved in the remodeling pathway of PAF biosynthesis. The unregulated production of structural analogs of PAF by non-specific oxidative reactions has expanded this superfamily of signaling molecules to include “PAF-like” lipids whose mode of action is identical to that of authentic PAF. The action of members of this family is mediated by the PAF receptor, a G protein-coupled membrane-spanning molecule that can engage multiple signaling pathways in various cell types. Inappropriate activation of this signaling pathway is associated with many diseases in which inflammation is thought to be one of the underlying features. Inactivation of all members of the PAF superfamily occurs by a unique class of enzymes, the PAF acetylhydrolases, that have been characterized at the molecular level and that terminate signals initiated by both regulated and unregulated PAF production.

Isolation and structural elucidation of biologically active phospholipids from Scytonema julianum (cyanobacteria).

The role of platelet-activating factor (PAF) as a mediator appeared in rather primitive organisms like protozoans and was maintained in more evolved organisms. No reports exist for the presence of PAF or PAF analogues - or even compounds that exhibit PAF-like activity - in cyanobacteria, even though they belong to a a group of organisms at a low evolutionary level where the content of alkylacyl forms of ether lipids is expected to be high. In addition, cyanobacteria serve as a rich source of novel bioactive metabolites. In the present study the total lipids of a strain of Scytonema julianum, a filamentous cyanobacterium isolated from a Greek cave, were separated into neutral lipids and phospholipids, the latter being further fractionated by HPLC. Each phospholipid fraction was tested in vitro for its ability to inhibit PAF-, arachidonic acid- and ADP-induced washed-rabbit-platelet aggregation and/or to cause platelet aggregation. Two types of phospholipids causing platelet aggregation were detected and shown to be an acetylsphingomyelin and an acylacetylglycerol phosphoacetylated glycolipid. The existence of the sphingomyelin analogues is very important, since ceramides, cerebrosides and related lipids are intracellular second messengers. The identification of the phosphoglycoglycerolipid demonstrates a new type of lipid in cyanobacteria, namely one that exhibits a biological activity very similar to that of PAF. Its presence reinforces the concept that PAF is a member of a large family of lipid mediators, apparently having different physiological roles in prokaryotic and eukaryotic organisms. In addition, Scytonema julianum contains a phosphatidylcholine (C(16:0)/(18:2)), even though bacteria in general seldom contain choline-containing phosphoacylglycerols.

Block of the background K(+) channel TASK-1 contributes to arrhythmogenic effects of platelet-activating factor.

Platelet-activating factor (PAF), an inflammatory phospholipid, induces ventricular arrhythmia via an unknown ionic mechanism. We can now link PAF-mediated cardiac electrophysiological effects to inhibition of a two-pore domain K(+) channel [TWIK-related acid-sensitive K(+) background channel (TASK-1)]. Superfusion of carbamyl-PAF (C-PAF), a stable analog of PAF, over murine ventricular myocytes causes abnormal automaticity, plateau phase arrest of the action potential, and early afterdepolarizations in paced and quiescent cells from wild-type but not PAF receptor knockout mice. C-PAF-dependent currents are insensitive to Cs(+) and are outwardly rectifying with biophysical properties consistent with a K(+)-selective channel. The current is blocked by TASK-1 inhibitors, including protons, Ba(2+), Zn(2+), and methanandamide, a stable analog of the endogenous lipid ligand of cannabinoid receptors. In addition, when TASK-1 is expressed in CHO cells that express an endogenous PAF receptor, superfusion of C-PAF decreases the expressed current. Like C-PAF, methanandamide evoked spontaneous activity in quiescent myocytes. C-PAF- and methanandamide-sensitive currents are blocked by a specific protein kinase C (PKC) inhibitor, implying overlapping signaling pathways. In conclusion, C-PAF blocks TASK-1 or a closely related channel, the effect is PKC dependent, and the inhibition alters the electrical activity of myocytes in ways that would be arrhythmogenic in the intact heart.

Platelet-activating factor increases prostaglandin E 2 release from astrocyte-enriched cortical cell cultures

The phospholipid mediator platelet-activating factor (PAF) increased the release of prostaglandin E 2 (PGE 2) from astrocyte-enriched cortical cell cultures in a concentration- and time-dependent manner. The nonhydrolyzable PAF analog methylcarbamyl-PAF (mc-PAF), the PAF intermediate lyso-PAF, and arachidonic acid (AA) also produced this effect. In contrast, phosphatidlycholine (PC) and lyso-PC, lipids that are structurally similar to PAF and lyso-PAF, had no effect on PGE 2 production, suggesting that PAF-induced PGE 2 release is not the consequence of nonspecific phospholipid-induced membrane perturbation. Antagonism of intracellular PAF binding sites completely abolished the ability of mc-PAF and lyso-PAF to mobilize PGE 2, and attenuated the AA effect. Antagonism of the G-protein-coupled PAF receptor in plasma membranes had no significant effect on mc-PAF, lyso-PAF or AA-induced PGE 2 release. Based on the present findings, we hypothesize that intracellular PAF is a physiologic stimulus of PGE 2 production in astrocytes.

Platelet-activating factor induces permeability transition and cytochrome c release in isolated brain mitochondria.

Platelet-activating factor (PAF), a potent bioactive phospholipid implicated in neuronal excitotoxic death, was assessed as a mediator of brain mitochondrial dysfunction. Carbamyl PAF, a non-hydrolyzable PAF analog, added to neurons in culture resulted in decreased mitochondrial membrane potential (DeltaPsi(M)) as measured by the DeltaPsi(M)-sensitive fluorophore 5,5', 6,6'-tetrachloro-1, 1', 3,3'-tetraethylethylbenzimidazolo-carbocyanide iodide (JC-1). To investigate whether PAF has a direct effect on the mitochondria, the mediator was added to rat brain mitochondria preparations and an increase in the permeability of the mitochondrial membrane, termed permeability transition (PT), and cytochrome c release were measured. We report that PAF causes both dose-dependent PT and cytochrome c release from isolated mitochondria. Furthermore, the selective PAF antagonist tetrahydro-4,7,8,10 methyl-1 (chloro-2 phenyl)-6 (methoxy-4 phenyl-carbamoyl)-9 pyrido [4',3'-4,5] thieno [3,2-f] triazolo-1,2,4 [4,3-a] diazepine-1,4 (BN50730), which has affinity for intracellular binding sites, and the peripheral benzodiazepine receptor ligands 7-chloro-5- [4'-chlorophenyl]-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and 1-(-2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), inhibit PAF induction of PT and cytochrome c release. These results suggest that PAF excitotoxicity involves, at least in part, alterations of the mitochondrial membrane.

Influence of acyl and plasmalogenic analogs of platelet activating factor on chemotaxis of human leukocytes in vitro and their inflammatory and antiinflammatory activity in vivo.

The effects of platelet activating factor (PAF) and its cell analogs 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphocholine (1-alkenyl-PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF) on chemotaxis of human leukocytes in vitro and their inflammatory and antiinflammatory activities in vivo were studied. Both analogs stimulated chemotaxis of human leukocytes in agarose gel. PAF and 1-alkenyl-PAF induced rat paw edema in the range of doses 0.1-10 and 10-100 micro g per paw, respectively. Paw edema induced by 1-acyl-PAF (10-100 micro g per paw) was more pronounced than that induced by PAF or 1-alkenyl-PAF. The latter also exhibited significant antiinflammatory effect by inhibiting PAF- or carrageenan-induced rat paw edema, and this effect exceeded that of dexamethasone. In these models of inflammation 1-acyl-PAF did not exhibit any antiinflammatory activity. The data suggest that PAF is not the only cell phospholipid mediating inflammation--its cell analogs, 1-acyl-PAF and 1-alkenyl-PAF, may also be involved into the inflammatory response. Possible interrelationships between cellular synthesis of 1-acyl-PAF, its formation in oxidized LDL, biological effects of lysolecithin, and penetration of LDL into the arterial wall are discussed.

Absorption and effects of 3-(N-phenylamino)-1,2-propanediol esters in relation to toxic oil syndrome.

Toxic Oil Syndrome (TOS) was an epidemic disease related to the consumption of rapeseed oil denatured with aniline that made its sudden appearance in Spain in 1981. The fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP), which is a chemical class of by-products resulting from the reaction of aniline with oil components, have shown a strong association with TOS-related oils. These compounds also show some structural similarities to platelet-activating factor (PAF). In search of a toxic agent that could explain the widespread systemic effects observed in TOS patients, we investigated the intestinal absorption and biotransformation of the different PAP esters found in TOS-related oil samples and the possible pathophysiological effect of these mediators and their metabolic products if acting as PAF analogs. Results indicate that PAP esters are absorbed in the gastrointestinal tract and are distributed and stored in different organs, particularly in the liver and brown adipose tissue. PAP in these organs showed different patterns of fatty acids, indicating the ability of the gastrointestinal tract to modify the fatty acid composition of the parent PAP. Thus, the fatty acid profile of the PAP esters found in intestine appears to be related to the type of oil used as vehicle. Some of these PAP esters, when a long acyl chain was present in the sn-1 position of the molecule, showed an inhibitory effect on the PAF synthesis. This is an important observation in line with the systemic nature of the disease.

Effects of platelet-activating factor on cytokine production by human uterine cervical fibroblasts.

Platelet-activating factor (PAF), a lipid that acts as a potent proinflammatory mediator, is involved in several reproductive processes including parturition. To investigate the effects of PAF on expression of various cytokines by cultured human uterine cervical fibroblasts obtained at term prior to labour, Northern blot analyses and enzyme-linked immunosorbent assays were performed. C-PAF, a stable analogue of PAF, increased expression of interleukin-6 and -8 mRNA in a dose-dependent manner (10(-10) to 10(-8) mol/l of C-PAF), and the expression peaked within 4 h. The corresponding protein concentrations were increased in culture media. Monocyte chemoattractant protein-1 mRNA showed marked induction by 10(-8) mol/l of C-PAF; this peaked by 4 h and was followed by an increase in the protein concentration. Another cytokine, RANTES (regulated upon activation, normal T cell expressed and secreted) showed marked mRNA induction by 10(-8) mol/l of C-PAF, and continued to increase in a time-dependent manner until 24 h. The protein concentration was correspondingly increased in the medium. The PAF-induced cytokine production was abolished by co-incubation with WEB 2170, a specific PAF receptor antagonist. PAF may stimulate local production of cytokines which may induce migration of leukocytes and accelerate collagenolysis in the uterine cervix, thus contributing to cervical ripening during parturition.

Inhibition of human platelet aggregation by amides and ester of salicylic acid with platelet-activating factor analogs.

The influence of acetyl salicylic acid (ASA) derivatives with platelet-activating factor (PAF) lipid analogs on PAF-induced human platelet aggregation has been studied. It was found that the ASA amide with an ethanolamine plasmalogen PAF analog (1-0-alk-1'-enyl-2-acetyl-sn-glycero-3-phospho-(N-2'-acetoxybenzoyl)ethanolamine) and the ASA ester with a choline plasmalogen PAF analog (1-0-alk-1'-enyl-2-(2'-acetoxybenzoyl)-sn-glycero-3-phosphocholine) at concentrations of 10-7-10-6 M effectively inhibit PAF-induced aggregation of human platelets. In contrast to these compounds, the ASA amide with an alkyl PAF analog (1-0-alkyl-2-acetyl-sn-glycero-3-phospho-(N-2'-acetoxybenzoyl)ethanolamine) did not inhibit PAF-induced platelet aggregation. As possible mechanisms of action of the studied compounds, the blockade of PAF-receptor and cyclooxygenase inhibition are proposed.

Platelet-activating Factor (PAF)-dependent Transacetylase and Its Relationship with PAF Acetylhydrolases

Platelet-activating Factor (PAF)-dependent Transacetylase and Its Relationship with PAF Acetylhydrolases

Regulation of the biosynthesis of acyl analogs of platelet-activating factor by purinergic agonist in endothelial cells

We have previously shown that platelet-activating factor (PAF)-dependent transacetylase (TA) contains three catalytic activities, namely PAF: lysophospholipid TA (TA L), PAF: sphingosine TA (TA S) and PAF acetylhydrolase. It serves as a modifier of PAF actions by producing different lipid signal molecules. The TA L activity is involved in the biosynthesis of acyl analogs of PAF (acyl-PAF, 1-acyl-2-acetyl- sn-glycero-3-phosphocholine, acylacetyl-GPC) in agonist-stimulated endothelial cells. In the present investigation, we have studied the mechanism(s) by which the TA activity is regulated in ATP-treated endothelial cells. We have demonstrated that ATP, and thiol-modifying agents with ATP, specifically regulate only the TA L part of the TA activities.

Spectrophotometric assay for serum platelet-activating factor acetylhydrolase activity

We developed a spectrophotometric assay for serum platelet-activating factor acetylhydrolase (PAF-AH, EC 3.1.1.47.) activity using a platelet-activating factor (PAF) analogue with a 4-nitrophenyl group as substrate. PAF-AH hydrolyzes the sn-2 position of the substrate [1-myristoyl-2-( p-nitrophenylsuccinyl)phosphatidylcholine], producing p-nitrophenyl succinate. This liberation was spectrophotometrically monitored and the activity determined from the change in absorption. The assay does not require radioisotopes and is applicable to an automatic analyzer. Utilizing this assay with an automatic analyzer, it is possible to measure the activities of thousands of samples in a few hours with excellent precision (CV 0.5%, n=30) and high correlation ( r=0.979, n=100) with the results of a conventional radioisotopic assay. The assay should be particularly useful for clinical diagnostics.

Modulation of PAF production by incorporation of arachidonic acid and eicosapentaenoic acid in phospholipids of human leukemic monocyte-like cells THP-1

Stimulated leukocytes generate platelet-activating factor (PAF) from membrane 1- O-alkyl-2-acyl-sn-glycerophosphocholine through hydrolysis of fatty acid and subsequent acetylation at the sn2 position of glycerol. Since the enzymes involved in the hydrolysis step of PAF biosynthesis have relative selectivity for arachidonic acid (AA), the fatty acid composition of PAF precursors might modulate PAF production. We studied the effect of AA and eicosapentaenoic acid (EPA) incorporation on PAF biosynthesis, by measuring the incorporation of [ 3H]acetate, in Ca 2+ ionophore (A23187)-stimulated human leukemic monocyte-like cells, THP-1. Supplementation of THP-1 with AA (25 μM, 1 week) or EPA (25 μM, 1 week) led to their efficient incorporation, in comparable quantities and with similar distributions, into phosphatidylcholine and phosphatidylethanolamine, and to a lesser extent into phosphatidylinositol. THP-1 cells supplemented with AA or with EPA synthetized similar amounts of PAF and of acyl analog of PAF under resting condition. However, AA-supplemented cells responded to A23187 stimulation by important raises of PAF (+125.71%) and of acyl analog of PAF (+381.75%) productions, whereas the same stimulation had little effect or no effect at all in cells supplemented with EPA. These results show that both EPA and AA may influence PAF production through their incorporation into PAF precursors, indicating that PAF production might be modulated by the fatty acid composition of its precursors.

52] Synthesis of inflammatory signal transduction species formed during ozonation and/or peroxidation of tissue lipids

Lipid peroxidation and ozonation produce oxidized lipids that are bioactive and can act as signal transducers. The synthesis and isolation of these oxidized lipid products in pure form will allow the testing of hypotheses regarding their role as potential relay molecules. Of special interest are oxidized lipids derived from 1,2-diacyl- sn -glycero-3-phosphocholine that feature a shortened acyl chain on the second position ending with an aldehyde function. These platelet-activating factor analogs are not only produced during ozonation but also during the peroxidation of 1,2-diacyl- sn -glycero-3-phosphocholines. Another class of biologically active lipids is the 1-hydroxy-l-hydroperoxyalkanes formed in vivo from the addition of water to the carbonyl oxides that are intermediates in the ozonation of unsaturated lipids in aqueous media. 1-Hydroxy-1-hydroperoxyalkanes are conveniently generated in situ from the hydrolysis of bis(1-hydroxyalkyl) peroxides. Bis(1-hydroxyalkyl)peroxides are not themselves direct lipid ozonation products, but are stable crystalline compounds that slowly hydrolyze to the corresponding 1-hydroxy-1-hydroperoxyalkanes and aldehydes. This chapter reports the syntheses of 1-palmitoyl-2-[3-(8-octanoyl)-5-(1-octyl)-l,2,4-trioxolane]- sn -glycero-3-phosphocholine (the Criegee ozonide of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), 1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphocholine, 1-palmitoyl-2-(1-hydroxy-1-hydroperoxynonanoyl)- sn -glycero-3-phosphocholine, bis(1-hydroxyheptyl) peroxide, and bis(1-hydroxynonyl)peroxide (the percursors of the corresponding 1-hydroxy-1-hydroperoxyalkanes).

Role of the Platelet-activating Factor in Inflammation

The platelet-activating factor (PAF) is an endogenous lipid mediator derived from membrane phospholipids. PAF was discovered in 1972 as a fluid-phase mediator released from IgE-stimulated basophils that induce the aggregation of rabbit platelets. Biological activity relevant to inflammatory reaction has also been demonstrated, subsequent to identification of the structure of PAF as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in 1979. The activity includes activation of leukocytes, increased vascular permeability and contraction of smooth muscle. In the search for antagonists from synthetic analogues of PAF and natural products, a number of antagonists that act on cell surface receptors have been found. A c-DNA for a PAF receptor was cloned first by Shimizu et al. in 1991. PAF is inactivated by the enzyme, PAF-acetylhydrolase, which removes the sn-2 acetyl group of PAF. Discovery of the deficiency of this enzyme, reported by Miwa et al. in 1988, prompted attention to its relationship with diseases such as bronchial asthma. The molecular cloning of intracellular- and plasma-type PAF-acetylhydrolases was achieved in 1994 and 1995, respectively. The isolation of these genes made possible elucidation of the role of PAF in inflammatory reactions on a molecular basis. Recent progress in the study of this factor by a genetical approach is discussed in detail.

A metabolic path for the degradation of lysophosphatidic acid, an inhibitor of lysophosphatidylcholine lysophospholipase, in neuronal nuclei of cerebral cortex

Neuronal nuclei isolated from rabbit cerebral cortex were found to be enriched in an NEM-insensitive lysophosphatidic acid (lysoPA) phosphohydrolase activity. LysoPA is an inhibitor of the nuclear lysophosphatidylcholine (lysoPC) lysophospholipase, and by preserving lysoPC levels, lysoPA boosted the nuclear production of the acyl analogue of platelet-activating factor by promoting the acetylation of lysoPC (Baker and Chang, Mol. Cell Biochem., 1999, in press). The nuclear phosphohydrolase converts lysoPA to 1-monoacylglycerol, and thus eliminates this lysoPA inhibition of lysoPC lysophospholipase. The nuclear lysoPA phosphohydrolase specific activity was more than three times that observed for the nuclear lysoPA lysophospholipase (Baker and Chang, Biochim. Biophys. Acta 1438 (1999) 253–263) and represents a more active route for nuclear lysoPA removal. The neuronal nuclear lysoPA phosphohydrolase was inhibited at acidic pH, and also inhibited by calcium ions. The 1-monoacylglycerol product of the phosphohydrolase is rapidly degraded by neuronal monoacylglycerol lipase, an enzyme some sevenfold more active than the phosphohydrolase and sensitive to inhibition by arachidonoyl trifluoromethyl ketone (AACOCF 3). Both acidic pH and free fatty acid inhibited the lipase. In the absence of AACOCF 3, production of fatty acid from lysoPA substrate could be largely attributed to the sequential actions of the nuclear phosphohydrolase and lipase. This facilitates fatty acid recycling back into phospholipid by lysophospholipid acylation when ATP levels are restored following periods of brain ischemia. At relatively low concentrations, sphingosine-1-phosphate, and alkylglycerophosphate were the most effective phosphohydrolase inhibitors while phosphatidic acid, alkylacetylglycerophosphate and ceramide were without effect. LysoPA is an interesting regulatory molecule that can potentially preserve lysophosphatidylcholine within the nuclear membrane for use in acetylation reactions. Thus conditions relevant to brain ischemia such as falling pH, falling ATP concentrations, rising fatty acid and intracellular calcium levels may, by slowing this metabolic path for lysoPA loss, promote the production of acyl PAF and contribute to the increased levels of the acetylated lipids noted in ischemia.

Stereospecific Synthesis of Functionalized Ether Phospholipids

A new stereospecific synthesis of functionalized alkyl ether phospholipids is reported. The synthesis is based upon the following: (1) the use of (R)-glycidyl tosylate as a chiral glycerol precursor; (2) the opening of a boron trifluoride catalyzed epoxide ring to introduce the functionalized sn-1-alkyl substituents; (3) the role of tetrahydropyranyl in protecting the sn-2-glycerol position; and (4) the elaboration of the sn-3-carbinol function, via the base hydrolysis of the acetoxy intermediate, obtained from the displacement of the toluenesulfonyl group of the substrate in dipolar aprotic media. Phosphorylation, using two different methods, has led to the development of two major classes of alkyllysophospholipids. For preparation of “modulator-phospholipid” analogues, the substituted glycerol is coupled with 2,2,2-trichloro-tert-butyl phosphodichloridite and an N-protected amino acid ester, while elaboration of the phosphocholine headgroup of the target platelet-activating factor (PAF) analogues is achieved via the 2-chloro-2-oxo-1,3,2-dioxaphospholane/trimethylamine sequence. The synthesis provides rapid and efficient access to both types of phospholipids: (1) construction of the functionalized/substituted glycerol skeleton is achieved in a straightforward four-step sequence in better than 50% overall yield, and (2) phosphitylation or phosphorylation of the respective glycerol intermediates relies on reagents that require minimal use of protecting groups. The phospholipid compounds prepared include (1) the first synthetic analogue exhibiting modulator activity in conjunction with the glucocorticoid-receptor complex and (2) an sn-1-(ω-amino)alkyl derivative of PAF, suitable for introduction of chain-terminal spectroscopic labels for biological and physicochemical studies to elucidate the mechanism of action of this highly potent alkyl ether phospholipid. The synthetic methods described herein have a great deal of flexibility, thus providing convenient general routes to a wide range of alkyl ether phospholipids.

Interleukin‐1 beta activates expression of cyclooxygenase‐2 and inducible nitric oxide synthase in primary hippocampal neuronal culture: Platelet‐activating factor as a preferential mediator of cyclooxygenase‐2 expression

Interleukin-1 beta (IL-1β) is an inflammatory cytokine whose expression is elevated in brain during seizures, ischemia, and injury. Expression of IL-1β and its receptor can also be observed in normal brain. Platelet-activating factor (PAF) is also a dual mediator that promotes neuronal plasticity responses as well as inflammation. We have determined the role of PAF in the regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes by IL-1β in rat primary hippocampal cultures. As assessed by reverse transcriptase/polymerase chain reaction (RT/PCR), recombinant mouse IL-1β (1 nM) led to an induction of COX-2 mRNA which peaked at 2 hours, declined to baseline levels by 4 hours, began to rise again by 6 hours, and remained elevated at 24 hours post-treatment. iNOS mRNA was also induced, but unlike COX-2, its abundance peaked at 4 hours and decreased by 6 hours to a plateau lasting through 24 hours. Pretreatment with PAF antagonist BN50730 blocked induction of COX-2 mRNA by 2-hour IL-1β treatment, and 2-hour treatment with the PAF analog mcPAF mimicked the effects of IL-1β on COX-2 mRNA levels. Following injury, synaptic plasticity changes may be affected by IL-1β-PAF-COX-2 neuronal signaling. J. Neurosci. Res. 58:593–598, 1999. © 1999 Wiley-Liss, Inc.