Platelet-activating Factor Receptor Agonist Research Articles

Platelet-activating Factor Receptor Agonists Mediate Xeroderma Pigmentosum A Photosensitivity

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

Loss of the platelet activating factor receptor in mice augments PMA-induced inflammation and cutaneous chemical carcinogenesis

Although platelet-activating factor (PAF) is a well-known acute inflammatory mediator, little is known regarding the role of PAF in chronic inflammation. Phorbol esters are known to stimulate PAF production. Moreover, the ability of repeated applications of phorbol esters to induce a sustained inflammatory response is crucial to their tumorigenic activity. We therefore examined whether PAF acts as a mediator of phorbol ester-induced inflammation and tumorigenesis. While PAF receptor knockout mice (PAFR(-/-)) showed an expected but modest reduction in the acute inflammatory response to phorbol 12-myristate 13-acetate (PMA), these mice exhibited a surprising increase in inflammation following chronic PMA application. This increased inflammation was documented by a number of findings that included: increased skin thickness, increased myeloperoxidase activity and expression and increased expression of known inflammatory mediators. Interestingly, vehicle-treated PAFR(-/-) mice also exhibited modest increases in levels of inflammatory markers. This suggests that the platelet activating factor receptor (PAFR) acts to suppress chronic inflammation in response to other stimuli, such as barrier disruption. The idea that chronic PAFR activation is anti-inflammatory was documented by repetitive topical PAFR agonist administration that resulted in reduced myeloperoxidase activity in skin. We next utilized a 7,12-dimethylbenz(a)anthracene/PMA carcinogenesis protocol to demonstrate that PAFR(-/-) mice exhibit significantly increased tumor formation and malignant progression compared with wild-type control mice. These studies provide evidence for two important, unexpected and possibly interrelated pathological roles for the PAFR: first, the PAFR acts to suppress PMA-induced chronic inflammation; secondly, the PAFR acts to suppress neoplastic development in response to chemical carcinogens.

Ultraviolet B Radiation of Human Skin Generates Platelet‐activating Factor Receptor Agonists

Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production. In addition to cytokines, such as tumor necrosis factor-alpha (TNF-alpha), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous in vitro studies in keratinocytes or epithelial cell lines have demonstrated that UVB-mediated production of PAF agonists is due primarily to the pro-oxidative effects of this stimulant, resulting in the nonenzymatic production of modified phosphocholines (oxidized glycerophosphocholines). The current studies use human skin to assess whether UVB irradiation generates PAF-receptor agonists, and the role of oxidative stress in their production. These studies demonstrate that UVB irradiation of human skin results in PAF agonists, which are blocked by the antioxidant vitamin C and the epidermal growth factor receptor inhibitor PD168393. Inasmuch as UVB-generated PAF agonists have been implicated in animal model systems as being involved in photobiologic processes including systemic immunosuppression and cytokine (TNF-alpha) production, these studies indicate that this novel activity could be involved in human disease.

Requirement of conserved amino acid residues for ER‐export and recycling of platelet‐activating factor receptor

In the transmembrane domains (TMs) of G‐protein coupled receptors, several amino acid residues are conserved. Here, we demonstrate that a conserved proline, Pro247, in TM6 of platelet‐activating factor receptor (PAFR) is critical for endoplasmic reticulum (ER)‐export and recycling after agonist‐induced internalization. Alanine‐substituted mutants of the conserved residues of PAFRs, including P247A, were accumulated in the ER. Because a PAFR antagonist, Y‐24180, acted as a pharmacological chaperone to rescue ER‐retention, this retention is due to misfolding of PAFR. mc‐PAF, a PAFR agonist, did not increase surface expression of P247A, even if another ER‐retained mutant, D63A, was trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc‐PAF‐treated P247A‐expressing cells, suggesting that P247A was trafficked to the cell surface by mc‐PAF, thereafter internalized, but not recycled. Deficient recycling was confirmed with a sortase‐A‐mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for the intracellular trafficking of PAFR. Supported in part by a Grants‐in‐aid from Ministry of Education, Culture, Sports, Science, and Technology of Japan

Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro(247), in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.

Platelet‐activating factor‐stimulated production of reactive oxygen species in ovarian granulosa cells from periovulatory follicles

The platelet-activating factor (PAF) is a proinflammatory lipid present in the fluid of ovarian Graafian follicle. Ovarian blockage of the PAF receptor (PAFr) reduces ovulations in the rat whereas underlying mechanism is poorly understood. Mural granulosa cells (MGC) were mechanically isolated from the theca interna of bovine periovulatory follicle. The mRNA abundance for PAFr, progesterone receptor and cyclooxygenase-2 were measured by real-time PCR. Cytosolic calcium (Ca2+) concentration was assayed by microscopy using Fura-2 AM as indicator, 8-isoprostaglandin F(2alpha) (8-isoPGF(2alpha)) by an ELISA kit. Fluorescent products arising from intracellular oxidation of hydroethidine (HE) and dihydrorhodamine (DHR) were quantified by flow cytometry. The cells expressed PAFr mRNA and PAFr protein and responded to cPAF (nonhydrolyzable form of PAF) with a pulsating increase in Ca2+, demonstrating functional PAFr. Elevation of Ca2+ was reversed by WEB-2086, an inverse PAFr agonist. cPAF elevated the level of 8-isoPGF(2alpha) in the medium of MGC cultured with luteinizing hormone (LH). cPAF alone had no significant influence on the oxidation of HE and DHR, or 8-isoPGF(2alpha) level. In MGC from vital periovulatory follicle, PAF and LH signaling plays an important role in regulating the production of excessive oxidants. Blockage of PAFr seems to interfere with these regulatory processes essential for ovulation.

Ultraviolet B Radiation Generated Platelet-Activating Factor Receptor Agonist Formation Involves EGF-R-Mediated Reactive Oxygen Species

Recent studies have implicated the lipid mediator platelet-activating factor (PAF) in UVB-mediated systemic immunosuppression known to be a major cause for skin cancers. Previously, our group has demonstrated that UVB irradiation triggers the production of PAF and oxidized glycerophosphocholines that act as PAF-receptor (PAF-R) agonists. The present studies explored the mechanisms by which UVB generates PAF-R agonists. UVB irradiation of human epidermal KB cells resulted in both increased levels of reactive oxygen species (ROS) and PAF-R agonistic activity. Pretreatment of KB cells with antioxidants vitamin C and N-acetylcysteine or the pharmacological inhibitor PD168393 specific for the epidermal growth factor receptor all inhibited UVB-induced ROS as well as PAF-R agonists, yet had no effect on fMLP-mediated PAF-R agonist production. In addition, in vivo production of PAF-R agonists from UVB-irradiated mouse skin was blocked by both systemic vitamin C administration and topical PD168393 application. Moreover, both vitamin C and PD168393 abolished UVB-mediated but not the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine-mediated immunosuppression as measured by the inhibition of delayed type contact hypersensitivity to the chemical dinitrofluorobenzene. These studies suggest that UVB-induced systemic immunosuppression is due to epidermal growth factor receptor-mediated ROS which results in PAF-R agonist formation.

Platelet Activation by Low Concentrations of Intact Oxidized LDL Particles Involves the PAF Receptor

Mitochondrial depolarization aids platelet activation. Oxidized LDL (oxLDL) contains the medium length oxidatively truncated phospholipid hexadecyl azelaoyl-lysoPAF (HAz-LPAF) that disrupts mitochondrial function in nucleated cells, so oxLDL may augment platelet activation. Flow cytometry showed intact oxLDL particles synergized with subthreshold amounts of soluble agonists to increase intracellular Ca2+, and initiate platelet aggregation and surface expression of activated gpIIb/IIIa and P-selectin. oxLDL also induced aggregation and increased intracellular Ca2+ in FURA2-labeled cells by itself at low, although not higher, concentrations. HAz-LPAF, alone and in combination with substimulatory amounts of thrombin, rapidly increased cytoplasmic Ca2+ and initiated aggregation. HAz-LPAF depolarized mitochondria in intact platelets, but this required concentrations beyond those that directly activated platelets. An unexpectedly large series of chemically pure truncated phospholipids generated by oxidative fragmentation of arachidonoyl-, docosahexaneoyl-, or linoleoyl alkyl phospholipids were platelet agonists. The PAF receptor, thought to effectively recognize only phospholipids with very short sn-2 residues, was essential for platelet activation because PAF receptor agonists blocked signaling by all these medium length phospholipids and oxLDL. Intact oxLDL particles activate platelets through the PAF receptor, and the PAF receptor responds to a far wider range of oxidized phospholipids in oxLDL than anticipated.

Augmentation of UVB Radiation-Mediated Early Gene Expression by the Epidermal Platelet-Activating Factor Receptor

UVB radiation (UVB) is a known inducer of many biological changes in human skin, and triggers the production of glycerophosphocholines that act as platelet-activating factor (PAF) agonists. To gain a better insight into the role of the epidermal PAF receptor (PAF-R) in UVB-mediated gene expression, Affymetrix oligonucleotide microarrays were used to compare mRNA expression in the PAF-R-negative epithelial cell line KB-expressing PAF-Rs (KBP) with that in KB cells transduced with a vector control (KBM). Total RNA was isolated from KB cells 1 hour after treatment with a PAF-R agonist or UVB irradiation. Treatment of KBP with PAF agonist resulted in altered expression of 220 genes, including cytokines and growth factors. UVB irradiation of KB cells resulted in an increased expression of genes in both cell types. A panel of genes including cytokines CCL20 (MIP3alpha) and tumor necrosis factor-alpha (TNF-alpha) were upregulated selectively in KBP cells and are also selectively upregulated in response to PAF agonist. Consistent with these in vitro findings, UVB irradiation resulted in increased levels of epidermal CCL20 and TNF-alpha mRNA in wild-type over PAF-R-deficient mice in vivo. These studies provide evidence that the epidermal PAF-R can modulate UVB-mediated early gene expression.

Activation of platelet‐activating factor receptor in SZ95 sebocytes results in inflammatory cytokine and prostaglandin E2 production

Platelet-activating factor (PAF) is a group of phosphocholines with various biological effects mediated by the PAF receptor (PAF-R). Activation of the epidermal PAF-R induces the expression of inflammatory mediators, including cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). The upregulation of COX-2 expression has been shown to be involved in sebocyte proliferation, sebaceous gland inflammation and carcinogenesis. The present study was designed to investigate whether PAF-R activation could induce the expression of COX-2 and production of PGE(2), as well as secretion of the inflammatory cytokine, interleukin-8 (IL-8), in the immortalized sebaceous gland cell line SZ95. Using calcium mobilization studies, we first confirmed that PAF can signal through PAF-R in SZ95 sebocytes. We then found that the production of IL-8 was induced following treatment with PAF-R agonist, however blocked by a specific PAF-R antagonist. Induction of COX-2 expression and increased PGE(2) production were observed in SZ95 sebocytes after PAF-R activation. Finally, it was demonstrated that the production of PGE(2), induced by PAF-R activation and mediated by COX-2 expression, was blocked following PAF-R antagonism in SZ95 sebocytes. These studies suggest that SZ95 sebocytes express functional PAF-Rs and PAF-Rs are involved in regulating the expression of inflammatory mediators, including COX-2, PGE(2) and IL-8.

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.

Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids

Lysophosphatidylcholine is an abundant component of plasma and oxidized LDL that displays several biological activities, some of which may occur through the platelet-activating factor (PAF) receptor. We find that commercial lysophosphatidylcholine, its alkyl homolog (lyso-PAF), and PAF all induce inflammation in a murine model of pleurisy. Hydrolysis of PAF to lyso-PAF by recombinant PAF acetylhydrolase abolished this eosinophilic infiltration, implying that lyso-PAF should not have displayed inflammatory activity. Saponification of lyso-PAF or PAF acetylhydrolase treatment of lyso-PAF or lysophosphatidylcholine abolished activity; neither lysolipid should contain susceptible sn-2 residues, suggesting contaminants account for the bioactivity. Lyso-PAF and to a lesser extent lysophosphatidylcholine stimulated Ca2+ accumulation in 293 cells stably transfected with the human PAF receptor, and this was inhibited by specific PAF receptor antagonists. Again, treatment of lyso-PAF or lysophosphatidylcholine with recombinant PAF acetylhydrolase, a nonselective phospholipase A2, or saponification of lyso-PAF destroyed the PAF-like activity, a result incompatible with lyso-PAF or lysophosphatidylcholine being the actual agonist. We conclude that neither lyso-PAF nor lysophosphatidylcholine is a PAF receptor agonist, nor are they inflammatory by themselves. We suggest that PAF or a PAF-like mimetic accounts for inflammatory effects of lysophosphatidylcholine and lyso-PAF. —Marathe, G. K., A. R. Silva, H. C. de Castro Faria Neto, L. W. Tjoelker, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre. Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids.

Evidence for Involvement of the Epidermal Platelet-Activating Factor Receptor in Ultraviolet-B-Radiation-Induced Interleukin-8 Production

Ultraviolet B radiation has been shown to generate cutaneous inflammation in part through inducing oxidative stress and cytokine production in human keratinocytes. Amongst the proinflammatory cytokines synthesized in response to ultraviolet B radiation is the potent chemoattractant interleukin-8. Though the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and keratinocytes express PAF receptors linked to cytokine biosynthesis, it is not known whether PAF is involved in ultraviolet-B-induced epidermal cell cytokine production. These studies examined the role of the PAF system in ultraviolet-B-induced epidermal cell interleukin-8 biosynthesis using a novel model system created by retroviral-mediated transduction of the PAF-receptor-negative human epidermal cell line KB with the human PAF receptor. Treatment of PAF-receptor-expressing KB cells with the metabolically stable PAF receptor agonist carbamoyl-PAF resulted in increased interleukin-8 mRNA and protein, indicating that activation of the epidermal PAF receptor was linked to interleukin-8 production. Ultraviolet B irradiation of PAF-receptor-expressing KB cells resulted in significant increases in both interleukin-8 mRNA and protein in comparison to ultraviolet-B-treated control KB cells. Pretreatment with PAF receptor antagonists inhibited both carbamoyl-PAF-induced and ultraviolet-B-induced interleukin-8 production in the PAF-receptor-positive cells, but not in control KB cells. Similarly, treatment of the PAF-receptor-expressing primary cultures of human keratinocytes or the human epidermal cell line A-431 with carbamoyl-PAF or ultraviolet B radiation resulted in interleukin-8 production that was partially inhibited by PAF receptor antagonists. These studies suggest that the epidermal PAF receptor may be a pharmacologic target for ultraviolet B radiation in skin and thus may act to augment ultraviolet-B-mediated production of cytokines such as interleukin-8.

Neuronal platelet-activating factor receptor signal transduction involves a pertussis toxin-sensitive G-protein.

In most nonneural systems, platelet-activating factor (PAF) receptor effects are mediated by G-proteins that are often pertussis toxin-sensitive. The activation of pertussis toxin-sensitive G-proteins linked to PAF receptors results in the mobilization of intracellular calcium, at least in part, through the second messenger inositol triphosphate. We have sought to determine if a pertussis toxin-sensitive G-protein is involved in the PAF receptor-mediated phenomena of growth cone collapse and of synaptic enhancement in primary neuronal culture. Using infrared differential interference contrast microscopy and patch-clamp recording techniques, pertussis toxin, but not the inactive B oligomer of the toxin, was found to block both the growth cone collapse and the enhanced synaptic release of excitatory transmitter induced by a nonhydrolyzable PAF receptor agonist, making it likely that Go, Gq, or Gi is the G-protein transducer of PAF receptors in primary neurons. We believe that PAF acts directly on neuronal receptors, which are linked to pertussis toxin-sensitive G-proteins, on the tips of developing neurites, and on presynaptic nerve terminals, leading to growth cone collapse and enhanced synaptic release of transmitter.

Augmentation of ultraviolet B radiation-induced tumor necrosis factor production by the epidermal platelet-activating factor receptor.

Ultraviolet B radiation (UVB) has been shown to damage human keratinocytes in part by inducing oxidative stress and cytokine production. Indeed, UVB-induced production of the pro-inflammatory and cytotoxic cytokine tumor necrosis factor alpha (TNF-alpha) has been implicated in the epidermal damage seen in response to acute solar radiation. Though the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and keratinocytes express PAF receptors linked to cytokine biosynthesis, it is not known whether PAF is involved in UVB-induced epidermal cell cytokine production. These studies examined the role of the PAF system in UVB-induced epidermal cell TNF-alpha biosynthesis using a novel model system created by retroviral-mediated transduction of the PAF receptor-negative human epidermal cell line KB with the human PAF receptor (PAF-R). Treatment of PAF-R-expressing KB cells with the metabolically stable PAF-R agonist carbamoyl-PAF resulted in increased TNF-alpha mRNA and protein, indicating that activation of the epidermal PAF-R was linked to TNF-alpha production. UVB irradiation of PAF-R-expressing KB cells resulted in significant increases in both TNF-alpha mRNA and protein in comparison to UVB-treated control KB cells. However, UVB treatment up-regulated cyclooxygenase-2 mRNA levels to the same extent in both PAF-R-expressing and control KB cells. Pretreatment with the antioxidant vitamin E or the PAF-R antagonists WEB 2086 and A-85783 inhibited UVB-induced TNF-alpha production in the PAF-R-positive but not control KB cells. These studies suggest that the epidermal PAF-R may be a pharmacological target for UVB in skin.

Oxidative Stress Can Activate the Epidermal Platelet-Activating Factor Receptor

Platelet-activating factor (1-alkyl-2-acetyl-glycero-phosphocholine) is a lipid mediator that has been implicated in keratinocyte function and cutaneous inflammation. Keratinocytes both synthesize platelet-activating factor and express functional platelet-activating factor receptors linked to calcium mobilization. Oxidative stress to various cells including keratinocytes can also result in the mobilization of intracellular Ca2+, a known stimulus for platelet-activating factor biosynthesis. The ability of the epidermal platelet-activating factor receptors to modulate oxidant-induced signaling was investigated using a unique model system created by retroviral-mediated transduction of the platelet-activating factor receptor-negative epithelial cell line KB with the platelet-activating factor receptor. Treatment of KB cells with the lipid pro-oxidant tert-butyl hydroperoxide induced transient increases in intracellular Ca2+ in a concentration-dependent fashion. Expression of the platelet-activating factor receptor in KB cells lowered the threshold for tert-butyl hydroperoxide-induced Ca2+ flux by an order of magnitude (10 microM in control KB versus 1 microM in KB cells expressing the platelet-activating factor receptors) and increased the peak change in intracellular Ca2+ concentration in response to this lipid hydroperoxide. This augmentation of tert-butyl hydroperoxide-induced Ca2+ mobilization was inhibited by pretreatment with the two competitive platelet-activating factor receptor antagonists CV-6209 and WEB 2086, as well as by the antioxidants vitamin E and 1,1,3,3-tetramethyl-2-thiourea. KB cells synthesized platelet-activating factor and the platelet-activating factor receptor agonist 1-palmitoyl-2-acetyl-glycero-phosphocholine in response to tert-butyl hydroperoxide treatment, suggesting the augmentation of oxidative stress-induced signaling seen in platelet-activating factor receptor-expressing cells was due in part to endogenous platelet-activating factor biosynthesis. These studies suggest involvement of the epidermal platelet-activating factor receptors in oxidant-mediated signaling.

Expression of the Platelet-activating Factor Receptor Results in Enhanced Ultraviolet B Radiation-induced Apoptosis in a Human Epidermal Cell Line

Recent studies have demonstrated that ultraviolet B radiation (UVB) damages human keratinocytes in part by inducing oxidative stress and cytokine production. Severe UVB damage to the keratinocyte can also result in apoptosis or programmed cell death. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to epidermal cell damage and epidermal cells express PAF receptors, it is not known whether PAF is involved in UVB-induced epidermal cell apoptosis. These studies examined the role of the PAF system in UVB-induced epidermal cell apoptosis using a novel model system created by retroviral-mediated transduction of the PAF receptor-negative human epidermal cell line KB with the human PAF receptor (PAF-R). Expression of the PAF-R in KB cells did not affect base-line growth or apoptosis, yet resulted in a decrease in the lag time between treatment of the cells and the induction of apoptosis following irradiation with 400 J/m2 UVB. This effect was inhibited by pretreatment with the PAF-R antagonists WEB 2086 and A-85783, confirming involvement of the PAF-R in this process. At lower doses (100-200 J/m2) of UVB, only KB cells that expressed the PAF-R became apoptotic. Treatment of PAF-R-expressing KB clones with the metabolically stable PAF-R agonist 1-hexadexyl-2-N-methylcarbamoyl-3-glycerophosphocholine (CPAF) alone did not induce apoptosis but augmented the degree of apoptosis observed if CPAF was used in combination with lower doses (200 J/m2) of UVB irradiation. Interestingly, UVB irradiation was found to stimulate PAF synthesis only in PAF-R-expressing KB cell clones. The antioxidants N-acetyl cysteine, 1,1,3,3-tetramethyl-2-thiourea, and vitamin E inhibited both UVB-induced PAF biosynthesis as well as the augmentation of UVB-induced apoptosis in PAF-R-expressing KB clones, suggesting the possibility that UVB stimulates the production of oxidized lipid species with PAF-R agonistic activity in this model system. Thus, these studies indicate that a component of UVB-induced epidermal cell cytotoxicity can be modulated by PAF-R activation through the production of PAF and PAF-like species.

Platelet-Activating Factor Receptor Stimulation Disrupts Neuronal MigrationIn Vitro

LIS-1 is a gene whose hemi-deletion causes the human neuronal migration disorder Miller-Dieker lissencephaly. It encodes a subunit of a brain platelet-activating factor (PAF) acetylhydrolase, an enzyme that inactivates PAF by hydrolyzing the acetyl moiety in the sn2 position of this phospholipid. Because PAF receptor activation has been shown to affect the developing neuronal cytoskeleton, we have hypothesized that a role for PAF in neurodevelopment is that of a modulator of neuroblast movement (a cytoskeletal function) and that an aberrant regulation of PAF could lead to an early arrest in migration. This report examines the effects of the nonhydrolyzable PAF receptor agonist methyl carbamyl PAF (mc-PAF) on the unidirectional in vitro migration of granule cells from cerebellar cell reaggregates on a laminin substrate. Bath treatment with mc-PAF yields a dose-dependent decrease in granule cell migration compared with controls. This effect can be blocked by the simultaneous bath application of BN 52021 and trans-BTD, PAF receptor-specific antagonists. Although mc-PAF minimally inhibited neurite growth, its primary effect was on somal movement along preextended neurites. These experiments suggest that the stimulation of neuronal PAF receptors could be one crucial step for the regulation of neuroblast migration and that disturbed PAF catabolism during neurodevelopment could contribute to the neuronal migration defects observed in Miller-Dieker lissencephaly.

Elvax as a slow-release delivery agent for a platelet-activating factor receptor agonist and antagonist

The ability of the slow-release polymer, Elvax, to deliver a large number of different molecules to distinct brain regions has been well-documented. However, there are currently no reports of Elvax-mediated delivery of ether phospholipids, whose lipid structure renders them difficult to solubilize and detect under experimental conditions. In order to potentially examine the role of platelet-activating factor (PAF) receptors in the brain in vivo, we tested the ability of Elvax to release the ether phospholipid, PAF receptor agonist, methyl-carbamyl platelet-activating factor (mc-PAF), and the ginkolide, PAF receptor antagonist, BN 52021. Mc-PAF and BN 52021 containing Elvax sections (200 μm) prepared in methylene chloride were assayed for release characteristics with a rabbit platelet aggregation bioassay. We measured a slow, sustained release of mc-PAF and BN 52021 from Elvax in vitro over a 5 day period. Similar in vivo mc-PAF and BN 52021 release kinetics were determined. Therefore, we believe that this novel method of slow-release delivery of PAF receptor agonists and antagonists as measured with platelet aggregation is viable and offers a simple, sensitive and inexpensive method for potential in vivo studies of the roles of PAF and its receptors in neural systems.

Piperbetol, methylpiperbetol, piperol A and piperol B: a new series of highly specific PAF receptor antagonists from Piper betle.

Piperbetol, methylpiperbetol, piperol A, and piperol B, isolated from Piper betle, selectively inhibited the washed rabbit platelet aggregation induced by platelet activating factor (PAF) in a concentration-dependent manner. The IC50 values of piperbetol, methylpiperbetol, piperol A, piperol B, and ginkgolide B were about 18.2, 10.6, 114.2, 11.8, and 4.8 mumol/l, respectively. The inhibitory potency of ginkgolide B was about 2.8, 1.2, 22.8, and 1.4 times higher than those of piperbetol, methylpiperbetol, piperol A, and piperol B. The concentration-response curve of PAF-induced platelet aggregation was shifted to the right by 50 mumol/l of piperbetol, methylpiperbetol, piperol A, piperol B, and ginkgolide B. The EC50 of PAF was increased by these compounds from 1.5 nmol/l to 14.3, 23.1, 2.4, 20.6, and 47.2 nmol/l, respectively. The compounds also inhibited the binding of [3H]-PAF to washed rabbit platelets with IC50 values of 8.7, 5.3, 88, 6.2, and 1.8 mumol/l. Correlating with the inhibitory potency for platelet aggregation, the inhibitory potency of ginkgolide B for binding of PAF was about 3.8, 1.9, 48, and 2.4 times higher than those of piperbetol, methylpiperbetol, piperol A, and piperol B. However, the aggregation of washed rabbit platelets induced by threshold ADP and arachidonic acid were unaffected by piperbetol, methylpiperbetol, piperol A, and piperol B. Furthermore, piperbetol, methylpiperbetol, piperol A, and piperol B had no effects on the cAMP contents in rest washed rabbit platelets. In conclusion, these data indicate that piperbetol, methylpiperbetol, piperol A, and piperol B are effective PAF receptor antagonists in vitro.