Stress-induced platelet-activating factor synthesis in human neutrophils

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent inflammatory mediator produced by cells in response to physical or chemical stress. The mechanisms linking cell injury to PAF synthesis are unknown. We used liquid chromatography-tandem mass spectrometry to investigate stress-induced PAF synthesis in human neutrophils. PAF synthesis induced by extracellular pH 5.4 correlated with the activation of a stress-activated kinase, p38 mitogen-activated protein kinase (MAPK), and was blocked by the p38 MAPK inhibitor SB 203580. A key enzyme of PAF synthesis, acetyl-CoA:lysoPAF acetyltransferase, which we have previously shown is a target of p38 MAPK, was also activated in an SB 203580-sensitive fashion. Another MAPK pathway, extracellular signal-regulated kinase-1/2 (ERK-1/2), was also activated. Surprisingly, the pharmacological blockade of the ERK-1/2 pathway with PD 98059 did not block, but rather enhanced, PAF accumulation. Two unexpected actions of PD 98059 may underlie this phenomenon: an augmentation of stress-induced p38 MAPK phosphorylation and an inhibition of PAF catabolism. The latter effect did not appear to be due to a direct inhibition of PAF acetylhydrolase. Finally, similar results were obtained using another form of cellular stress, hypertonic sodium chloride. These data are consistent with a model in which stress-induced PAF accumulation is regulated positively by p38 MAPK and negatively by ERK-1/2. Such a model contrasts with the PAF accumulation induced by other forms of stimulation, which we and others have found is up-regulated by both p38 MAPK and ERK-1/2.     

Intracellular localization of platelet-activating factor synthesis in human neutrophils

Human neutrophils were homogenized and fractionated on a continuous sucrose gradient to assess the subcellular location of acetyl-CoA: lyso-PAF acetyltransferase and of newly synthesized PAF (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Acetyltransferase activity showed two subcellular locations in resting neutrophils. One of them cofractionated with plasma membrane and endoplasmic reticulum markers, whereas another major location corresponded to a region of the gradient enriched in tertiary granules. No PAF was detected in resting neutrophils, but PAF synthesis was induced by cell stimulation with ionophore A23187. Most of the newly synthesized PAF was found cell-associated, showing a bimodal subcellular distribution similar to that found for acetyltransferase activity in activated cells. PAF and acetyltransferase were located in a light membrane fraction, enriched in plasma membrane and endoplasmic reticulum, and in an ill-defined region of the gradient between the specific and azurophilic granules in A23187-stimulated cells. These data support the involvement of the acetyltransferase pathway in the synthesis of PAF induced by ionophore A23187, and demonstrate the synthesis and accumulation of newly synthesized PAF in a light membrane fraction as well as in an intracellular dense organelle upon neutrophil activation.     

Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases

Human neutrophils (PMN) are potentially a major source of platelet-activating factor (PAF) produced during inflammatory responses. The stimulated synthesis of PAF in PMN is carried out by a phospholipid remodeling pathway involving three enzymes: acetyl-CoA:lyso-PAF acetyltransferase (acetyltransferase), type IV phospholipase A(2) (cPLA(2)) and CoA-independent transacylase (CoA-IT). However, the coordinated actions and the regulatory mechanisms of these enzymes in PAF synthesis are poorly defined. A23187 has been widely used to activate the remodeling pathway, but it has not been shown how closely its actions mimic those of physiological stimuli. Here we address this important problem and compare responses of the three remodeling enzymes and PAF synthesis by intact cells. In both A23187- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN, acetyltransferase activation is blocked by SB 203580, a p38 MAP kinase inhibitor, but not by PD 98059, which blocks activation of the ERKs. In contrast, either agent attenuated cPLA(2) activation. Correlating with these results, SB 203580 decreased stimulated PAF formation by 60%, whereas PD 98059 had little effect. However, the combination of both inhibitors decreased PAF formation to control levels. Although a role for CoA-IT in PAF synthesis is recognized, we did not detect activation of the enzyme in stimulated PMN. CoA-IT thus appears to exhibit full activity in resting as well as stimulated cells. We conclude that the calcium ionophore A23187 and the receptor agonist fMLP both act through common pathways to stimulate PAF synthesis, with p38 MAP kinase regulating acetyltransferase and supplementing ERK activation of cPLA(2).     

Salmonella typhimurium porins stimulate platelet-activating factor synthesis by human polymorphonuclear neutrophils.

Porins, a family of hydrophobic proteins located in the outer membrane of cell-wall of Gram-negative bacteria, were shown to stimulate the synthesis and release of platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine mediator of inflammation and endotoxic shock produced by polymorphonuclear neutrophils. PAF synthesis was independent either from contamination by LPS or generation of TNF. Experiments with labeled precursors demonstrated that PAF was synthesized via the remodeling pathway that involves acetylation of 1-O-alkyl-sn-glyceryl-3-phosphorylcholine generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins, indeed, induced a sustained PLA2-dependent mobilization of [14C]arachidonic acid that was inhibited by p-bromodiphenacylbromide. p-Bromodiphenacylbromide, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase. The addition of 2-lyso-PAF restored PAF synthesis. The activity of acetyl CoA:2-lyso-PAF acetyltransferase was transiently increased in porin-stimulated PMN and the [3H]acetyl group was incorporated in the synthetized PAF after cell preincubation with [3H]acetyl CoA. The activation of PAF synthesis by porins as well as its release were dependent on extracellular Ca2+. Porins by forming trans-membrane channels determined a sustained influx of 45Ca2+ into the cytosol. As shown by inhibitors of Ca(2+)-calmodulin complexes, calmodulin mediated the Ca(2+)-dependent activation of enzymes involved in PAF synthesis.     

Differential fMet-Leu-Phe- and platelet-activating factor-induced signaling toward Ral activation in primary human neutrophils.

We have measured the activation of the small GTPase Ral in human neutrophils after stimulation with fMet-Leu-Phe (fMLP), platelet activating factor (PAF), and granulocyte macrophage-colony stimulating factor and compared it with the activation of two other small GTPases, Ras and Rap1. We found that fMLP and PAF, but not granulocyte macrophage-colony stimulating factor, induce Ral activation. All three stimuli induce the activation of both Ras and Rap1. Utilizing specific inhibitors we demonstrate that fMLP-induced Ral activation is mediated by pertussis toxin-sensitive G-proteins and partially by Src-like kinases, whereas fMLP-induced Ras activation is independent of Src-like kinases. PAF-induced Ral activation is mediated by pertussis toxin-insensitive proteins, Src-like kinases and phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase is not involved in PAF-induced Ras activation. The calcium ionophore ionomycin activates Ral, but calcium depletion partially inhibits fMLP- and PAF-induced Ral activation, whereas Ras activation was not affected. In addition, 12-O-tetradecanoylphorbol-13-acetate-induced activation of Ral is completely abolished by inhibitors of protein kinase C, whereas 12-O-tetradecanoylphorbol-13-acetate-induced Ras activation is largely insensitive. We conclude that in neutrophils Ral activation is mediated by multiple pathways, and that fMLP and PAF induce Ral activation differently.     

Granulocyte-macrophage colony-stimulating factor is a stimulant of platelet-activating factor and superoxide anion generation by human neutrophils.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied for its ability to stimulate the synthesis and release of the inflammatory mediator platelet-activating factor (PAF) from human neutrophils as measured by bioassay and incorporation of [3H]acetate into PAF. GM-CSF stimulated the synthesis but not the release of PAF from neutrophils. PAF synthesis took place in a time- and concentration-dependent manner, was dependent on a pertussis toxin-sensitive G protein and could be inhibited by antibodies to GM-CSF. On the other hand, pre-incubation of neutrophils with GM-CSF followed by stimulation with the bacterial tripeptide formylmethionylleucylphenylalanine caused PAF synthesis and release. The effect of GM-CSF was qualitative and not simply the result of larger amounts of PAF being synthesized since similar amounts were generated in response to the calcium ionophore A23187 but no released PAF could be detected. In functional studies GM-CSF stimulated superoxide anion generation from neutrophils with a time and dose relationship that paralleled PAF synthesis. In addition, the serine protease inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone, which inhibits PAF synthesis, reduced PAF accumulation as well as superoxide generation, raising the possibility of a causal relationship between cell-associated PAF and cell activation. These results identify PAF as a direct product of GM-CSF stimulation in neutrophils where it may play a role in signal transduction and demonstrate that PAF is released only after subsequent neutrophil stimulation. The selective release of PAF may play a role in regulating and amplifying the inflammatory response.     

Granulocyte-macrophage colony-stimulating factor increases the synthesis of leukotriene B4 by human neutrophils in response to platelet-activating factor. Enhancement of both arachidonic acid availability and 5-lipoxygenase activation.

Granulocyte-macrophage CSF (GM-CSF) primes human neutrophils for increased functional responsiveness to a variety of inflammatory agonists. In the present report, we have investigated the effect of human GM-CSF on the ability of platelet-activating factor (PAF) to induce the synthesis of 5-lipoxygenase products in human neutrophils. Human neutrophils stimulated with PAF in the range of 10(-5) to 10(-7) M for 15 min released small quantities of leukotriene B4 and its omega-oxidation products, 20-OH- and 20-COOH-leukotriene B4 in amounts that were detectable by enzyme immunoassay. Preincubation of normal peripheral blood neutrophils with human rGM-CSF enhanced the synthesis of the 5-lipoxygenase products in a time- and dose-dependent manner. Treatment with GM-CSF enabled their detection in response to lower concentrations of PAF (greater than or equal to 10(-9) M). The PAF receptor antagonist BN52021 inhibited the synthesis of 5-lipoxygenase products by GM-CSF-treated neutrophils in response to PAF. In addition to its effect on PAF-induced leukotriene synthesis, GM-CSF also augmented intracellular calcium mobilization by PAF. This observation prompted us to examine the effect of GM-CSF on two calcium-dependent events that are essential for leukotriene synthesis, arachidonic acid liberation, and 5-lipoxygenase activation. GM-CSF by itself, did not directly activate either of these two processes, however, it consistently and markedly enhanced the ability of PAF to do so. These results indicate that preincubation of peripheral blood neutrophils with GM-CSF enhances the ability of PAF to stimulate leukotriene synthesis by increasing both arachidonic acid availability and 5-lipoxygenase activation in response to PAF. These observations provide additional evidence of an important role for GM-CSF in the modulation of inflammatory responses to endogenous agonists through enhancement of the production of potent cellular inflammatory mediators such as leukotrienes.     

Calmodulin antagonists inhibit formation of platelet-activating factor in stimulated human neutrophils

Conversion of native, 97-100 kDa rat liver microsomal HMG CoA reductase to membrane-bound 62 kDa and soluble 52-56 kDa catalytically active forms was catalyzed in vitro by the calcium-dependent, leupeptin- and calpastatin-sensitive protease calpain-II purified from rat liver cytosol. Cleavage of the native 97-100 kDa reductase was enhanced by pretreatment (inactivation) of microsomes with ATP(Mg2+) and liver reductase kinase (compared to protein phosphatase-pretreated controls). This was reflected in a loss of the 97-100 kDa species and an increase in the soluble 52-56 kDa species (total enzyme activity and specific immunoblot recovery).     

Enhancement by staurosporine of platelet-activating factor formation in N-formyl peptide-challenged human neutrophils is mediated by intracellular platelet-activating factor binding sites.

Staurosporine potentiates the formation of platelet-activating factor (PAF) and causes a sustained elevation of intracellular Ca2+ ([Ca2+]i). WEB 2086, a specific PAF-receptor antagonist, inhibits both potentiation of PAF formation and elevation of [Ca2+]i by 78% and 65%, respectively. Moreover, the PAF produced by FMLP and/or Staurosporine was completely retained in the cell. This suggests that the effect of staurosporine in FMLP-stimulated neutrophils may be mediated by the action of endogenously produced PAF, which in turn leads to an increase in [Ca2+]i and PAF formation. We conclude that PAF is the major product of human neutrophils which reacts via specific intracellular PAF binding sites to stimulate the phospholipase A2, and its synthesis is under control of a staurosporine-sensitive protein kinase.     

The release of a platelet-activating factor by stimulated rabbit neutrophils.

Normal rabbit peripheral blood neutrophils released a platelet-activating factor upon stimulation by opsonized zymosan. The liberation was Ca++ dependent and the time course of release was closely associated with phagocytosis. The material extracted into chloroform and exhibited an identical mobility by thin layer chromatography to basophil-derived, IgE-stimulated, platelet-activating factor (PAFb). It was similar to PAFb in its effect on platelets in both aggregation and release but was distinguished from ADP, thrombin, arachidonic acid, and thromboxanes. This factor appears to be responsible for some previously reported neutrophil-platelet interactions.     

Phospholipid remodeling/generation in Giardia: the role of the Lands cycle.

Recent results suggest that Giardia is able to carry out deacylation/reacylation reactions (the Lands cycle) to generate new phospholipids, effectively bypassing the de novo synthesis of the entire phospholipid molecule. The successful operation of this deacylation/reacylation cycle is important for Giardia because this protozoan parasite possesses limited lipid synthesis ability. This article discusses how Giardia might use the Lands cycle to alter phospholipids acquired from the host during its colonization in the human small intestine.     

Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids.     

Reversible activation/inactivation of the deacylation/acylation cycle in rat liver microsomes

Rat liver microsomes incorporate [¹⁴C]palmitoyl CoA into membrane phospholipids via the deacylation/acylation cycle. This activity is reversibly inactivated/activated by treatment of the microsomes with ATP, MgCl₂, and 105,000g supernatant or with 105,000g supernatant alone. These observations suggest that the acylation cycle is controlled by a mechanism involving phosphorylation/dephosphorylation. As the pool of lysolecithin in the membranes is not altered by conditions increasing incorporation of palmitoyl CoA into phospholipid, it is probable that the site of regulation of deacylation/acylation is at the acyltransferase rather than the phospholipase.     

Human endothelial cells are target for platelet-activating factor. II. Platelet-activating factor induces platelet-activating factor synthesis in human umbilical vein endothelial cells.

Platelet-activating factor (PAF), a phospholipid mediator with broad and potent biologic activities, is synthesized by several inflammatory cells including endothelial cells (EC). PAF is also an effective stimulating agent for EC leading to increased cell permeability and adhesivity. We examined the synthesis of PAF in human umbilical cord vein EC after stimulation of EC with PAF or with its nonmetabolizable analog 1-O-alkyl-2-N-methyl-carbamyl-sn-glycero-3-phosphocholine (C-PAF). PAF (1 to 100 nM) induced a dose- and time-dependent increase of PAF synthesis as detected by [3H]acetate incorporation into PAF fraction. Stimulation of PAF synthesis occurred via activation of the "remodeling pathway" as the 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase was dose-dependently increased after PAF treatment. The de novo pathway of PAF synthesis was not activated under these conditions. C-PAF was able to mimic the effect of authentic PAF on [3H] acetate incorporation. The inactive metabolite lyso-PAF (100 nM) had no influence on PAF synthesis in EC. CV-3988, BN 52021, and WEB 2086, potent and specific antagonists of PAF suppressed PAF effects on the remodeling pathway completely. The PAF- and C-PAF-induced [3H]PAF remained 93% cell-associated and was not degraded up to 10 min after stimulation. Characterization of the [3H]acetate-labeled material co-migrating with authentic PAF revealed that a significant proportion (approximately 57%) was actually 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. PAF-induced PAF synthesis might be an important mechanism for amplifying original PAF signals and potentiating adhesive interactions of circulating cells with the endothelium.     

Selective deacylation of arachidonate-containing ethanolamine-linked phosphoglycerides in stimulated human neutrophils

The involvement of the ethanolamine-linked phosphoglyceride fraction (PE) in neutrophil signal transduction is suggested by the stimulus-induced release of arachidonic acid from PE (Chilton, F. H., and Connell, T. R. (1988) J. Biol. Chem. 263, 5260-5265) and by the synthesis of acetylated PE species, predominantly 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alkenylacetyl-GPE; Tessner, T. G., and Wykle, R. L. (1987) J. Biol. Chem. 262, 12660-12664) in stimulated cells. In the studies reported here, we investigated the relationship between arachidonic acid release from PE and generation of the lysophospholipid precursor required in the biosynthesis of alkenylacetyl-GPE. In order to follow these reactions, we prelabeled neutrophils with 1-O-[3H]alk-1'-enyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (alkenyl-acyl-GPE). We also followed the hydrolysis of endogenous PE by analysis as the dinitrophenyl derivative using a high pressure liquid chromatography method we developed. Our results coupled with those of Chilton et al. (Chilton, F. H., Ellis, J. M., Olson, S. C., and Wykle, R. L. (1984) J. Biol. Chem. 259, 12014-12019) indicate that in human neutrophils the metabolism of alkenylacyl-GPE and alkylacyl-sn-glycero-3-phosphocholine (GPC) are strikingly similar with regard to arachidonate metabolism. When added to neutrophils, both 1-O-[3H]alkenyl-2-lyso-GPE and 1-O-[3H]alkyl-2-lyso-GPC are acylated predominantly with arachidonic acid, and the resulting arachidonoyl-containing phospholipids are extensively deacylated upon stimulation. However, hydrolysis of PE in the neutrophil differs from hydrolysis of choline-containing phosphoglycerides in that stimulation leads to a greater accumulation of the ethanolamine-linked lysophospholipid. A comparison of the molecular species of endogenous PE (based on molar concentrations measured as the dinitrophenyl derivative) from resting and stimulated neutrophils indicated that only those species which contain arachidonate are significantly hydrolyzed.     

Effect of arachidonic acid reacylation on leukotriene biosynthesis in human neutrophils stimulated with granulocyte-macrophage colony-stimulating factor and formyl-methionyl-leucyl-phenylalanine

Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B(4) and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca(2+)](i) induced by thimerosal in neutrophils stimulated with GM-CSF/fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca(2+)](i) was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A(2)alpha activation.     

Studies on the mechanism of platelet-activating factor production in GM-CSF primed neutrophils: involvement of protein synthesis and phospholipase A2 activation

GM-CSF regulates the growth of hemopoietic progenitor cells, enhances the responsiveness of mature PMN and primes these cells for synthesis of leukotrienes and PAF in response to secondary stimuli. The biochemical requirements for PAF production in GM-CSF primed PMN was examined using different metabolic inhibitors. GM-CSF stimulates uridine incorporation into RNA and inhibitors for RNA and protein synthesis decrease PAF synthesis in our model. This suggests a role for gene expression and de novo synthesis of proteins in the action of GM-CSF. Different PLA2 inhibitors, including a 9 amino-acid peptide derived from a conserved region of the calpactin superfamily, decrease PAF production, indicating that in GM-CSF primed PMN the chemotactic peptide fMLP triggers lipid mediator synthesis by activating PLA2.     

Enhancement of phorbol ester-induced protein kinase activity in human neutrophils by platelet-activating factor

We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipid-dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from gamma-32P-ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 +/- 9 pmoles 32P/10(7)PMN/minute in cells treated with 20 ng/ml PMA compared to 267 +/- 24 pmoles 32P in cells exposed to PMA and 10(-6)M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10(-7) M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calcium-dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.     

Evidence for a platelet-activating factor receptor on human lymphoblastoid B cells: activation of the phosphatidylinositol cycle and induction of calcium mobilization

In this report we demonstrate evidence which strongly suggests that a receptor for platelet-activating factor (PAF) exists on a lymphoblastoid B cell line, LA350. PAF ranging in concentration from 10(-6)-10(-9)M initiated the incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PI) with no change in phosphatidylethanolamine (PE) and phosphatidylcholine (PC) over baseline. Lyso-PAF, the inactive precursor, at 10(-7)M had no effect on membrane phospholipid metabolism. In addition, PAF from 10(-6)-10(-8)M when added to Fura-2 containing B cells induced a rapid and significant rise of calcium within the cell, with lyso-PAF having no effect. These data suggest that PAF binds to a receptor on B cells and induces the hydrolysis of PI and a subsequent increase of intracellular calcium.     

Phospholipase D activation by platelet-activating factor, leukotriene B4, and formyl-methionyl-leucyl-phenylalanine in rabbit neutrophils. Phospholipase D activation is involved in enzyme release.

Lipid chemoattractants, such as platelet-activating factor and leukotriene B4, as well as the peptide chemoattractant FMLP, were found to stimulate [3H]phosphatidic acid ([3H]PA) formation in 1-O-[3H]octadecyl-lyso platelet-activating factor-labeled rabbit neutrophils. The stimulation of [3H]PA formation appears to result from the activation of phospholipase D (PLD), because in the presence of ethanol, chemoattractant stimulation produced [3H]phosphatidylethanol, the characteristic compound produced by PLD at the expense of [3H]PA formation. The PLD activation by all chemoattractants tested was primed by cytochalasin B and revealed a similar time dependence. However, lipid chemoattractants were less potent as compared with FMLP, and the maximal stimulation by the former was lower than that by the latter. From these results, it is concluded that the mechanism of PLD activation by lipid chemoattractants is similar to, but different from, that by FMLP. Cytochalasin B stimulated degranulation and [3H]PA formation in agonist-stimulated neutrophils, and their stimulations were well correlated. Ethanol inhibited both agonist-stimulated [3H]PA formation and degranulation in a concentration-dependent manner, but the inhibition in degranulation was much less than that in [3H]PA formation. These results suggest that PLD activation is involved in degranulation, but another signaling pathway may also be required for full stimulation of degranulation. When the radiolabeled neutrophils were stimulated by chemoattractants for 5 min, 1,2-[3H]diglyceride was found to accumulate. The accumulation was inhibited by either ethanol or the phosphatidate phosphohydrolase inhibitor propranolol, which indicates that PA produced by PLD can be converted to 1,2-diglyceride by phosphatidate phosphohydrolase. Under these conditions, propranolol did not inhibit degranulation stimulated by chemoattractants. These results indicate that PA produced by PLD is more important than its metabolite diglyceride for the degranulation of rabbit neutrophils.