Arachidonic Acid Mobilization Research Articles

Chitosan-induced phospholipase A 2 activation and arachidonic acid mobilization in P388D 1 macrophages

We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose- and time-dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan-induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A 2 (PLA 2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA 2 inhibitor 3-(3-acetamide)-1-benzyl-2-ethylindolyl-5-oxy-propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage-activating factor that elicits AA mobilization in P388D 1 macrophages by a mechanism involving the participation of two distinct phospholipases A 2.

The role of eicosanoids in tumor growth and metastasis.

Mobilization of esterified arachidonic acid (AA) from membrane glycerolipid pools represents the key regulatory step in cellular responses to various stimuli, such as growth factors, cytokines, chemokines and circulating hormones. Released AA is metabolized via the cyclo-oxygenase (COX 1 and COX 2), lipoxygenase (LOX) or P450-dependent epoxygenase pathways to generate eicosanoids. In addition to their normal biological activities, such as stimulation of mitogenesis and cellular motility, eicosanoids have also been postulated to contribute to tumorigenesis and to the progression of certain tumor cells. Various AA metabolites have been implicated in a variety of growth-related signaling pathways involving ras (Han et al. 1991), interferon-α, epithelial growth factor (EGF), cyclic adenosine monophosphate, protein kinase C (PKC; Hannigan and Williams 1991; Peppelenbosch et al. 1993; Tang et al. 1995a), mitogen-activated kinases (Rao et al. 1988) and fos (Danesch et al. 1994). Numerous studies have demonstrated a strong correlation between growth-factor-promoted cell proliferation and generation of various COX products, primarily prostaglandins (Nolan et al. 1988).

Sphingosine 1-phosphate induces arachidonic acid mobilization in A549 human lung adenocarcinoma cells

In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca 2+-independent phospholipase A 2 (iPLA 2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA 2 activities.

Regulation of Arachidonic Acid Mobilization in Lipopolysaccharide-activated P388D1 Macrophages by Adenosine Triphosphate

Murine P388D(1) macrophages exhibit a delayed prostaglandin biosynthetic response when exposed to bacterial lipopolysaccharide (LPS) for prolonged periods of time that is dependent on induction of the genes coding for Group V secretory phospholipase A(2) and cyclooxygenase-2. We herein report that LPS-induced arachidonic acid (AA) metabolite release in P388D(1) macrophages is strongly attenuated by the P2X(7) purinergic receptor antagonists periodate-oxidized ATP and pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid, and this is accompanied by suppression of the expression of both Group V secretory phospholipase A(2) and cyclooxygenase-2. The effect appears to be specific for LPS, because the P2 purinergic receptor antagonists do not affect P388D(1) cell stimulation by other stimuli such as platelet-activating factor or the Ca(2+) ionophore A23187. Moreover, extracellular nucleotides are found to stimulate macrophage AA mobilization with a pharmacological profile that implicates the participation of the P2X(7) receptor and that is inhibited by periodate-oxidized ATP. Collectively these results demonstrate coupling of the P2X(7) receptor to the AA cascade in P388D(1) macrophages and implicate the participation of this type of receptor in LPS-induced AA mobilization.

Biosynthesis and turnover of anandamide and other N-acylethanolamines in peritoneal macrophages

Polyunsaturated N-acylethanolamines (NAEs), including anandamide (20:4 n−6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca 2+ ionophore A23187 had no effect on the production of NAE or its precursor N-acylphosphatidylethanolamine ( N-acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N-acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of 18O from H 2 18O-containing media into the amide carbonyls of both NAE and N-acyl PE demonstrated a rapid, constitutive turnover of both lipids.

Ca2+-Permeable AMPA Receptors Induce Phosphorylation of cAMP Response Element-Binding Protein through a Phosphatidylinositol 3-Kinase-Dependent Stimulation of the Mitogen-Activated Protein Kinase Signaling Cascade in Neurons

Ca(2+)-permeable AMPA receptors may play a key role during developmental neuroplasticity, learning and memory, and neuronal loss in a number of neuropathologies. However, the intracellular signaling pathways used by AMPA receptors during such processes are not fully understood. The mitogen-activated protein kinase (MAPK) cascade is an attractive target because it has been shown to be involved in gene expression, synaptic plasticity, and neuronal stress. Using primary cultures of mouse striatal neurons and a phosphospecific MAPK antibody we addressed whether AMPA receptors can activate the MAPK cascade. We found that in the presence of cyclothiazide, AMPA caused a robust and direct (no involvement of NMDA receptors or L-type voltage-sensitive Ca(2+) channels) Ca(2+)-dependent activation of MAPK through MAPK kinase (MEK). This activation was blocked by GYKI 53655, a noncompetitive selective antagonist of AMPA receptors. Probing the mechanism of this activation revealed an essential role for phosphatidylinositol 3-kinase (PI 3-kinase) and the involvement of a pertussis toxin (PTX)-sensitive G-protein, a Src family protein tyrosine kinase, and Ca(2+)/calmodulin-dependent kinase II. Similarly, kainate activated MAPK in a PI 3-kinase-dependent manner. AMPA receptor-evoked neuronal death and arachidonic acid mobilization did not appear to involve signaling through the MAPK pathway. However, AMPA receptor stimulation led to a Ca(2+)-dependent phosphorylation of the nuclear transcription factor CREB, which could be prevented by inhibitors of MEK or PI 3-kinase. Our results indicate that Ca(2+)-permeable AMPA receptors transduce signals from the cell surface to the nucleus of neurons through a PI 3-kinase-dependent activation of MAPK. This novel pathway may play a pivotal role in regulating synaptic plasticity in the striatum.

Regulation of Prostaglandin F2α by the Specific Inhibition of Secretory Type II Phospholipase A2: Implications for the Management of Premature Labour

Arachidonic acid mobilisation by phospholipase A₂ (PLA₂) and subsequent prostaglandin synthesis is thought to be a pivotal event in the onset and/or maintenance of human labour. The purpose of this study was to examine the efficacy of a monoclonal human type II PLA₂ antibody (10 B2) in reducing prostaglandin F_2α (PGF_2α) production in Chinese hamster ovary (CHO) cells (that overexpress human recombinant type II PLA₂) and in human placental explants. 10 B2 caused a concentration-dependent inhibition of PLA₂ activity (p < 0.00001) and PGF_2α release (p < 0.01) by CHO cells. 1 μM of 10 B2 inhibited PLA₂ activity (p < 0.00001) and PGF_2α production (p < 0.0001) in human placental explants. The latter effect was significantly reversed by the addition of arachidonic acid (30 μM; p < 0.01). On the basis of these findings, it is proposed that 10 B2 inhibits PGF_2α production by interfering with the extracellular activity of type II PLA₂.

Contribution of Type II PLA 2 to prostaglandin formation: A study using a Type II PLA 2 specific inhibitor SB 203347

Arachidonic acid (AA) mobilization by phospholipase A 2 (PLA 2) and subsequent prostaglandin synthesis is considered to be a pivotal event in inflammation. The purpose of this study was to assess the efficacy of a Type II PLA 2 specific inhibitor, SB 203347, in reducing prostaglandin production in Type II PLA 2-transfected Chinese hamster ovary (CHO) cells and in human placenta. In both experimental models uilised, Type II PLA 2 represents the principal isozyme contributing to total PLA 2 enzymatic activity. PLA 2 enzymatic activity released into cell culture media and placental explant media was quantified by radiolabelled substrate assay [ 14C-phosphatidylethanolamine (PE)]. Immunoreactive prostaglandin F 2α (PGF 2α) concentrations were determined by radioimmunoassay. SB 203347 (at 0.1–10 μM final concentration) inhibited PLA 2 enzymatic activity released by Zn ++-activated CHO cells by up to 60% ( P < 0.0001). The concentration of PGF 2α present in culture media was concomitantly reduced by up to 90% ( P < 0.0001). Similar results were observed for human placental explants. Treatment of human placental explants with SB 203347 (1 μM final concentration) significantly reduced PLA 2 enzymatic activity recovered in media after 24 h incubation ( P < 0.0001; n = 10). Incubation media PGF 2α concentrations were also reduced by 60% ( P < 0.00001). The addition of endogenous arachidonic acid (30 μM final concentration) significantly attenuated SB 203347-inhibition of PGF 2α release ( P < 0.01). The data obtained in this study are consistent with the hypothesis that Type II PLA 2 contributes to the liberation of arachidonic acid for prostanoid formation in human placenta and in cells that abundantly express this isozyme.

Role of prostaglandin H synthase isoforms in murine ear edema induced by phorbol ester application on skin

Topical application of TPA to a murine ear induced an edema that was accompanied by eicosanoid biosynthesis and an early enhancement of prostaglandin H synthase 2 (PGHS-2) expression. PGHS-2 induction may be correlated with the time-course of TPA-induced edema formation. Treatment with drugs that inhibit AA mobilization such as dexamethasone or manoalide or inhibitors of leukotriene formation such as zileuton or baicalein, reduced TPA-induced edema development and PGHS-2 levels. On the other hand, arachidonic acid (AA) application on the murine ear induced rapid expression of PGHS-2. This effect was not reproduced by other fatty acids such as oleic, linoleic, eicosatetraynoic or eicosapentaenoic acids. PGHS-2 expression induced by AA application was independent of PGHS and lipoxygenase metabolite synthesis. However, topical application of PGE 2 on skin induced PGHS-2 overexpression. This study suggests that AA release and/or subsequent metabolism by PGHS may be involved in the induction of PGHS-2 expression in murine TPA- and AA-induced ear oedema.

Regulation of Delayed Prostaglandin Production in Activated P388D1 Macrophages by Group IV Cytosolic and Group V Secretory Phospholipase A2s

Group V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6-24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2 (cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2 revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2 expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2.

N-ethylmaleimide-stimulated arachidonic acid release in human platelets

Treatment of human platelets with the alkylating agent N-ethylmaleimide (NEM) induces arachidonic acid release. The effect was time- and dose-dependent. NEM-stimulated arachidonic acid mobilisation could be prevented by pretreating platelets with the cytosolic phospholipase A 2 (cPLA 2)-specific inhibitor arachidonyltrifluoromethyl ketone. Moreover, the tyrosine kinase inhibitor genistein was able to significantly inhibit arachidonic acid mobilisation. NEM-stimulated release of arachidonic acid appears to be a Ca 2+-dependent mechanism, as shown by the observation that arachidonic acid mobilisation was significantly reduced by platelet treatment with EGTA and abolished by preloading platelets with the intracellular chelator 1,2- bis ( o-aminophenoxy) ethane- N, N, N′, N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM). In Fura-2-loaded platelets, NEM was able to significantly increase the intracellular Ca 2+ level. The Ca 2+ elevation was significantly reduced in the presence of EGTA and suppressed by cell treatment with BAPTA/AM. Arachidonic acid released by NEM produced a significant increase in reactive oxygen species (ROS) intracellular levels, which was partially inhibited by diphenyleneiodonium and almost completely suppressed by 5,8,11,14-eicosatetraynoic acid. In conclusion, the results in this study demonstrate that NEM stimulates arachidonic acid release by cPLA 2 activation through intracellular Ca 2+ elevation. In addition, tyrosine specific protein kinases seem to be involved in arachidonic acid release. ROS was also shown to be formed during arachidonic acid metabolisation.

Determination of phospholipase A2s expression in mast cells by reverse-transcriptase polymerase chain reaction.

The release of arachidonic acid (AA) from membrane phospholipids by phospholipase A2 (PLA2) is a critical step in the generation of lipid mediators by mast cells. AA released by PLA2 activity is metabolized to bioactive compounds, such as prostaglandins, thromboxane, and leukotrienes. During the last decade, several mammalian PLA2s have been shown to play a role in the mobilization of AA from phospholipids. At least ten different PLA2 types have been described (,). In general, these consist of secretory, cytosolic, or membrane bound activities.

Secretory Type II PLA2Immunoreactivity and PLA2Enzymatic Activity in Human Gestational Tissues Before, During and After Spontaneous-onset Labour at Term

Arachidonic acid mobilization by phospholipase A2(PLA2) and subsequent prostaglandin synthesis is thought to be a pivotal event in the onset and/or maintenance of human labour. The purpose of this study was to quantify secretory PLA2(sPLA2) and non-secretory PLA2enzymatic activity and type II sPLA2immunoreactivity in human gestational tissues before, during and after labour of spontaneous onset. Placental tissue and fetal membranes were collected from women before, during and after spontaneous-onset labour at term and stored at −80°C. PLA2activity in supernatants was quantified by radiolabelled substrate assay (14C-phosphatidylethanolamine) with and without 12.5mmdithiothreitol (DTT) to separate enzymatic activity contributed by secretory and non-secretory PLA2components. Immunoreactive type II PLA2in supernatants was determined by a monoclonal, non-competitive sandwich ELISA. Total PLA2enzymatic activity in amnion, choriodecidua and placenta was not significantly different before, during and after labour (n=18–20). Likewise, non-secretory enzymatic activity was not significantly different before (n=9), during (n=10) and after labour (n=9) in any of the three types of gestational tissue examined. Although immunoreactive type II PLA2was significantly higher in the placenta (P<0.01) compared to amnion and choriodecidua, there was no significant difference in immunoreactive type II PLA2within each tissue group according to labour status (n=18–20). Overall, no change in PLA2secretory or non-secretory enzymatic activity or immunoreactive type II PLA2could be detected throughout the peripartum period.

Anandamide synthesis is induced by arachidonate mobilizing agonists in cells of the immune system

The hypothesis that the capability of agents to mobilize arachidonic acid (AA) could predict increased anandamide (ANA) synthesis in a macrophage cell line has been examined. Lipopolysaccharide (LPS), platelet-activating factor (PAF) and cannabinoids such as Δ 9-tetrahydrocannabinol (THC) and anandamide were all found to be agonists for the release of AA and led to increased ANA synthesis in RAW264.7 mouse macrophage cells. Nitric oxide, in contrast, stimulated AA release without raising ANA levels. ANA stimulation of its own synthesis indicates the existence of a positive feedback mechanism. The possible involvement of the CB2 receptor in THC-mediated AA release and ANA synthesis is addressed using the antagonist SR144528. ANA synthesis is also increased by the combination of calcium ionophore and indomethacin, suggesting that ANA is metabolized by a cyclooxygenase in this system. The data imply that ANA could play a role in the response of the immune system to cannabinoids and bacterial endotoxins and that AA mobilization is a predictor for increased ANA synthesis.

Induction by interleukin-1β peptide of prostaglandin E 2 formation via enhanced prostaglandin H synthase-2 expression in 3T6 fibroblasts

Several synthetic interleukin-1 (IL-1) peptides were tested in vivo for pyrogenic activity and in vivo for their ability to stimulate prostaglandin production. Only the IL-1β fragment (208–240) enhanced body temperature, although both IL-1β (208–240) and IL-1α (223–250) stimulated prostaglandin E 2 (PGE 2) production in vitro. We report here that the IL-1β fragment (208–240) did not have the capacity to induce arachidonic acid (AA) mobilization by 3T6 fibroblasts. However, this peptide was able to increase the expression of the inducible prostaglandin H synthase isoform (PGHS-2; EC 1.14.99.1.), which is related to its ability to stimulate prostaglandin E 2 synthesis.

IL-13 increases the cPLA2 gene and protein expression and the mobilization of arachidonic acid during an inflammatory process in mouse peritoneal macrophages

Pretreatment of mouse peritoneal macrophages with interleukin-13 (IL-13) potentiates the mobilization of arachidonic acid (AA) and the production of HETEs but does not affect the production of cyclooxygenase metabolites triggered by the suboptimal concentration of an inflammatory agonist (opsonized-zymosan). Cycloheximide suppresses these effects of IL-13 suggesting that de novo protein synthesis is involved. Indeed, IL-13 induces a time-dependent increase in the levels of cytolosic PLA2 (cPLA2) protein and mRNA. This study demonstrates a new pathway for IL-13 to modulate the inflammatory process in macrophages via modifications of cPLA2 expression and subsequent AA mobilization.

Developmental maturation of baboon placental trophoblast: expression of messenger ribonucleic acid and protein levels of cytosolic and secretory phospholipases A2.

Although the human placenta at term exhibits high levels of phospholipase A2 (PLA2) enzyme activity, our understanding of the ontogenesis, regulation, and specific roles of placental phospholipases A2 remains relatively incomplete. Using the baboon as the experimental model, the present study determined whether the levels of the messenger ribonucleic acid (mRNA) for the cytosolic (cPLA2) and/or type IIa, nonpancreatic secretory (sPLA2) enzymes are developmentally regulated and modulated by glucocorticoid treatment. Total RNA was extracted from whole villous placenta obtained on days 60 (early; n = 3), 100 (mid; n = 3), and 165 (late; n = 4) of gestation (term = day 184) from untreated baboons and on day 100 (n = 4) after maternal administration on days 60-99 of betamethasone (3 mg/day). The complementary DNA to cPLA2 recognized a single 2.9-kb mRNA transcript in both baboon and human placenta. Mean (+/-SE) levels of cPLA2 mRNA, expressed, in arbitrary units as a ratio to that of beta-actin, were similar at early (0.19 +/- 0.05) and midgestation (0.34 +/- 0.17) and increased (P < 0.005) 10-fold (2.53 +/- 0.53) by late gestation. Levels of cPLA2 protein were also greater (P < 0.05) on day 165 (2.6 +/- 0.3 arbitrary units) than on day 60 (0.6 +/- 0.2). Like that in the human, the baboon placenta contained very high levels of a single 0.9-kb mRNA transcript for sPLA2. In contrast to that of cPLA2, normalized levels of sPLA2 mRNA were similar at all three time points and were associated with high levels of sPLA2 protein throughout gestation. Treatment with betamethasone increased (P < 0.02) cPLA2 mRNA levels on day 100 by over 4-fold, but had no effect on sPLA2 mRNA levels. Additional studies indicated that the mRNAs for sPLA2 and cPLA2 were detected in an enriched fraction of nontrophoblast cells isolated by collagenase dispersion and Percoll density centrifugation. The mRNA for cPLA2 was also expressed in cytotrophoblast and syncytiotrophoblast cell fractions. Collectively, these findings indicate that the baboon placenta expresses mRNA and protein for both the cytosolic and secretory PLA2 enzymes, and that expression of cPLA2 is developmentally regulated and modulated by glucocorticoids. We previously demonstrated an estrogen-dependent developmental increase in placental expression of specific components of the progesterone steroidogenic pathway during the second half of baboon pregnancy. The current findings indicate that the developmental increase in placental function also includes expression of at least one specific PLA2 enzyme controlling arachidonic acid mobilization and eicosanoid synthesis.

Functional coupling between secretory phospholipase A2 and cyclooxygenase-2 and its regulation by cytosolic group IV phospholipase A2.

Secretory phospholipase A2 (sPLA2) is the major effector involved in arachidonic acid (AA) mobilization and prostaglandin E2 (PGE2) production during stimulation of P388D1 macrophages with the inflammatory stimuli bacterial lipopolysaccharide and platelet-activating factor. We herein demonstrate that PGE2 in stimulated P388D1 cells is accounted for by the inducible cyclooxygenase (COX)-2. COX-1, though present, appears not to participate significantly in stimulus-induced PGE2 production in P388D1 macrophages. Reconstitution experiments utilizing exogenous recombinant sPLA2 demonstrate that activation of the sPLA2 at the plasma membrane is highly dependent on previous activation of the cytosolic phospholipase A2 (cPLA2). Collectively these results demonstrate (i) that functional coupling exists between sPLA2 and COX-2 in activated cells, (ii) the critical role that cPLA2 plays in lipid mediator production, and (iii) that there is crosstalk between cPLA2 and sPLA2 in the cell.

Convulxin Induces Platelet Activation by a Tyrosine-Kinase-Dependent Pathway and Stimulates Tyrosine Phosphorylation of Platelet Proteins, Including PLCγ2, Independently of Integrin αIIbβ3

Convulxin (Cvx) is a well-characterized platelet aggregating glycoprotein isolated fromCrotalus durissus terrificusandC. d. cascavellavenoms. In the present report we show that Cvx induces tyrosine phosphorylation of human platelet proteins, including phospholipase C-γ 2 (PLCγ2), and also stimulates [3H]arachidonic acid ([3H]AA) mobilization, pleckstrin phosphorylation, and an increase in the cytosolic Ca2+concentration ([Ca2+]in) due to both Ca2+entry and internal Ca2+mobilization. Staurosporine, a potent protein kinase inhibitor, and genistein, a specific inhibitor of protein tyrosine kinases (PTK), were used to evaluate the role of protein tyrosine phosphorylation (PTP) in the signal transduction evoked by Cvx. Staurosporine and genistein inhibited in a dose-dependent manner platelet aggregation induced by Cvx. Both inhibitors significantly blocked to near basal levels breakdown of phosphatidylinositol 4,5-bisphosphate from [myo-2-3H]inositol-labeled platelets and the production of [3H]AA metabolites from [3H]AA-labeled platelets after challenge with Cvx. Cvx provokes an increase in [Ca2+]inin Fura-2-loaded platelets that was abolished by concentrations of staurosporine which also inhibited Cvx-induced platelet aggregation. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelets proteins with molecular masses of 40, 72/74, 78/80, 105, 120, and 145 kDa, followed by dephosphorylation. Furthermore, Cvx stimulates a rapid tyrosyl phosphorylation of a 145-kDa molecular mass protein that was identified as PLCγ2. PTP induced by Cvx was not inhibited when platelets were stimulated in the presence of indomethacin, apyrase, EDTA, or RGDS peptide. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and is independent of aggregation or fibrinogen binding to the integrin αIIbβ3. On the other hand, the dephosphorylation step is inhibited by RGDS peptide or EDTA, suggesting that integrin αIIbβ3is envolved in this step. The profile obtained with Cvx resembles that obtained in platelets adherent to an immobilized ligand, such as immobilized collagen, in which PTP is independent on integrin αIIbβ3. Thus, we suggest that Cvx is an example of a protein with adhesion molecule-like properties; i.e., it is an adhesin. In conclusion, our results show that Cvx induces multiple signaling pathways in platelets via a PTK-dependent pathway involving PLCγ2 tyrosyl phosphorylation, with the subsequent platelet responses. Cvx is unique among platelet soluble agonists because under test tube stirring conditions it induces a PTP profile independently of integrin αIIbβ3.

Leukotriene B4 Activates the NADPH Oxidase in Eosinophils by a Pertussis Toxin-Sensitive Mechanism That Is Largely Independent of Arachidonic Acid Mobilization

Experiments were designed to investigate whether leukotriene (LTB4) receptors can couple directly to phospholipase A2 (PLA2) in guinea pig eosinophils and the role of endogenous arachidonic acid (AA) in LTB4-induced activation of the NADPH oxidase. LTB4 (EC50 approximately 16 nM) and AA (EC50 approximately 6 microM) generated hydrogen peroxide (H2O2) in a concentration-dependent manner and at an equivalent maximum rate (5-6 nmol/min/10(6) cells). LTB4 stimulated PLA2 over a similar concentration range that activated the NADPH oxidase, although kinetic studies revealed that the release of [3H]AA (t1/2 approximately 2 s) preceded H2O2 generation (t1/2 > 30 s). Pretreatment of eosinophils with pertussis toxin abolished the increase in inositol(1,4,5)trisphosphate mass, [Ca2+]c, [3H]AA release, and H2O2 generation evoked by LTB4. Qualitatively identical results were obtained in eosinophils in which phospholipase C (PLC) was desensitized by 4beta-phorbol 12,13-dibutyrate with the exception that [3H]AA release was largely unaffected. Additional studies performed with the protein kinase C inhibitor, Ro 31-8220, and under conditions in which Ca2+ mobilization was abolished, provided further evidence that LTB4 released [3H]AA independently of signal molecules derived from the hydrolysis of phosphatidylinositol(4,5)bisphosphate by PLC. Pretreatment of eosinophils with the PLA2 inhibitor, mepacrine, abolished LTB4-induced [3H]AA release at a concentration that inhibited H2O2 by only 36%. Collectively, the results of this study indicate that agonism of LTB4 receptors on guinea pig eosinophils mobilizes AA by a mechanism that does not involve the activation of PLC. In addition, although LTB4 effectively stimulated PLA2, a central role for AA in the activation of the NADPH oxidase was excluded.