Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that Do and Do not mobilize calcium. Novel role of mitogen-activated protein kinase pathways in cytosolic phospholipase A2 regulation.

The 85-kDa cytosolic phospholipase A(2) (cPLA(2)) mediates agonist-induced arachidonic acid release and eicosanoid production. Calcium and phosphorylation on Ser-505 by mitogen-activated protein kinases (MAPKs) regulate cPLA(2). Arachidonic acid release and eicosanoid production induced by stimuli that do (A23187, zymosan) or do not (phorbol myristate acetate (PMA), okadaic acid) mobilize calcium were quantitatively suppressed in cPLA(2)-deficient mouse peritoneal macrophages. The contribution of MAPKs to cPLA(2)-mediated arachidonic acid release was investigated. Both extracellular signal-regulated kinases (ERKs) and p38 contributed to cPLA(2) phosphorylation on Ser-505. However, although ERK inhibition did not affect A23187-induced arachidonic acid release, it suppressed zymosan-, PMA-, and okadaic acid-induced arachidonic acid release under conditions where phosphorylation of cPLA(2) on Ser-505 was unaffected. This indicates an additional regulatory mechanism for the ERK pathway. A role for transcriptional regulation is suggested by data showing that cycloheximide and actinomycin D inhibited arachidonic acid release induced by zymosan, PMA and, okadaic acid but not by A23187. Our results show that MAPK pathways contribute to arachidonic acid release in macrophages through alternative mechanisms in addition to their ability to phosphorylate cPLA(2) on Ser-505 and suggest a role for new protein synthesis.

Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.

Regulation of arachidonic acid release and cytosolic phospholipase A2 activation.

The 85-kDa cytosolic PLA2 (cPLA2) mediates agonist-induced arachidonic acid release in many cell models, including mouse peritoneal macrophages. cPLA2 is regulated by an increase in intracellular calcium, which binds to an amino-terminal C2 domain and induces its translocation to the nuclear envelope and endoplasmic reticulum. Phosphorylation of cPLA2 on S505 by mitogen-activated protein kinases (MAPK) also contributes to activation. In macrophages, zymosan induces a transient increase in intracellular calcium and activation of MAPK, which together fully activate cPLA2 and synergistically promote arachidonic acid release. There are alternative pathways for regulating cPLA2 in macrophages because PMA and okadaic acid induce arachidonic acid release without increasing calcium. The baculovirus expression system is a useful model to study cPLA2 activation. Sf9 cells expressing cPLA2 release arachidonic acid to either A23187 or okadaic acid. cPLA2 is phosphorylated on multiple sites in Sf9 cells, and phosphorylation of S727 is preferentially induced by okadaic acid. However, the phosphorylation sites are non-essential and only S505 phosphorylation partially contributes to cPLA2 activation in this model. Although okadaic acid does not increase intracellular calcium in Sf9 cells, calcium binding by the C2 domain is necessary for arachidonic acid release. A23187 and okadaic acid activate cPLA2 by different mechanisms, yet both induce translocation to the nuclear envelope in Sf9 cells. The results demonstrate that alternative regulatory pathways can lead to cPLA2 activation and arachidonic acid release.

The role of calcium and phosphorylation of cytosolic phospholipase A2 in regulating arachidonic acid release in macrophages.

Arachidonic acid release is induced in macrophages with diverse agonists including calcium ionophores, phorbol myristate acetate (PMA), okadaic acid, and the phagocytic particle, zymosan, and correlates with activation of cytosolic phospholipase A2 (cPLA2). The role of calcium and phosphorylation of cPLA2 in regulating arachidonic acid release was investigated. Zymosan induced a rapid and transient increase in [Ca2+]i. This in itself is not sufficient to induce arachidonic acid release since ATP and platelet activating factor (PAF), agonists that induce transient calcium mobilization in macrophages, induced little arachidonic acid release. Unlike zymosan, which is a strong activator of mitogen-activated protein kinase (MAPK), ATP and PAF were weak MAPK activators and induced only a partial and transient increase in cPLA2 phosphorylation (gel shift). However, ATP or PAF together with colony stimulating factor-1 (CSF-1) synergistically stimulated arachidonic acid release. CSF-1 is a strong MAPK activator that induces a rapid and complete cPLA2 gel shift but not calcium mobilization or arachidonic acid release. Arachidonic acid release was more rapid in response to CSF-1 plus ATP or PAF than zymosan and correlated with the time course of the cPLA2 gel shift. Although low concentrations of ionomycin induced a lower magnitude of calcium mobilization than ATP, the response was more sustained resulting in arachidonic acid release. A23187 and ionomycin induced weak MAPK activation, and a partial and transient cPLA2 gel shift. The MAPK kinase inhibitor, PD 98059 suppressed A23187-induced MAPK activation and cPLA2 gel shift but had little effect on arachidonic acid release. These results indicate that in macrophages a transient increase in [Ca2+]i and sustained phosphorylation of cPLA2 can act together to promote arachidonic acid release but neither alone is sufficient. A sustained increase in calcium is sufficient for inducing arachidonic acid release. However, PMA and okadaic acid induce arachidonic acid release without increasing [Ca2+]i, although resting levels of calcium are required, suggesting alternative mechanisms of regulation.

Phospholipase A2 from plasma of patients with septic shock is associated with high-density lipoproteins and C3 anaphylatoxin: some implications for its functional role.

Phospholipase A2 (PLA2) activity was purified 12,544-fold with a 13% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtration, and reverse-phase f.p.l.c. Gel-filtration chromatography of plasma omitting high-ionic-strength buffer revealed a molecular mass different from that of purified PLA2 and co-elution with apolipoprotein A-I peaks, which suggests its association with high-density lipoproteins (HDL). N-terminal analysis of the enzyme activity protein band, electroblotted from a SDS-acrylamide gel and with an assessed molecular mass of 19 kDa, showed an identical sequence to that of alpha-chain of human C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA2 linked side-by-side. Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from fibroblasts transfected with the coding sequence of human group-II PLA2, and because the addition of C3-derived anaphylatoxins from human serum inhibited the activity of this recombinant PLA2, it was considered that C3a-related peptides behave as inhibitors of group-II PLA2. The enzyme showed optimal activity on [14C]oleate-labelled autoclaved E. coli, on synthetic phosphatidylethanolamine, and on [3H]arachidonate-labelled membranes of the monoblast cell line U937, but it did not show any activity on the release of [3H]arachidonate from pre-labelled human polymorphonuclear leukocytes (PMNs). In short, PLA2 from plasma of sepsis patients shows unique associations with other plasma proteins which may influence its functional properties. The association with C3-related peptides shows an inhibitory effect on the enzyme activity, whereas the association with HDL might influence its environment and/or its interaction with cells. The study of the catalytic properties shows a prominent effect on bacterial phospholipids, synthetic phosphatidylethanolamine, and membranes from U937 monoblasts, but not on synthetic phosphatidylcholine or on PMNs, even when these cells were maintained in culture to allow spontaneous apoptosis and became a good substrate for pancreatic type PLA2.

Effect of immunological stimulation on the production of platelet-activating factor by rat peritoneal cells: its relevance to anaphylactic reactions.

The production of platelet-activating factor (PAF) by rat peritoneal cells was studied using as stimuli either monoclonal IgE, IgG1 or IgG2b anti-DNP (2,4-dinitrophenyl), and DNP-BSA. Peritoneal cells sensitized in vitro with any of these antibodies at concentrations higher than 10 nM and challenged with 1 microM DNP-BSA produced PAF. PAF production was also elicited by preformed IgE/ and IgG2b/DNP-BSA immune complexes, preferentially at a large antigen/antibody ratio. The production of PAF was unrelated to the activation of mast cells, since it occurred in populations depleted of mast cells by adherence to plastic dishes. Moreover, the release of [3H]serotonin from IgE-sensitized mast cells showed a time-course more rapid than PAF production and occurred in cells sensitized with IgE at concentrations lower than those required for PAF formation. In contrast, peritoneal cells sensitized with IgG1 and IgG2b failed to release [3H]serotonin. Rat peritoneal cells showed a significant ability to catabolize PAF by intracellular PAF-acetylhydrolase in view of both the amounts of enzyme activity assayed in cellular homogenates, and the 15-fold increase on controls of PAF quantities detected in peritoneal cells treated with phenylmethylsulfonyl fluoride (PMSF), a known inhibitor of PAF-acetylhydrolase. The PAF activity produced upon PMSF addition showed a retention time on reverse-phase HPLC which suggests structural identity to PAF produced by either immunological challenge or ionophore A23187. These data suggest that PAF formed during rat passive anaphylaxis reactions depends on the activation of mononuclear phagocytes. This production may be triggered by two types of low affinity receptors: Fc epsilon RII/CD23 and Fc gamma R. The ability of peritoneal cells to catabolize PAF by intracellular acetylhydrolase seems unaffected by immunological stimulation.

The role of platelet-activating factor and peptidoleukotrienes in the vascular changes of rat passive anaphylaxis.

1. The role of platelet-activating factor (PAF) and peptidoleukotrienes as putative mediators of some of the vascular changes triggered by antigen was investigated in rats passively sensitized with monoclonal anti-DNP (2,4-dinitrophenyl) IgE. 2. Lethal anaphylaxis with respiratory distress, systemic hypotension, detachment of the intestinal mucosa, leukopenia and extravasation of protein-rich plasma was observed after antigen challenge of rats sensitized with partially purified monoclonal IgE at concentrations of 15 mg protein kg-1. 3. Analysis of the peritoneal fluid obtained after i.v. challenge with DNP-BSA (bovine serum albumin) showed the presence of significant amounts of PAF (101 +/- 8 pg/rat), whereas this mediator was undetectable in control animals. Leukotriene D4 was the predominant peptidoleukotriene that could be recovered after antigen challenge, and showed an extremely high concentration (92 +2- 15 ng/rat) as compared to PAF levels. 4. Extravasation of protein-rich plasma was observed shortly after challenge and reached a maximum at 30 min. Treatment of animals with i.v. PCA 4248 (1-2 mg kg-1) and WEB 2086 (1 mg kg-1), two chemically unrelated compounds which are antagonists of the PAF-receptor, produced a significant reduction of the extravasation of protein-rich plasma. 5. The same degree of protection could be afforded by MK-886, an inhibitor of leukotriene biosynthesis. Combined treatment with WEB 2086 and MK-886 provided greater inhibition of protein-rich plasma extravasation than either compound alone. PCA 4248 was also found to inhibit in a dose-dependent manner the systemic hypotension observed upon DNP-BSA challenge.6. These data indicate that the lipid mediators PAF and peptidoleukotrienes are major effectors of the vascular disturbances observed in rat passive IgE-mediated anaphylaxis.

Metabolism of platelet-activating factor in human haematopoietic cell lines. Differences between myeloid and lymphoid cells.

The binding and metabolism of platelet-activating factor (PAF) was studied in human cell lines resembling myeloid cells (HL60 and U937) and B and T lymphocytes (Daudi and Jurkat). All of the cell lines were found to bind and catabolize exogenous [3H]PAF in a time- and temperature-dependent manner. PAF binding could also be demonstrated in isolated membrane fractions, which provides further evidence of the existence of true membrane receptors. Myeloid cell lines contained numbers of receptors at least 10-fold higher than in lymphoid cell lines. Biosynthesis of PAF upon challenge by ionophore A23187 could be demonstrated in HL60 and U937 cells. In contrast, lymphoid cell lines were unable to produce PAF. Incubation with [14C]acetate showed incorporation of the label into three main fractions: neutral lipids, phosphatidylcholine and PAF, but the distribution of the label varied depending on the cell line. Significant incorporation into phosphatidylcholine was observed in uninduced myeloid cell lines. A phospholipase A2 acting on 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine and an acetyl-CoA:lyso-PAF acetyltransferase were expressed in the HL60 cell line and showed variations in specific activity with granulocytic differentiation. In contrast, these enzyme activities were not expressed in Daudi and Jurkat cell lines. These data indicate (1) the occurrence of PAF binding and catabolism in both myeloid and lymphoid cell lines; (2) the restriction of PAF biosynthesis to myeloid cell lines, especially HL60 cells; (3) the occurrence of differentiation-elicited changes in the specific activities of the enzymes involved in PAF biosynthesis by the remodelling pathway; and (4) the central role played by the disposal of lyso-PAF, a product of the phospholipase A2 reaction, in PAF biosynthesis.

1,4-Dihydropyridines, a new class of platelet-activating factor receptor antagonists: in vitro pharmacologic studies.

PCA 4233 [2-(phenylthio)ethyl-5-ethoxycarbonyl-2,4,6-trimethyl- 1,4-dihydropyridine-3-carboxylate] and PCA 4248 [2-(phenylthio) ethyl-5-methoxycarbonyl-2, 4, 6-trimethyl-1, 4-dihydropyridine-3-carboxylate], two compounds developed from a series of 1,4-dihydropyridines that lack pharmacologic effects on voltage-operated calcium channels, were found to block selectively rabbit operated calcium channels, were found to block selectively rabbit and human platelet aggregation and secretion, and binding of [3H]-labeled platelet-activating factor (PAF) to human platelet and polymorphonuclear PAF receptors. Rabbit platelet aggregation was tested with 1.9 nM PAF, i.e., a concentration producing maximal response, and was completely blocked with 10 microM PCA 4233 and 3 microM 4248 (IC50 values, 2.55 and 1.05 microM, respectively). Human platelet aggregation in platelet-rich plasma was studied with 1 microM PAF, a concentration that caused a response comparable with that of 1.9 nM PAF in rabbit platelets. The IC50 of PCA 4248 for ATP release under these conditions was 3.6 microM. PCA 4248 behaved as a competitive and selective antagonist in [3H]serotonin secretion studies on rabbit platelets, since it displaced rightwards log dose-response curves and lacked any effect on thrombin- and ionophore A23187-induced platelet secretion. A pA2 value of 7.5 was obtained from Schild plots on [3H]serotonin secretion studies. PCA 4248 also produced a dose-dependent inhibition of [3H]PAF binding to human platelets and to human polymorphonuclear leukocytes. These data indicate that PCA 4233 and PCA 4248 belong to a new class of selective PAF-receptor antagonists.

Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. The role of lyso-PAF disposal and free arachidonic acid.

Theophylline and 1-methyl-3-isobutylxanthine (MIX), compounds that block eicosanoid formation and modulate phospholipase A2 activity, inhibited in a dose-dependent manner the formation of both leukotriene B4 (LTB4) and platelet-activating factor (PAF) by human polymorphonuclear leucocytes (PMN) in response to ionophore A23187. Theophylline and MIX lacked any inhibitory effect on acetyl-CoA: lyso-PAF acetyltransferase activity, which is the rate-limiting step for PAF biosynthesis in PMN. The effect of theophylline and MIX on PAF formation could be reversed by incubating the cells in the presence of 1-10 microM exogenous lyso-PAF. Incubation of PMN homogenates in the presence of unsaturated non-esterified fatty acids resulted in dose-dependent inhibition of the acetyltransferase. This effect was linked to the presence of a free carboxyl group, since both arachidonic acid methyl ester and palmitoyl-arachidonoyl phosphatidylcholine lacked inhibitory activity. This inhibitory effect was also dependent on the number of double bonds, since arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5) displayed maximal effect. Kinetic analysis showed that the effect of arachidonic acid was consistent with competitive inhibition, with a Ki value of about 19 microM. Oxidative metabolites of arachidonic acid showed a lesser inhibitory effect with the following order of potency: arachidonic acid greater than 15-HETE (15-hydroxy-6,8,11,14-eicosatetraenoic acid) greater than LTB4 greater than 5-HETE (5-hydroxy-6,8,11,14-eicosatetraenoic acid) greater than lipoxin A4. Examination of enzymes involved in CoA-dependent acylation revealed a low activity of both arachidonoyl-CoA synthetase and arachidonoyl-CoA: lyso-PAF arachidonoyltransferase. These data indicate a strong influence on PAF biosynthesis of the products of the phospholipase A2 reaction, with lyso-PAF disposal being a critical event for PAF formation, and unsaturated fatty acids acting as feed-back inhibitors. The conversion of arachidonic acid via oxidative metabolism into less active inhibitors of acetyl-CoA:lyso-PAF acetyltransferase seems to be an additional mechanism of modulation of this enzyme activity, linked to the function of lipoxygenases. Finally, the enzyme activities involved in arachidonoyl-CoA-dependent acylation of lyso-PAF show a low efficiency in capturing arachidonic acid.

Occupancy of platelet receptors for platelet-activating factor in patients with septicemia.

The possible involvement of platelet-activating factor (PAF) in the pathogenesis of endotoxemia, was investigated by using a binding assay to patients' platelets, complemented with the extraction and chemical characterization of PAF obtained from patients' platelets. Platelets from 12 human volunteers had 281 +/- 63 freely accessible high affinity binding sites (PAF-receptors) per platelet; whereas this number was of 49 +/- 37 PAF-receptors per platelet, n = 14 samples, P less than 0.01, in a group of 13 patients with positive blood culture. A group of patients with respiratory or cardiovascular disturbances and negative blood culture had 253 +/- 74, accessible receptors per platelet (n = 19 samples from 16 patients, P less than 0.01 as compared to septic patients, which was not significantly different when compared to control individuals). Patients with sepsis possessed significant amounts of PAF associated to their platelets, whereas this mediator could not be isolated from platelets of patients with respiratory or cardiovascular disturbances and negative blood culture, nor from platelets of control individuals. PAF was also assayed in whole blood samples and found at high concentrations in sepsis patients. These data indicate that occupancy of PAF receptors in combination with high amounts of platelet-associated PAF, is a common finding in patients with sepsis.

Role of complement-derived peptides in thrombocytopenia elicited by soluble aggregates of immunoglobulin G in the rat.

The variations in platelet counts upon intravenous challenge with soluble aggregates of IgG were assessed in normal rats. A time- and dose-dependent thrombocytopenia, followed by recovery to preinfusion values after 30 minutes was observed. Rats injected with immune aggregates showed an increase in plasma levels of immunoreactive thromboxane B2, however, this increase was delayed as compared with the peak level of the thrombocytopenia. Previous treatment of rats with either indomethacin or aspirin, inhibited thromboxane B2 release, but did not affect thrombocytopenia. Pretreatment of the animals with BN 52021, a potent antagonist of platelet-activating factor binding to its receptor, also failed to block thrombocytopenia. Complement depletion by prior treatment with cobra venom factor, caused a significant reduction of the thrombocytopenia, whereas DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, an inhibitor of carboxypeptidase N, potentiated the thrombocytopenia elicited by submaximal doses of either IgG aggregates or a homogeneous preparation of rat anaphylatoxin containing C5a. In addition, rats challenged with doses of IgG aggregates higher than 5 mg/kg showed a massive complement consumption coincident with the onset of thrombocytopenia. "In vitro" aggregation/secretion experiments with rat platelets showed little platelet-stimulating activity either by aggregated IgG through the Fc receptor or through the CR1 receptor. By contrast, a preparation of rat serum anaphylatoxins containing C5a, showed a high platelet-secreting activity. These data suggest that a complement-derived peptide(s), most probably C5a, is one of the effector substances for platelet activation in response to soluble aggregates of IgG.

Biosynthesis of platelet-activating factor in glandular gastric mucosa. Evidence for the involvement of the 'de novo' pathway and modulation by fatty acids.

The biosynthesis of platelet-activating factor (PAF), a phospholipid autocoid with potent ulcerogenic properties that is produced in secretory exocrine glands by physiological secretagogues, was assessed in microsomal preparations of glandular gastric mucosa. For this purpose, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase (EC 2.3.1.67); the enzymes of the 'de novo' pathway: 1-O-alkyl-2-lyso-sn-glycero-3-phosphate (alkyl-lyso-GP):acetyl-CoA acetyltransferase and 1-O-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-choline cholinephosphotransferase (EC 2.7.8.16); and some enzymes involved in the catabolism of PAF and lyso-PAF were assayed. Only the enzymes of the 'de novo' pathway and small amounts of PAF acetylhydrolase, phospholipase A2 and a lysophospholipase D acting on either lipids could be detected in the gastric preparations, whereas lyso-PAF:acetyl-CoA acetyltransferase activity was undetectable. The specific activity of alkyl-lyso-GP:acetyl-CoA acetyltransferase in the gastric mucosa was about one-tenth of that found in spleen microsomes and its apparent Km for acetyl-CoA was 454 microM compared with 277 microM in spleen microsomes. Glandular mucosa homogenates contained preformed PAF at a concentration of 2.7 +/- 0.7 ng equivalents of PAF (hexadecyl)/mg of protein. When gastric microsomes were incubated with micromolar concentrations of fatty acids (arachidonic, palmitic and oleic) prior to the assay of dithiothreitol (DTT)-insensitive cholinephosphotransferase, a dose-dependent reduction in the formation of PAF was observed, arachidonic acid being the most potent inhibitor, followed by linoleic acid (only tested on spleen microsomes) and oleic acid. By contrast, 1,2-diolein and phosphatidylcholine (dipalmitoyl) showed no or little effect. These results indicate that glandular gastric mucosa can produce PAF through the 'de novo' pathway, and that fatty acids, especially unsaturated, can reduce that synthesis by modulating the expression of DTT-insensitive cholinephosphotransferase.