Tumour necrosis factor (cachectin) induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells.

Tumour necrosis factor (TNF) is an important mediator of endotoxin-induced vascular collapse and other inflammatory reactions. Eicosanoids have been implicated in the pathogeensis of these responses. In order to explore further the potential interactions between TNF and eicosanoid metabolism in eliciting vascular responses, we studied the effects of TNF on the bovine endothelial cell line CPAE. TNF induced cellular retraction observed by light microscope. This morphological change was monitored by the passage of iodinated protein A between adjacent cells and by release of [3H]arachidonic acid metabolites from cells. Both the morphological and functional responses were abrogated by inhibition of eicosanoid synthesis with BW755c. The release of [3H]arachidonic acid metabolites appeared to be mediated by a transient increase in phospholipase A2 activity. Phospholipase C activity was not affected by TNF. The maximal increase in phospholipase A2 activity occurred at 5 min following the addition of TNF. Phospholipase A2 activation, [3H]arachidonic acid-metabolite synthesis and passage of iodinated protein A, required both RNA and protein synthesis and were associated with an increase in the synthesis of a recently described phospholipase A2-activating protein. The Bordetella pertussis toxin, islet-activating protein, also inhibited the increase in phospholipase A2 activity, the release of [3H]arachidonic acid metabolites and the passage of iodinated protein A, suggesting that the TNF receptor-ligand interaction resulting in cellular retraction, phospholipase A2 activation and eicosanoid synthesis, is coupled through the Ni guanine nucleotide regulatory protein in these cells.     

Defining the role of protein kinase c in calcium-ionophore-(A23187)-mediated activation of phospholipase A2 in pulmonary endothelium.

We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 microM and 2.5 microM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At concentrations of 5-12.5 microM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose-dependent activation of membrane-bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5-12.5 microM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4 alpha-phorbol 12 beta-myristate 13 alpha-acetate (4 alpha-PMA) and an active phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (4 beta PMA). Neither 4 alpha-PMA nor 4 beta-PMA affected basal arachidonic acid release. 4 alpha-PMA also did not augment the effects of A23187. In contrast, 4 beta-PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A23187. Under these conditions, sphingosine completely inhibits the stimulatory effects of 4 beta-PMA on protein kinase C translocation, phospholipase A2 and arachidonic acid release. Thus, at low doses (1 microM and 2.5 microM) A23187 increases phospholipase A2 activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane-bound protein kinase C with 4 beta-PMA causes a synergistic increase in phospholipase A2 activity and arachidonic acid release. At higher doses (5-12.5 microM), A23187 acts in large part by stimulating protein kinase C translocation. Overall, our results indicate that activating membrane-bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A2 activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium.     

Arachidonic acid releasing systems in pig aorta endothelial cells

Endothelial cells synthesize prostacyclin both from platelet-derived endoperoxides and from the arachidonic acid released from its intracellular stores. The mechanisms controlling this release does not appear to be mediated through phospholipid methylation but by means of phosphoinositide hydrolysis. As yet two possible mechanisms have so far been proposed to regulate arachidonic acid release in a number of cellular systems: phospholipase C-controlled phospholipase A2 activity or phospholipase C-diglyceride lipase system. The results presented here show that using phospholipases inhibitors is not a reliable strategy to study arachidonic acid release in cultures of endothelial cells. Our data also strongly suggest that the release of prostacyclin may be accounted in these cells for by a phospholipase C-diglyceride lipase system.     

Inhibition of prostacyclin synthesis in endothelial cells by methylisobutylxanthine is not mediated through elevated cAMP level

Methylisobutylxanthine (MIX) raised cAMP levels and inhibited prostacyclin synthesis and arachidonic acid release in endothelial cells from both pig aorta and human umbilical vein. These effects were reversible and dose dependent on MIX concentrations. Dibutyryl cAMP (3 mM) alone did not inhibit prostacyclin synthesis or arachidonic acid release. When added with MIX, dibutyryl cAMP did not enhance the inhibition elicited by MIX. MIX inhibited the formation of lysophospholipids, 1,2-diacylglycerol and phosphatidic acid in bradykinin-stimulated pig endothelial cells, suggesting that the inhibition of prostacyclin synthesis resulted from an apparent inhibition of both phospholipase A2 and phospholipase C. Other phosphodiesterase inhibitors, theophylline and mopidamole, also raised cAMP levels and inhibited arachidonic acid release. However, there was no correlation between cAMP levels and these inhibitions. Forskolin, an adenylate cyclase activator, elevated intracellular cAMP levels with no apparent inhibition on prostacyclin synthesis. We conclude that the inhibitory effect of MIX on phospholipase A2 and phospholipase C is probably through mechanisms other than the elevation of the cAMP level.     

Characterization of arachidonic acid-induced apoptosis

Tumor necrosis factor (TNF) can induce apoptosis in a number of different cell types. This response often depends on the activity of cytosolic phospholipase A₂ (cPLA₂), which catalyzes the release of arachidonic acid from the sn-2 position of membrane phospholipids. In this study, we investigate the ability of arachidonic acid itself to cause cell death. We show that in assays with 10% fetal bovine serum (FBS) arachidonic acid will not kill, nor does act synergistically with TNF. In contrast, by lowering the concentration of FBS to 2% it is possible to use arachidonic acid to induce cell death. Arachidonic acid-induced cell death was judged to be apoptotic based on morphology and the cleavage of poly (ADP) ribose polymerase. Arachidonic acid was able to kill all cell lines tested including two human melanoma-derived cell lines, and susceptibility to arachidonic acid was not influenced by adenovirus gene products that control susceptibility to TNF. Finally, we show that arachidonic acid is unique among 20 carbon fatty acids for its ability to induce apoptosis and that several other unsaturated, but not saturated fatty acids can also induce apoptosis.     

Differential effects of aspirin and dexamethasone on phospholipase A2 and C activities and arachidonic acid release from endothelial cells in response to bradykinin and leukotriene D4

The CPAE bovine endothelial cell line may be stimulated to produce eicosanoids. Leukotriene D4 increased the release of arachidonic acid primarily by activating phospholipase A2 while bradykinin activated the phospholipase C pathway. Cells pretreated with dexamethasone, a phospholipase A2 inhibitor, no longer responded to stimulation by LTD4 but did release arachidonic acid when treated with bradykinin. Aspirin blocked bradykinin-stimulated production of arachidonic acid but left the response to LTD4 unaffected. We conclude that these cells produce eicosanoids by activation of both PLA2 and PLC, and that the two different methods of arachidonic acid release can be distinguished by using the common anti-inflammatory drugs aspirin and dexamethasone.     

A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7

Arachidonoyl-hydrolyzing phospholipase A2 plays a central role in providing substrate for the synthesis of the potent lipid mediators of inflammation, the eicosanoids, and platelet-activating factor. Although Ca2+ is required for arachidonic acid release in vivo and most phospholipase A2 enzymes require Ca2+ for activity in vitro, the role of Ca2+ in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase A2 from the macrophage-like cell line, RAW 264.7, exhibits Ca2(+)-dependent association with membrane. The intracellular distribution of the enzyme was studied as a function of the Ca2+ concentration present in homogenization buffer. The enzyme was found almost completely in the 100,000 x g soluble fraction when cells were homogenized in the presence of Ca2+ chelators and there was a slight decrease in soluble fraction activity when cells were homogenized at the level of Ca2+ in an unstimulated cell (80 nM). When cells were homogenized at Ca2+ concentrations expected in stimulated cells (230-450 nM), 60-70% of the phospholipase A2 activity was lost from the soluble fraction and became associated with the particulate fraction in a manner that was partly reversible with EGTA. Membrane-associated phospholipase A2 activity was demonstrated by [3H]arachidonic acid release both from exogenous liposomes and from radiolabeled membranes. With radiolabeled particulate fraction as substrate, this enzyme hydrolyzed arachidonic acid but not oleic acid from membrane phospholipid, and [3H]arachidonic acid was derived from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase A2 is regulated by Ca2+: in an unstimulated cell phospholipase A2 is found in the cytosol; upon receptor ligation the cytosolic Ca2+ concentration increases, and the enzyme becomes membrane-associated which facilitates arachidonic acid hydrolysis.     

Oligomers of prostaglandin B1 inhibit arachidonic acid mobilization in human neutrophils and endothelial cells

The previous paper (Biochim. Biophys. Acta 1006 (1989) 272-277) has demonstrated that oligomers of prostaglandin B1 are effective in vitro inhibitors of a wide range of both cell-derived and extracellular phospholipases A2. The present study has investigated the effects of prostaglandin oligomers on agonist-stimulated phospholipase activity on intact human cells. PGBx, an oligomer (n = 6) or PGB1, and PGB-trimer inhibit as much as 95% of the A23187-stimulated release of arachidonic acid from human neutrophils. The effect is dose-dependent, with an IC50 of 4-5 microM; near maximal inhibition is obtained with as little as 1 min of preincubation with PGB-trimer. Consistent with its role as a phospholipase A2 inhibitor, PGB-trimer also inhibits the A23187-stimulated incorporation of [3H]acetate into platelet-activating factor. PGBx and PGB-trimer also inhibit the release of arachidonic acid from human umbilical vein endothelial cells stimulated with histamine, thrombin, or ionophore A23187; inhibition of the basal or unstimulated turnover of both arachidonic acid and oleic acid is also observed. Inhibition by PGB-trimer can be blocked by simultaneous addition of 50 microM albumin; cells preincubated with PGB-trimer are not affected by albumin. Furthermore, removal of exogenous PGB-trimer prior to challenge with A23187 does not reverse the inhibition of either endothelial cells and neutrophils. Thus, prostaglandin B1 oligomers are taken up by human neutrophils and vascular endothelial cells and serve as potent inhibitors of arachidonic acid mobilization. One mechanism for the pharmacological effects of PGBx may be inhibition of cell-associated and extracellular phospholipase A2.     

Arachidonic Acid Liberated by Diacylglycerol Lipase Is Essential for the Release Mechanism in Chromaffin Cells from Bovine Adrenal Medulla

Chromaffin cells from bovine adrenal medulla secrete catecholamines on stimulation with acetylcholine. In addition to the activation of the phosphatidylinositol cycle, arachidonic acid is generated, which was thought to be the result of phospholipase A2 activation. We have demonstrated in isolated plasma membranes of these cells that arachidonic acid is generated by a two-step reaction of diacylglycerol and monoacylglycerol lipase splitting diacylglycerol, which originates from the action of phospholipase C on phosphatidylinositols. No phospholipase A2 activity could be detected in plasma membranes so far. External addition of arachidonic acid increases the release in the absence and in the presence of agonist. Inhibition of the diacylglycerol lipase by RHC 80267 suppresses the catecholamine release, which is restored on addition of arachidonic acid. This effect, however, is reversed by lipoxygenase inhibitors, indicating that it is not arachidonic acid itself, but one of its lipoxygenase products, that is essential for inducing exocytosis.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

A calcium-independent phospholipase activity insensitive to bromoenol lactone mediates arachidonic acid release by lindane in rat myometrial cells.

Arachidonic acid release is an important regulatory component of uterine contraction and parturition, and previous studies showed that lindane stimulates arachidonic acid release from myometrium. The present study partially characterized the enzyme activity responsible for lindane-induced arachidonic acid release in myometrial cells. Lindane released arachidonic acid from cultured rat myometrial cells in concentration- and time-dependent manners. This release was primarily from phosphatidylcholine and phosphatidylinositol, and was independent of intracellular and extracellular calcium. In cells prelabeled with [3H]arachidonic acid, 85% of radiolabel was recovered as free arachidonate and only 5% was recovered as eicosanoids. Pretreatment with the antioxidants Cu, Zn-superoxide dismutase, alpha-tocopherol or Trolox did not significantly modify lindane-induced arachidonic acid release. Pretreatment of cells with the phosphatidylcholine-specific phospholipase C inhibitor D609, phosphatidylinositol-specific phospholipase C inhibitor ET-18-OCH3, or an interrupter of the phospholipase D pathway (ethanol) did not suppress lindane-induced arachidonic acid release. Although these results are consistent with calcium-independent phospholipase A2 activation by lindane, the calcium-independent phospholipase A2 inhibitor bromoenol lactone failed to inhibit lindane-induced arachidonic acid release in myometrial cells, even though bromoenol lactone effectively blocked arachidonic acid release in neutrophils. These results suggest that myometrial cells express a novel, previously unidentified phospholipase that is arachidonate-specific, calcium-independent, insensitive to bromoenol lactone, insensitive to reactive oxygen species activation, shows substrate preference for phosphatidylcholine and phosphatidylinositol, and is stimulated by lindane. Moreover, the data show that the overwhelming majority of arachidonic acid released remains as arachidonate, but that lindane does not significantly inhibit metabolism of arachidonate to eicosanoids.     

Signal transduction by bFGF, but not TGF beta 1, involves arachidonic acid metabolism in endothelial cells.

We investigated the stimulation of early cellular events resulting from the interaction of the growth factor basic FGF (bFGF) and of the growth inhibitor transforming growth factor beta-type 1 (TGFβ1), with their specific receptors on bovine endothelial cells. At mitogenic concentrations, bFGF stimulated the rapid release of arachidonic acid and its metabolites from (³H)-arachidonic acid labeled cells. When arachidonic acid metabolism was stimulated by addition of the calcium ionophore A23187, the effect of bFGF was amplified. Nordihydroguaïaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, decreased the mitogenic effect of bFGF, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, was ineffective. These findings suggest that metabolism of arachidonic acid to lipoxygenase products may be necessary for the mitogenic effect of bFGF. Basic FGF did not stimulate the production of inositol phosphates from cells labelled with myo-(2-³H)-inositol nor did it induce calcium mobilization, as measured by fura-2 fluorescence, indicating that bFGF does not activate phosphoinositide specific phospholipase C in endothelial cells, but rather, that bFGF-induced arachidonic acid metabolism is mediated by another phospholipase. TGFβ1, which inhibits basal and bFGF-induced endothelial cell growth, had no effect on arachidonic acid matabolism and inositol phosphate formation and did not prevent bFGF-induced arachidonic acid metabolism. These results suggest that the inhibitory action of TGFβ1 on endothelial cell growth occurs through different mechanisms.     

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.     

Resistance to killing by tumor necrosis factor in an adipocyte cell line caused by a defect in arachidonic acid biosynthesis

We have found that TA1-R6, which are resistant to the cytotoxic effects of tumor necrosis factor (TNF) in the presence of cycloheximide (Reid, T. R., Torti, F., and Ringold, G. M. (1989) J. Biol. Chem. 264, 4583-4589), have reduced ability to release arachidonic acid (20:4) from membrane phospholipids in response to either TNF or the calcium ionophore A23187 treatment. However, no defect in the activity of phospholipase A2, the principal enzyme responsible for the release of 20:4 from phospholipids, was observed in these cells. Detailed biochemical characterization of these TNF-resistant cells has revealed that these cells are unable to synthesize 20:4 endogenously because of a defect in delta 6-desaturase, the rate-limiting enzyme of 20:4 biosynthesis. This deficiency leads to a marked decrease in the steady-state levels of 20:4 present in choline-containing phospholipid (PC) and ethanolamine-containing phospholipid (PE). The TA1-R6 cells, however, are capable of incorporating exogenous 20:4 into PC and PE, and when loaded in such manner they become significantly more sensitive to the cytotoxic effects of TNF in the presence of cycloheximide. Therefore, the release of arachidonic acid from phospholipids appears to be a critical element in the signaling pathway utilized by TNF and is essential to the rapid cytotoxic response elicited by TNF in the absence of protein synthesis in wild-type TA1 cells.     

Phosphatidylcholine metabolism in human endothelial cells: modulation by phosphocholine

Phosphatidylcholine is the principal phospholipid in mammalian tissues, and a major source for the production of arachidonic acid. In this study, the effect of exogenous phosphocholine, a precursor of phosphatidylcholine biosynthesis, on the metabolism of phosphatidylcholine in human umbilical vein endothelial cells was investigated. Incubation of endothelial cells with exogenous phosphocholine at concentrations of 1 to 5 mM was found to inhibit choline uptake and its subsequent incorporation into phosphatidylcholine. Phosphocholine appeared to inhibit choline uptake in a competitive manner. Since phosphatidylcholine is metabolized mainly by the action of phospholipase A₂, with the release of arachidonic acid and other fatty acids, the effect of phosphocholine on arachidonic acid release in endothelial cells was also examined. The induction of arachidonic acid release by ATP was enhanced in cells treated with 1 mM phosphocholine. In vitro assays of phospholipase A₂ activity in cells incubated with phosphocholine, however, did not produced any significant change in the activity of this enzyme. The results of this study show that phosphocholine modulates the biosynthesis and catabolism of phosphatidylcholine in an indirect manner.     

Signal transduction by tumor necrosis factor alpha is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3-E1.

Transmembrane signalling mechanisms of tumor necrosis factor alpha (TNF alpha) were examined with special reference to the involvement of G-protein, in intact and permeabilized murine osteoblast-like cells. TNF alpha stimulated the release of 3H radioactivity from intact cells labeled with [3H]arachidonic acid within 10 min in a dose dependent manner and the production of lyso forms of phospholipids, an event presumably mediated through the activation of phospholipase A2. Production of cAMP and inositol 1,4,5-trisphosphate was not affected by TNF alpha. Pretreatment of the cells with pertussis toxin inhibited the liberation of [3H]arachidonate. GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) reduced the binding affinity of [125I]TNF alpha to beta-escin-permeabilized cells. The addition of TNF alpha together with an unhydrolyzable analog of GTP, GTP gamma S, to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid led to a release of the 3H radioactivity. The production of prostaglandin E2 (PGE2) was markedly stimulated by TNF alpha in a dose over 100 ng/ml, with a latent time of about 3 h, and the stimulation was abolished by pretreatment with pertussis toxin. The time and dose requirements for this process differed from those for the possible activation of phospholipase A2, thereby indicating that other process(es) in addition to the activation of phospholipase A2 may be responsible for the enhanced production of PGE2. The activity of cyclooxygenase (i.e. the combined activities of prostaglandin endoperoxide syntase and PGH2-PGE2 isomerase) was stimulated by TNF alpha with much the same time and dose requirements as for the production of PGE2, and the activation was found to be due to the increased amount of the enzyme, as assessed by a Western blot analysis with anti-cyclooxygenase antibody. This process was also sensitive to pertussis toxin. Therefore, receptors for TNF alpha in MC3T3-E1 cells apparently couple to G-protein sensitive to pertussis toxin and the coupling regulates the activations of phospholipase A2 and the de novo synthesis of cyclooxygenase.     

Chemotactic peptide stimulation of arachidonic acid release in HL60 cells, an interaction between G protein and phospholipase C mediated signal transduction

The mechanism of phospholipase A2 activation by chemotactic peptide was investigated in human promyelocytic HL60 cells. N-Formyl-methionyl-leucyl-phenylalanine (fMetLeuPhe) and the non-hydrolyzable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induced arachidonic acid release in permeabilized and metabolically inhibited HL60 cells, a preparation in which calcium was buffered and inositol phospholipid hydrolysis was inhibited. Inositol phosphate generation and arachidonic acid were shown to be temporally dissociated. These results suggest that receptor-dependent phospholipase C activity is not required for fMetLeuPhe to induce arachidonic acid release. However, fMetLeuPhe effects were highly calcium-dependent and inhibition of phospholipase C reduced fMetLeuPhe stimulation of arachidonic acid release even in the permeabilized cell preparation. We conclude that although phospholipase A2 activation is linked to the fMetLeuPhe receptor independent of phospholipase C, actions of phospholipase C to mobilize calcium and release diacylglycerol may be important to phospholipase A2 activation in the intact cell.     

Effect of streptokinase on prostacyclin synthesis and phospholipase activity in cultured pulmonary artery endothelial cells

In this study, we examined the effects of streptokinase on arachidonic acid release and prostacyclin biosynthesis in cultured bovine pulmonary artery endothelial cells. When intact cells were incubated with streptokinase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident without any cellular damage at all concentrations used (1-10,000 units/ml). Streptokinase also caused a marked release of arachidonic acid. It induced rapid phospholipid hydrolysis, resulting in the release of up to 15% of incorporated [3H]arachidonic acid into the medium. After the addition of streptokinase, degradation of phosphatidylcholine and phosphatidylethanolamine was observed and lysophosphatidylcholine and lysophosphatidylethanolamine were produced. We also observed a transient rise in diacylglycerol after the addition of streptokinase. To test for phospholipase C activity, the release of incorporated [3H]choline, [3H]inositol and [3H]ethanolamine into the culture medium was determined. The level of radioactive inositol showed an increase, but the changes in choline and ethanolamine were comparatively small. An increase in inositol was detectable within 1 min after streptokinase addition and peaked after 15 min. Inositol phosphate and inositol trisphosphate were released, and these releases were suppressed by the addition of neomycin (50 microM). These results suggest that streptokinase stimulates phospholipase A2 and C activity, and that prostacyclin biosynthesis is subsequently increased in cultured endothelial cells.