Stimulation of arachidonic acid release by guanine nucleotide in saponin-permeabilized neutrophils: evidence for involvement of GTP-binding protein in phospholipase A2 activation

Addition of a guanine nucleotide analog, guanosine 5'-O-(thiotriphosphate) (GTP gamma S)(1-100 microM) induced release of [3H]arachidonic acid from [3H]arachidonate-prelabeled rabbit neutrophils permeabilized with saponin. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced arachidonate release was enhanced by GTP gamma S, Ca2+, or their combination. Ca2+ alone (up to 100 microM) did not effectively stimulate lipid turnover. However, the combination of fMLP plus GTP gamma S elicited greater than additional effects in the presence of resting level of free Ca2+. The addition of 100 microM of GTP gamma S reduced the Ca2+ requirement for arachidonic acid liberation induced by fMLP. Pretreatment of neutrophils with pertussis toxin resulted in the abolition of arachidonate release and diacylglycerol formation. Neomycin (1 mM) caused no significant reduction of arachidonate release. In contrast, about 40% of GTP gamma S-induced arachidonate release was inhibited by a diacylglycerol lipase inhibitor, RHC 80267 (30 microM). These observations indicate that liberation of arachidonic acid is mediated by phospholipase A2 and also by phospholipase C/diacylglycerol lipase pathways. Fluoride, which bypasses the receptor and directly activates G proteins, induced arachidonic acid release and diacylglycerol formation. The fluoride-induced arachidonate release also appeared to be mediated by these two pathways. The loss of [3H]arachidonate was seen in phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These data indicate that a G protein is involved between the binding of fMLP to its receptor and activation of phospholipase A2, and also that the arachidonic acid release is mediated by both phospholipase A2 and phospholipase C/diacylglycerol lipase.     

Analysis of prostaglandins formed from endogenous and exogenous arachidonic acid in homogenates of human reproductive tissues

Isotope-labelled arachidonic acid has been used to study in vitro formation of prostaglandins and other products in mammalian tissue. Quantitative conclusions about cyclooxygenase activity have been drawn from such studies. However, arachidonic acid is present in all tissues, free and esterified, and therefore it can be expected that endogenous arachidonate would interfere with transformation of the radioactive exogenous substrate. (1-14C)-labelled arachidonate was, therefore, incubated with homogenates of various human tissues (amnion, chrorion, placenta and myometrium), and the two molecular forms, 12C and 14C, of arachidonic acid as well as of prostaglandin E2 and prostaglandin F2 alpha were quantitated, before and after 30 min of incubation, using gas chromatography-mass spectrometry with multiple ion detection. The results demonstrate a substantial release of arachidonic acid into the medium during incubation. There was no correlation between either the initial concentration of [12C]arachidonic acid and initial concentration of [12C]prostaglandin E2 or the percent increase of those compounds during incubation. The net formation of [12C]prostaglandin E2 and [14C]prostaglandin E2 from endogenous and exogenous precursor, respectively, were also very different. The study shows that by simply incubating (1-14C)-labelled arachidonic acid in tissue homogenates and measuring the amount of radioactivity transformed into various prostaglandins only qualitative conclusions can be drawn.     

Protein kinase C-independent activation of a novel nonspecific phospholipase C pathway by phorbol myristate acetate releases arachidonic acid for prostaglandin synthesis in MC3T3-E1 osteoblasts

The effects of phorbol myristate acetate, an activator of protein kinase C, on the release of [³H]arachidonic acid and prostaglandin synthesis were studied in an osteoblast cell line (MC3T3-E1). Phorbol myristate acetate (20 uM) liberated 16 and 55% of the [³H]arachidonate in prelabeled phosphatidylinositol and phosphatidylethanolamine, respectively, and evoked a 19-fold stimulation in the synthesis of prostaglandin E₂. Phorbol myristate acetate doubled the cellular mass of 1,2-diacylglycerol and stimulated the liberation of [³H]arachidonate from the diacylglycerol pool in prelabeled cells. The diacylglycerol lipase inhibitor RHC 80267 blocked 75–80% of the phorbol ester-promoted (total) cellular liberation of [³H]arachidonic acid and production of prostaglandin E₂. In comparison, the release of [³H]arachidonate from phosphatidylethanolamine (but not phosphatidylinositol) was only partially antagonized (to the same degree) by the PLA₂ inhibitor p-bromophenacylbromide and the protein kinase C inhibitor Et-18-OMe. PMA-induced formation of diacylglycerol or synthesis of PGE₂ was not affected by the prior inhibition of protein kinase C. Therefore, we have shown a novel pathway for the liberation of arachidonic acid in osteoblasts involving the nonspecific hydrolysis of phosphatidylinositol and phosphatidylethanolamine by phospholipase C followed by the deesterification of diacylgycerol. This pathway can be activated by a phorbol ester through a protein kinase C-independent mechanism.     

Tumor necrosis factor-alpha stimulates phosphatidylinositol breakdown by phospholipase C to coordinately increase the levels of diacylglycerol, free arachidonic acid and prostaglandins in an osteoblast (MC3T3-E1) cell line

The effects of (human recombinant) tumor necrosis factor-alpha on phosphatidylinositol breakdown, release of 1,2-diacylglycerols, mobilization of arachidonate from diacylglycerol and prostaglandin synthesis were examined in a model osteoblast cell line (MC3T3-E1). Tumor necrosis factor-alpha (10 nM) caused a specific (30%) decrease in the mass of phosphatidylinositol (and no other phospholipids) within 30 min of exposure. Tumor necrosis factor-alpha doubled the rate of incorporation of [32P]orthophosphoric acid into phosphatidylinositol, indicating that the turnover of inositol phosphate was enhanced, and increased the content of diacylglycerol in parallel with phosphatidylinositol breakdown. The cytokine (10-50 nM; 4 h) also promoted a specific release of 24-34% of the [3H]arachidonate from prelabeled phosphatidylinositol, a release of 80% of the 3H-fatty acid from the diacylglycerol pool, and a 30-fold increase in the synthesis of prostaglandin E2. The tumor necrosis factor-alpha induced liberation of [3H]arachidonate from diacylglycerol, cellular arachidonate release and the synthesis of prostaglandin E2 were each blocked by an inhibitor of diacylglycerol lipase, the compound RHC 80267 (30 microM). Therefore, we conclude that, in the MC3T3-E1 cell line, tumor necrosis factor-alpha activates a phosphatidylinositol-specific phospholipase C (phosphatidylinositol inositolphosphohydrolase; EC 3.1.4.3) to release diacylglycerol, and increases the metabolism of diacylglycerol to liberate arachidonate for prostaglandin synthesis.     

A23187 and protein kinase C activators stimulate phosphatidylinositol metabolism and prostaglandin synthesis in a human lung cancer cell line

Activation of cell phospholipase, release of arachidonic acid and stimulation of prostaglandin synthesis were studied in a newly described human tumor cell line (Lu-65). In the Lu-65 tumor cell line, the calcium ionophore A23187 (2 microM) caused a 100% increase in the release of 3H-arachidonic acid and a 7-fold increase in the synthesis of prostaglandin E2. 1-oleoyl, -2-acetyl-glycerol (100 microM) increased arachidonate release and prostaglandin E2 synthesis by 100%. A23187 and the protein kinase C activators, 1,2-dioctanoyl-glycerol and 1-oleoyl, -2-acetyl-glycerol, decreased the specific radioactivity of 3H-arachidonate in phosphatidylinositol by 37% and 57%, respectively. The effects of A23187 were blocked in Ca2+-free media or in the presence of the phospholipase A2 inhibitor, p-bromophenacyl bromide, while those of 1-oleoyl, -2-acetyl-glycerol were not. The data provide evidence in a human tumor cell line for calcium/phospholipase A2-dependent and independent pathways for arachidonic acid release, both of which preferentially hydrolyze phosphatidylinositol.     

Vinca alkaloids inhibit conversion of arachidonic acid to thromboxane by human platelet microsomes: comparison with other microtubule-active drugs

The Vinca alkaloid vinblastine causes dose-dependent inhibition of malondialdehyde formation and aggregation in activated human platelets as a result of inhibition of arachidonic acid metabolism via the thromboxane pathway (Brammer, J.P., Kerecsen, L. and Maguire, M.H. (1982) Eur. J. Pharmacol. 81, 577). The nature of the inhibition by vinblastine has been investigated with human platelet microsomes, measuring conversion of arachidonic acid to malondialdehyde and thromboxane B2 via spectrophotometric assay and RIA, respectively, determining arachidonate oxygenation by monitoring oxygen consumption, and identifying metabolites formed from [1-14C]arachidonic acid. Vinblastine was compared with other Vinca alkaloids and with structurally unrelated microtubule-active drugs. Vinca alkaloids were unique in causing dose-dependent inhibition of both malondialdehyde and thromboxane B2. Order of potency was vinblastine = vincristine = vindesine greater than leurosine greater than vinepidine. Inhibition of malondialdehyde and thromboxane B2 by 50 microM vinblastine was at least 60%. Microsomal cyclooxygenase was not inhibited by 200 microM vinblastine. Inhibition by vinblastine of [1-14C]arachidonic acid conversion to thromboxane B2 was associated with a 4-fold increase in prostaglandin E2 formation. Thromboxane B2, but not malondialdehyde, formation was inhibited by colchicine less than nocodazole much less than vinblastine. Results indicate that microsomal thromboxane synthetase is inhibited by Vinca alkaloids and other tubulin-binding drugs, and suggest that the action of vinblastine in inhibiting thromboxane synthesis, aggregation and release in intact platelets is not dependent upon its antimicrotubular actions.     

Inhibition of prostaglandin E2 synthesis by a blocker of epithelial chloride channels

Arginine-vasopressin (AVP) elicits a variety of responses in cultured rat mesangial cells, among them stimulation of prostaglandin biosynthesis and activation of Cl- channels. AVP produced an 11-fold increase over basal levels in prostaglandin E2 release from cultured mesangial cells. This response was completely inhibited by 25 microM indomethacin and 82 +/- 5% inhibited by 25 microM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) which is a potent blocker of epithelial Cl- channels. The IC50 for NPPB inhibition of prostaglandin E2 release was 8 microM. Indomethacin and NPPB at 25 microM also inhibited AVP-stimulated cellular accumulation of prostaglandin E2 by 98% and 79 +/- 7% respectively. The inhibitory effect of NPPB was not due to interference with the cellular response to AVP since at 50 microM it did not block AVP-stimulated release of arachidonate metabolites from cells metabolically labeled with [3H]-arachidonic acid. It is suggested that NPPB inhibition of prostaglandin E2 synthesis is at the cyclooxygenase level on the basis of its structural similarity to the fenamic acid type of cyclooxygenase inhibitors.     

Platelet activating factor. Stimulation of the lipoxygenase pathway in polymorphonuclear leukocytes by 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (AAGPC) triggered the release of [3H]arachidonate but not [14C]stearate from cellular phospholipids in cytochalasin B-treated rabbit polymorphonuclear leukocytes. Concentrations of AAGPC up to 20 nM caused a dose-dependent release and subsequent metabolism of the released [3H]arachidonic acid. Most of the release of the [3H]arachidonate had taken place within the first 2 min of stimulation. Phosphatidylinositol and phosphatidylcholine served as the sources of [3H]arachidonate with about 50% of the label coming from each pool. Challenge of cytochalasin B-treated polymorphonuclear leukocytes with AAPGC led to the production of [3H]hydroxyeicosatetraenoic acids and [3H]dihydroxyeicosatetraenoic acids. No significant production of [3H]prostaglandins or [3H]thromboxanes was detected. AAGPC also caused a dose-dependent degranulation of cytochalasin B-treated rabbit polymorphonuclear leukocytes as shown by the release of beta-glucuronidase and lysozyme. Both the AAGPC-stimulated production of arachidonate metabolites and the degranulation response were blocked by eicosatetraynoic acid and non-dihydroguaiaretic acid at similar inhibitor concentrations. These findings suggest the bioactions of AAGPC on polymorphonuclear leukocytes may be mediated by the release of arachidonic acid and the production of mono- and dihydroxyeicosatetraenoic acids.     

Inhibition of microcystin-induced release of cyclooxygenase products from rat hepatocytes by anti-inflammatory steroids

We showed previously that exposure to microcystin causes eicosanoid release. That study was extended further to test the effect of glucocorticoids on microcystin-induced release of [14C]arachidonic acid and its metabolites from rat hepatocytes previously treated with [14C]arachidonic acid. Release of total radioactivity was 4-fold greater from hepatocytes after 2-hr incubation with 1 microM microcystin than after incubation with control medium. Fluocinolone pretreatment decreased the microcystin-induced synthesis and release of prostacyclin by 24 +/- 2.6% (P less than 0.05) and thromboxane B2 by 39 +/- 3% (P less than 0.025). Treatment of hepatocyte cultures with either microcystin (1 microM) or steroids had no effect on cell viability or total cell protein. Total radioactivity released into the incubation medium was not affected by glucocorticoid alone. Under these conditions, the quantities of both prostaglandin F2 alpha and prostaglandin E2 released were not significantly different when control and microcystin-treated cultures were compared. The half-maximal inhibition (IC50) values obtained from the dose-response data for the inhibition of arachidonic acid release by steroids were comparable with normal cortisol levels in humans. Dose-response curves gave the following rank order of inhibitory potency: fluocinolone greater than dexamethasone greater than hydrocortisone. These results suggest that glucocorticoid therapy might be beneficial in microcystin toxicosis.     

Lysophosphatidic acid potentiates the thrombin-induced production of arachidonate metabolites in platelets

Concentrations (1 to 20 microM) of 1-oleoyl-lysophosphatidic acid which alone do not affect platelet metabolism of arachidonic acid, do augment the effects of suboptimal concentrations of thrombin on the formation of [14C]phosphatidic acid and the production of [14C]arachidonate metabolites from platelets prelabeled with [14C]arachidonate. The effect on [14C]phosphatidate occurs with concentrations of thrombin (0.1 unit/ml) which are lower than those (0.2 unit/ml) needed to observe the effects on [14C]arachidonate metabolites. The effect of 1-oleoyl-lysophosphatidic acid (10 microM) plus thrombin (0.2 unit/ml) on the formation of phosphatidic acid temporally precedes the production of arachidonate metabolites consistent with a sequential activation of phosphatidylinositol-specific phospholipase C and phospholipase A2 activities. Preincubation of platelets with (32P)orthophosphate shows that the phosphatidic acid formed by 1-oleoyl-lysophosphatidic acid (10 microM) plus thrombin (0.2 unit/ml) is derived from phosphatidylinositol. The Ca2+-ionophoretic properties of lysophosphatidic acid might explain the accumulation of phosphatidic acid since Ca2+ prevents the conversion of phosphatidic acid to phosphatidylinositol. That effect of lysophosphatidic acid is inhibited by prostacyclin, possibly through a cyclic-AMP-mediated effect on calcium homeostasis.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Alpha 1-adrenergic stimulation of arachidonic acid release and metabolism in a rat thyroid cell line. Mediation of cell replication by prostaglandin E2

The rat thyroid cell line, FRTL-5, expresses an alpha 1-adrenergic receptor when exposed to thyrotropin. We have found that occupation of this alpha 1-adrenergic receptor by norepinephrine stimulated the release of [3H]arachidonic acid from prelabeled cells. Arachidonic acid was metabolized primarily to prostaglandin E2 and to much smaller amounts of 11-hydroxy-5,8,11,13-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, prostaglandin D2, and thromboxane B2. Synthesis of all these metabolites was inhibited by the cyclooxygenase inhibitor indomethacin. When FRTL-5 cells were starved of thyrotropin for 24 h, norepinephrine nearly doubled [3H]thymidine uptake into DNA. Cyclooxygenase inhibitors inhibited norepinephrine-stimulated thymidine uptake by 60-70%. Of several arachidonic acid metabolites tested, none was able to stimulate thymidine uptake directly in the presence of indomethacin. Prostaglandin E2, however, was able to restore [3H]thymidine uptake when added together with norepinephrine in the presence of indomethacin. Thus, occupation of an alpha 1-adrenergic receptor in a functional rat thyroid cell line leads to arachidonic acid release. Subsequent metabolism of the arachidonic acid by the cyclooxygenase pathway leads to synthesis of prostaglandin E2, which mediates a norepinephrine-stimulated activity related to cell replication.     

Tocopherol analogs suppress arachidonic acid metabolism via phospholipase inhibition.

alpha-Tocopherol and three derivatives in which the phytol chain is modified or deleted were examined for their effect on cultured keratinocyte arachidonic acid metabolism. 2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC), in which the phytol chain is replaced by a methyl group, inhibited basal, bradykinin (BK)- and A23187-stimulated prostaglandin E2 (PGE2) synthesis with an apparent Ki of 1.3 microM. The Ki of the analogue with six carbon atoms in the side chain (C6) was 5 microM while that of the C11 analogue was 10 microM. No effect of alpha-tocopherol was observed. The mechanism of inhibition was studied using PMC. The effect of PMC on phospholipase and cyclooxygenase activity was assayed using stable isotope mass measurements of PGE2 formation, which assesses arachidonate release and cyclooxygenase metabolism simultaneously. BK-stimulated formation of PGE2, derived from endogenous phospholipid, was decreased 60% by 5 microM PMC and eliminated by 50 microM PMC, compared with controls. No difference in PGE2 formed from exogenous arachidonic acid was observed, indicating no effect of PMC on cyclooxygenase activity. In contrast, no effect of 5 microM PMC was observed on BK-stimulated [3H]arachidonic acid release from prelabeled cultures. The capacity of PMC to inhibit phospholipase activity in vitro was also assessed. PMC inhibited hydrolysis of phospholipid substrate by up to 60%. These results suggest that alpha-tocopherol analogues with alterations in the phytol chain inhibit eicosanoid synthesis by preferential inhibition of phospholipase.     

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.     

Disturbance of pulmonary prostaglandin metabolism in fetuses of alloxan-diabetic rabbits

[14C]Arachidonic acid conversion in lung homogenates of 28-day fetuses from control and alloxan-diabetic rabbits was studied. The major metabolites were 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and prostaglandin E2. Small amounts of 6-ketoprostaglandin F1 alpha, prostaglandin F2 alpha, and thromboxane B2 were also observed. Lung homogenates from fetuses of alloxan-diabetic rabbits convert significantly less [14C]arachidonic acid to prostaglandin E2, whereas all other metabolites were present in similar quantities compared to fetuses of non-diabetic rabbits. These studies suggest that the decreased arachidonic acid conversion to prostaglandin E2 could be partially responsible for the functional delay of lung maturation in offspring of alloxan-diabetic rabbits.     

Differential sensitivity of arachidonic acid release and 1,2-diacylglycerol formation to pertussis toxin, GDP beta S and NaF in saponin-permeabilized human platelets: possible evidence for distinct GTP-binding proteins involving phospholipase C and A2 activation

Human platelets labeled with [3H]arachidonic acid and permeabilized with saponin produced [3H]1,2-diacylglycerol (DG) by phospholipase C and released [3H]arachidonate by phospholipase A2, when activated with thrombin. Thrombin-induced arachidonate liberation was almost completely inhibited with pretreatment of pertussis toxin (10 micrograms/ml), whereas DG formation was decreased by only 20-40% in the toxin-treated platelets. Although guanosine 5'-o-(2-thiodiphosphate) (GDP beta S) suppressed arachidonate release and DG production in a dose-dependent manner, the half maximal inhibition required less than 10 microM for arachidonate release but more than 100 microM for DG production. Moreover, the dose-response effects of NaF on arachidonate release and DG formation were different. These results indicate that arachidonate release and DG formation are differently affected by these agents acting on guanine nucleotide binding proteins (G-proteins), suggesting that the distinct G proteins modulate the activity of phospholipase C and phospholipase A2.     

Arachidonic acid metabolites regulate interleukin-1 production

We have investigated the role of arachidonic acid metabolites in the regulation of interleukin-1 production by murine peritoneal macrophages. Indomethacin a potent inhibitor of prostaglandin synthesis caused a dose-dependent augmentation of lipopolysaccharide induced interleukin production (up to 7-fold at 5 microM). In contrast, lipoxygenase inhibitors, nordihydroguarietic acid and nafazatrom had no effect at doses that did not significantly decrease prostaglandin synthesis. Added to lipopolysaccharide stimulated cultures, PGE2 was also augmented by indomethacin but unlike lipopolysaccharide treated cultures was suppressed by nordihydroguarietic acid. These data suggest that arachidonate metabolites may be potent autoregulators of macrophage interleukin-1 production.     

A mutant HSDM1C1 fibrosarcoma line selected for defective eicosanoid precursor uptake lacks arachidonate-specific acyl-CoA synthetase

Mutagenesis followed by suicide with highly radioactive tritiated arachidonic acid has been used to select for mouse fibrosarcoma (HSDM1C1) cells defective in eicosanoid precursor uptake. Survivors of the selection were screened by replica plating and autoradiographic assay of [3H]arachidonate esterification; a mutant cell line, EPU-1, was established. EPU-1 cells contain one-third as much arachidonate as normal HSDM1C1 cells. The mutant lacks arachidonate-specific acyl-CoA synthetase, which accounts for decreased arachidonate uptake. EPU-1 exhibits enhanced turnover of arachidonoyl- but not linoleoyl-phosphatidylcholine. Bradykinin-induced arachidonate release and prostaglandin E2 synthesis are decreased in EPU-1. Thus, arachidonoyl-CoA synthetase is required for arachidonate homeostasis in HSDM1C1 cells.     

Stimulatory effects of cholera toxin on arachidonic acid metabolism in Chinese hamster ovary cells

Cholera toxin (CT) stimulated the release of arachidonic acid (AA) from Chinese hamster ovary cells with no apparent lag period. CT-induced release of [3H]AA or its metabolites was dose dependent during a 4-hr period of toxin exposure with a minimum effective dose of 0.1 ng/ml. CT-induced release of [3H]AA metabolites began within 15 min of toxin addition and became maximal after approximately 5 hr. Neither CT-A subunit nor CT-B subunit alone caused [3H]AA release. Furthermore, [3H]AA release was not caused by addition of dibutyryl cAMP to the culture medium, indicating that the observed effect of CT on arachidonate metabolism appeared to be independent of cAMP. The effect of CT on AA metabolism is proposed as a possible mechanism leading to the synthesis of prostaglandin E and fluid secretion during cholera.