The role of arachidonate lipoxygenase in the release of SRS-A from guinea-pig chopped lung

The immunological release of SRS-A was investigated in guinea-pig chopped lung. A number of unsaturated fatty acids, all of which are substrates for arachidonate lipoxygenase were found to potentiate the release of SRS-A. This potentiation was enhanced by indomethacin, a cyclo-oxygenase inhibitor, and completely reversed by nordihydroguaiaretic acid (NDGA) and eicosatetraynoic acid (ETA) which inhibit lipoxygenase. This suggests that some aspect of arachidonate lipoxygenase action stimulates release of SRS-A and that release of SRS-A is increased by redirection of arachidonic acid (AA) metabolism via the lipoxygenase pathway (Hamberg, 1976). However, although exogenous ¹⁴C-AA increased SRS-A output it was not incorporated into SRS-A.     

The mechanism of enhancement by fatty acid hydroperoxides of anaphylactic mediator release.

Indomethacin augmented the release of histamine and SRS-A but abolished synthesis of TxB2. Compound CLI that inhibited both cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism did not augment release of anaphylactic mediators. 13-HPLA enhanced mediator release from lungs in which arachidonic acid metabolism was blocked by compount CLI. Thus, it is concluded that 13-HPLA enhances mediator release not by altering the balance of arachidonic acid metabolites, e.g. by inhibiting synthesis of prostacyclin, but by a direct effect on lung mast cells. A corollary to this conclusion is that the fatty acid hydroperoxide (HPETE) formed by lipoxygenase from arachidonic acid may also augment the release of anaphylactic mediators. Thus, the enhancement of mediator release by indomethacin may be attributed to increased synthesis of HPETE following inhibition of cyclo-oxygenase.     

Albumin redirects platelet eicosanoid metabolism toward 12(S)-hydroxyeicosatetraenoic acid

Albumin is a major determinant of eicosanoid formation, affecting autacoids important in cell-cell interactions. We delineated three mechanisms by which albumin controlled platelet eicosanoid formation: 1) Albumin diverted free arachidonate toward 12-lipoxygenation. 2) Albumin enhanced release of arachidonate from phospholipids. 3) Albumin inhibited incorporation of arachidonate from the medium into platelet phospholipids. 12(S)-Hydroxyheptadecatrienoic acid (12-HHTrE) formation was reduced 70% by albumin as compared to that formed in albumin-free medium. In sharp contrast, formation of 12(S)-hydroxyeicosatetraenoic acid (12-HETE), the platelet lipoxygenase product, was much less influenced by albumin. Moreover, 12-HETE production in the presence of albumin was markedly increased and prolonged after aspirin treatment. These data suggested that albumin redirected released endogenous arachidonate from cyclooxygenase to lipoxygenase. Therefore, the metabolic fate of arachidonate present in the medium of stimulated platelets was studied by adding tracer [3H]arachidonate 30 sec before thrombin. Albumin increased arachidonate metabolism by lipoxygenase 7-fold as compared to albumin-free controls, while cyclooxygenation increased 2.7-fold. Redirection of eicosanoid metabolism by albumin toward lipoxygenase products constitutes a heretofore undescribed and potentially important physiological role for albumin. In vitro utilization of albumin may reflect in vivo events in thrombosis and hemostasis more accurately than previous studies without albumin could appreciate.     

Growth hormone releasing factor (GRF) increases free arachidonate levels in the pituitary: a role for lipoxygenase products

GRF, a specific stimulator of GH release, increased in a concentration- and time-dependent manner pituitary [3H]-arachidonate levels in vitro. This effect was antagonized by 100 nM somatostatin. Exogenous arachidonate also stimulated GH release in vitro. Quinacrine, a phospholipase A2 inhibitor, reduced both basal and GRF-stimulated free arachidonate levels as well as GH release. The cyclooxygenase inhibitor indomethacin was ineffective, while BW755c, which also inhibits the lipoxygenase pathway, produced a further increase in the levels of the fatty acid stimulated by GRF and potently reduced GH release. These results provide additional evidence for the involvement of arachidonate metabolism in the hormone-releasing effect of GRF at the somatotroph.     

Arachidonic acid metabolites regulate the secretion of atrial natriuretic peptide in cultured rat atrial cardiocytes.

Prostaglandins F2 alpha and E2 increase release of immunoreactive (irANP) in primary cultures of rat atrial cardiocytes. This effect is independent of cell density in the cultures and does not appear to operate through a cAMP-dependent mechanism. Studies that probed the PGF2 alpha effect with a number of different pharmacological antagonists suggest that it is tied to a calmodulin-dependent step. This latter effect does not appear to be related to increased calcium entry through voltage-gated channels in the plasma membrane nor to mobilization of ryanodine-sensitive intracellular calcium pools. Inhibitors of the lipoxygenase pathway, a second avenue of arachidonate metabolism, resulted in a decrease in irANP release from cultured atrial or ventricular cardiocytes. Leukotriene C4, a lipoxygenase product, had a modest effect to promote irANP release over a 24-h period. However, pretreatment of anesthetized rats with nordihydroguarietic acid, a lipoxygenase inhibitor, had no effect on stretch-dependent release of irANP from the heart in vivo. These findings suggest that the prostaglandins represent the more important group of arachidonate metabolites in regulating irANP release physiologically.     

Effect of arachidonic acid and the chemotactic factor F-Met-Leu-Phe on cation transport in rabbit neutrophils

A detailed examination of the effects of exogenous arachidonate on cation metabolism in rabbit neutrophils was undertaken. Arachidonic acid stimulates the movement of ⁴⁵Ca into and out of the neutrophils with a net result, in the presence of extracellular calcium, of increasing the steady-state level of ⁴⁵Ca. Arachidonate also increases the uptake of ²²Na. These effects of arachidonate are specific to these cations, concentration-dependent, and sensitive to lipoxygenase inhibitors. At the concentrations used in this study arachidonate does not influence the permeability of human erythrocytes to ⁴⁵Ca. Furthermore, both arachidonic acid and F-Met-Leu-Phe release calcium from a previously unexchangeable intracellular pool and the effect of the two stimuli are not additive. Arachidonic acid-dependent, but not F-Met-Leu-Phe-dependent, calcium release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release calcium from the same pool(s) by separate mechanisms. The results summarized above are consistent with the hypothesis that one or more arachidonate metabolites are involved in the mechanism underlying the chemotactic factor induced permeability changes in rabbit neutrophils.     

Modulation of Cell-Substrate Adhesion by Arachidonic Acid: Lipoxygenase Regulates Cell Spreading and ERK1/2-inducible Cyclooxygenase Regulates Cell Migration in NIH-3T3 Fibroblasts

Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute approximately 25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion-signaling pathway, wherein lipoxygenase oxidation generates leukotriene metabolites regulating the spreading stage of cell adhesion, whereas ERK 1/2-induced cyclooxygenase synthesis results in oxidation of a later release, generating prostaglandin metabolites regulating the later migration stage.     

A re-interpretation of lipoxygenase-dependent insulin release: which metabolites of arachidonic acid, or none?

There are considerable data implicating a pancreatic islet 12-lipoxy-genase in glucose-induced insulin secretion. This enzyme traditionally is conceived as converting unesterified arachidonic acid to "free" hydroperoxyeicosatetraenoic acid and metabolites thereof. However, studies employing the provision of exogenous metabolites of arachidonic acid to islet tissue fail to identify convincingly the mediator of insulin release. It is proposed that the islet lipoxygenase directly peroxidizes unsaturated fatty acids esterified within membrane phospholipids, leading to changes in ion flux and enzyme activity (particularly phospholipase A2) at the membrane level. The release of unesterified metabolites of arachidonate, although reflecting islet lipoxygenase activity, may be an epiphenomenon.     

Effects of lipoxygenase and cyclooxygenase inhibitors on glucose-stimulated insulin secretion from the isolated perfused rat pancreas

Some of the metabolites of arachidonic acid formed in the lipoxygenase and cyclooxygenase pathways stimulate insulin release. We studied the relative importance of each of these pathways in the modulation of glucose-induced insulin release by using inhibitors of arachidonate metabolism. Perfusion of the isolated rat pancreas with two chemically different inhibitors of cyclooxygenase, flurbiprofen and sodium salicylate, markedly inhibited prostaglandin E2 release, but had little effect on glucose-induced insulin release or on potentiation of insulin release caused by prior exposure to glucose. On the other hand, nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, not only inhibited both phases of glucose-induced insulin release but also abolished the potentiation effect. These effects of NDGA prevailed, when it was administered together with flurbiprofen, which caused profound inhibition of prostaglandin E2 release. We conclude that 1) lipoxygenase pathways play a dominant role in glucose-stimulated insulin release, and 2) endogenous lipoxygenase metabolites influence the potentiating effect of glucose on the release of insulin in response to a subsequent stimulation.     

Contribution of lipoxygenase and cyclooxygenase metabolites in the modulation of cyclic nucleotides in the guinea pig myometrium. Differential effects of carbachol and ionophore A23187

Accumulation of cyclic GMP in estradiol-treated immature guinea pig myometrium was enhanced by carbachol, ionophore A23187, unsaturated fatty acids and their hydroperoxides. Cyclic AMP content was elevated only by arachidonic acid, A23187 and PGI2. Eicosatetraynoic acid (TYA), but not indomethacin prevented all cyclic GMP responses. The effects of A23187 and arachidonate on cyclic AMP were accompanied by a parallel increase (2-3 fold) in the generation of PGI2 by the myometrium. Both events were similarly reduced by indomethacin, TYA, 15-hydroperoxyarachidonic acid and tranylcypromine, suggesting that PGI2 was involved. Omission of Ca2+ or addition of mepacrine or p-bromophenacylbromide abolished the stimulatory effects of A23187 and carbachol on cyclic GMP as well as the A23187-induced elevations in both PGI2 and cyclic AMP generation. Thus, with both exogenous arachidonate as well as with endogenous fatty acid, released through an apparent phospholipase A2-induced activation process, the lipoxygenase pathway was associated with an activation of the cyclic GMP system and the cyclooxygenase pathway, via PGI2 generation, with an activation of the cyclic AMP system. Carbachol failed to alter both cyclic AMP content and the release of PGI2 suggesting a cholinergic receptor-mediated fatty acid release process, selectively coupled to the lipoxygenase route.     

Induction of plasminogen activator by 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore: Suppression by inhibitors of fatty acid lipoxygenase

HeLa cells incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and rat basophilic leukemia (RBL-1) cells incubated with calcium ionophore, showed increased levels of the protease plasminogen activator. These treatments have previously been shown to stimulate the cellular metabolism of arachidonic acid. The induction of plasminogen activator in both cell types was inhibited in a dose-dependent manner by 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, two compounds known to inhibit arachidonate metabolism via lipoxygenases. In contrast, indomethacin, which selectively inhibits arachidonate metabolism via cyclooxygenase, was inactive. The levels of four enzyme markers in HeLa cells were unchanged by treatment with TPA plus the lipoxygenase inhibitors, indicating that the inhibitors did not exert their effects on plasminogen activator via general cell toxicity. HeLa cells preincubated with [³H]arachidonate and subsequently challenged with TPA produced small amounts of material with the chromatographic mobilities and resistance to indomethacin expected of hydroxylated fatty acids derived via lipoxygenase. RBL-1 cells have been shown previously to produce leukotrienes and other lipoxygenase metabolites when treated with calcium ionophore. Plasminogen activator in HeLa cells was stimulated by up to 2.5-fold by incubation with 0.5–2 μg/ml 5-hydroxyeicosatetraenoic acid. Our results suggest that the induction of plasminogen activator in HeLa and RBL-1 cells is not mediated by prostaglandins or thromboxanes, but may be mediated or modulated by arachidonate metabolites derived via a lipoxygenase pathway.     

Role of exogenous arachidonate in the biosynthesis of 5-lipoxygenase metabolites of arachidonic acid by human polymorphonuclear leukocytes induced by the calcium ionophore A23187

By using high performance liquid chromatography with simultaneous detection of unlabeled and radiolabeled product of lipoxygenase oxidation of arachidonic acid, the mechanism of exogenous arachidonate involvement in leukotriene synthesis in human neutrophils induced by the Ca2+ ionophore A23187 was studied. It was found that after addition of labeled arachidonate the specific radioactivity of the reaction product (leukotriene B4) does not change on a time scale, i.e., the free arachidonic acid exchange between the cell and extracellular space is a very rapid process. Exogenous arachidonic acid was found to be the substrate of the lipoxygenase reaction which acts in parallel with the endogenous one. The dependence of specific radioactivity of leukotriene B4 in added arachidonic acid concentration is described by a hyperbolic curve with saturation. When exogenous arachidonate is used at a concentration of 10.8 +/- 3.9 microM, that of intracellular arachidonic acid increases twofold at the expense of the exogenously added acid.     

Effect of vitamin E enrichment on arachidonic acid release and cellular phospholipids in cultured human endothelial cells

The effect of (R,R,R)-alpha-tocopherol on agonist-stimulated arachidonate release and cellular lipids was investigated in cultured human umbilical cord endothelial cells. Endothelial cells in culture incorporate added tocopherol in a dose-dependent manner at both physiological (23.2 microM) or pharmacological (92.8 microM) concentrations which were well tolerated by the cells, as judged by unaltered cell number and viability. Two experiments were conducted in which cells were either incubated with (R,R,R)-alpha-tocopherol followed by labelling with [1-14C]arachidonic acid or they were labelled with arachidonate followed by incubation with tocopherol. Irrespective of the sequence of incubation with arachidonate and tocopherol, (R,R,R)-alpha-tocopherol-enriched cells released significantly more labelled arachidonate when stimulated with thrombin (2.5 U/ml) or ionophore A23187 (1 microM) for 10 min. The magnitude of [1-14C]arachidonate release was higher from ionophore A23187 stimulation than from thrombin stimulation, but the trend of increased arachidonate release in tocopherol-enriched cells was the same. Results from these studies demonstrate that (R,R,R)-alpha-tocopherol can stimulate arachidonate release in human endothelial cells. This observation is in direct contrast to the role of tocopherol, which has been shown to inhibit platelet and cardiac phospholipase A2 activity in rats, and to reduce thrombin-stimulated thromboxane release in rat platelets.     

Anandamide and 2-arachidonoylglycerol inhibit fatty acid amide hydrolase by activating the lipoxygenase pathway of the arachidonate cascade

Treatment of intact human neuroblastoma CHP100 cells with anandamide (arachidonoylethanolamide, AEA) or 2-arachidonoylglycerol (2-AG) inhibits intracellular fatty acid amide hydrolase (FAAH). This effect was not associated with covalent modifications of FAAH, since specific inhibitors of farnesyltransferase, kinases, phosphatases, glycosyltransferase or nitric oxide synthase were ineffective. Electrophoretic analysis of (33)P-labelled proteins, Western blot with anti-phosphotyrosine antibodies, and glycan analysis of cellular proteins confirmed the absence of covalent modifications of FAAH. The inhibition by AEA was paralleled by an increased arachidonate release, which was not observed upon treatment of cells with linoleoylethanolamide, palmitoylethanolamide, or oleoylethanolamide. Moreover, cell treatment with AEA or 2-AG increased the activity of cyclooxygenase and 5-lipoxygenase, and the hydro(pero)xides generated from arachidonate by lipoxygenase were shown to inhibit FAAH, with inhibition constants in the low micromolar range. Consistently, inhibitors of 5-lipoxygenase, but not those of cyclooxygenase, significantly counteracted the inhibition of FAAH by AEA or 2-AG.     

Activation of soluble guanylate cyclase by arachidonic acid and 15-lipoxygenase products

The activity of soluble guanylate cyclase can be increased by exposure of the enzyme to arachidonic acid or to some oxidized metabolites of the fatty acid. We have tried to determine whether activation of the enzyme by arachidonate requires that the fatty acid be converted to an oxidized metabolite, either by a possible trace contaminant of a lipoxygenase or by guanylate cyclase itself, which contains a heme moiety. Soluble guanylate cyclase purified from bovine lung was activated 4-6-fold by arachidonic acid. This activation was not dependent on the presence of oxygen in the incubation medium. No detectable metabolites of arachidonic acid were formed during incubation with soluble guanylate cyclase. Addition of soybean lipoxygenase to the incubation did not increase activation by arachidonic acid. The inhibitors of lipoxygenase activity, nordihydroguaiaretic acid and eicosatetraynoic acid, had direct effects on soluble guanylate cyclase and interfered with its activation by arachidonate, whereas another lipoxygenase inhibitor, BW 755 C, did not. The data suggest that arachidonic acid increases the activity of guanylate cyclase by direct interaction with the enzyme rather than by being converted to an active metabolite.     

Effects of platelet-activating factor on the release of arachidonic acid and prostaglandins by rabbit iris smooth muscle. Inhibition by calcium channel antagonists.

Addition of physiological concentrations (10(-12)-10(-8)M) of platelet-activating factor (PAF) to rabbit iris muscle induced a rapid release (in 15s) of prostaglandin (PG)E2 and 6-oxo-PGF1 alpha, measured by radioimmunoassay and rapid release of 14C-labelled arachidonate and PGE2 in muscle prelabelled with [14C]arachidonic acid, measured by radiochromatography. These PAF actions are concentration- and time-dependent. The effect of PAF on PG release is not mediated through the cyclo-oxygenase pathway. The studies on the properties and mechanism of arachidonate release from phosphatidylinositol and other phospholipids in prelabelled irides by PAF suggest the involvement of a phospholipase A2. This conclusion is supported by the findings: (a) that both the removal of arachidonate and formation of lysophosphatidylinositol, from phosphatidylinositol, by PAF occur concomitantly in a time-dependent manner, (b) that Ca2+ is required for the agonist-induced release of arachidonate and PGE2, and (c) that in contrast to the rapid release of [3H]myo-inositol phosphates by carbachol and other Ca2+-mobilizing agonists previously reported in the iris muscle [Akhtar & Abdel-Latif (1984) Biochem. J. 224, 291-300], PAF (10(-12)-10(-8)M) did not appreciably enhance the release of [14C]myo-inositol phosphates and 32P labelling of phosphatidate and phosphatidylinositol in this tissue. Ca2+-channel antagonists, such as nifedipine, verapamil, diltiazem and manganese inhibited PAF-induced arachidonate and PGE2 release in a dose-dependent manner. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not increase the release of arachidonate and PGE2. The ability of Ca2+ antagonists to inhibit arachidonate release by PAF in this tissue probably reflects interference with PAF binding to its receptor. The PAF-induced release of arachidonate and PGE2 occur independently of the cyclo-oxygenase and lipoxygenase pathways. Whether the PAF-induced release of arachidonate and PG in the iris muscle is involved in the pathogenesis of inflammatory and/or physiological reactions in the eye, and how much the inhibitory effects of Ca2+-entry blockers on the PAF actions contribute to the therapeutic use of these drugs, remain to be established.     

Decreased prostaglandin production by cholesterol-rich macrophages

The regulation of prostaglandin production by macrophages enriched in cholesterol was examined. Mouse peritoneal macrophages were incubated for 18 h with 25 micrograms/ml of human acetyl-LDL (low density lipoprotein) and trace amounts of labeled arachidonic acid. After cholesterol enrichment, the cells were incubated with phorbol 12-myristate 13-acetate (PMA), calcium ionophore, or zymosan to stimulate endogenous arachidonic acid metabolism. A high performance liquid chromatography profile of the eicosanoids released revealed no qualitative differences between unmodified and modified macrophages. Cholesterol-rich cells, however, released less prostacyclin (PGI2) and prostaglandin E2 (PGE2) compared to unmodified cells, and products from the lipoxygenase pathway became the predominant metabolites. A decrease in the synthesis of PGI2 and PGE2 by cholesterol-rich macrophages was confirmed by radioimmunoassay and radiolabeled experiments. The activity of prostaglandin synthetase was modestly increased in the cholesterol-modified macrophages compared to controls. As an estimation of phospholipase activity, the release of labeled arachidonic acid from membrane phospholipids, however, was significantly decreased in cholesterol-rich macrophages. The phosphatidylinositol fraction was particularly resistant to arachidonate release in response to calcium ionophore and PMA in the modified cells. The measurement of membrane phospholipid fatty acid composition before and after calcium ionophore supported the observation that less arachidonate was released by cholesterol-enriched cells in response to the ionophore. Based on these observations, we propose that prostaglandin synthesis from endogenous arachidonate stores is decreased in the cholesterol-rich macrophage. A decrease in agonist-induced activation of the phospholipase activity is proposed as a mechanism for this effect.     

Identification of lipoxygenase products from arachidonic acid metabolism in stimulated murine eosinophils

The presence of arachidonic acid lipoxygenase pathways in murine eosinophils was demonstrated by the isolation and identification of several lipoxygenase products from incubations of these cells. The most abundant arachidonate metabolite from murine eosinophils stimulated with ionophore A23187 and exogenous arachidonic acid was 12-S-hydroxyeicosatetraenoic acid (12-S-HETE), and the next most abundant was 15-HETE. Two families of leukotrienes were also recovered from these incubations. One family comprised the hydrolysis products of leukotriene A4, and the other included products derived from the 14,15-oxido analog of leukotriene A4 (14,15-leukotriene A4). Two double oxygenation products of arachidonate were also identified. These compounds were a 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and a 5,12-dihydroxyeicosatetraenoic acid (5,12-diHETE). Eosinophil stimulation promoter is a murine lymphokine which enhances the migration of eosinophils. When murine eosinophils were incubated with eosinophil stimulation promoter in concentrations sufficient to produce a migration response, a 2-3-fold increase in the production of 12-HETE was observed compared to unstimulated cells. Coupled with the recent demonstration that arachidonic acid lipoxygenase inhibitors suppress the migration response to eosinophil stimulation promoter and that 12-HETE induces a migration response, this observation provides further evidence in support of the hypothesis that eosinophil stimulation promoter stimulation of eosinophils results in the generation of lipoxygenase products which modulate the migratory activity of the cells.     

The effect of eicosapentaenoic acid on leukotriene B production by human neutrophils

Eicosapentaenoic acid, which is a major fatty acid in fish oil, previously has been shown to competitively inhibit the cyclooxygenase-catalyzed metabolism of arachidonic acid in platelets. In the present study the effect of eicosapentaenoic acid on the production of leukotriene B via the lipoxygenase pathway in human neutrophils was examined. Eicosapentaenoate was incorporated into complex lipids of neutrophils at the same rate as arachidonate; release of the two homologous fatty acids in response to calcium ionophore A23187 was equivalent and both fatty acids were metabolized to a leukotriene B. The products derived from eicosapentaenoic acid were identified as leukotriene B5 and its stereoisomers. Eicosapentaenoate was a less favorable substrate for leukotriene B5 synthesis (94 ng/10(7) cells/5 min at 20 microM exogenous fatty acid) than arachidonate was for leukotriene B4 (401 ng under the same conditions). However, eicosapentaenoate or an oxygenated product inhibited arachidonate metabolism since at equimolar concentrations of eicosapentaenoate and arachidonate leukotriene B4 production was decreased by 68%. The inhibitory effect occurred at the level of leukotriene A hydrolase. The biological activity of eicosapentaenoate -derived products was tested; leukotriene B5 was found to have only approximately 10% of the potency of leukotriene B4 in inducing the aggregation of neutrophils, and the stereoisomers of leukotriene B5 were inactive. These data suggest that diets enriched in eicosapentaenoic acid affect neutrophils by decreasing the quantity of leukotriene B and by the production of a less potent leukotriene.     

Arachidonic acid evokes epithelium-dependent relaxations in canine airways

Responses to arachidonate were examined in rings with and without epithelium of lobar, segmental, and subsegmental canine bronchi. Arachidonate evoked epithelium-dependent relaxations, which were less pronounced in subsegmental bronchi and abolished by indomethacin and meclofenamate. Nordihydroguairetic acid (NDGA) and nafazatrom reduced epithelium-dependent relaxations only in lobar but unmasked epithelium-independent relaxations to arachidonate in all bronchi. Prostaglandin E2 and prostacyclin relaxed all tissues similarly. In lobar bronchi without epithelium, basal release of prostaglandin E2 was reduced by indomethacin but unaffected by NDGA. Arachidonate augmented prostaglandin E2 release more in subsegmental than in lobar bronchi with epithelium; in bronchi without epithelium the rise was absent (lobar) or attenuated (subsegmental). Arachidonate augmented the release of 6-ketoprostaglandin F1 alpha more in lobar bronchi with than without epithelium; this was inhibited by indomethacin, but not NDGA. Thus arachidonate releases prostaglandin E2 (possibly produced by cyclooxygenase inaccessible to inhibitors and activated by lipoxygenase products) but not prostacyclin from the epithelium. Heterogeneity in response to arachidonate is not due to different sensitivity to, or production of, prostaglandins.