Conversion of arachidonic acid to lipoxygenase products by human fetal tissues

[14C]Arachidonic acid was converted to several lipoxygenase products by homogenates of human fetal tissues as determined by thin-layer chromatography. The net conversions of [14C]arachidonic acid to radiolabeled lipoxygenase products were high (greater than or equal to 5%) in the case of fetal liver and brain, and low (less than or equal to 2%) in the case of fetal adrenal, heart, and kidney.     

Possible involvement of lipoxygenase products of arachidonic acid pathway in ovulation

The possible involvement of products of the lipoxygenase pathway of arachidonic acid cascade in ovulation was tested by intrabursal injection of nordihydroguaiaretic acid (NDGA); 5, 8, 11-eicosatriynoic acid (5, 8, 11-ETYA), 3 amino-1-(3 trifluromethyphenyl)-2-pyrazoline hydrochloride (BW755c) and (FPL 55712). All these drugs reduced the number of ova released from the treated ovaries in a dose-dependent manner, without affecting ovulation from contralateral ovaries. NDGA was most potent since it completely blocked ovulation from the treated ovaries in 17/38 rats receiving a dose higher than 0.15 mg/bursa. This effect of NDGA cannot be ascribed to its inhibition of ovarian PGE synthesis. Conversion of labeled arachidonic acid via the lipoxygenase pathway by preovulatory rat follicles was demonstrated by TLC chromatography. Collectively, these results suggest the involvement of products of lipoxygenase pathway of arachidonic acid in ovulation in the rat.     

Modulation of insulin secretion by lipoxygenase products of arachidonic acid. Relation to lipoxygenase activity of pancreatic islets

Glucose (16.7 mM)-induced insulin secretion from isolated pancreatic islets of rats was inhibited by nordihydroguaiaretic acid (NDGA), 1-phenyl-3-pyrazolidinone (phenidone), 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline (BW755C), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 2,6-di-tert-butyl-4-methylphenol (BHT). Indomethacin and aspirin, however, failed to inhibit the glucose-induced insulin secretion but rather tended to enhance it. The glucose-induced insulin secretion was inhibited by 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) (50 microM), 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) (100 microM), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) (100 microM), but not by 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) (100 microM). Exogenous 5-HETE (10 microM) induced significant insulin secretion in a low glucose (3.3 mM) medium. Racemic 5-HETE also showed insulinotropic effect in a concentration-dependent manner with the concentrations 20 microM or above, whereas 12-HETE, 15-HETE, 15-HPETE, 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid, 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid, 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2 alpha failed to induce insulin secretion. Although significant insulin release was observed with arachidonic acid (greater than or equal to 100 microM), reduce cell viability was evident at 200 microM. When the 10,000 X g supernatant of isolated pancreatic islet homogenate was incubated with [3H]arachidonic acid at 37 degrees C in the presence of GSH and Ca2+, and the labeled metabolites then extracted with ethyl acetate and subjected to reverse phase high pressure liquid chromatography, several radioactive peaks, coeluted with authentic 15-, 12-, and 5-HETE, were observed. The radioactive peaks were completely suppressed by the addition of either NDGA, BW755C, or phenidone into the medium. The results support our contention i.e. the involvement of lipoxygenase product(s) in the secretory mechanism of insulin, and further suggest that 5-lipoxygenase system may play a role.     

Involvement of lipoxygenase products of arachidonic acid metabolism in bovine luteal function

A series of experiments was conducted to determine the effects of lipoxygenase products of arachidonic acid (AA) metabolism on the function of the bovine corpus luteum (CL). In the first experiment, reaction products of soybean lipoxidase-AA were added to dispersed bovine luteal cells in increasing concentrations. These lipoxygenase products resulted in a dose-related reduction in the biosynthesis of progesterone and 6-keto-prostaglandin (PG)F1 alpha, while the synthesis of PGF2 alpha was unaffected. In a second experiment, the addition of 5-hydroxyeicosatetraenoic acid (5-HETE), a specific lipoxygenase product, again resulted in a reduction in progesterone and 6-keto-PGF1 alpha, with no change in PGF2 alpha synthesis. Extremely high endogenous concentrations of 5-HETE were measured in luteal tissues (36 +/- 17 to 46 +/- 13 ng/10(6) cells) in a third experiment. In the fourth experiment, an inhibitor of the lipoxygenase pathways, nordihydroguaiaretic acid (NDGA) infused into the uterine lumen twice daily on Days 14-18 of the estrous cycle delayed luteolysis and resulted in lengthened estrous cycles (27.2 +/- 0.3 vs 21.5 +/- 1.0 days for controls, p less than 0.05). Thus, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism delays luteolysis, possibly by removing the preferential inhibition of PGF1 alpha biosynthesis caused by 5-HETE and other products of the lipoxygenase system. Collectively, these results suggest that products of the lipoxygenase pathway are involved in luteolysis in normal heifers.     

Chemokinetic activity of arachidonic acid lipoxygenase products on leuocyctes of different species

A number of hydroperoxy (HPETE) and hydroxy (HETE) products of the lipoxygenase pathway of arachidonic acid metabolism are chemotactic and chemokinetic for human neutrophils. We have investigated the relative chemokinetic potency of some of these products on human, rat and rabbit neutrophils. The most potent lipoxygenase product studied was 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-diHETE), which was maximally chemokinetic and chemotactic between 0.1 and 1.0ng/ml for the three species. The 5, 11 and 12-HPETEs and HETEs were chemokinetic, but less active by at least two orders of magnitude, for human and rabbit neutrophils at concentrations between 0.1 and 10μg/ml. 15-HPETE and 15-HETE were inactive on human leucocytes, and none of the monosubstituted products studied were chemokinetic for rat neutrophils. These results indicate that 5,12-diHETE may be an important mediator in the local accumulation of leucocytes in the inflammatory response.     

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.     

A role for lipoxygenase metabolites of arachidonic acid in porcine ovulation

Prostaglandins, products of arachidonic acid via the cyclooxygenase pathway, are essential to the porcine ovulatory process in that inhibition of their synthesis results in ovulation failure. Studies in the rat have shown that ovulation is also preceded by a rise in three ovarian hydroxyeicosatetraenoic acids, products of the lipoxygenase pathway, and inhibition of this pathway also inhibits ovulation. Experiments were designed, using a pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-treated prepuberal gilt model, to measure pre-ovulatory changes in follicular fluid concentrations of 15-hydroxyeicosatetraenoic acid (15-HETE), and to compare the effects of indomethacin and nordihydroguaiaretic acid (NDGA) on ovulation in the pig and on 15-HETE and prostaglandin F₂α synthesis both in vivo and in vitro. Follicular fluid concentrations of 15-HETE were elevated significantly just prior to the expected time of ovulation (40 h after hCG). When indomethacin (10 mg) was injected into the ovarian stalk at 24 h after hCG, follicular fluid concentrations of both 15-HETE and prostaglandin F₂α were lower (P<0.01) than controls at 40 h and ovulation rate was suppressed (P<0.01). When NDGA (5 mg) was administered in the same manner, ovulation rate was suppressed (P<0.01), but the levels of 15-HETE and prostaglandin F₂α were not altered. Synthesis of 15-HETE by cultured granulosa and theca interna cells was reduced by the presence of NDGA (1 mg/ml), whereas indomethacin (100 ng/ml) lowered 15-HETE production in theca interna cells only. These results clearly demonstrate that indomethacin can block the lipoxygenase as well as the cyclooxygenase pathways, depending on the dose used, and suggest that lipoxygenase metabolites of arachidonic acid are involved in the ovulatory process in the pig.     

Pitfalls in the use of arachidonic acid oxidation products to assign lipoxygenase activity in cancer cells

Arachidonic acid (AA) reaction with cyclooxygenase (COX) and lipoxygenases (LOX) yield eicosanoids that can mediate prostate cancer proliferation and enhance both tumour vascularization and metastasis. Increasingly measurement of eicosanoids with liquid chromatography is employed to implicate LOX activity in different biological systems and in particular link LOX activity to the progression of cancer in experimental models. This study demonstrates that simply identifying patterns of eicosanoid regio-isomerism is insufficient to designate LOX activity in prostate cancer cells and the analysis must include complete stereochemical assignment of the various isomers in order to validate the assignment of LOX activity.     

Stimulus-specificity of the chemotactic deactivation of human neutrophilis by lipoxygenase products of arachidonic acid

Preincubation of human neutrophils with chemotactic concentrations of 5(S)-hydroxy-eicosatetraenoic acid (5-HETE) or 5(S), 12(R)-dihydroxy-6, 14 cis-8, 10 trans-eicosatetraenoic acid (leukotriene B₄) induces a state of preferential chemotactic unresponsiveness to the homologous factor, termed deactivation, and less suppression of the responses to other chemotactic stimuli. The ratio of the concentration required for maximal chemotactic deactivation of neutrophils to that which stimulates chemotaxis optimally is greater for 5-HETE and leukotriene B₄ than for peptide and protein factors. In contrast to other chemotactic factors, 5-hydroperoxy-eicosatetraenoic acid (5-OOHETE) induces neutrophil chemotactic deactivation that is independent of the nature of the subsequent stimulus and is more slowly reversible after elimination of the fluid-phase deactivating factor. The unique characteristics of the chemotactic deactivation of human neutrophils by 5-OOHETE may be attributable in part to its endogenous metabolism to potent deactivating factors or to covalent derivatization of subcellular structures of the neutrophils by the highly reactive 5-OOHETE.     

Stimulus-specificity of the chemotactic deactivation of human neutrophils by lipoxygenase products of arachidonic acid

Preincubation of human neutrophils with chemotactic concentrations of 5(S)-hydroxy-eicosatetraenoic acid (5-HETE) or 5(S), 12(R)-dihydroxy-6,14 cis-8,10 trans-eicosatetraenoic acid (leukotriene B4) induces a state of preferential chemotactic unresponsiveness to the homologous factor, termed deactivation, and less suppression of the responses to other chemotactic stimuli. The ratio of the concentrations required for maximal chemotactic deactivation of neutrophils to that which stimulates chemotaxis optimally is greater for 5-HETE and leukotriene B4 than for peptide and protein factors. In contrast to other chemotactic factors, 5-hydroperoxy-eicosatetraenoic acid (5-OOHETE) induces neutrophil chemotactic deactivation that is independent of the nature of the subsequent stimulus and is more slowly reversible after elimination of the fluid-phase deactivating factor. The unique characteristics of the chemotactic deactivation of human neutrophils by 5-OOHETE may be attributable in part to its endogenous metabolism to potent deactivating factors or to covalent derivatization of subcellular structures of the neutrophils by the highly reactive 5-OOHETE.     

Edema from cyclooxygenase products of endogenous arachidonic acid in isolated lung

We infused A23187, a calcium ionophore, into the pulmonary circulation of dextran-salt-perfused isolated rabbit lungs to release endogenous arachidonic acid. This led to elevations in pulmonary arterial pressure and to pulmonary edema as measured by extravascular wet-to-dry weight ratios. The increase in pressure and edema was prevented by indomethacin, a cyclooxygenase enzyme inhibitor, and by 1-benzylimidazole, a selective inhibitor of thromboxane (Tx) A2 synthesis. Transvascular flux of 125I-albumin from vascular to extravascular spaces of the lung was not elevated by A23187 but was elevated by infusion of oleic acid, an agent known to produce permeability pulmonary edema. We confirmed that A23187 leads to elevations in cyclooxygenase products and that indomethacin and 1-benzylimidazole inhibit synthesis of all cyclooxygenase products and TxA2, respectively, by measuring perfusate levels of prostaglandin (PG) I2 as 6-ketoprostaglandin F1 alpha, PGE2, and PGF2 alpha and TxA2 as TxB2. We conclude that release of endogenous pulmonary arachidonic acid can lead to pulmonary edema from conversion of such arachidonic acid to cyclooxygenase products, most notably TxA2. This edema was most likely from a net hydrostatic accumulation of extravascular lung water with an unchanged permeability of the vascular space, since an index of permeability-surface area product (i.e., transvascular albumin flux) was not increased.     

The effect of trinitrobenzene sulfonic acid on gut-derived smooth muscle cell arachidonic acid metabolism: role of endogenous prostanoids

The contribution of smooth muscle cells as a potential source of eicosanoid production during inflammatory states remains to be elucidated. We investigated the effect of trinitrobenzene sulfonic acid (TNB), a known pro-inflammatory agent, on jejunal smooth muscle cell eicosanoid production. Human gut-derived smooth muscle cells (HISM) were incubated with TNB for 1 hour. Additionally, some cells were preincubated with either dimethylthiourea, or indomethacin for 1 hour before exposure to identical concentrations of TNB. Incubation with TNB led to significant increases in PGE(2) and 6-keto PGF-1(alpha) release, but not leukotriene B(4) release; responses which were both inhibited by dimethylthiourea and indomethacin treatment. Our results suggest that gutderived smooth muscle cells may represent an important source of proinflammatory prostanoids but not leukotrienes during inflammatory states of the intestine. The inhibition of prostanoid activity by thiourea may be mediated by suppression of cyclooxygenase activity in this cell line.     

Profiling assay for lipoxygenase products of linoleic and arachidonic acid by gas chromatography-mass spectrometry.

A method for determination of the lipoxygenase products of linoleic acid (9- and 13-hydroxyoctadecadienoic acid; 9-HODE, 13-HODE) and of arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acid; 5-, 8-, 9-, 11-, 12-, and 15-HETE) is described. The method combines solid-phase extraction, derivatization to the corresponding fully hydrogenated methylester/trimethylsilylether derivatives and capillary gas chromatography coupled with electron impact mass spectrometry. Each regioisomeric HODE and HETE shows a unique pair of mass spectrometric fragment ions originating from fission of the fatty acid carbon chain at the hydroxylated position. The carboxyl-terminal fragment is used for quantification relative to a carboxyl-18O2-labeled analogue added as internal standard and the methyl-terminal fragment is monitored for confirmation. The assay can be extended for quantification of the complete hydroxylation profile of linoleic and arachidonic acid. Applications of this assay are demonstrated for the quantification of HODEs and HETEs in normal, hyperplastic, and neoplastic mouse epidermis. In mouse epidermis papilloma, the tissue levels of 8- and 12-HETE were found to be increased by one to two orders of magnitude compared to levels in normal epidermis.     

A role for the lipoxygenase pathway of arachidonic acid metabolism in glucose- and glucagon-induced insulin secretion

Although the cylo-oxygenase pathway of arachidonic acid (AA) metabolism inhibits glucose-stimulatedinsulin release throught synthesis of prostaglandins, very little attention has been given to the effects of lipoxygenase pathway products on beta cell function. We have examined the effects of two structurally-dissimilar lipoxygenase inhibitors on insulin release from mono-layer-cultured rat islet cell. Both nordihydroguaiaretic acid (NDGA, 20–50 μM) and BW755c (100–250μM) caused a dose-responsive inhibition of glucose-induced insulin release. This inhibitory effect occurred despite concomitant inhibition of prostaglandin E synthesis. Lipoxygenase inhibitors also impeded cyclic AMP accumulation. Insulin and cyclic AMP release induced by glucagon were also blunted. These studies suggest the hypothesis that AA released in or near the beta cell is metabolized to lipoxygenase product(s) which have feed-forward properties important to glucose- and glucagon-stimulated cyclic nucleotide accumulation and insulin release.     

Effects of arachidonic acid and its cyclo-oxygenase and lipoxygenase products on lymphatic vessel contracility in vitro

Lymphatic vessels exhibit rhythmical contractility $\text{in vivo}$ and $\text{in vitro}$ and this activity appears ti regulate lymph flow. A technique for measuring the cicurlar muscle contractions of isolated bovine mesenteric lymphatic vessel segments has been devised and utilized to study the pharmacological properties of these vessels. Non-contracting lympahtic vessels can be induced to contract rhythmically with a variety of mediators, the most potent being a stable PGH₂ analogue (compound U46619), and the leukotrienes B₄, C₄ and D₄ (threshold concentrations in the nanomolar range). Prostagladin F_2α, noradrenaline, serotonin and histamine also elicited rhythmical activity but much higher concentrations were required. PGE₂ and PGE₁ were potent inhibitors of spontaneous contractions or those induced with U46619. In keeping with the diverse pharmacological effects of the metabolites of arachidonic acid, the addition of arachidonate to an isolated lymphatic vessel generated both stimulatory and inhibitory activities.It is concluded that arachidonic acid products (produced in the lymphatic vessel or entering the vessel in lymph draining the tissues) regulate lymph flow through their effects on lymphatic smooth muscle.     

Mechanism of action of glucocorticoid-induced immunoglobulin production: role of lipoxygenase metabolites of arachidonic acid

Glucocorticoids stimulate polyclonal immunoglobulin (Ig) production in cultures of human peripheral blood lymphocytes. The mechanism of action of glucocorticoids in this system, and indeed in any physiologic system, is unknown. Because glucocorticoids stimulate the production of phospholipase A2-inhibitory glycoproteins, we investigated whether glucocorticoids stimulate polyclonal Ig production by inhibition of arachidonic acid metabolism. Nonspecific lipoxygenase/cyclooxygenase inhibitors stimulate polyclonal Ig production in a manner similar to the effect of glucocorticoids, whereas specific cyclooxygenase inhibitors actually inhibit Ig production. Two specific 5-lipoxygenase inhibitors, with little or no activity against cyclooxygenase or other lipoxygenases, also stimulate Ig production. The dose-response effect of all of these drugs on Ig production was similar to the dose response of inhibition of 5-lipoxygenase. Leukotriene B4 (LTB4) added in low concentrations (10(-10)M) on days 1, 2, and 3 of a culture eliminated the stimulatory effect of glucocorticoids or 5-lipoxygenase inhibitors, whereas LTC4, LTD4, prostaglandin E, or 5-hydroxyeicosatetraenoic acid had no effect. These results suggest that the relevant action of glucocorticoids in stimulating Ig production might be in preventing endogenous arachidonic acid metabolism, perhaps the endogenous production of LTB4.     

Evidence against a role for lipoxygenase-derived products of arachidonic acid in the lamb ductus arteriosus

The effects of leukotrienes, the leukotriene antagonist FPL55712 (sodium 7-(3-(4-acetyl-3-hydroxy-2-propyl-phenoxy)-2-hydroxypropoxy)-4-oxo-8-propyl-4H-1-benzopyran-2-carboxylate), and inhibitors of arachidonate lipoxygenase and cyclo-oxygenase (compound BW755C, 3-amino-1-(m-(trifluoromethyl)-phenyl)-2-pyrazoline; ETYA, 5,8,11,14-eicosatetraynoic acid) were studied in an isolated preparation of ductus arteriosus from mature foetal lambs. Leukotrienes (LT) C4 and D4 produced a modest relaxation of the ductus but only at the highest concentrations tested (10(-7) to 10(-6) M) and under hypoxic conditions (PO2, 6--9 Torr (1 Torr = 133.322 Pa)). LTB4 had no effect at any concentration tested. BW755C (10(-6) to 10(-5) M) and FPL55712 (10(-5) M) contracted the hypoxic ductus; however, their action was abolished by pretreatment of the tissue with the cyclooxygenase inhibitor indomethacin (2.8 x 10(-6) M). Indomethacin-treated preparations were also unresponsive to ETYA 3 x 10(-5) M. The contraction of hypoxic tissues to either BW755C or FPL55712 increased further upon raising the oxygen tension of the medium (PO2 591--691 Torr). These findings indicate that leukotrienes and allied compounds formed from lipoxygenase-catalysed reactions do not contribute to prenatal patency of the ductus and are unlikely to have a role in its closure at birth. It is also confirmed that prostaglandin E2 is essential for keeping the vessel patent in the foetus.     

Effect of inhibitors of arachidonic acid metabolism on mitogenesis in human lymphocytes: possible role of thromboxanes and products of the lipoxygenase pathway.

Although it is already known that prostaglandins inhibit lymphocyte responses to mitogens the role of other products of arachidonic acid (AA) metabolism has not previously been investigated. Various inhibitors of AA metabolism were studied for their effects on mitogenesis in human lymphocytes, including imidazole, benzylimidazole, N-0164, L-8027, 5, 8, 11, 14 eicosatetraynoic acid, nordihydroguaiaretic acid, indomethacin, and aspirin. Selective or partially selective inhibitors of thromboxane synthesis, such as imidazole, benzylimidazole, N-0164, and L-8027 inhibited the mitogenic response at concentrations that also substantially affect thromboxane B2 synthesis in platelet-free lymphocyte preparations. Since indomethacin failed to reverse the inhibition by imidazole or N-0164, it is probably due to decreased thromboxane synthesis per se rather than secondary increases in prostaglandin synthesis. Eicosatetraynoic acid and nordihydroguaiaretic acid were more effective inhibitors of mitogenesis than of thromboxane synthesis. Since these agents also affect the lipoxygenase pathway, it is possible that part of their action is at this level. Thus, in addition to the inhibitory effects of prostaglandins on mitogenesis, other products of AA metabolism may promote the response.     

Alveolar macrophage lipoxygenase products of arachidonic acid: isolation and recognition as the predominant constituents of the neutrophil chemotactic activity elaborated by alveolar macrophages

Human immune interferon preparations have anticellular activity on human cell lines (WISH and HEp-2). This anticellular activity copurified with the human immune interferon and appears to be a function of the immune interferon molecule. On the basis of a unit of antiviral activity, purified human immune interferon had about 20 and 100 times more anticellular activity than purified fibroblast or leukocyte interferon, respectively. The possible implications of this finding in the treatment of human neoplasia are discussed.