Effects of protein deficiency on the metabolism of arachidonic acid by rat pleural polymorphonuclear leukocytes

The effects of protein deficiency on the biosynthesis of metabolites of arachidonic acid by rat pleural polymorphonuclear leukocytes stimulated with calcium ionophore were investigated. The major products of metabolism by lipoxygenase in these cells were leukotriene B4 and 5-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas the major cyclooxygenase products were thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. At high substrate concentrations (100 microM), the formation of all products by polymorphonuclear leukocytes was lower for protein-deficient rats than for controls. Similar results were obtained when products synthesized from endogenous substrate were measured, except that there was no change in the amount of 5-hydroxy-6,8,11,14-eicosatetraenoic acid formed. The biosynthesis of prostaglandins E2 and F2 alpha by homogenates of rat kidney medulla was reduced as a result of protein deficiency. Acetylsalicylic acid inhibited the formation of cyclooxygenase products and stimulated the formation of lipoxygenase products by polymorphonuclear leukocytes. Protein deficiency did not alter the effects of acetylsalicylic acid on the biosynthesis of these products, although at any given concentration the amounts of products formed were less with protein-deficient rats than with rats fed control diets.     

Determination of leukotriene B4, 6-keto-prostaglandin F1 alpha and thromboxane B2 in peritoneal lavage fluid of sham-operated and adrenalectomized rats

In macrophages, isolated from the peritoneal fluid of rats, after activation, formation of metabolites of arachidonic acid occurs both by the cyclooxygenase and lipoxygenase pathways. The cells of normal animals produce mainly cyclooxygenase products. After adrenalectomy, a considerable increase occurs in the formation of lipoxygenase products, and less in those of the cyclooxygenase (1). In the experiments described here, the effect of adrenalectomy on the presence of leukotriene B4 (LTB4), 6-keto-PGF1 alpha and thromboxane B2 (TxB2) in the peritoneal fluid is determined.     

Increase in the formation of leukotriene B4 and other lipoxygenase products in peritoneal macrophages of adrenalectomized rats

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined. After isolation, the cells were incubated with [1-14C]arachidonic acid and the calcium ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the controls are 6-keto-prostaglandin F1 alpha, thromboxane B2 and 12-HETE. One peak represents 5,12-di-HETE. Smaller amounts of prostaglandin F2 alpha, prostaglandin E2, prostaglandin D2, leukotriene B4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of leukotriene B4, 15-HETE and 12-HETE. The increase in the prostaglandins is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, 6-keto-prostaglandin F1 alpha and thromboxane B2 are produced in higher amounts than leukotriene B4. After adrenalectomy, the formation of leukotriene B4 is much more increased than that of 6-keto-prostaglandin F1 alpha. These effects are most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid-induced peptide with phospholipase A2 inhibitory activity in adrenalectomized animals.     

Metabolism of icosa-5,11,14-trienoic acid in human platelets and the inhibition of arachidonic acid metabolism in human platelets by icosa-5,8,14-triynoic and icosa-5,11,14-triynoic acids

Two fatty acids differing from arachidonic acid in lacking one of the internal double bonds (20:35,8,14 and 20:35,11,14) and their 1-C14 and acetylenic analogues were synthesized. 20:35,8,14 was not metabolized by human platelets but 20:35,11,14 yielded a small amount (1.5% conversion) of two hydroxy fatty acids in a three (11-hydroxy-5,12,14-icosatrienoic acid) to one (15-hydroxy-5,11,13-icosatrienoic acid) proportion. Indomethacin inhibited formation of both hydroxy fatty acids indicating that they are produced via cyclooxygenase. Both ethylenic acids were weak inhibitors of cyclooxygenase (substrate 20 microM arachidonic acid) (ID50: 8.8 microM 20:35,8,14; 11.2 microM 20:35,11,14) but were inactive against lipoxygenase (ID50 greater than 100 microM). Similarly, both acetylenic analogues were poor inhibitors of lipoxygenase (ID50: 23.4 microM 20:35,8,14; 47.8 microM 20:35,11,14) but although 20:35,8,14 was inactive against cyclooxygenase (ID50 greater than 100 microM) the 20:35,11,14 was a potent inhibitor (ID50: 0.35 microM). The results are interpreted on the basis that hydrogen removal by the lipoxygenase is from C10 and by the cyclooxygenase from C13 but only in 20:35,11,14 are these hydrogens (C13) located at the center of a 1,4 cis cis pentadiene system (ethylenic) or a 1,4 pentadiyne system (acetylenic).     

Characterization of CGS 8515 as a selective 5-lipoxygenase inhibitor using in vitro and in vivo models

CGS 8515 inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 synthesis in guinea pig leukocytes (IC50 = 0.1 microM). The compound did not appreciably affect cyclooxygenase (sheep seminal vesicles), 12-lipoxygenase (human platelets), 15-lipoxygenase (human leukocytes) and thromboxane synthetase (human platelets) at concentrations up to 100 microM. CGS 8515 inhibited A23187-induced formation of leukotriene products in whole blood (IC50 values of 0.8 and 4 microM, respectively, for human and rat) and in isolated rat lung (IC50 less than 1 microM) in vitro. The selectivity of the compound as a 5-lipoxygenase inhibitor was confirmed in rat whole blood by the 20-70-fold separation of inhibitory effects on the formation of leukotriene from prostaglandin products. Ex vivo and in vivo studies with rats showed that CGS 8515, at an oral dose of 2-50 mg/kg, significantly inhibited A23187-induced production of leukotrienes in whole blood and in the lung. The effect persisted for at least 6 h in the ex vivo whole blood model. CGS 8515, at oral doses as low as 5 mg/kg, significantly suppressed exudate volume and leukocyte migration in the carrageenan-induced pleurisy and sponge models in the rat. Inhibitory effects of the compound on inflammatory responses and leukotriene production in leukocytes and target organs are important parameters suggestive of its therapeutic potential in asthma, psoriasis and inflammatory conditions.     

Naturally occurring conjugated octadecatrienoic acids are strong inhibitors of prostaglandin biosynthesis

Fatty acids from natural sources (mostly seed oils) were isolated and assayed for their effect on the bioconversion of arachidonic acid into prostaglandin E2, using sheep vesicular gland microsomes. Homologues and isomers of the naturally occurring fatty acids, obtained by chemical modification and/or organic synthetic methods, were also tested. Two very active cyclooxygenase inhibitors were discovered, namely jacarandic acid (8Z, 10E, 12Z-octadecatrienoic acid), isolated from Jacaranda mimosifolia, the concentration which gives 50% inhibition ([I]50) being 2.4 microM and the synthetic 8Z, 10E, 12E-octadecatrienoic acid, having an [I]50 of 1.0 microM. Under the conditions of the assay (75 microM substrate), earlier described potent inhibitors showed the following [I]50's: indomethacin: 1.3 microM; 9,12-octadecadiynoic acid: 1.3 microM, 8Z, 12E, 14Z-eicosatrienoic acid: 2.7 microM; 5,8,11,14-eicosatetraynoic acid: 4.4 microM. At a concentration of about half that of the substrate, the following naturally occurring fatty acids revealed inhibition ([I]50): columbinic acid (29 microM), calendulic acid (31 microM), liagoric acid (31 microM), ximenynic acid (39 microM), crepenynic acid (40 microM) and timnodonic acid (43 microM). Other fatty acids, and some of the above acids, were converted themselves more or less rapidly, mostly into conjugated monohydroxy fatty acids.     

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.     

Effects of stilbene derivatives on arachidonate metabolism in leukocytes

The effects of various alpha-phenylcinnamic acid derivatives (i.e., alpha-(3,4-dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid, alpha-(3,4-dihydroxyphenyl)-4-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3, 4-dihydroxycinnamic acid) synthesized from 3,4-dihydroxyphenyl acetic acid and hydroxy-benzaldehyde, and 3,3',4-trihydroxystilbene obtained by decarboxylation of alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid on rat peritoneal polymorphonuclear leukocyte lipoxygenase and cyclooxygenase activities were studied. 3,3',4-Trihydroxystilbene was found to inhibit the 5-lipoxygenase product, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HETE), and cyclooxygenase products, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2; its concentrations for 50% inhibition (IC50) were 0.885 +/- 0.016 microM for the leukocyte lipoxygenase product, 5-HETE, 7.70 +/- 0.104 microM for the formations of HHT and 7.96 +/- 0.143 microM for the formation of thromboxane B2. Alpha-(3,4-Dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3,4-dihydroxycinnamic acid also inhibited the formations of 5-HETE, HHT and thromboxane B2, although less strongly. Their IC50 values were, respectively, 91.3 +/- 3.62 microM, 947.5 +/- 28.7 microM, 453.3 +/- 229.3 microM and 148.8 +/- 50.6 microM for the formation of 5-HETE, 894.0 +/- 5.57 microM, 792.5 +/- 15.9 microM, greater than 1000 microM and 925.0 +/- 7.64 microM for the formation of HHT and 941.0 +/- 18.0 microM, 825 +/- 14.4 microM, greater than 1000 microM and 932.7 +/- 3.93 microM for the formation of thromboxane B2.     

Unsaturated fatty acids as second messengers of superoxide generation by macrophages

Chemically elicited guinea pig peritoneal exudate macrophages respond by superoxide (O2-) production to a large number of unrelated stimulants. It has been found that 8 out of 10 stimulants also induce arachidonic acid (20:4) liberation and thromboxane synthesis. The elicitation of O2- production by most stimulants was reduced or totally suppressed by three procedures that inhibit the activity of endogenous phospholipases: the use of drug p-bromophenacyl bromide, elevation of the cellular cyclic AMP level, and the removal of extracellular Ca2+. O2- production in response to concanavalin A, wheat germ agglutinin, and fMet-Leu-Phe were exquisitely sensitive to inhibition of phospholipase activity. Exogenously applied 20:4 as well as other unsaturated fatty acids (linolenic, linoleic, and oleic) induced massive and instantaneous O2- production in a dose-dependent manner. Saturated fatty acids (stearic) and methyl esters of unsaturated acids were inactive. Lysophosphoglycerides were also inactive. Incubation of macrophages with inhibitors of cyclooxygenase or lipoxygenase did not prevent the elicitation of O2- production by stimulants or fatty acids. On the contrary, O2- formation was enhanced by indomethacin and indomethacin by itself was capable of evoking O2- generation. Treatment of 20:4 with soybean lipoxygenase did not abolish its capacity to induce O2- production; native and lipoxygenase-treated 20:4 exhibited similar dose-response ratios. Purified 15-hydroxyeicosatetraenoic acid also elicited O2- production by macrophages with a potency comparable to but not exceeding that of 20:4. Equimolar amounts of prostaglandin E2 were inactive. These findings suggest that liberation of unsaturated fatty acid (principally, 20:4) from membrane phospholipids, as a consequence of phospholipase activation, is a necessary step in the elicitation of an oxidative burst in macrophages. O2- generation is stimulated by unesterified 20:4 and, possibly, by certain metabolites of 20:4. It appears that the lipoxygenase pathway may generate metabolites with stimulating capacity while the cyclooxygenase pathway is abortive.     

Catecholamines decrease leukotriene B4 and increase thromboxane B2 synthesis in A23187-stimulated human whole blood.

Catecholamines (adrenaline, dopamine, isoprenaline, noradrenaline) and caffeic acid (catecholic compound without adrenergic receptor activity) decreased leukotriene (LT)B4 synthesis in A23187-stimulated human whole blood. Salbutamol, a non-catecholic beta 2-adrenergic agonist, did not influence LTB4 synthesis. Catecholamines stimulated thromboxane (TX)B2 synthesis with a concomitant inhibition of LTB4 synthesis; caffeic acid and salbutamol did not stimulate TXB2 synthesis. These results, obtained in A23187-stimulated whole blood, which also takes into account the complex interaction between different cell types, are similar to our previous results with polymorphonuclear leukocytes. Catecholamines show an opposite effect on lipoxygenase and cyclooxygenase pathways, which may give rise to a marked change in LT/TX ratio in physiological or pathological conditions where sufficient concentrations of catecholamines are present.     

Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol- 2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes.

We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-i ndol-2- yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or formyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its omega-oxidation products, and 6-trans-isomers with IC50 values of 2.8-4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 microM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its omega-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5-11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3-0.9, 3.7-5.3, and 8.5-17.3 nM, respectively. In neutrophils primed with granulocyte--macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7-8.7 nM. At the concentration of 1 microM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.     

The effect of essential fatty acid deficiency on eicosanoid production in the inflamed rat colonic mucosa

Eicosanoids are potent mediators of inflammation and are synthesized in increased quantity in active ulcerative colitis. To elucidate the role of prostaglandin E2, thromboxane A2, prostaglandin I2, and leukotriene B2 in acute chemical colitis induced by 4% acetic acid, we utilized an animal model which has a deficiency of arachidonic acid, the precursor of eicosanoids due to an essential fatty acid deficient diet. Forty-eight hours after colitis was induced, mucosal synthesis of the cyclooxygenase products, prostaglandin E2, thromboxane A2, and prostaglandin I2, was significantly decreased in essential fatty acid deficient rats compared to normal controls. However, the 5-lipoxygenase product, leukotriene B4, was not different between groups. The decrease in cyclooxygenase products did not correlate with any change in the severity of colonic inflammation as assessed by gross morphology, histology, or myleoperoxidase activity. Thus inhibition of formation of the cyclooxygenase products of arachidonate metabolism does not appear to improve the degree of inflammation under the experimental conditions employed in this study.     

A formyl peptide contracts guinea pig lung: role of arachidonic acid metabolites

We studied the role of cyclooxygenase and lipoxygenase products of arachidonic acid metabolism in mediating N-formyl-methionyl-leucyl-phenylalanine- (FMLP) induced contractions of guinea pig lung parenchymal strips. The cyclooxygenase inhibitors indomethacin (10(-5) M) and aspirin (3 X 10(-5) to 10(-4) M), the lipoxygenase inhibitor nordihydroguaiaretic acid (10(-5) to 3 X 10(-5) M), and the combined cyclooxygenase/lipoxygenase inhibitors 1-phenyl-3-pyrazolidinone (Phenidone) (3 X 10(-5) to 3 X 10(-4) M) and BW 755C (10(-5) to 10(-4) M) each caused a decrease in the maximum force induced by FMLP (Fmax) and an increase in the concentration of FMLP required to produce 50% of Fmax (EC50). The thromboxane synthesis inhibitor imidazole (3 X 10(-3) M) also decreased Fmax. The leukotriene D4 receptor antagonist FPL 55712 (5.7 X 10(-6) to 1.9 X 10(-5) M) increased the EC50 for FMLP, whereas desensitization of lung parenchymal strips to leukotriene B4 by pretreatment with this leukotriene (10(-7) M) had no effect on FMLP-induced contraction. After exposure to FMLP (10(-6) M), guinea pig lung produced (as determined by high-performance liquid chromatography and radioimmunoassay) leukotrienes C4 and B4, thromboxane A2 (as measured by its stable degradation product thromboxane B2), and prostaglandin F2 alpha. Lung strips not exposed to FMLP showed no evidence of leukotriene production. We conclude that thromboxane A2 and leukotriene C4 generated in response to FMLP mediate a substantial fraction of the force induced by this peptide in guinea pig lung parenchymal strips.     

Endogenous suppression of neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes

Phospholipase A2 activity was measured in homogenized and acid-extracted human polymorphonuclear leukocytes using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate. In whole homogenate and in the supernatant and particular fractions separated by centrifugation at 150,000 X g, phospholipase activity was barely detectable (1-4 pmol/h per 10(6) cell equivalents). By contrast, acid extracts of these fractions contained over 10-times as much phospholipase activity in the dialyzed supernatants (20-300 pmol/h per 10(6) cell equivalents), whereas phospholipase inhibitor(s) were found in the sediment. The acid-solubilized phospholipase A2 activity was absolutely Ca2+-dependent and optimal at pH 7.0-7.5 with 1.0 mM added Ca2+. Addition of the resuspended sediment of the acid extract dose-dependently suppressed phospholipase activity in the supernatant; less than equivalent amounts were sufficient to inhibit 95%. Suppressor activity was lipid-extractable. After thin layer chromatography of lipid extracts, the bulk of inhibitory activity was recovered from the free fatty acid region. Analysis of the fatty acids by gas liquid chromatography showed that 63% were unsaturated. All unsaturated fatty acids tested were potent inhibitors of phospholipase A2 activity (IC50 3-10 microM). Oleoyl-CoA, hydroxyeicosatetraenoic acids and leukotriene D4 were also inhibitory, while methyl oleate, saturated fatty acids and the prostaglandins E2 and F2 alpha had no effect. These in vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

Activation of a 15-lipoxygenase/leukotriene pathway in human polymorphonuclear leukocytes by the anti-inflammatory agent ibuprofen

Human peripheral blood polymorphonuclear leukocytes (PMNs) metabolized [14C]arachidonic acid predominantly by lipoxygenase pathways. The major products were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-HETE. These and other lipoxygenase products, including their derived leukotrienes, have been implicated as mediators of inflammatory and allergic reactions. In human platelets, the nonsteroidal anti-inflammatory drug ibuprofen inhibited production of the cyclooxygenase product thromboxane B2 (I50 = 65 microM), whereas the lipoxygenase product 12-HETE was not appreciably affected even at 5 mM ibuprofen. The 5-lipoxygenase of human PMNs (measured by 5-HETE formation) was inhibited by ibuprofen but was about six times less sensitive (I50 = 420 microM) than the platelet cyclooxygenase. The unexpected observation was made that the human PMN 15-lipoxygenase/leukotriene pathway was selectively activated by 1-5 mM ibuprofen. Metabolites were identified by ultraviolet spectroscopy, by radioimmunoassay, or by retention times on high pressure liquid chromatography in comparison with authentic standards. The major product was 15-HETE; and in all of 19 donors tested, 15-HETE formation was stimulated up to 20-fold by 5 mM ibuprofen. Other identified products included 12-HETE and 15- and 12-hydroperoxyeicosatetraenoic acid. Activation of the 15-lipoxygenase by ibuprofen occurred within 1 min and was readily reversible. The effects of aspirin, indomethacin, and ibuprofen on the PMN 15-lipoxygenase were compared in six donors. Ibuprofen produced an average 9-fold stimulation of the enzyme, whereas aspirin and indomethacin resulted in an average 1.5- and 2-fold enhancement, respectively.     

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: possible pitfall of using the calcium ionophore A23187 to stimulate 5' lipoxygenase

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5' lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5' lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B4 by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC50 1.6 X 10(-4)M) was approximately 100 times less potent than BW755C (IC50 1.7 X 10(-6)M) at inhibiting leukotriene B4 synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 X 10(-6)M). The data obtained using STZ as stimulus are consistent with previous in vivo studies and indicate that benoxaprofen is a relatively selective inhibitor of cyclo-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5' lipoxygenase for evaluating inhibitors of eicosanoid synthesis.     

Selective inhibitors of platelet lipoxygenase: 4,7,10,13-eicosatetraynoic acid and 5,8,11,14-henicosatetraynoic acid

Nine acetylenic acids were evaluated to determine what structural features are required for selective inhibition of platelet lipoxygenase. Both 4,7,10,13-icosatetraynoic acid and 5,8,11,14-henicosatetraynoic acid inhibited the synthesis of 12-L-hydroxy-5,8,10,14-icosatetraenoic acid (HETE) more than 95 percent without significantly altering the production of either thromboxane B2 or 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The ID50 concentrations (microM) for inhibiting the synthesis of thromboxane B2 and HETE were respectively 51 and 0.46 with 4,7,10,13-icosatetraynoic acid while similar concentrations of 64 and 0.31 were found for 5,8,11,14-henicosatetraynoic acid.     

The involvement of phospholipid:diacylglycerol acyltransferases in triacylglycerol production

We have characterized three CoA-independent types of enzyme, phospholipases, phospholipid:diacylglycerol acyltransferases (PDATs) and cholinephosphotransferases, responsible for the removal of unusual fatty acids from phosphatidylcholine (PC) in microsomal preparations from developing oil seeds. The metabolism of sn-2-[(14)C]acyl-PC was monitored in microsomal preparations from various oilseeds having either medium-chain, acetylenic, epoxy or hydroxy fatty acids as their major fatty acids in the oil. The results indicate that PDAT plays a major role in removing ricinoleic acid and vernolic acid from phospholipids in Ricinus communis and Crepis palaestina seeds, respectively. However, vernolic, crepenynic and capric acids are primarily removed from phospholipids by phospholipases in Euphorbia lagascae, Crepis rubra and elm seeds, respectively. Further, we show that significant PDAT activity is also present in vegetative tissues of Arabidopsis thaliana.     

Arachidonic acid, 12- and 15-hydroxyeicosatetraenoic acids, eicosapentaenoic acid, and phospholipase A2 induce starfish oocyte maturation

In starfish oocyte maturation (meiosis reinitiation) is induced by the natural hormone 1-methyladenine (1-Me-Ade). This paper shows that arachidonic acid (AA) induces oocyte maturation at concentrations above 0.5 microM. This maturation shares many characteristics with 1-MeAde-induced maturation: same kinetics, same required contact time, same stimulations of protein phosphorylation and sodium influx. Although calcium facilitates the AA-induced but not the 1-MeAde-induced maturation, AA, like 1-MeAde, does not stimulate the uptake of calcium. Calcium does not facilitate the uptake of AA by oocytes. Out of 36 different fatty acids (saturated and unsaturated), only eicosatetraenoic (AA) and eicosapentaenoic acids were found to mimic 1-MeAde. Calcium-dependent phospholipases A2 from bee venom and Naja venom also induce maturation (0.1-1 unit/ml) when added externally to the oocytes. Phospholipase A2 inhibitors (quinacrine, bromophenacylbromide) block maturation; inhibition is reversed by increasing the 1-MeAde concentration and only occurs during the hormone-dependent period. AA is usually metabolized through oxidation by cyclooxygenase or lipoxygenase. Cyclooxygenase inhibitors (acetylsalicylic acid, indomethacin, tolazoline) do not block maturation; prostaglandins E2, D2, F2 alpha, I2, and thromboxane B2 do not induce meiosis reinitiation. On the other hand, lipoxygenase inhibitors (quercetin, butylated hydroxytoluene, and eicosatetraynoic acid) block 1-MeAde-induced maturation; although leukotrienes (A4, B4, C4, D4, E4) have no effects on oocytes, two other lipoxygenase products, 12- and 15-hydroxyeicosatetraenoic acids (and their corresponding hydroperoxy-) induce oocyte maturation (around 1 microM). The possible mode of action of the fatty acids inducing oocyte maturation is discussed.