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.     

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.     

Eicosanoid-mediated increase in glucose and lactate output as well as decrease and redistribution of flow by complement-activated rat serum in perfused rat liver

Rat serum, in which the complement system had been activated by incubation with zymosan, increased the glucose and lactate output, and reduced and redistributed the flow in isolated perfused rat liver clearly more than the control serum. Heat inactivation of the rat serum prior to zymosan incubation abolished this difference. Metabolic and hemodynamic alterations caused by the activated serum were dose dependent. They were almost completely inhibited by the cyclooxygenase inhibitor indomethacin and by the thromboxane antagonist 4-[2-(4-chlorobenzesulfonamide)-ethyl]-benzene-acetic acid (BM 13505), but clearly less efficiently by the 5'-lipoxygenase inhibitor nordihydroguaiaretic acid and the leukotriene antagonist N-(3-[3-(4-acetyl-3-hydroxy-2-propyl-phenoxy)-propoxy]-4-chlorine-6-meth yl- phenyl)-1H-tetrazole-5-carboxamide sodium salt (CGP 35949 B). Control serum and to a much larger extent complement-activated serum, caused an overflow of thromboxane B2 and prostaglandin F2 alpha into the hepatic vein. It is concluded that the activated complement system of rat serum can influence liver metabolism and hemodynamics via release from nonparenchymal liver cells of thromboxane and prostaglandins, the latter of which can in turn act on the parenchymal cells.     

Differential inhibition of thromboxane B2 and leukotriene B4 biosynthesis by two naturally occurring acetylenic fatty acids

The seed oil of the plant Ixiolaena brevicompta is a rich source of crepenynic acid (octadec-cis-9-en-12-ynoic acid), which has been linked with extensive sheep mortalities in Western New South Wales and Queensland, Australia. A number of acetylenic fatty acids have been found to interfere with lipid and fatty acid metabolism and inhibit cyclooxygenase and lipoxygenase enzymes in a variety of tissues. We have investigated the effects of crepenynic acid and ximenynic acid (octadec-trans-11-en-9-ynoic acid) on leukotriene B4 and thromboxane B2 production in rat peritoneal leukocytes and compare them with non-acetylenic compounds linoleic and ricinoleic acids. In concentrations ranging from 10 to 100 microM linoleic acid and ricinoleic acid had only minimal effects on leukotriene B4 and thromboxane B2 production in ionophore-stimulated cells. Ximenynic acid gave dose-dependent inhibition of leukotriene B4, thromboxane B2 and 6-ketoprostaglandin F1 alpha production. Ximenynic acid appears to be a more effective inhibitor of leukotriene B4 than crepenynic acid with an IC50 of 60 microM compared to 85 microM. On the other hand, crepenynic acid is a much more effective inhibitor of the cyclooxygenase products, having an IC50 for thromboxane B2 of less than 10 microM. Both acetylenic fatty acids inhibited phospholipase activity in these cells by 40-50% at a concentration of 100 microM but had no inhibitory effect at 10 microM. These results indicate that crepenynic acid and ximenynic acid differentially inhibit the cyclooxygenase and lipoxygenase products of stimulated leukocytes, and that at high doses of these fatty acids the effect on these products may be partially due to inhibition of phospholipase A2.     

Prostaglandin thromboxane, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid production by ionophore-stimulated rat serosal mast cells.

Production of several metabolites of arachidonic acid by purified rat serosal mast cells in response to stimulation with the ionophore A23187 was assessed by stable isotope dilution assay using gas chromatography-mass spectrometry. Compounds quantified were prostaglandins D2, E2, F2 alpha, 6-keto-F1 alpha, thromboxane B2, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid. Mast cells incubated at 37 degrees C for 30 min without ionophore produced measurable quantities of all metabolites assayed. 4 microM A23187 resulted in substantial increased synthesis of all metabolites compared to control cells. Of the metabolites quantified, prostaglandin D2 and prostacyclin were the major products derived from arachidonic acid in ionophore-stimulated rat mast cells.     

Release of prostaglandins and monohydroxy and trihydroxy metabolites of linoleic and arachidonic acids by adult and fetal aortae and ductus arteriosus

The conversion of arachidonic acid (20:4) to prostaglandins by vascular tissue is important in the adult because of the antithrombotic effect of prostacyclin and in the fetus because of the vasodilatory effect of prostaglandin (PG) E2 on the ductus arteriosus. We have shown that vascular tissue converts various polyunsaturated fatty acids to monohydroxy and trihydroxy metabolites derived from hydroperoxides, which may be involved in regulating prostaglandin synthesis. We have now measured the amounts of these hydroperoxide metabolites, as well as those of prostaglandins, released from slices of rat, rabbit and bovine aortae, as well as from fetal calf aorta and ductus arteriosus. The major oxygenated polyunsaturated fatty acid metabolite formed by rat and bovine blood vessels was 6-oxo-PGF1 alpha. Fetal calf aorta and ductus arteriosus produced about five times as much 6-oxo-PGF1 alpha as adult bovine aorta. Much smaller amounts of the cyclooxygenase products, PGE2, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11-hydroxy-20:4), and 15-hydroxy-20:4, were released by aortae. Small amounts of the lipoxygenase product, 12-hydroxy-20:4, were also detected. Substantial amounts of free and esterified monohydroxy and trihydroxy metabolites of linoleic acid (18:2) were detected, especially in rat and rabbit aortae. Rabbit aorta, which had low cyclooxygenase activity, formed more oxygenated 18:2 metabolites than 20:4 metabolites. Indomethacin did not inhibit the formation of the 18:2 metabolites, indicating that cyclooxygenase was not involved. Neither exogenous 13-hydroxy-18:2 nor trihydroxyoctadecenoic acid was incorporated to a large extent into lipids from vascular endothelial or smooth muscle cells, suggesting that the esterified 18:2 oxygenation products had arisen mainly via direct oxygenation of lipids.     

Macrophage eicosanoid formation is stimulated by platelet arachidonic acid and prostaglandin endoperoxide transfer

The present study was designed to determine whether platelets transfer arachidonic acid or prostaglandin endoperoxide intermediates to macrophages which may be further metabolized into cyclooxygenase products. Adherent peritoneal macrophages were prepared from rats fed either a control diet or an essential fatty acid-deficient diet, and incubated with a suspension of washed rat platelets. Macrophage cyclooxygenase metabolism was inhibited by aspirin. In the presence of a thromboxane synthetase inhibitor, 7-(1-imidazolyl)heptanoic acid, immunoreactive 6-ketoprostaglandin F1 alpha formation was significantly increased 3-fold. Since this increase was greater (P less than 0.01) than that seen with either 7-(1-imidazolyl)heptanoic acid-treated platelets or aspirin-treated macrophages alone, these results indicate that shunting of endoperoxide from platelets to macrophages may have occurred. In further experiments, macrophages from essential fatty acid-deficient rats were substituted for normal macrophages. Essential fatty acid-deficient macrophages, depleted of arachidonic acid, produced only 2% of the amount of eicosanoids compared to macrophages from control rats. When platelets were exposed to aspirin, stimulated with thrombin, and added to essential fatty acid-deficient macrophages, significantly more immunoreactive 6-ketoprostaglandin F1 alpha was formed than in the absence of platelets. This increased macrophage immunoreactive 6-ketoprostaglandin F1 alpha synthesis, therefore, must have occurred from platelet-derived arachidonic acid. These data indicate that in vitro, in the presence of an inhibition of thromboxane synthetase, prostaglandin endoperoxides, as well as arachidonic acid, may be transferred between these two cell types.     

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

Hydrogen peroxide inhibits alveolar macrophage 5-lipoxygenase metabolism in association with depletion of ATP

We have previously shown that the biologically important reactive oxygen metabolite hydrogen peroxide (H2O2) stimulates arachidonic acid (AA) release and thromboxane A2 synthesis in the rat alveolar macrophage. We have now investigated the effects of H2O2 on alveolar macrophage 5-lipoxygenase metabolism. H2O2 failed to stimulate detectable synthesis of leukotriene B4, leukotriene C4, or 5-hydroxyeicosatetraenoic acid (5-HETE) as determined by reverse-phase high performance liquid chromatography (RP-HPLC) and sensitive radioimmunoassays (RIAs). This was not explained by oxidative degradation of leukotrienes by H2O2 at the concentrations used. Moreover, RIA and RP-HPLC analyses demonstrated that H2O2 dose-dependently inhibited synthesis of leukotriene B4, leukotriene C4, and 5-HETE induced by the agonists A23187 (10 microM) and zymosan (100 micrograms/ml), over the same concentration range at which it augmented synthesis of the cyclooxygenase products thromboxane A2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. Four lines of evidence suggested that H2O2 inhibited alveolar macrophage leukotriene and 5-HETE synthesis by depleting cellular ATP, a cofactor for 5-lipoxygenase. 1) H2O2 depleted ATP in A23187- and zymosan-stimulated alveolar macrophages with a dose dependence very similar to that for inhibition of agonist-induced leukotriene synthesis. 2) The time courses of ATP depletion and inhibition of leukotriene B4 synthesis by H2O2 were compatible with a rate-limiting effect of ATP on leukotriene synthesis in H2O2-exposed cultures. 3) Treatment of alveolar macrophages with the electron transport inhibitor antimycin A prior to A23187 stimulation depleted ATP and inhibited leukotriene B4 and C4 synthesis to equivalent degrees, while thromboxane A2 production was spared. 4) Incubation with the ATP precursors inosine plus phosphate attenuated both ATP depletion and inhibition of leukotriene B4 and C4 synthesis in alveolar macrophages stimulated with A23187 in the presence of H2O2. Our results show that H2O2 has the capacity to act both as an agonist for macrophage AA metabolism, and as a selective inhibitor of the 5-lipoxygenase pathway, probably as a result of its ability to deplete ATP. Depletion of cellular energy stores by oxidants generated during inflammation in vivo may be a means by which the inflammatory response is self-limited.     

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.     

Characterization of the major polypeptide chains of reduced bovine thyroglobulin

The metabolism of bioreactive lipid mediators was studied in two types of activated macrophages (M phi). We compared the capacity of resident and activated M phi to release, upon a zymosan challenge, cyclooxygenase and lipoxygenase products as well as PAF-acether (platelet-activating factor) and its 2-lyso precursor. Activated M phi were obtained from mice injected intraperitoneally either with nonviable C74 streptococci (St-M phi) or with trehalose dimycolate, a defined immunostimulant isolated from Mycobacterium tuberculosis (TDM-M phi). Both activated populations exhibited common features: conversion of endogenous [14C]arachidonic acid into prostaglandin E2 and thromboxane A2 rather than into prostaglandin I2 and low biosynthesis of PAF-acether, probably due to an impairment of the acetylation step. However, contrary to St-M phi, TDM-M phi did not display a marked overall reduction of arachidonate metabolism. In addition, as compared to resident M phi, TDM-M phi presented a ratio of thromboxane B2/6-ketoprostaglandin F1 alpha 30-fold higher, a better conversion of leukotriene C to leukotriene D and a higher capacity to release the PAF-acether they synthesize. These macrophages thus seem to be valuable tools for studying the formation of mediators and for determining specific markers of an activated state.     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Leukotrienes increase glucose and lactate output and decrease flow in perfused rat liver

In isolated perfused rat liver leukotriene C4 and D4 but not B4 and E4 enhanced glucose and lactate output and lowered perfusion flow similar to the thromboxane A2 analogue U46619, extracellular ATP and prostaglandin F2 alpha. The kinetics of the metabolic changes caused by leukotriene C4 and D4 resembled those effected by U46619 and ATP but not those elicited by prostaglandin F2 alpha; the kinetics of the hemodynamic changes were similar only to those caused by U46619. The results show that leukotrienes could be important modulators of hepatic metabolism and hemodynamics and point to a complex intra-organ cell-cell communication between non-parenchymal and parenchymal cells.     

Contribution of macrophages to immediate hypersensitivity reaction

The interaction of mast cells with other leukocytes during immediate hypersensitivity reactions was tested by in vivo and in vitro experiments. Intraperitoneal challenge of passively sensitized rats with antigen caused the production of peptidoleukotrienes, leukotriene (LT)B4, thromboxane (TX)B2, and 6-keto-prostaglandin (PG) F1 alpha in the peritoneal cavity. Pretreatment of the rats with thioglycollate i.p. markedly changed the amount of eicosanoids formed. When polymorphonuclear leukocytes were the predominant cell type in the peritoneal exudate, both LTC4 and 6-keto-PGF1 alpha were decreased by 75% each and TXB2 by 50%. When elicited macrophages were predominant, there was an additional reduction in LTC4 by 68% as compared with 18 hr after thioglycollate treatment, but no additional change in the other arachidonic acid metabolites. In vitro antigen challenge of passively sensitized mouse bone marrow-derived mast cells caused the release of LTC4, LTB4, 6-trans-LTB4, 5-hydroxyeicosatetraenoic (5-HETE), and TXB2. Exposure to antigen of these mast cells in the presence of resident peritoneal macrophages markedly altered eicosanoid formation. Early in the time course (2 to 15 min), macrophages markedly enhanced all 5-lipoxygenase products. However, later in the time course (30 to 120 min), these products were decreased. This decrease was reversed by catalase and superoxide dismutase, which suggests the involvement of oxygen radicals. These active oxygen species also seemed to be generated by mast cells, because these enzymes caused an increase in 5-lipoxygenase products when mast cells were challenged alone. RIA of cyclooxygenase products showed that mast cells released only TXB2 when stimulated with antigen. When they were stimulated in the presence of macrophages, TXB2 and also PGE2 and 6-keto-PGF1 alpha were synthesized. Therefore, macrophages probably contribute the PGE2 and 6-keto-PGF1 alpha. Because the same amount of TXB2 was generated whether macrophages were present or not, the mast cells seem to be the major source of this compound. These data indicate that macrophages and possibly polymorphonuclear leukocytes participate in immediate hypersensitivity reactions.     

Differential alteration of lipoxygenase and cyclooxygenase metabolism by rat peritoneal macrophages induced by endotoxin tolerance

Altered macrophage arachidonic acid metabolism may play a role in endotoxic shock and the phenomenon of endotoxin tolerance induced by repeated injections of endotoxin. Studies were initiated to characterize both lipoxygenase and cyclooxygenase metabolite formation by endotoxin tolerant and non-tolerant macrophages in response to 4 different stimuli, i.e. endotoxin, glucan, zymosan, and the calcium ionophore A23187. In contrast to previous reports of decreased prostaglandin synthesis by tolerant macrophages, A23187-stimulated immunoreactive (i) leukotriene (LT)C4/D4 and prostaglandin (PG)E2 production by tolerant cells was greater than that by non-tolerant controls (p less than 0.001). However, A23187-stimulated i-6-keto-PGF1 alpha levels were lower in tolerant macrophages compared to controls. Stimulation of prostaglandin and thromboxane (Tx)B2 synthesis by endotoxin or glucan was significantly less in tolerant macrophages compared to controls (p less than 0.05). iLTC4/D4 production was not significantly stimulated by endotoxin or glucan, but was stimulated by zymosan in the non-tolerant cells. Synthesis of iLTB4 by control macrophages was stimulated by endotoxin (p less than 0.01). These results demonstrate that arachidonic acid metabolism via the lipoxygenase and cyclooxygenase pathways in macrophages is differentially altered by endotoxin tolerance.     

Prolonged lipopolysaccharide inhibits leukotriene synthesis in peritoneal macrophages: mediation by nitric oxide and prostaglandins

Resident rat peritoneal macrophages synthesize a variety of prostanoids and leukotrienes from arachidonic acid. Overnight treatment with lipopolysaccharide (LPS) induces the synthesis of cyclooxygenase-2 (COX-2) and an altered prostanoid profile that emphasizes the preferential conversion of arachidonic acid to prostacyclin and prostaglandin E2. In these studies, we report that exposure to LPS also caused a strong suppression of 5-lipoxygenase but not 12-lipoxygenase activity, indicated by the inhibition of synthesis of both leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE), but not of 12-HETE. Inhibition of 5-lipoxygenase activity by LPS was both time- and dose-dependent. Treatment of macrophages with prostaglandin E2 partially inhibited leukotriene synthesis, and cyclooxygenase inhibitors partially blocked the inhibition of leukotriene generation in LPS-treated cells. In addition to COX-2, nitric oxide synthase (NOS) was also induced by LPS. Treatment of macrophages with an NO donor mimicked the ability of LPS to significantly reduce leukotriene B4 synthesis. Inhibition of NOS activity in LPS-treated cells blunted the suppression of leukotriene synthesis. Inhibition of both inducible NOS and COX completely eliminated leukotriene suppression. Finally, macrophages exposed to prolonged LPS demonstrated impaired killing of Klebsiella pneumoniae and the combination of NOS and COX inhibitors restored killing to the control level. These results indicate that prolonged exposure to LPS severely inhibits leukotriene production via the combined action of COX and NOS products. The shift in mediator profile, to one that minimizes leukotrienes and emphasizes prostacyclin, prostaglandin E2 and NO, provides a signal that reduces leukocyte function, as indicated by impaired killing of Gram-negative bacteria.     

Products, kinetics, and substrate specificity of homogeneous thromboxane synthase from human platelets: development of a novel enzyme assay

Homogeneous thromboxane synthase from human platelets converted prostaglandin H2 (PGH2) to thromboxane A2 (measured as thromboxane B2, TxB2), 12(L)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and malondialdehyde (MDA) in equimolar amounts under a variety of experimental conditions. PGG2 was transformed to MDA and corresponding 15- and 12-hydroperoxy products. PGH1 was enzymatically transformed into 12(L)-hydroxy-8,10-heptadecadienoic acid (HHD) and PGH3 into TxB3 and 12(L)-hydroxy-5,8,10,14-heptadecatetraenoic acid (delta 14-HHT) as earlier reported for solubilized and partially purified thromboxane synthase preparations. The ratio of thromboxane to C17 hydroxy fatty acid formation was 1:1 with PGG2, PGH2, and PGH3 as substrates. These results confirm and extend earlier observations with partially purified enzyme that the three products are formed in a common enzymatic pathway (Diczfalusy, U., Falardeau, P., and Hammarstr?m, S. (1977) FEBS Lett. 84, 271-274). A convenient spectrophotometric assay for thromboxane synthase activity measuring the ultraviolet light absorption of the C17 hydroxy acid formed (e.g., HHT) was developed. The validity of the assay was determined employing specific inhibitors for thromboxane synthase. The substrate specificity of thromboxane synthase was determined using this assay. PGG2 and PGH3 showed Vmax and KM values similar to those of PGH2. The KM value of PGH1 was also identical to that of PGH2 but the Vmax value PGH1 was more than twice as high as that of PGH2.     

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.     

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.     

Formation and metabolism of leukotriene C4 in macrophages exposed to bacterial lipopolysaccharide

Lipopolysaccharide (10 micrograms/ml) was found to stimulate resident mouse peritoneal macrophages to produce leukotriene C4 (36 +/- 1.3 ng/10(6) cells, SEM, n = 20) within 16 h. Spontaneous synthesis in control cultures without lipopolysaccharide was less than 1.6 ng/10(6) cells. Leukotriene C4 was characterized by reversed-phase high-performance liquid chromatography, ultraviolet spectrometry and radioimmunoassay. When the macrophages, prelabeled with [3H]arachidonic acid, were treated with lipopolysaccharide radioactivity was incorporated into leukotriene C4. The amount produced varied with the method of macrophage preparation and incubation conditions and was dependent on the amount of lipopolysaccharide added (0.5-60 micrograms/ml), on cell counts and on the incubation time (4-16 h). The released leukotriene C4 was converted to a compound identified as a C6-cysteinylleukotriene, indicating metabolism of the leukotriene by the macrophages. Parallel determinations of prostaglandins E2 and F2 alpha by radioimmunoassay demonstrated that leukotriene C4 and prostaglandin E2 are formed by mouse peritoneal macrophages to a similar degree.