On the structure of slow reacting substance of anaphylaxis: Evidence of biosynthesis from arachidonic acid

The mononuclear cells in peritoneal washings from normal rats can be induced to produce large amounts of slow reacting substance of anaphylaxis by incubation with 10 mM cysteine in the presence of the calcium ionophore A-23187. This production of slow reacting substance could be inhibited by the addition of non-steroidal anti-inflammatory drugs, e.g., indomethacin, ibuprofen and flurbiprofen. Furthermore, mediator production was inhibited by eicosatetraynoic acid, the substrate analog of arachidonic acid, and by 9,11-azoprosta-5,13-dienoic acid (AzO analog 1), a structural analog of the prostaglandin endoperoxide, PGH₂, which is known to inhibit thromboxane synthesis. Relatively high concentrations of hydrocortisone acetate inhibited mediator production; this inhibition could be partly reversed by the addition of arachidonic acid or to a lesser extent by eicosatrienoic acid. Preliminary results suggest that a small fraction of the ³H-labeled arachidonic acid which was taken up by these cells in vitro was associated with slow reacting substance. We postulate that slow reacting substance of anaphylaxis may be derived from a prostaglandin endoperoxide which is formed during the oxidation of arachidonic acid by the prostaglandin fatty acid cyclooxygenase.     

Flavonoids: potent inhibitors of arachidonate 5-lipoxygenase

Various flavonoids were found to be relatively selective inhibitors of arachidonate 5-lipoxygenase which initiates the biosynthesis of leukotrienes with the activity of slow reacting substance of anaphylaxis. Cirsiliol (3',4',5-trihydroxy-6,7-dimethoxyflavone) was most potent, and the enzyme partially purified from rat basophilic leukemia cells was inhibited by 97% at a concentration of 10 microM (IC50, about 0.1 microM). 12-Lipoxygenases from bovine platelets and porcine leukocytes were also inhibited but at higher concentrations (IC50, about 1 microM), and fatty acid cyclooxygenase purified from bovine vesicular gland was scarcely affected. The compound at 10 microM suppressed by 99% the immunological release of slow reacting substance of anaphylaxis from passively sensitized guinea pig lung (IC50, about 0.4 microM).     

The autoxidation of arachidonic acid: formation of the proposed SRS-A intermediate.

The air oxidation of 5,8,11,14-eicosatetraenoic [arachidonic] acid and its methyl ester is reported. A mixture of hydroperoxy arachidonic acid products was obtained from the oxidation and subsequent separation of the mixture by high pressure liquid chromatography led to pure hydroperoxides. One of these hydroperoxides, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, is a proposed intermediate in the biosynthesis of slow reacting substance of anaphylaxis.     

Lipid metabolism in cultured cells. Activators of endogenous thromboxane A2 synthesis in cultured lung fibroblasts

Thromboxane A2 (rabbit aorta-contracting substance) is a proaggregatory vasoconstrictive, oxygenated metabolite of arachidonic acid which was originally discovered in guinea pig lung perfusates during antigen-induced anaphylaxis. The specific stimuli which activate synthesis and the cellular source in the lung remain undefined. In order to study pulmonary thromboxane A2 (TXA2) synthesis, a cultured lung cell model has been used. Monolayer cultures of human diploid embryonic lung fibroblast (WI-38) metabolized exogenously supplied [14C]arachidonic acid to TXA2 as well as prostaglandin E2. Both were unequivocally identified by gas chromatography/mass spectrometry. Cellular phospholipids were labeled by preincubating cultures overnight with [14C]arachidonic acid. Release of thromboxane A2 into the culture fluid from these prelabeled cultures was stimulated by two phospholipase activating agents, mellitin and the calcium ionophore A23187. The lung cells also released TXA2 and prostaglandin in a dose-dependent fashion when treated with thrombin but not when exposed to trypsin. Bradykinin, an anaphylactic mediator in vivo, was a potent TXA2 releasing agent in this in vitro system whereas histamine was inactive. In addition, anaphylactic shock perfusates from guinea pig lung were shown to contain a factor (other than bradykinin) which activates fibroblasts TXA2 synthesis in these cultured lung cells. These experiments indicate that the lung fibroblast is probably a source of pulmonary thromboxane in vivo and that the cultured lung cell system described here is a useful model for defining the complex interactions of mediators of anaphylaxis and asthma.     

Arachidonic acid metabolism by cultured mesothelial cells. Different transformations of exogenously added and endogenously

The capacity of cultured mesothelial cells to produce prostaglandins from both exogenous an endogenous arachidonic acid has been investigated. Incubations with labelled [1-14C]arachidonic acid and [1-14C]prostaglandin endoperoxide H2 indicated the formation of prostacyclin and prostaglandin E2. Evaluation of the transformation of endogenously released arachidonic acid, however, could only confirm the production of prostacyclin.     

Activation mechanism of prostaglandin endoperoxide synthetase by hemoproteins

The mechanism of the activation of prostaglandin endoperoxide synthetase by hemeproteins was investigated using the enzyme purified from bovine seminal vesicle microsomes. At pH 8, the maximal enzyme activities with methemoglobin (2 microM), indoleamine 2,3-dioxygenase (2 microM), and metmyoglobin (2 microM) were 70%, 42%, and 15% of that with 1 microM hematin. Apomyoglobin and apohemoglobin inhibited the enzyme activities caused by hemoproteins as well as that caused by hematin. The inhibition was removed by the addition of excess hematin. The dissociation of heme from hemoproteins was demonstrated by trapping the free heme with human albumin or to a DE-52 column. The dissociation of heme from methemoglobin was facilitated by increasing concentrations of arachidonic acid. The amount of heme dissociated from hemoproteins (methemoglobin, metmyoglobin, and indoleamine 2,3-dioxygenase) in the presence of arachidonic acid correlated with their stimulatory effects on the prostaglandin endoperoxide synthetase activity. Horseradish peroxidase and beef liver catalase, the hemes of which were not dissociated in the presence of arachidonic acid, were ineffective in activating prostaglandin endoperoxide synthetase. Spectrophotometric titration of prostaglandin endoperoxide synthetase with hematin demonstrated that the enzyme bound hematin at the ratio of 1 mol/mol with an association constant of 0.6 x 10(8) M-1. From these results, we conclude that hemoproteins themselves are ineffective in activating prostaglandin endoperoxide synthetase and free hematin dissociated from the hemoproteins by the interaction of arachidonic acid is the activating factor for the enzyme.     

Effects of thromboxane A2 on lymphocyte proliferation

The main cyclooxygenase-dependent arachidonic acid derivatives produced by monocytes and macrophages have been shown to be thromboxane A2 and prostaglandin E2. The immunomodulatory effects of thromboxane A2 were examined using a specific thromboxane synthase inhibitor (dazoxiben), a thromboxane A2 analog (U46619), and a thromboxane A2 receptor blocker (BM13.177). Dazoxiben inhibited lymphocyte proliferation in response to mitogens (PHA and OKT3), but also reoriented cyclic endoperoxide metabolism towards the production of prostaglandin E2. Prostaglandin E2 has been shown previously to inhibit mitogen-induced lymphocyte proliferation. U46619, a stable thromboxane A2 analog, slightly enhanced lymphocyte responses to mitogens in the presence of dazoxiben and in the presence of a cyclooxygenase inhibitor (indomethacin). This occurred at concentrations of U46619 which are probably supraphysiological in view of the short half-life of natural thromboxane A2. Finally, the thromboxane A2 receptor blocker BM13.177 did not have any effect on mitogen-induced lymphocyte proliferation. It is concluded that thromboxane A2 has no or minimal modulatory effects on lymphocyte proliferative responses to mitogens and that the effect of thromboxane A2 synthase inhibition is rather due to reorientation of cyclic endoperoxide metabolism, resulting in increased prostaglandin E2 production.     

Stimulated production of slow reacting substances by mercaptans from ionophore A 23187-induced mononuclear cells: mercaptan structure-activity studies.

When isolated mononuclear cells from the peritoneal cavity of the rat are challenged with the ionophore, A 23187, slow reacting substance (SRS) is produced. SRS production is markedly enhanced by the addition of mercaptans to the incubations. In confirmation of previously published reports, this enhancement is dependent on the duration of a preincubation of the cells with the mercaptans before addition of the ionophore, a two minute preincubation being optimal. Quantitative structure-activity studies revealed that a variety of mercapto carboxylic acids, where the mercapto group was one or two carbon atoms removed from the carboxyl group, were all active in enhancing SRS production and the enhancement followed parallel dose response curves. A 4-mercapto acid, while weakly active, had a distinctly different dose response curve and cysteamine, which lacks a carboxyl function, was inactive. Preliminary characterization of the products of the incubations produced in the presence of different mercaptans has revealed that, while they all qualitatively meet the criteria of stability to alkali, lability in acid and destruction by arylsulfatase which are associated with slow reacting substance of anaphylaxis, there are quantitative differences in stability which suggest that the products may not be identical.     

The interaction between lipid peroxidation and prostaglandin synthesis in rabbit kidney-medulla slices.

Lipid peroxidation induced by ascorbic acid and Fe2+ was inhibited by mepacrine (phospholipase A2 inhibitor) and aspirin (prostaglandin cyclo-oxygenase inhibitor) in rabbit kidney-medulla slices. Moreover, ascorbic acid and Fe2+ potentiated the inhibitory effect on prostaglandin E2 formation by mepacrine, but they had no influence on prostaglandin E2 production decreased by aspirin. Lipid peroxidation induced by ascorbic acid and Fe2+ appears to be affecting the activity of prostaglandin endoperoxide synthase. These results suggest that lipid peroxidation is connected closely with the prostaglandin-generating system, and it has the potential to modulate the turnover of arachidonic acid and prostaglandin synthesis.     

Flurbiprofen: highly potent inhibitor of prostaglandin synthesis.

Flurbiprofen, 2-(2-fluoro-4-biphenylyl)propionic acid, inhibited the formation of prostaglandin E2 from arachidonic acid by bovine seminal vesicular microsomes. It was found that flurbiprofen was an approx. 12.5-fold better inhibitor than indomethacin by comparison of their I50 values. It was suggested that the inhibition of prostaglandin synthesis by flurbiprofen might be due to the inhibition of the endoperoxygenase which catalyzed conversion of arachidonic acid to cyclic endoperoxide. Other carboxylic acid compounds such as aspirin, ibuprofen and indomethacin showed the same type of inhibition as flurbiprofen. In contrast, phenylbutazone which was a pyrozolone derivative inhibited the formation of prostaglandin E2, but not affected the endoperoxygenase reaction. The kinetic studies for inhibition of prostaglandin E2 synthetase indicated that flurbiprofen competitively inhibited prostaglandin E2 synthesis, just like indomethacin. The Ki values were estimated to be 0.128 micron for flurbiprofen and 3.18 micron for indomethacin.     

Prostaglandins and renin release: I. Stimulation of renin release from rabbit renal cortical slices by PGI2.

Prostaglandins have been shown to be involved in the mechanism of renin secretion in a variety of situations. Both arachidonic acid and prostaglandin endoperoxide have been shown to release renin from cortical slices and to be converted to PGI2 by cortical microsomes. In the present studies PGI2 was found to cause a time dependent increase in renin release from rabbit renal cortical slices, a system isolated from any indirect effects that result from the administration of prostaglandins in vivo. The stimulation was linear up to 30 minutes and effective over a range of concentrations from 10(7 M to 10(-5) M. At similar concentrations 6-keto-prostaglandin F1alpha was not active on these slices. Thus, it is proposed that PGI2 exerts a direct effect on the release of renin from cortical cells and may be the mediator of arachidonate or prostaglandin endoperoxide stimulated renin secretion.     

Leukotriene C4: The major lipoxygenase metabolite of arachidonic acid in dog spleen

Slices of dog spleen converted [¹⁴C]-arachidonic acid (AA) to a polar material which conjugated with [³H]-glutatione. Nordihydroguaiaretic acid (NDGA) and 5,8,11,14, Eicosatetraynoic acid (ETYA) but not indomethacin, inhibited the conversion of [¹⁴C]-arachidonic acid by the spleen slices into the polar material indicating that it is derived through the lipoxygenase pathway. Physicochemical analysis of the polar metabolite of arachidonic acid after thin-layer chromatography and high pressure liquid chromatography revealed that it has chemical properties identical to authentic leukotriene C₄ standard (LTC₄). The biological activity of the purified material was found to be similar to the slow reacting substance of anaphylaxis (SRS-A), viz, it caused contraction of the guinea-pig ileum which was abolished by FPL-55172, a specific SRS-A receptor antagonist. These data suggest that dog spleen slices convert arachidonic acid through lipoxygenase pathway into a polar material that appears to be identical to LTC₄.     

Slow reacting substance of anaphylaxis, SRS-A: Assignment of the stereochemistry

We have recently described the structure elucidation of slow reacting substance of anaphylaxis (SRS-A) from lung and of a slow reacting substance (SRS) from basophilic leukaemia cells as 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid. The stereochemistry of this molecule has now been shown to be 5(S)-hydroxy-6(R)-cysteinylglycinyl-7,9-trans-11,14-cis-eicosatetraenoic acid by comparison of the synthetic and natural products and their derivatives using mass spectrometric and HPLC chromatographic techniques. The synthetic and natural compounds are also indistinguishable by their pharmacological properties, their conversion by soybean lipoxygenase, and their UV spectra.     

Induction of prostacyclin formation by sodium n-butyrate in a cloned epithelial liver cell line

The effect of sodium n-butyrate on prostaglandin synthesis in cultured cells was examined. Exposure of BC-90 cells, a clone of an epithelial rat liver cell line, to 1 mM sodium n-butyrate for 40 h induced prostacyclin production. Prostacyclin synthesis was proved by demonstrating: (1) production of labeled 6-ketoprostaglandin F1 alpha by treating [14C]arachidonic acid pre-labeled cells with calcium ionophore A23187, (2) production of unstable substance that inhibited adenosine diphosphate-induced platelet aggregation, and (3) conversion of [14C]arachidonic acid to 6-ketoprostaglandin F1 alpha in homogenates of n-butyrate-treated cells. Untreated control cells showed negligible prostaglandin synthesis. Untreated cell homogenates did not convert [14C]arachidonic acid to any prostaglandins, but they converted [14C]prostaglandin H2 to prostacyclin. Induction of prostacyclin production by n-butyrate was also demonstrated with cells that had been treated with acetylsalicylic acid before n-butyrate treatment in acetylsalicylic acid-free medium. Incorporation of [3H]acetylsalicylic acid by sodium n-butyrate-treated cells increased in accordance with treatment time, while that of untreated cells did not change during culture. There was no difference in the phospholipase A2 activities of n-butyrate-treated and -untreated cells. From these findings, the possibility that n-butyrate induced prostacyclin in BC-90 cells through induction of fatty acid cyclooxygenase activity is discussed.     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid synthesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3, 6, or 30 micrograms/ml) was placed under cranial windows in newborn pigs that had been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentrations, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dose-dependent increases of PGE2 in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF1 alpha and TXB2 only increased at the highest concentration of arachidonic acid (30 micrograms/ml). Cerebral ischemia did not decrease the conversion of any concentration of arachidonic acid to PGE2, 6-keto-PGF1 alpha, or TXB2. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies

Prostaglandin endoperoxide synthase transforms arachidonic acid to prostaglandin H2 via prostaglandin G2. The enzyme purified from bovine vesicular gland was given to mice as antigen, and monoclonal antibodies were raised by the hybridoma technique. Two species of the monoclonal antibody recognizing different sites of the enzyme were utilized to establish a peroxidase-linked immunoassay of prostaglandin endoperoxide synthase. Fab' fragment of one of the antibodies was prepared and conjugated to horseradish peroxidase. The conjugate was then bound to prostaglandin endoperoxide synthase, and the labeled enzyme was precipitated by the addition of the other antibody. The peroxidase activity of the immunoprecipitate correlated linearly with the amount of prostaglandin endoperoxide synthase. This sensitive and convenient method to determine the enzyme amount rather than the enzyme activity was utilized to extensively screen the amount of prostaglandin endoperoxide synthase in various bovine tissues. In addition to vesicular gland, platelets and kidney medulla previously known as rich enzyme sources, the immunoenzymometric assay demonstrated a high content of the enzyme in various parts of alimentary tract and a low but significant amount of enzyme in some parts of brain.     

Solubilization and partial purification of prostaglandin endoperoxide synthetase of rabbit kidney medulla.

The microsomes of rabbit kidney medulla converted arachidonic acid into prostaglandin E2 in the presence of hemoglobin, tryptophan and glutathione as activators. When themicrosomal suspension was treated with 1% Tween 20, a solubilized enzyme was obtained which catalyzed the conversion of arachidonic acid to prostaglandins G2 and H2. The solubilized enzyme was adsorbed to and then eluted from an omega-aminooctyl Sepharose 4B column, resulting in about 10-fold purification over the microsomes. The partially purified enzyme produced predominantly prostaglandin G2 in the presence of hemoglobin, while prostaglandin H2 was produced in the presence of both hemoglobin and tryptophan. The stimulation of prostaglandin endoperoxide formation was also observed with other heme and aromatic compounds. Prostaglandin H2 synthesis was inhibited by a variety of compounds including non-steroidal anti-inflammatory drugs, thiol compounds and prostaglandin analogues with a thiol group(s).     

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Dexamethasone stimulates arachidonic acid conversion to prostaglandin E2 in human amnion cells

The purpose of this investigation was to study the mechanism of stimulation of PGE2 output from human amnion epithelial cells by the synthetic glucocorticoid dexamethasone. Cells incubated in serum-free pseudo-amniotic fluid produced very low levels of PGE2, even when arachidonic acid (1 microM) was present. Pretreatment of cells with dexamethasone (50 nM) for 21 h increased the PGE2 output 6- to 7-fold in 2-h incubations only in the presence of arachidonic acid. The RNA synthesis inhibitor, actinomycin D (1 microgram/ml), and the protein synthesis inhibitor, cycloheximide (40 micrograms/ml), each blocked dexamethasone-stimulated arachidonic acid conversion to PGE2. The time course of these events suggests that dexamethasone first initiates RNA synthesis. Acetylsalicylic acid, a specific and irreversible blocker of prostaglandin endoperoxide H synthase (cyclooxygenase), was used to determine whether dexamethasone could stimulate new enzyme synthesis. Cells treated first with acetylsalicylic acid (30 min) then dexamethasone (22 h) produced as much PGE2 in response to 1 microM arachidonate as did cells exposed to dexamethasone only. Exposing cells to acetylsalicylic acid after dexamethasone completely eliminated PGE2 output. These data suggest that dexamethasone stimulates the synthesis of prostaglandin endoperoxide H synthase.