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Tumor-derived exosomes can modulate the cancer microenvironment and induce metastatic spread. Exosomes may carry enzymes for leukotriene (LT) biosynthesis, but the role of exosomal LTs has not been studied in cancer. We isolated exosomes and malignant cells from pleura exudates from 14 patients with non-small cell lung cancer. Lipidomic profiles, migration and apoptosis were determined. Both exosomes and primary cancer cells contained γ-glutamyl transpeptidase 1 (GGT-1) and avidly transformed exogenous LTC4 to pro-tumorigenic LTD4, for the cells to levels 100-fold above their endogenous CysLT production. This suggests that cancer cells promote their own survival via LTD4 if supplied with LTC4, which in the exudates was produced by monocytic cells. Furthermore, exosomes promoted migration of cancer cells, which was counteracted by the CysLT1 antagonist montelukast. Montelukast also induced apoptosis of cancer cells, and this was partially inhibited by exosomes. Our results demonstrate how cancer cells and exosomes, together with monocytic cells in lung cancer tissue, can produce high amounts of LTD4, to stimulate cancer cell migration and survival. This suggests that part of the pro-metastatic effect of exosomes is mediated by the leukotriene machinery, further supporting the use of CysLT1 antagonists for lung cancer therapy.

5-Lipoxygenase (5-LOX) catalyzes two steps in the biosynthesis of leukotrienes (LTs), lipid mediators of inflammation derived from arachidonic acid. In this review we focus on 5-LOX biochemistry including 5-LOX interacting proteins and regulation of enzyme activity. LTs function in normal host defense, and have roles in many disease states where acute or chronic inflammation is part of the pathophysiology, as briefly summarized at the end of this chapter. This article is part of a Special Issue entitled “Oxygenated metabolism of PUFA: analysis and biological relevance".

The role of basic science in the development of health care has received more and more attention. In my own area of research involving the so-called eicosanoids, there are many examples of how studies of structure and function of small molecules, as well as proteins and genes, have led to new therapeutic agents for treatment of a variety of diseases. In most of the cases, the discoveries have resulted in the recognition of novel therapeutic targets amenable to modulation by small molecules. However, there are also examples in which the molecular mechanisms of actions of drugs, discovered by phenotypic screening, have been elucidated. The majority of the examples in this article consist of approved drugs; however, in some cases, ongoing developments of potential therapeutics are cited.

5-Lipoxygenase (5-LO) catalyzes two steps in biosynthesis of leukotrienes (LTs), a group of lipid mediators of inflammation derived from arachidonic acid (AA). LT antagonists are used in treatment of asthma; more recently a potential role also in atherosclerosis has raised considerable interest. Furthermore, possible effects of 5-LO metabolites in relation to tumorigenesis have emerged. Thus, an understanding of the biochemistry of this lipoxygenase has potential implications for treatment of various diseases.

Microsomal prostaglandin E synthase type 1 (mPGES-1) converts prostaglandin endoperoxides, generated from arachidonic acid by cyclooxygenases, into prostaglandin E2. This enzyme belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral membrane proteins, and because of its link to inflammatory conditions and preferential coupling to cyclooxygenase 2, it has received considerable attention as a drug target. Based on the high resolution crystal structure of human leukotriene C4 synthase, a model of mPGES-1 has been constructed in which the tripeptide co-substrate glutathione is bound in a horseshoe-shaped conformation with its thiol group positioned in close proximity to Arg-126. Mutation of Arg-126 into an Ala or Gln strongly reduces the enzyme's prostaglandin E synthase activity (85-95%), whereas mutation of a neighboring Arg-122 does not have any significant effect. Interestingly, R126A and R126Q mPGES-1 exhibit a novel, glutathione-dependent, reductase activity, which allows conversion of prostaglandin H2 into prostaglandin F2alpha. Our data show that Arg-126 is a catalytic residue in mPGES-1 and suggest that MAPEG enzymes share significant structural components of their active sites.

5-Lipoxygenase (5-LO) catalyzes the first two steps in the biosynthesis of leukotrienes, a group of pro-inflammatory lipid mediators derived from arachidonic acid. Leukotriene antagonists are used in the treatment of asthma, and the potential role of leukotrienes in atherosclerosis, another chronic inflammatory disease, has recently received considerable attention. In addition, some possible effects of 5-LO metabolites in tumorigenesis have emerged. Thus, knowledge of the biochemistry of this enzyme has potential implications for the treatment of various diseases. Recent advances have expanded our understanding of the regulatory mechanisms underlying the expression and control of 5-LO activity. With regard to the control of enzyme activity, many of these findings focus on the N-terminal domain of 5-LO.

This review article focuses on two aspects regarding 5-lipoxygenase. First, mechanisms for activation of the enzyme. Second, the involvement of 5-lipoxygenase and leukotrienes in atherosclerosis.

Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 µmol min–1 mg–1) and high kcat/Km (310 mM–1 s–1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 µmol min–1 mg–1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 µmol min–1 mg–1), 5-hydroperoxyeicosatetraenoic acid (0.043 µmol min–1 mg–1), and 15-hydroperoxy-PGE2 (0.04 µmol min–1 mg–1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 µmol min–1 mg–1). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1. (Less)

Coactosin was first isolated from Dictyostelium discoideum and, as reported, human coactosin-like protein (CLP) was identified in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. A mouse CLP (mCLP) cDNA clone was identified among EMBL/GenBank EST sequences. The derived amino acid sequence (142 residues) was 95.1% identical with human CLP. Here, we also show that mCLP interacts with actin and 5LO in the two-hybrid system. High-speed cosedimentation assays and GST-binding assays confirmed these protein interactions. In chemical cross-linking experiments, one molecule of mCLP was covalently linked to either one subunit of actin or one molecule of 5LO. The mCLP-F-actin and mCLP-5LO associations were pH-insensitive and Ca(2+)-independent. However, association with actin was best observed at low salt concentrations, while association with 5LO was favored by salt, indicating different binding characteristics.

The specific binding of leukotriene B4 to its receptor on human leukocyte membranes was enhanced in a dose-dependent manner by metal ions. Mg2+ and Ca2+ were the most potent followed by Mn2+ and Co2+. These divalent cations increased receptor density and did not affect the binding affinity. Addition of the same divalent cations to a partially purified receptor prepared by precipitation with polyethylene glycol produced a greater effect, whereby both binding affinity and receptor density were significantly increased. Zn2+ inhibited activity in both preparations by decreasing the density of receptors. In the membrane preparation, binding activity was decreased by Na+ and increased by K+, while Li+ had no effect. However, in the partially purified receptor preparation, these monovalent cations all increased the binding activity. Our findings show that purification of the leukotriene B4 receptor modifies its response to various monovalent and divalent cations, perhaps by removing certain cellular components involved in receptor regulation.

Induction of 5-LO activity in DMSO differentiated HL-60 cells, by the serum protein TGF beta, was partially dependent on the presence of serum lipids (Steinhilber, D., Hoshiko, S., Grunewald, J., Rådmark, O., and Samuelsson, B. (1993) Biochim. Biophys. Acta 1178, 1-8). Here we demonstrate that the serum lipid fraction can be substituted by picomolar concentrations of 1,25-dihydroxyvitamin D3 (VD3). A high concentration of VD3 (24 nM) gave a 4-fold induction of 5-LO mRNA, a 14-fold increase in 5-LO protein, and a 38-fold upregulation of the 5-LO activity of intact HL-60 cells after differentiation in the presence of DMSO and serum proteins for 4 days. Also VD3 alone gave a substantial upregulation of 5-LO protein expression and activity. On the other hand, TGF beta alone was a poor inducer of the 5-LO pathway, the presence of a differentiation inducer (retinoic acid, DMSO or VD3) was required. The most prominent induction of 5-LO protein expression and activity in cell homogenates and intact cells was observed when VD3 and TGF beta were combined.

5-Lipoxygenase (5-LO) activity in differentiating HL-60 cells was upregulated by a heat stable protein in human serum. Hematopoietic cytokines were evaluated for this effect on 5-lipoxygenase activity, none of the compounds tested could replace serum. However, TNFα or GM-CSF were able to augment the effect of heat treated serum, giving 5-LO activities that (per cell) were higher than for granulocytes isolated from peripheral blood. The serum factor primarily upregulated 5-LO activity. The amounts of 5-LO protein, or mRNA for 5-LO or 5-lipoxygenase activating protein (FLAP), were less affected.

Purified human leukotriene A4 hydrolase is shown to contain 1 mol of zinc per mol of enzyme, as determined by atomic absorption spectrometry. The enzyme is inhibited dose-dependently by the chelating agents 8-hydroxy-quinoline-5-sulfonic acid, and 1,10-phenanthroline with KI values of about 2 and 8 x 10(-4) M, respectively, whereas dipicolinic acid and EDTA are ineffective in this respect. The inhibition by 1,10-phenanthroline is time-dependent, and at a concentration of 5 mM, 50% inhibition of enzyme (3 x 10(-7) M) occurs after about 15 min. The zinc atom of leukotriene A4 hydrolase can be removed by dialysis against 1,10-phenanthroline which results in loss of enzyme activity. The catalytic activity is almost completely restored by the addition of stoichiometric amounts of Zn2+ or Co2+.

The metabolism of arachidonic acid by washed human platelets was investigated. [1-14C]Arachidonic acid was extensively converted to [1-14C]12-hydroxyeicosatetraenoic acid. In addition, several minor labeled products were formed with a considerably lower specific activity, indicating their preferential formation from endogenous substrate. These were dihydroxy metabolites of arachidonic acid with conjugated triene structures and were identified as 14,15-dihydroxyeicosatetraenoic acid (three isomers) and 8,15-dihydroxyeicosatetraenoic acid (three isomers). The identification was based on comparison with reference compounds with respect to chromatographic properties, characteristic UV spectra, and mass spectrometry of several derivatives. Bradykinin (10(-8)-10(-5) M) was found to enhance the formation of all these compounds. In addition, the monohydroxy acid fraction was found to contain 15-hydroxyeicosatetraenoic acid. The present investigation thus demonstrates the occurrence of a 15-lipoxygenase in human platelets in addition to the previously known 12-lipoxygenase.

A nomenclature for thromboxanes is proposed in which the parent unsubstituted compound is thrombane and the corresponding carboxylic acid is thrombanoic acid.

The following labeled compounds were isolated and identified after incubation of 8,11,14-eicosatrien [1-14C] oic acid with human platelets: 12-L-hydroxy-8,10,14-eicosatrienoic acid, 8,11,12-trihydroxy-9,14-eicosadienoic acid, 8,9,12-trihydroxy-10,14-eicosadienoic acid, 12-L-hydroxy-8,10-heptadecadienoic acid, prostaglandin E1, prostaglandin D1, and 8-(1-hydroxy-3-oxopropyl)-9,12-dihydroxy-10-heptadecenoic acid (thromboxane B1).

A slow reacting substance, produced by murine mastocytoma cells, has been shown to have the structure 5(S)-hydroxy-6(R)- S -glutathionyl-7,9,11- trans -14- cis -eicosatetraenoic acid (11- trans leukotriene C, previously referred to as leukotriene C-2) by ultraviolet spectroscopy, amino acid analyses, lipoxygenase conversion and comparisions with a synthetic compound of known structure and stereochemistry.

Leukotriene C, a “Slow Reacting Substance” (SRS) from mouse mast cell tumors, was earlier shown to be a derivative of 5-hydroxy-7,9,11,14-eicosatetraenoic acid with a cysteine containing substituent in thioether linkage at C-6 (Murphy, R.C., Hammarstrom, S., Samuelsson, B.: Proc. Natl. Acad. Sci. USA, 76 , 4275–4279 (1979)). The substituent has now been identified as γ-glutamylcysteinylglycine (glutathione).

Arachidonic acid, linolenic acid and 14 different oxygenated fatty acid derivatives were tested as activators of human protein kinase C in vitro using histone as substrate. Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid) activated the kinase in the presence of calcium at 30 fold lower concentration (1 μM) than did arachidonic acid or 1,3-dioleoylglycerol. The methyl ester of lipoxin A and the free acids of leukotriene B 4 as well as two lipoxin B isomers were without effect. In contrast, linolenic acid, leukotriene C 4 , certain mono- and dihydroxylated eicosanoids and one lipoxin B isomer had stimulatory effects, albeit at higher concentrations. The substrate specificity of protein kinase C activated by lipoxin A proved to be different from that of the phosphatidylserine or phorbol ester activated kinase. Results of the present study suggest that arachidonic acid derived oxygenation products, in particular lipoxin A, may serve as intracellular activators of protein kinase C.

A preparation of human leukocytes was incubated with arachidonic acid. Two new dihydroxy acids with conjugated triene structures, were isolated and characterized as 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-leukotriene B4) and 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene B4).

The prostaglandin endoperoxide prostaglandin H 2 (15-hydroxy-9α,11α-peroxidoprosta-5,13-dienoic acid) inhibits basal and hormone-stimulated adenylate cyclase in fat cell ghosts. This inhibition by prostaglandin H 2 has been found to be antagonized by GTP and Gpp(NH)p. Dose response studies have shown GTP and Gpp(NH)p to be maximally effective at 3.3 μM, the lowest concentration tested. Although the system is exceedingly sensitive to modulation by GTP or Gpp(NH)p, UTP, CTP, GMP, and cyclic GMP did not antagonize the antihormone activity of prostaglandin H 2 . Kinetic studies indicate that the GTP or Gpp(NH)p antagonism of prostaglandin H 2 is observable on initial rates of cyclic AMP synthesis, and persists throughout the adenylate cyclase measurements. Preincubation of fat cell ghosts with GTP followed by washing and resuspension results in a prostaglandin H 2 -sensitive adenylate cyclase system. However, the same preincubation experiment with Gpp(NH)p produces an irreversible antagonism of the prostaglandin H 2 inhibition of hormone-stimulated adenylate cyclase. It is suggested that prostaglandin H 2 stabilizes that fat cell adenylate cyclase system in a state that is resistant to hormone stimulation, and GTP or Gpp(NH)p overcome this stabilization.

Binding sites for [3H]LTC4 were observed in crude membrane preparations of rat central nervous system tissue. Equilibrium binding studies indicated one high affinity [3H]LTC4 binding site with a KD of 31.4 +/- 3.4 nM for whole brain preparations. The binding was highly specific for [3H]LTC4 and could be inhibited by the SRS-A antagonist FPL 55172. Specific binding was increased with both mono- and di-valent ions. Regional distribution studies revealed a three-fold difference in binding capacity within different regions of the brain with the highest binding capacity in the brainstem (94.1 +/- 6.9 fmol/mg of protein) and the lowest in the hypothalamus (29.6 +/- 12.8 fmol/mg of protein). In addition, weak low capacity binding was observed for [3H]LTB4 and [3H]LTE4, while no saturable binding was observed for [3H]LTD4. The order of selectivity in inhibiting [3H]LTC4 binding was LTC4 much greater than LTD4 = LTE4 greater than LTB4.

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B4(LTB4). 6- trans -LTB4, 12- epi -6- trans -LTB4, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohyroxyeicosatetraenoic acids (i.e., 5-HETE) and w-oxidation products (i.e., 20-COOH LTB4) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 uM), LTB4 but not 5-HETE formation was impaired. (1-14C) Arachidonate-labeled platelets exposed to MSU released (1-14C)-arachidonate. (14C)-12 HETE, (14C)-HHT and (14C)-thromboxane B2. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB4.

The human leukocyte 5-lipoxygenase is a unique enzyme in its class because of its involvement in the synthesis of the biologically active leukotrienes. Furthermore, unlike most other lipoxygenases, this enzyme requires multiple stimulatory factors for maximal activity. These include Ca2+, ATP, and three non-dialyzable cellular components, two cytosolic, and one membrane-associated. The mechanism of action of these factors is not yet well understood; however, a Ca2+-dependent association of the enzyme and one of the cytosolic factors with the membrane has been demonstrated. These findings suggest that stimulation of the leukocyte, resulting in an increased intracellular Ca2+ concentration, may result in the translocation of the enzyme and the factor to a membrane site, thereby facilitating the interaction of these two proteins with other enzymes involved in the 20:4 metabolic cascade. The development of a better understanding of these processes should not only help to define the biochemical basis for the regulation of leukotriene formation, but should also yield valuable information concerning the more general aspects of stimulus-response coupling in the leukocyte.

Addition of 15L-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) to human leukocytes led to the formation of a novel series of compounds containing four conjugated double bonds. The yield of tetraenes was increased approx. 100-fold when ionophore A23187 (5 μM) was added simultaneously with 15-HPETE. The structure of the major tetraene was established by physical methods as well as by chemical degradation and found to be 5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid.

An unstable epoxide, leukotriene A4 (5(S)- trans -5,6-oxido-7,9- trans -11,14- cis -eicosatetraenoic acid), was earlier proposed to be an intermediate in the conversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C4. In the present work synthetic leukotriene A4 was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C4 and 11- trans -leukotriene C4 were the main products with SRS activity. It was not established whether the 11- trans -compound was formed by isomerization at the leukotriene A4 or C4 stage.

Lipoxin A and lipoxin B (LXA and LXB) are formed from the oxygenation of arachidonic acid by interactions between the 5- and 15-lipoxygenases of human leukocytes. Each compound displays highly stereospecific biological actions. Here, we present a computational description of the following compounds: lipoxin A, (5S,6R,15S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; 11-trans-lipoxin A, (5S,6R,15S)-trihydroxy-7,9,11,13-trans-eicosatetraenoic acid; lipoxin B, (5S,14R,15S)-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid; and 8-trans-lipoxin B, (5S,14R,15S)-trihydroxy-6,8,10,12-trans-eicosatetraenoic acid. The analyses considered van der Waals energy, electrostatic interactions, torsional potential, and alterations in electrostatic forces. Additional analyses were carried out with each of the four compounds forming complexes with one calcium ion. Each compound gave very different conformers. Both lipoxin A and lipoxin B can form globular conformations, while their all-trans isomers form rigid extended structures. When complexes with each of these compounds and one calcium ion were examined (i.e., (LXA)2Ca: (11-trans-LXA)2Ca), both LXA and LXB formed several flexible conformations including crumpled, wrapped or extended conformations. In this situation, LXA showed a higher probability than LXB to wrap around one Ca2+. In contrast, the two all-trans isomers always lead to extended conformations. Results from the present study illustrate that changes in the stereochemistry of LXA and LXB lead to unique conformations which may underlie the different biological actions of these compounds. Moreover, they indicate that the conformations of eicosanoids can change while in aqueous or hydrophobic environments (i.e., biomembranes).

Leukotriene A was assigned the structure 5(S)- trans -5,6-oxido-7,9- trans -11,14- cis -eicosatetraenoic acid by the enzymatic conversion of a synthetic product of known stereochemistry into the naturally occurring isomer of 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid in human polymorphonuclear leukocytes.

Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 microM) to human neutrophils led to the formation of both lipoxin A4 and lipoxin B4 as well as a novel 5,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A4 and lipoxin B4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA4 (7-cis-11-trans-LXA4). LXA4, 11-trans-LXA4, 7-cis-LXA4 and 7-cis-11-trans-LXA4 all evoked dose-dependent (0.1-10 microM) contractions of the guinea pig lung strip, whereas 6-cis-LXB4 and 6-cis-8-trans-LXB4 relaxed this preparation. LXA4 and 7-cis-LXA4 were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.

A radioimmunoassay was developed for a mono-O-methyl derivative of thromboxane B2. The antibodies showed high specificity for this compound and cross reacted only 1.2% with thromboxane B2 and less than 0.1% with prostaglandins and prostaglandin metabolites. The method had a sensitivity of 7 picog. The radioimmunoassay was employed in studies where thromboxane A2 was generated in human platelets and immediately converted into mono-O-methyl thromboxane B2 by treatment of the sample with a large volume of methanol. In some of the experiments, thromboxane B2 was simultaneously measured by a separate radioimmunoassay. Using these two assays it was demonstrated that thromboxane A2 could be detected only during the earlier stages of the platelet aggregation, whereas thromboxane B2 rapidly reached a constant level. In a separate experiment, the half-life of thromboxane A2 in buffer was found to be 32.5±2.5 (S.D.) sec at 37°C; the compound was more stable at lower temperatures. The t 1 2 for thromboxane A2 was also considerably longer in plasma.

Summary Oxyphenbutazone was oxidized when incubated with acetone powder prepared from sheep vesicular gland microsomes or with lipoxygenase at pH 4 or 5. Oxidation also occurred at pH 8 or 9, if arachidonate or linoleate was added to either of the incubation mixtures. The oxygenated product was found to be identical with 4-hydroxyoxyphenbutazone, which was synthesized and analyzed by gas liquid chromatography and mass spectrometry. The oxygenated compound was not an inhibitor of prostaglandin biosynthesis.

now in real terms than ever it has been in the past. In such circumstances the failure of academic pharmacologists and chemists to work together must be lamented. Talent is wasted when not put to best use. In universities and Schools of Pharmacy up and down the country the possibility of collaboration between the two disciplines is going unregarded and in this respect the fault must lie at the pharmacologist’s door. If the chemist is not made aware of the presence of a motivated pharmaco- logist in his vicinity actively seeking the novel compounds he is making and urgently wishing to effect a collaboration it is unlikely that the chemist will make the first move himself. With the work that needs to be done such pharmacologicail dereliction cannot be afforded. The time for insularity is past.

Ten years have elapsed since the discovery and structural elucidation of the leukotriene compounds (1-5). The leukotrienes constitute a group of potent biologically active arachidonic acid-derived metabolites which are implicated as mediators of immediate hypersensitivity reactions and inflammation. They were first discovered in leukocytes and were determined to contain a conjugated triene structure, thus the derivation of their name (6). The cysteinyl-containing leukotrienes (LTC,, LTD4, and LTE,),' which together make up the slow reacting substance of anaphylaxis, are synthesized primarily by eosinophils, mast cells, and macrophages. They stimulate contraction of various smooth muscle cells, promote bronchial mucous secretion, and induce extravasation of plasma proteins by increasing microvascular permeability. Leukotriene B, (LTB,), synthesized predominantly by neutrophils, is capable of stimulating circulating leukocytes to adhere to the vascular endothelium and induces extravasation and chemotaxis of polymorphonuclear leukocytes in nanomolar concentrations. At higher concentrations, LTBI induces polymorphonuclear leukocyte degranulation, superoxide generation, and can augment natural killer cell activity. Several reviews on the structure, biosynthesis, and biological actions of the leukotrienes have been written (7-13). It is the aim of the present review to concentrate on the advances made in recent years, primarily through molecular biology studies, on the reaction pathway leading to production of LTB,. 5-Lipoxygenase

The vasodepressor actions of the cyclic endoperoxides PGG 2 and PGH 2 were compared with those of their products PGD 2 and PGE 2 using anaesthetised normotensive and genetically hypertensive rats. Given into the aortic arch of normotensives PGE 2 was approximately 6 times more potent than PGH 2 and 11 times more potent than PGG 2 and PGD 2 . Hypertensive animals were 1.5–10 times more sensitive than normotensives to the depressor effects of PGG 2 and PGH 2 , but their sensitivity to either PGD 2 or PGE 2 was similar. Thus in hypertensives the endoperoxides may be converted more readily to PGE 2 and other products. In both types of rat PGG 2 and PGH 2 given intravenously were as active or more active than after intra-arterial administration. Low but not high intravenous doses of PGE 2 were less effective than intra-arterial. Therefore PGG 2 and PGH 2 may be converted more readily to more active products during passage through the lungs but whereas small doses of PGE 2 are almost completely eliminated large doses may saturate pulmonary removal mechanisms.

Human leukocyte 5-lipoxygenase (EC 1.13.11.12) is unique among the human lipoxygenase not only in its requirement for free ionized calcium, but also in its regulation by a membrane-associated stimulatory factor, the 100,000 x g pellet. In the present study, phosphatidylcholine (PC) vesicles, in the absence of 100,000 x g pellet, exhibited a dose-dependent stimulatory activity on the 5-lipoxygenase, which was at least as effective as the 100,000 x g pellet. Furthermore, the enzyme was activated by isolated human neutrophil plasma membranes and to a lesser degree by endoplasmic reticulum. The chemoattractant peptide fMet-Leu-Phe (0.1 microM), GTP (10 microM), toxin from bacterium Bordetella pertussis (islet activating protein, 5 micrograms/ml) and their various combinations were unable to modulate the enzymatic activity of the 5-lipoxygenase. Stimulation of the 5-lipoxygenase by relatively low levels of free ionized calcium was observed both in the presence of the pellet and PC vesicles: maximal stimulation was seen at about 10 microM Ca2+. The human leukocyte leukotriene A4 synthase activity also exhibited a similar requirement for free calcium ions. The present study indicates that the membrane-associated stimulatory factor of the human leukocyte 5-lipoxygenase may be replaced by PC vesicles. Moreover, the 5-lipoxygenase and leukotriene A4 synthase activities require significantly lower Ca2+ levels for maximal activation than has been reported previously.

Das aus Cyclopentadien und Acrylsauremethylester gebildete Addukt (I) wird uber das nach gangigen Verfahren herge = stellte Addukt (II) in den Aldehyd (IIIa) ubergefuhrt, aus dem nach Wittig der Vinyl= ather (IIIb) erhalten wird.

Slow reacting substance (SRS), produced by rat peritoneal mononuclear cells after stimulation with ionophore A23187, consists of two main components ( Bach, M.K. et al. (1979) J. Immunol. 122, 160–165). One of these components was recently identified as leukotriene C-1. The other component has now been identified as leukotriene D.

Human erythrocytes transformed leukotriene A4 into leukotriene B4. Metabolism was proportional to the erythrocyte concentration, even at subphysiological levels (0.08-4 X 10(9) erythrocytes/ml). Comparative metabolic studies excluded the possibility that leukotriene B4 originated from trace amounts of polymorphonuclear leukocytes or platelets present in the purified erythrocyte suspensions. For example, suspensions of isolated platelets (100-500 X 10(6) cells/ml) failed to convert leukotriene A4 into leukotriene B4; and conversion by suspensions of isolated polymorphonuclear neutrophils was insufficient to account for the amounts of leukotriene B4 formed by erythrocytes. Leukotriene B4 formation was maximal within 2 min and substrate concentration dependent. Enzymatic activity originated from a 56 degrees C labile nondialyzable (Mr greater than 30,000) soluble component in the 100,000 X g supernatant obtained from lysed erythrocytes. In contrast to the contemporary view, our results indicate that human erythrocytes are not metabolically inert in terms of eicosanoid biosynthesis. The role of human erythrocytes during inflammatory or pulmonary disorders deserves re-examination in this context.

The mechanism of the conversion of 8,11,14-eicosatrienoic acid into prostaglandin E1 (PGE1) and prostaglandin F1α (PGF1α) has been studied. Incubation of [13d-3H,3-14C]- and [13l-3H,3-14C]8,11,14-eicosatrienoic acids showed that the hydrogen removed from C-13 during the conversion into PGE1 and PGF1α has the l configuration. The conversion of [13l-3H,3-14C]8,11,14-eicosatrienoic acid into PGE1 is accompanied by a hydrogen isotope effect. No conversion of [2-14C]15l-hydroperoxy-8(cis),11(cis),13(trans)-eicosatrienoic acid or [2-14C]15l-hydroxy-8(cis), 11(cis),13(trans)-eicosatrienoic acid into PGE1 or PGF1α could be detected. Incubations of [9-3H,3-14C]8,11,14-eicosatrienoic acid revealed that PGF1α is not formed via PGE1 in the system used. The mechanistic implications of the results obtained are discussed. It is suggested that 11-peroxy-8,12,14-eicosatrienoic acid is the first intermediate in the conversion. The peroxy acid is cyclized into an endoperoxide, which is eventually transformed into PGE1 or PGF1α by independent reactions.

7α-Hydroxy-5,11-diketotetranor-prosta-1,16-dioic acid was identified as the major urinary metabolite after intravenous administration of tritium-labeled prostaglandin E2, prostaglandin E1, 11α-hydroxy-9,15-diketoprosta-5,13-dienoic acid, 11α-hydroxy-9,15-diketoprost-5-enoic acid, and 11α-hydroxy-9,15-diketoprostanoic acid to male and female subjects. Injected prostaglandin E2 was rapidly converted initially into 11α-hydroxy-9, 15-diketoprost-5-enoic acid. Kinetic parameters for these two compounds are given. A method for quantitative determination of the major urinary metabolite was developed. In three subjects, 7, 16, and 27 µg/24 hours were excreted.