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1 Slow-reacting substance of anaphylaxis (SRS-A) was produced by antigen challenge of passively sensitized human lung and actively sensitized guinea-pig lung. 2 A slow-reacting substance (SRS) was prepared from the peritoneal fluid of rats treated with calcium ionophore A23187. 3 These substances were extensively purified by charcoal adsorption, Sephadex G-15 gel filtration, ether extraction and reverse phase high pressure liquid chromatography. 4 The three substances are pharmacologically, chemically and chromatographically indistinguishable. 5 Our data suggest that the same SRS entities are released from a variety of tissues and that these acidic lipids may have a wider physiological significance than just anaphylaxis.

We have previously demonstrated that vasoactive intestinal peptide and calcitonin gene-related peptide inhibit leukotriene release from platelet activating factor-stimulated rat lung. We now report evidence that Peptide Histidine Isoleucine and Secretin, two naturally occurring peptides which are structurally homologous to vasoactive intestinal peptide, show a significant ability to inhibit leukotriene release in the same animal model. A preliminary hypothesis about the mechanism of this inhibition by the neuropeptides is discussed.

Slow reacting substances (leukotrienes C4, D4, E4) are synthesized in vivo by a combination of two previously unrelated pathways: lipoxygenase oxygenation of arachidonic acid and the glutathione detoxification pathway. Enzymes involved in the latter pathway (glutathione transferase [RX: glutathione R-transferase, EC 2.5.1.18]; gamma-glutamyltransferase [(5-glutamyl)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2] ) have been investigated in guinea pig lung and rat basophilic leukemia (RBL-1) cells. We report data on levels of enzymic activity both before and during the release of slow reacting substances. Both glutathione transferase and gamma-glutamyltransferase are present in significant quantities in guinea pig lung and RBL-1 cells. A model for the changes in gamma-glutamyltransferase during leukotriene release is proposed for the cell line, and differences from the guinea pig lung system are reported. Leukotriene C4 is converted to the more potent leukotriene D4 by the action of gamma-glutamyltransferase on guinea pig ileum during bioassay. gamma-Glutamyltransferase may represent a control feature in the biosynthesis of leukotriene D4, and thus be involved in leukotriene-induced bronchoconstriction in the lung.

Endometrium and myometrium were collected at hysterectomy from 21 women with measured menstrual blood loss. Eight women complained of dysmenorrhea and the remaining 13 had pain-free periods. Specimens were obtained throughout the menstrual cycle (menstrual, n = 5; follicular, n = 4; early luteal, n = 3; mid-luteal, n = 5; late luteal, n = 4). Leukotriene C4, leukotriene D4 and leukotriene E4 release were examined using a short-term incubation technique. Endometrial leukotriene release, which was always significantly greater than myometrial release, changed throughout the menstrual cycle and the highest concentrations were found during menstruation. Endometrial, but not myometrial, leukotriene concentrations were significantly higher in tissues obtained from women with a complaint of dysmenorrhoea compared with those in tissue from pain-free women. No correlation was found between leukotriene release in either endometrium or myometrium and menstrual blood loss (range 15-457 ml).

The immunological release of SRS-A was investigated in guinea-pig chopped lung. A number of unsaturated fatty acids, all of which are substrates for arachidonate lipoxygenase were found to potentiate the release of SRS-A. This potentiation was enhanced by indomethacin, a cyclo-oxygenase inhibitor, and completely reversed by nordihydroguaiaretic acid (NDGA) and eicosatetraynoic acid (ETA) which inhibit lipoxygenase. This suggests that some aspect of arachidonate lipoxygenase action stimulates release of SRS-A and that release of SRS-A is increased by redirection of arachidonic acid (AA) metabolism via the lipoxygenase pathway (Hamberg, 1976). However, although exogenous 14C-AA increased SRS-A output it was not incorporated into SRS-A.

Slow Reacting Substances have been produced from RBL-l cells by calcium ionophore A23187 and purified to homogeneity by high pressure liquid chromatography (HPLC). The structure of the major biologically active species has been determined by mass spectrometric examination of the intact molecule as a derivative, together with amino-acid analysis and sequence determination. The characteristic triene chromophore which we originally identified in immunologically generated SRS-A is present in RBL-l SRS, and we determine the structure of this SRS as the thio-substituted dipeptide, 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid.

The effect of four neuropeptides and acetylcholine on the release of leukotrienes LTC4, LTD4 and LTE4 from platelet activating factor-stimulated rat lung and ionophore A23187-stimulated guinea pig lung, as detected by the combined use of HPLC and radioimmunoassay, was studied. Both vasoactive intestinal peptide and calcitonin gene-related peptide were found to inhibit the release of leukotrienes in both preparations. This effect was most marked in platelet activating factor-stimulated rat lung, where inhibition of LTC4 release was more pronounced than either inhibition of LTD4 or LTE4 production. The effect of vasoactive intestinal peptide on LTC4 biosynthesis was dose-related in rat lung. Neither substance P nor beta-endorphin were found to inhibit leukotriene release in rat lung. Vasoactive intestinal peptide inhibition of leukotriene release is independent from its actions on the muscarinic receptor, since acetylcholine was found to have no effect in the same preparation.

Sputum extracts from 16 of 25 adult patients with CF produced a slow, sustained contraction of isolated guinea pig ileum which was partially inhibitable by the SRS-A antagonist FPL 55712. Following progressive purification and HPLC, the major ileum-contracting substance was identified as LTD4. LTC4 was also present but in smaller amounts. The sputum contained other SRS-A-like activity which was not inhibited by FPL 55712; this substance was of a smaller molecular size than LTD4. The presence of LTB4 was also established using HPLC and an assay of neutrophil chemotaxis. These studies raise the possibility that leukotrienes and other lipoxygenase products may contribute to the progressive lung disease characteristics of CF.

1 Rabbit aorta contracting substance (RCS; consisting mainly of thromboxane A2) and prostaglandin-like material were released from guinea-pig isolated perfused lungs by injection of slow reacting substance of anaphylaxis (SRS-A). 2 SRS-A was resistant to boiling and proteolytic enzymes and was therefore distinguished from rabbit aorta contracting substance releasing factor (RCS-RF). 3 The release of RCS and prostaglandin-like material by SRS-A was anatagonized by indomethacin (1 microgram/ml), betamethasone and dexamethasone (4 to 50 microgram/ml). 4 Imidazole (200 microgram/ml) inhibited the formation of thromboxane A2 but not that of prostaglandins. 5 The activity of SRS-A on guinea-pig ileum and its ability to release RCS and prostaglandins were destroyed by incubation with arylsulphatase (0.83 microgram to 1 mg/ml) and with lipoxidase (16.5 to 50 microgram/ml): SRS-A lost activity on incubation with bovine serum albumin (9 microgram/ml) due to protein binding.

We have recently described the structure elucidation of slow reacting substance of anaphylaxis S(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)-cysteinylghlycinyl-7,9-trans-11,14-ciseicosatetraenoic 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.

A radioimmunoassay for leukotriene D4 (LTD4) has been developed which exhibits sufficiently high sensitivity to be useful in conjunction with RP-HPLC in the detection of LTC4, LTD4 and LTE4 in physiological samples. The detection limit of the assay was approximately 240 amoles, using antiserum TG1 at a dilution of 6 X 10(3), with 50% displacement at 70 fmoles. Antiserum NW1, also at a dilution of 6 X 10(3), displayed a detection limit of 9 fmoles with 50% displacement at 100 fmoles. The two antisera have similiar crossreactivities, both manifesting useful affinities for LTE4 and LTC4, and low or negligible affinities for other arachidonic acid metabolites, or their derivatives. The radioimmunoassay was used to detect 1) LTC4, LTD4 and LTE4 released from perfused rat lung in response to platelet-activating factor (PAF) stimulation, 2) conversion of exogenous LTD4 to LTE4 in human blood, and 3) endogenous leukotrienes in human blood samples.

Leukotriene (LT) release stimulated by platelet activating factor was inhibited by vasoactive intestinal polypeptide (VIP) in an in vitro rat lung preparation. This was detected by HPLC and radioimmunoassay. LTC4, although the major species in the stimulatory model used, was not detected in peptide-treated preparations and LTD4 and LTE4 levels were considerably reduced.

For more than forty years since their discovery, the structure of a group of closely related materials known collectively as slow-reacting substances has been unknown. These substances are released from a variety of tissues in response to immunological or non-immunological stimulation. A slow-reacting substance is believed to be implicated in hypersensitivity reactions such as asthma; in order to fully understand its bronchoconstrictor role, the structural elucidation of these materials has been a necessary (albeit difficult) task. Studies on both immunologically generated slow-reacting substance of anaphylaxis (SRS-A) and other slow-reacting substances (SRSs) have indicated a precursor role for arachidonic acid in their biosynthesis; this, coupled with enzymic and chemical activity destruction data, gave an insight into the structure of these moieties. In order to define the structure of these materials homogeneous SRS-A was required; a purification scheme was developed relying on the high resolution separative capability of reverse-phase high pressure liquid chromatography, resulting in extensively purified SRS-A. It was then possible to demonstrate that SRS-A possessed a characteristic ultraviolet spectrum, allowing us for the first time to define a major structural moiety in the molecule (conjugated triene). To complement studies on, and to act as a model for the more pathologically relevant SRS-A, a slow-reacting substance was produced from rat basophilic leukaemia (RBL-1) cells. The structure of this biologically active species has been determined by mass spectrometric examination of the intact molecule as a derivative, together with analytical protein chemical studies, and shown to be the novel peptidolipid 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid.

The work that we have described had originally three main aims: (a) to design a new purification system for SRS-A from which we would obtain pure material for the structural analysis: (b) to define the functional groups in the pure material by spectrophotometric, chemical, and enzymic inactivation methods; and (c) to deduce the complete covalent structure by an accepted spectroscopic method capable of defining structure in atomic detail. These aims have been achieved. The structure of SRS-A, the physiologically more relevant example of the SRSs that were studied, because it was derived immunologically from an animal model of an acute hypersensitivity reaction, has been rigorously defined. Of paramount importance in the determination of this structure was the mass spectrometric analysis of the intact molecule. Degradative and comparative studies are not capable of unequivocally defining structure. For example, the mass spectrum clearly showed the absence of an amide or similar C-terminal blocking groups or, as has been suggested, a sulfone in the molecule; such conclusions could not be drawn from comparative chromatographic data even on multiple systems. Mass spectrometric analysis of the intact molecule could overcome these problems by allowing the complete covalent structure to be collated from the information obtained from each fragmentation. The use of stable isotopes and accurate mass measurement removed possible ambiguities in the interpretation, and the sensitivity and specificity of mass spectrometry made it the method of choice for the structural analysis.

The effect of five neuropeptides was tested on platelet activating factor (PAF)-stimulated peptidoleukotriene biosynthesis in rat lungs. Calcitonin and peptide histidine valine-42 (PHV-42) were found to inhibit leukotriene C4 (LTC4) and D4 (LTD4) biosynthesis to an extent similar to the previously reported effects of calcitonin gene-related peptide (CGRP) and peptide histidine isoleucine (PHI) respectively. Helodermin potently inhibited the biosynthesis of all three peptidoleukotrienes. Neuropeptide Y and galanin, both present in airways, enhanced the levels of LTD4 and had no effect on LTC4 or leukotriene E4 (LTE4) levels. The theoretical implications of these findings are discussed.

The effect of prostaglandins E1 and F1 alpha on peptidoleukotriene biosynthesis/release from rat chopped lung stimulated with platelet activating factor was studied. Prostaglandin E1, known to stimulate adenylate cyclase in airways, inhibited the biosynthesis of leukotrienes C4, D4 and E4 and total peptidoleukotrienes whereas prostaglandin F1 alpha, which has no effect on adenylate cyclase, did not exert any effect on total peptidoleukotriene release, though a small inhibition was found for leukotriene D4. Cyclic AMP itself inhibited peptidoleukotriene release from platelet activating factor-stimulated lung, suggesting that the effect of prostaglandin E1 is mediated by cyclic AMP.