Marwa N. Samhoun

The mechanism of action of leukotrienes C4 and D4 in guinea-pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat

The biological actions of pure slow-reacting substance of anaphylaxis (SRS-A) from guinea-pig lung, pure slow-reacting substance (SRS) from rat basophilic leukaemia cells (RBL-1) and synthetic leukotrienes C4 (LTC4) and D4 (LTD4) have been investigated on lung tissue from guinea pig, rabbit and rat. In the guinea pig, the leukotrienes released cyclo-oxygenase products from the perfused lung and contracted strips of parenchyma. The effects of SRS-A, SRS and LTD4 were indistinguishable. LTC4 and LTD4 had similar actions although LTD4 was more potent than LTC4. Indomethacin (1 microgram/ml) inhibited the release of cyclo-oxygenase products from perfused guinea-pig lung and caused a marked reduction in contractions of guinea-pig parenchymal strips (GPP) due to LTC4 and LTD4. The residual contraction of the GPP was abolished by FPL 55712 (0.5 - 1.0 microgram/ml). It appears, therefore, that a major part of the constrictor actions of LTC4 and LTD4 in guinea-pig lung are mediated by myotropic cyclo-oxygenase products, i.e. thromboxane A2 (TxA2) and prostaglandins (PGs). In rabbit and rat lung, however, SRS-A, SRS and the leukotrienes were much less potent in contracting parenchymal strips and there was little evidence of the release of cyclo-oxygenase products. FPL 55712 at a concentration of 1 microgram/ml failed to antagonise leukotriene-induced contractions.

STIMULATION OF ARACHIDONIC ACID METABOLISM AND GENERATION OF THROMBOXANE A2 BY LEUKOTRIENES B4, C4 AND D4 IN GUINEA‐PIG LUNG in vitro

1 Leukotriene C4 (LTC4), LTD4, slow‐reacting substance of anaphylaxis (SRS‐A) (from guinea‐pig lung), bradykinin (Bk) and arachidonic acid (AA) release thromboxane A2 (TxA2) and prostaglandin‐like materials from guinea‐pig isolated perfused lungs. 2 Release of TxA2 induced by LTC4 and LTD4 is inhibited by a thromboxane synthetase inhibitor, imidazole (2.9 mm). 3 Mepacrine (200 μm), a phospholipase inhibitor, inhibits release of TxA2 and prostaglandin‐like materials caused by SRS‐A and Bk but not that due to exogenous AA 4 Leukotrienes B4, C4 and D4 are approximately equipotent in inducing dose‐related contractions of guinea‐pig parenchymal strips (GPPs). 5 Leukotriene‐induced contractions of GPPs are greatly inhibited by imidazole (2.9 mm), carbox‐yheptylimidazole (24 μm) and mepacrine (400 μm). 6 FPL 55712 (1.9 μm), the SRS‐A antagonist, blocks contractions of GPPs induced by LTC4 and LTD4 but not those due to LTB4 or Bk. 7 Tachyphylaxis to LTB4 occurs in GPPs but not to LTC4 or LTD4. 8 These results suggest that in guinea‐pig lung in vitro, LTB4, LTC4 and LTD4 activate a phospholipase with subsequent generation of cyclo‐oxygenase products of which TxA2 plays an important role.

The combined use of isolated strips of guinea-pig lung parenchyma and ileum as a sensitive and selective bioassay for leukotriene B4

The biological effects of leukotriene (LT)B4 were compared, on a molar basis, with those of LTC4, LTD4, LTE4, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-oxo-PGF1 alpha, bradykinin (BK) and angiotensin II (Ang II) on isolated strips of guinea-pig lung parenchyma (GPP) and ileum smooth muscle (GPISM) superfused in series. LTB4 was similar to LTC4 and LTD4 on GPP, in relation to potency and contractions induced, but differed from LTE4 in being ten times more active and causing contractions of a much shorter duration of action on this tissue. However, unlike the other LTs, LTB4 produced contractions which were resistant to FPL 55712 (1.9 microM) and, when given repeatedly, caused tachyphylaxis in GPP. LTB4 was considerably more active on GPP than the other substances investigated. Further, PGD2, PGF2 alpha and PGI2 contracted GPP, the order of potency being PGD2 greater than PGF2 alpha approximately equal to PGI2, whereas PGE1 and PGE2 relaxed this tissue. In contrast to all other agonists tested which contracted GPISM, LTD4 displaying the highest activity, LTB4 was inactive on this tissue. 5-HETE and 6-oxo-PGF1 alpha were inactive on both GPP and GPISM. On the basis of differential effects of LTB4 on GPP and GPISM, this assay represents a simple and selective means to distinguish LTB4-like materials from other naturally-occurring substances likely to be generated in inflammatory fluids.

Actions and interactions of lipoxygenase and cyclo-oxygenase products in respiratory and vascular tissues

Actions of leukotrienes (LTs) B4, C4, D4 and E4 were found to be largely mediated via formation of cyclo-oxygenase products in guinea-pig isolated perfused lung and parenchymal strips. In contrast, LTs exerted a direct contractile effect on human isolated parenchymal and bronchial strips. An LT-like substance which had similar biological actions to LTD4 was generated from porcine and guinea-pig vascular tissue. The highest concentration of this material was formed by coronary and pulmonary arteries and the surrounding adventitia, as well as from the lung parenchyma.

Slow-reacting substances and their formation by a lipoxygenase pathway

Slow-reacting substances are formed from arachidonic acid by the action of a lipoxygenase, which leads to the formation of 5-hydroperoxy,6,8,11,14 eicosatetraenoic acid.The covalent structures of SRS-A from guinea-pig lung and SRS from RBL-1 cells have been determined by protein chemical analysis and electron impact mass spectrometry of a derivative of the intact molecules. The structures of SRS-A and SRS are identical, being 5-hydroxy-6-cysteinyl-glycinyl-7,9,11,14-eicosatetraenoic acid.SRSs may be formed by a combination of the metabolism of arachidonic acid by the lipoxygenase pathway and the glutathione detoxification pathway involving nucleophilic attack on 5,6-oxidoeicosatetraenoic acid.

Leukotriene F4: Comparison of its pharmacological profile with that of the other cysteinyl-containing leukotrienes in guinea-pig ileum and lung parenchyma in vitro

Abstract The biological effects of leukotriene (LT)F 4 were compared, on a molar basis, with those of LTC 4 , LTD 4 and LTE 4 on isolated superfused strips of guinea-pig ileum smooth muscle (GPISM) and lung parenchyma (GPP). LTF 4 was 1–2 orders of magnitude less active than the other leukotrienes on GPISM (LTD 4 > LTC 4 > LTE 4 > LTF 4 ) whereas, in the GPP, the activity of LTF 4 was comparable with that of LTE 4 , both leukotrienes being about one order of magnitude less active than LTC 4 or LTD 4 (LTC 4 =LTD 4 > LTE 4 =LTF 4 ). Further, LTF 4 caused protracted contractions of the GPP which were indistinguishable from those due to LTE 4 and of a much longer duration than responses elicited by either LTC 4 or LTD 4 . FPL 55712 (1.9μM) antagonised actions of LTF 4 in both tissue preparations. Indomethacin (2.8μM) inhibited contractions induced by LTF 4 in GPP indicating that part of the bronchoconstriction due to LTF 4 , like that elicited by the other leukotrienes, is mediated via release of cyclo-oxygenase products.

Structure elucidation and biosynthesis of slow reacting substances and slow reacting substance of anaphylaxis from guinea pig and human lungs

Abstract The structure of slow reacting substance of anaphylaxis (SRS-A) from lung is defined by advanced spectroscopic and protein chemical methods to be 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, CYS-GLY SRS-A. 12SRS-A is identical to the major SRS released non-immunologically from rat basophil leukaemia (RBL-1) cells whose structure we first defined as the dipeptidyl (CYS-GLY) peptidolipid.9 Human lung SRS-A is chromatographically indistinguishable from guinea-pig lung SRS-A, suggesting that this is also the CYS-GLY SRS-A structure.7 SRS-A is biosynthesized by a combination of lipoxygenase metabolism of arachidonic acid8,15 coupled with the glutathione detoxification pathway. Glutathione S- transferase and γ-glutamyl transferase levels in both the lung and isolated cell systems have been measured before, during and after immunological and non-immunological challenge. Enzyme levels rise significantly upon challenge and coincide with the release of SRS-A and SRS.

Relaxations of guinea-pig isolated trachea induced by platelet-activating factor are epithelial-dependent and are antagonised by WEB 2086.

Platelet-activating factor (PAF, 10-1000 pmol) induced dose-dependent relaxations of the basal tone of superfused strips of epithelium-intact guinea-pig trachea. Indomethacin (1.4 microM) completely inhibited and WEB 2086 (1 and 10 nM) effectively antagonised these relaxations. Following epithelial removal PAF evoked a single contraction. These results show that the PAF-induced relaxations of guinea-pig trachea are dependent on an intact epithelial layer and are mediated by a cyclo-oxygenase product.

Leukotrienes in the Lung and Cardiovascular System

The formation of leukotrienes (LTs) from arachidonic acid derived from phospholipids of the cell membrane is initially catalysed by 5-lipoxygenasel. Metabolism of the unstable epoxide LTA4 leads to the formation of LTB4 and the cysteinyl-containing LTs C4, D4 and E4. All these LTs have potent, although different, biological activities. LTB4 is a powerful chemotactic agent for leukocytes whereas LTs C4, D4 and E4 have potent smooth muscle stimulating actions and account for the biological activity of the allergic mediator previously known as slow-reacting substance of anaphylaxis (SRS-A)2. Leukotriene B4 has pro-inflammatory actions but little smooth muscle stimulating activity of its own whereas cysteinyl-containing LTs have potent actions in the cardiovascular system and in the airways in vitro and in vivo (see3,4).

Comparative Effects of Leukotrienes in Respiratory Tissue of Various Species

It is now recognised that leukotrienes (LTs) C4, D4 and their probable metabolite E4, which contain a sulphur linkage and amino acid side chain at C-6, collectively account for the biological activity of slow-reacting substance of anaphylaxis (SRS-A) obtained from various sources which, for many years, has been proposed as an important mediator of bronchoconstriction in man. These LTs display pronounced spasmogenic and vasoactive properties in various preparations and possess unprecedented potencies in respiratory smooth muscle in particularl.