Functional characterisation of receptors for cysteinyl leukotrienes in smooth muscle.

The cysteinyl leukotrienes (leukotriene C4, D4 and E4) have potent biological actions which significantly contribute to the airway obstruction in asthma. Several of these effects are blocked by drugs known as CysLT1-receptor antagonists. However, there are actions of leukotrienes which are not sensitive to these antagonists, suggesting the presence of additional receptor subtypes. It was the aim of this Thesis to extend the knowledge about receptors for cysteinyl leukotrienes. Three different isolated smooth muscle preparations kept in organ baths under non-flow conditions were characterised with respect to responsiveness to cysteinyl leukotrienes and sensitivity to purported CysLT1-receptor antagonists. In addition, the study involved evaluation of a leukotriene E4 analogue, BAY u9773, suggested to inhibit responses which cannot be blocked by CysLT1-receptor antagonists. These responses have provisionally been considered to be mediated by CysLT2-receptors. In the guinea pig ileum, BAY u9773 but not the selective CysLT1 receptor antagonist ICI 198,615 inhibited the contractile response to leukotriene C4 in a fashion suggesting competitive antagonism. In sheep trachealis muscle, BAY u9773 antagonised contractions induced by leukotriene C4 and leukotriene D4 in a similar manner, whereas ICI 198,615 did not. The observations support that leukotriene C4 in guinea pig ileum, and leukotriene C4 as well as leukotriene D4 in sheep trachealis muscle, mediated contractions via activation of CysLT2-receptors. In guinea pig lung parenchyma, the effects of BAY u9773 and conventional cysteinyl leukotriene receptor antagonists (ICI 198,615, FPL 55,712) were more complex. First, BAY u9773 evoked a contraction, which could be inhibited by antagonists of CysLT1- and TP-receptors. This suggested that BAY u9773 acted as an agonist at these two receptors. Second, pretreatment with BAY u9773 inhibited a distinct but relatively small component of the contractile response to leukotriene C4 and D4. The effects of BAY u9773 and ICI 198,615 were similar in guinea pig lung parenchyma. The findings suggest that the receptor mediating the major part of the contractile response to exogenous cysteinyl leukotrienes in guinea pig lung parenchyma was different from the currently defined CysLT2-receptor. Furthermore, the data suggested that BAY u9773 was a partial agonist at cysteinyl leukotriene receptors, which presumably contributed to its profile of activity as a combined CysLT1- and CysLT2-receptor antagonist. In addition to contracting guinea pig lung parenchyma, leukotriene C4 and lipoxin A4 also evoked release of thromboxane A2. This release was sensitive to CysLT1-receptor antagonists and contributed to part of the contractile response. Finally, the investigations included a characterisation of the role of leukotrienes in antigen-induced contractions of lung parenchyma from actively sensitised guinea pigs. Combination of antihistamines with CysLT1-receptor antagonists or inhibitors of leukotriene biosynthesis blocked the major component of the antigen-induced contraction. The findings are similar to those observed in isolated human bronchi and support that this model may be used to investigate mediator mechanisms of relevance to asthma.

Functional characterization of receptors for cysteinyl-leukotrienes in sheep trachealis muscle.

Cysteinyl-leukotrienes (CysLTs: LTC4, LTD4 and LTE4) are inflammatory mediators which significantly contribute to the airway obstruction in asthma. At least two distinct receptor subtypes exist for cysteinyl-leukotrienes, the CysLT1- and CysLT2-receptor. The purpose of this study was to test whether sheep trachealis muscle is a useful preparation for further characterization of CysLT2-receptors, previously implicated in contraction of human pulmonary veins. Leukotriene C4 and leukotriene D4 evoked contractile responses, leukotriene C4 being significantly more potent than leukotriene D4, whereas leukotriene E4 failed to elicit contractions. The response to leukotriene C4 exhibited tachyphylaxis upon repeated administration. There were no significant effects of epithelial denudation, NO-synthesis inhibition (L-NAME) or cyclooxygenase inhibition (indomethacin) on the responses to cysteinyl-leukotrienes or cholinergic agonists. Neither was responsiveness to different agonists changed by overnight storage. The responses to leukotriene C4 and leukotriene D4 were markedly potentiated when their metabolism was inhibited by S-hexyl glutathione and L-cysteine. The selective CysLT1-antagonist ICI 198,615 had no significant effect on these responses. However, the combined CysLT1- and CysLT2-antagonist BAY u9773 competitively antagonized leukotriene C4 and leukotriene D4 (pA2 values of 7.0 and 6.8 against leukotriene C4 and leukotriene D4, respectively). The findings support that leukotriene C4 and leukotriene D4 act predominantly on CysLT2-receptors in sheep trachea.

The cysteinyl-leukotriene receptor antagonist BAY u9773 is a competitive antagonist of leukotriene C4 in the guinea-pig ileum.

Two main classes of receptors exist for leukotrienes C4, D4 and E4, collectively named cysteinyl-leukotrienes (CysLTs). The CysLT1 receptor is blocked by currently available leukotriene antagonists, and the CysLT2 receptor is defined by the absence of selective antagonists. The contractile response to leukotriene C4 in guinea-pig ileum longitudinal muscle is resistant to CysLT1 receptor antagonists. However, the leukotriene E4 analogue BAY u9773 (6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z), 14(Z)-eicosatetraenoic acid) has recently been reported to inhibit CysLT2 responses. Therefore BAY u9773 was evaluated for antagonism of the effect of leukotriene C4 in the guinea-pig ileum longitudinal muscle. We found that BAY u9773 (0.3-10 microM) did not contract the preparation, but produced a concentration-dependent rightward shift in the concentration-response relation for leukotriene C4. Schild plot analysis yielded a slope which was not significantly different from unity and a pA2 value of 6.1. The inhibition of leukotriene C4 by BAY u9773 was not altered by antagonism of CysLT1 receptors by ICI 198,615 {[1-[[2-methoxy-4-[[(phenylsulfonyl)amino]carbonyl]-phenyl] methyl]-1H-indazol-6-yl]carbamic acid cyclopentyl ester}(100 nM). The CysLT1 receptor agonist, leukotriene E4 (1 microM), contracted the preparation but did not inhibit the contraction induced by leukotriene C4. Taken together, the antagonism exerted by BAY u9773 appeared unrelated to actions on CysLT1 receptors. In conclusion, BAY u9773 was a useful selective competitive antagonist of leukotriene C4, and the findings support the classification of the receptors for leukotriene C4 in the guinea-pig ileum as CysLT2.

Interactions between leukotrienes and histamine in the anaphylactic contraction of guinea pig lung parenchyma.

The aim of the study was to investigate if antigen-induced contraction of guinea pig lung parenchyma (GPLP) was an appropriate model for the study of antileukotriene drugs. Antileukotrienes have recently shown antiasthmatic effects in humans. Challenge of GPLP with a cumulatively increasing concentration of antigen evoked a graded contractile response. The antigen response could be divided into an immediate peak phase and a plateau phase of long duration. Histamine antagonism alone (mepyramine, H1, and metiamide, H2) had no effect on the response, whereas 5-lipoxygenase (5-lox) inhibitors (BAY x1005, MK-886 or BWA4C) depressed the plateau phase. When 5-lipoxygenase inhibition (BAY x1005 or MK-886) or cysteinyl-leukotriene receptor antagonism (ICI 198,615) was combined with histamine antagonism, there was a major attenuation of both components of the antigen response, leaving only a small residual response. In contrast, cyclooxygenase inhibition (diclofenac or indomethacin), antagonism of platelet-activating factor (WEB 2086) and thromboxane receptor antagonism combined with inhibition of thromboxane synthesis (BAY u3405 and CS-518) failed to inhibit the antigen response. In conclusion, cysteinyl-leukotrienes and histamine synergistically mediated the major part of the Schultz-Dale response in GPLP. The characteristics of GPLP anaphylaxis closely resembled those of antigen-challenged human bronci, supporting that antigen challenge of GPLP is a suitable model in experimental asthma research.