Pharmacological Characterization of Leukotriene Receptors

SVEN-ERIK DAHLÉN

Experimental Asthma and Allergy Research, National Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

	INTRODUCTION

TOP INTRODUCTION RECEPTORS FOR LEUKOTRIENE B4 RECEPTORS FOR CYSTEINYL-... CONCLUSIONS NOTE ADDED IN PROOF REFERENCES

Whereas inhibition of 5-lipoxygenase will result in global inhibition of the production of leukotrienes, antagonism of leukotrienes at their tissue receptors provides selective inhibition of actions mediated by distinct classes of receptors (Table 1). The different profiles of biological activities for leukotriene B₄ (LTB₄) and the cysteinyl-leukotrienes suggested that the two main classes of leukotrienes possessed different classes of receptors, and pharmacological investigations have indeed confirmed this. The receptors for LTB₄ are called BLT receptors whereas the receptors for cysteinyl-leukotrienes are designated CysLT receptors (1). With regard to antagonism of the CysLT receptors, studies support some heterogeneity and at least two main subgroups of receptors have therefore been proposed, CysLT₁ and CysLT₂. It is likely that there are more subdivisions to consider, but as the CysLT receptors remain to be cloned, this classification so far remains the framework for our understanding. Although there are experimental compounds that may block several of the known leukotriene receptors (Table 1), clinically useful drugs have so far been developed only against the main receptor for cysteinyl-leukotrienes in the human lung. This receptor is now called the CysLT₁ receptor but was previously known as the LTD₄ receptor. This article serves to give an overview of the leukotriene receptors, with particular attention to the virgin state of knowledge in this area.

	RECEPTORS FOR LEUKOTRIENE B4

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The experimental data have indeed established that LTB₄ acts at a specific receptor that now is designated the BLT receptor (1). For example, specific [³H]LTB₄ binding has been demonstrated in many tissues including human polymorphonuclear leukocytes (PMNs) (2, 3). The binding sites in PMNs were selectively inhibited by guanine nucleotides (4, 5) and structurally related metabolites displaced LTB₄ with a potency that correlated with their activities in chemotactic assays (6).

Activation of the BLT receptor is a potent stimulus for leukocytes, eliciting chemokinetic and chemotactic responses in vitro (7). In vivo, LTB₄ increases leukocyte rolling and adhesion to the venular endothelium, followed by their emigration into the extravascular space (8). During a short-lasting exposure to LTB₄, polymorphonuclear leukocytes are mainly recruited. With prolonged exposure to LTB₄, as presumably occurs when LTB₄ is formed in vivo, other granulocytes, including eosinophils, are found in tissues or exudates after challenge with LTB₄ (9). It has been shown that LTB₄ is a chemoattractant for interleukin 5-primed eosinophils (10), and LTB₄ may stimulate production of interleukin 5 in T lymphocytes (11).

In addition to effects on leukocyte adhesion and migration, LTB₄ stimulates secretion of superoxide anion and release of different granular constituents from leukocytes (12, 13). Among effects of LTB₄ on inflammatory cells, it has been observed that LTB₄ may affect expression of low-affinity receptors for IgE on B lymphocyte cell lines (14), and IgE synthesis induced by interleukin 4 (15). More recently, the observation that LTB₄ is an agonist for the nuclear transcription factor PPAR (peroxisome proliferator-activated receptor ) has created considerable interest (16). The finding may implicate a role for LTB₄ in the control of central events in lipid metabolism and inflammation, but also indicates the presence of a feedback loop that may respond to increased leukotriene production by enhanced leukotriene catabolism (17). The structure-activity relations for this effect of LTB₄ and the influence of antagonists of LTB₄ on the response remain to be learned, but the observations nevertheless point out the possibility that LTB₄ also has intracellular and nuclear targets, which may participate in long-term control of gene expression.

It is also established that LTB₄ has contractile activity in guinea pig lung parenchyma (18). The response is indirect, involving release of thromboxane A₂ (TXA₂) (19), and most likely also histamine (21). The response has so far not been observed in other smooth muscles, including human bronchi, but nevertheless indicates that certain tissues may contain elements with the ability to release spasmogenic mediators when exposed to LTB₄.

In a dog model, LTB₄ was found to increase airway reactivity to acetylcholine (22). In a study of normal volunteers, there was, however, no change in bronchial hyperresponsiveness to histamine after the inhalation of LTB₄ alone, or in combination with PGD₂ (23). Nor was there any direct bronchoconstrictor effect of inhaled LTB₄ (23). A subsequent study, however, observed that inhalation of LTB₄ by healthy human volunteers was followed by striking cellular changes in the airways, and possibly also by some plasma exudation (24). In a study in which LTB₄ was inhaled by a group of subjects with asthma, the lack of immediate bronchoconstrictive properties was confirmed as well as prominent acute effects on leukocyte traffic in the lung and blood (25).

Pharmacologic evidence has accumulated to suggest that BLT receptors are G protein coupled (4, 5), and it was possible to isolate the cDNA for a BLT receptor in retinoic acid-differentiated HL-60 cells (26). The cDNA encoded a 352-amino acid cell surface protein that was G protein coupled and mediated chemotaxis. Incidentally, this cDNA had previously been published but described as an orphan receptor possibly mediating chemoattractant responses (27). Northern blotting experiments of human tissues displayed a preferential expression of mRNA for the BLT receptor in PMNs (26). There was also some expression in the spleen and thymus, whereas most other examined tissues, including the lung, showed no or insignificant expression of message for the BLT receptor (26). As discussed in another article in this supplement, observations have stimulated the hypothesis that the BLT receptor may be involved in certain aspects of the host response to human immunodeficiency virus (HIV) infection (28).

It has been observed that chemotaxis, in particular, is often mediated at lower agonist concentrations of LTB₄ than those required for degranulation and superoxide generation (29). Ligand-binding experiments have also demonstrated the presence of low- and high-affinity binding sites (5, 30). However, when tested against competitive antagonists, similar dose ratios are produced for all effects of LTB₄ (17). Likewise, naturally occurring metabolites or synthetic analogs show similar displacement potencies for the low- and high-affinity binding sites (22). Therefore, there is currently no basis for definition of subclasses of receptors for LTB₄, and all available antagonists appear to block the effects of LTB₄ and its immediate metabolites at a common BLT receptor.

A number of selective and relatively potent antagonists of LTB₄ have been developed (31). A few compounds have entered into early clinical testing in humans. The drug candidate LY-293,111 (VML 295) has been found to inhibit LTB₄-induced neutrophil responses in vivo and allergen-induced PMN activation, but had no effect on allergen-induced early- or late-phase airway obstruction in patients with asthma (32). The results with LY-293,111 in patients with asthma argue against an important role for LTB₄ as a mediator in asthma, but do not exclude the possibility that LTB₄ may be involved in other pulmonary reactions.

	RECEPTORS FOR CYSTEINYL-LEUKOTRIENES

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The biological activities of the cysteinyl-leukotrienes (Table 1) are consistent with their role as central proinflammatory mediators. The potent spasmogenic activity of the cysteinyl-leukotrienes is well established. Thus, LTC₄ and LTD₄ are potent inducers of bronchoconstriction in guinea pig airways in vitro and in vivo (33, 34) and they cause contractions of isolated human bronchi (35). When injected intravenously in guinea pigs, LTC₄ and LTD₄ cause biphasic changes in blood pressure (33, 34). These two cysteinyl-leukotrienes also increase microvascular permeability (33, 34) resulting in exudation of plasma proteins in postcapillary venules (8). In the guinea pig, it was shown that LTC₄, LTD₄, and LTE₄ were each capable of inducing plasma exudation in the airways (38). Their effects were observed in all airway segments, ranging from the most peripheral small bronchi to trachea.

The biological effects of LTE₄ have generally been studied much less, perhaps because this leukotriene was found to be an incomplete and less potent agonist than LTC₄ and LTD₄ in guinea pig ileum (39). However, LTE₄ has been documented to possess a bronchoconstrictor activity in vitro and in vivo that is closely similar to that of LTC₄ and LTD₄ (40). It has also been observed that prolonged exposure to LTE₄ may produce enhancement of the responsiveness of smooth muscle to histamine (41, 42). Moreover, LTE₄ is a full agonist for contraction of human bronchi in vitro (43), and it is not significantly less potent than LTC₄ and LTD₄ (44).

In addition to the bronchospastic and vasoactive properties of the cysteinyl-leukotrienes (Table 1), it has been observed that LTC₄ and LTD₄ may stimulate mucous secretion in isolated animal and human airways (45). Experiments in isolated perfused hearts also disclosed a depressive effect on cardiac contractility (48, 49). The effect correlated with coronary vasoconstriction (44, 50), but a direct negative inotropic effect on the myocardium may also be involved (51). Additional effects with potential relevance to the role of cysteinyl-leukotrienes in asthma and pulmonary inflammation include increased infiltration of eosinophils into the airway mucosa of patients with asthma after inhalation of LTE₄ (52). Likewise, inhalation of LTD₄ was found to increase the number of eosinophils in induced sputum samples from patients with asthma (53). The capacity of cysteinyl-leukotrienes to promote eosinophil recruitment has been confirmed in experimental models (54, 55), although the mechanisms involved remain to be defined. There are also experimental data in vitro (56) and in vivo (57) suggesting that cysteinyl-leukotrienes may be involved in airway smooth muscle proliferation and remodeling.

Despite the current introduction of receptor antagonists for cysteinyl-leukotrienes as a new therapy in asthma, the structure of the receptors for cysteinyl-leukotrienes remains unknown. On the basis of primarily functional studies in smooth muscle assays, two main classes of receptors have been outlined (1) (Table 1). The CysLT₁ receptor is blocked by the class of drugs that currently is being used in the clinic for the treatment of asthma. In fact, it appears as if most effects of cysteinyl-leukotrienes in human airways are mediated by this CysLT₁ receptor (Table 1). Certain responses to cysteinyl-leukotrienes in vascular tissues from humans and in some animal smooth muscle assays are, however, resistant to available CysLT₁ antagonists. These responses are currently classified as being mediated by a CysLT₂ receptor (Table 1), but there is no selective antagonist available and the CysLT₂ receptor is mainly an operational concept. The evidence supporting the concept of these two main subclasses is discussed in greater detail below. It will also be evident that there are indications that subclasses may exist among both CysLT₁ and CysLT₂ receptors.

When the SRS-A antagonist FPL-55712 (58) was tested against LTC₄ and LTD₄ in guinea pig airway preparations, it was found that FPL-55712 was a competitive antagonist of LTD₄ but not LTC₄ (59, 60). In the guinea pig trachea, when the metabolic conversion of LTC₄ into LTD₄ was arrested, it was observed that LTC₄ not could be antagonized by FPL-55712 (59, 60) or subsequently developed antagonist of LTD₄ (61, 62). These observations supported the hypothesis that two different receptors for cysteinyl-leukotrienes, tentatively called the LTC₄ and LTD₄ receptors, existed. The findings produced with metabolic inhibitors also argued against the hypothesis that LTC₄ was bioactive only after having been transformed into LTD₄ (63).

However, when the influence of FPL-55712 on LTC₄ and LTD₄ also was examined in human bronchi in the presence of drugs that inhibit the metabolic conversion of LTC₄, it was discovered that FPL-55712 antagonized the effect of LTC₄ and LTD₄ in this tissue to the same extent (44). Human airways were thus different from guinea pig trachea or ileum, where LTC₄ and LTD₄ appeared to cause contractions by activation of different receptors. Subsequent studies with more potent antagonists have indeed confirmed that LTC₄ and LTD₄ act at the same receptor in human airways (64). Moreover, LTE₄ is also a full and potent agonist at the receptor for cysteinyl-leukotrienes in human bronchi (43, 44), and a selective antagonist such as ICI-204,219 (zafirlukast) produces an identical shift in the concentration-response curve for each of LTC₄, LTD₄, and LTE₄ (43).

It had been observed that LTC₄ and LTD₄ contracted human pulmonary vessels (36). When Labat and coworkers examined the effects of antagonists on contractions evoked by cysteinyl-leukotrienes in human pulmonary veins (65), they discovered that the responses were resistant to several potent compounds (ICI-198,615, MK-571, and SKF-104,353) belonging to the current class of antagonists. The agonist sensitivity was also different from that in the bronchial preparations, with LTE₄ being a comparatively weak agonist producing only a transient submaximal contraction of the human pulmonary vein. Responses to both LTC₄ and LTD₄, which were resistant to the current class of antagonists, had previously been reported in animal tissues such as ferret trachea (66), but these effects had not received as much attention as the antagonist-resistant effects of LTC₄ in guinea pig ileum (39) and trachea (59). The findings altogether suggest that there are at least two different patterns of atypical receptors for cysteinyl-leukotrienes, one being preferentially sensitive to LTC₄ (guinea-pig ileum and trachea) and the other mediating contractions to both LTC₄ and LTD₄ (human pulmonary vein and ferret trachea).

The effects of LTC₄ and LTD₄ on the human pulmonary vein were antagonized in an apparently competitive manner by the leukotriene analog BAY u9773 [5(S)-hydroxy-6(R)-(4'-carboxyphenylthio)-7,9-trans-11,14-cis-eicosatetraenoic acid] (39). The compound BAY u9773 has subsequently been found to be a competitive antagonist of atypical responses to LTC₄ or LTD₄ in guinea pig ileum (67) and trachea (68), sheep bronchus (67) and trachea (69), and ferret trachea (67). However, the compound also antagonizes the effects of cysteinyl-leukotrienes in preparations in which the responses are sensitive to the current class of antagonists (67). Therefore, BAY u9773 is not a selective antagonist of atypical responses to cysteinyl-leukotrienes, but has broader antagonistic activity than all other explored antagonists.

On the basis of the evidence thus discussed, the names CysLT₁ and CysLT₂ have therefore been introduced to describe responses that are sensitive and resistant, respectively, to the class of drugs currently being introduced in the clinic (Table 1) (1). There are tissues, such as guinea pig ileum and trachea, in which both types of receptors coexist, and there are tissues that seem to have homogeneous populations of receptors (Table 2). For example, CysLT₁ receptors predominate in human bronchi and rat lung, whereas CysLT₂ receptors appear to dominate in sheep trachea. However, there are tissues in which opposing and interdependent responses to cysteinyl-leukotrienes occur (70), implying that results with current CysLT₁-selective antagonists may underestimate the role of cysteinyl-leukotrienes in different responses.

The current classification introducing two main classes of receptors for cysteinyl-leukotrienes is a first step supported by the available evidence but is nevertheless likely to represent an oversimplification. There is, for example, a significant difference in the potency of the selective CysLT₁ antagonist ICI-198,615 between rat lung and guinea pig trachea (66), which in part could reflect species differences. However, in the guinea pig, it had already been found, in one of the first studies to explore the activity of the prototype of antagonist FPL-55712 against leukotrienes (60), that the response to LTD₄ in the ileum was most susceptible to blockade, whereas the trachea and in particular the lung parenchyma required considerably higher concentrations of the antagonist. Such differences within the same species have been observed in other studies, and may indicate the presence of subclasses of the receptors. The observation that the contraction response to LTD₄ in guinea pig lung parenchyma is poorly inhibited both by potent CysLT₁ antagonists such as ICI-198,615 as well as by the combined CysLT₁/CysLT₂ antagonist BAY u9773 (68, 71) raises the possibility of a third main subclass (CysLT₃).

Although specific binding sites for LTD₄ have been identified in many tissues (72, 73), including the human lung (74), radioligand-binding studies with LTC₄ in lung and other tissues have often shown less evident correlations between binding and functional responses (75). It has been shown that LTC₄ also binds effectively to a microsomal glutathione S-transferase (78, 79). In view of the recently discovered and emerging family of related proteins with the capacity to synthesize LTC₄ (80), it appears as if many enzymes and transport mechanisms display high affinity for LTC₄ and the other cysteinyl-leukotrienes. Such effects remain a challenge for future reseach and for the moment complicate interpretation of binding studies.

	CONCLUSIONS

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The development of selective antagonists of the CysLT₁ receptor has introduced a new class of drugs for the treatment of asthma and other inflammatory diseases. Despite such progress, the basic knowledge about the receptors for leukotrienes is fairly limited and there would seem to be major opportunities for exploration of the cell biology, molecular biology, and pharmacology of leukotriene receptors. Novel information in such areas would also be expected to create new targets for intervention with the leukotriene pathway.

	NOTE ADDED IN PROOF

TOP INTRODUCTION RECEPTORS FOR LEUKOTRIENE B4 RECEPTORS FOR CYSTEINYL-... CONCLUSIONS NOTE ADDED IN PROOF REFERENCES

During the preparation of this supplement, the cloning of the CysLT₁ receptor was reported by two groups (Lynch et al., Nature 1999;399:789-793 and Sarau et al., Mol. Pharmacol. 1999;56:657-663).

	Footnotes

Correspondence and requests for reprints should be addressed to Sven-Erik Dahlén, M.D., Ph.D., Experimental Asthma and Allergy Research, National Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden. E-mail: se.dahlen{at}imm.ki.se.

Acknowledgments: Supported by the Karolinska Institutet and by the following Swedish foundations: the Heart-Lung Foundation, the Association against Asthma and Allergy, the Medical Research Council (Project 71X-9071), and the Foundation for Health Care Sciences and Allergy Research.

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