Binding to Cysteinyl-Leukotriene Receptors

SIMONETTA NICOSIA, VALÉRIE CAPRA, SAULA RAVASI, and G. ENRICO ROVATI

Laboratory of Molecular Pharmacology, Institute of Pharmacological Sciences, University of Milan, Milan, Italy

	INTRODUCTION

TOP INTRODUCTION THE CLASSIFICATION AND... IDENTIFICATION OF CYSTEINYL-... NOTE ADDED IN PROOF REFERENCES

It is widely accepted that leukotrienes (LTs) exert their actions through high-affinity specific receptors, although only the LTB₄ receptor has been cloned so far (1). Given the differences between the two classes of LTs (LTB₄ versus LTC₄, LTD₄, and LTE₄, i.e., cysteine-containing leukotrienes or cysteinyl-LTs) in terms of both structure and profile of biological activity, it is not unexpected that the receptors for LTB₄ are totally different from those for cysteinyl-LTs and no cross- reactivity has been shown among the two classes.

The first hint of the existence of leukotriene receptors had been given even before their structure had been elucidated. FPL-55712 had been proposed as an antagonist of slow-reacting substance of anaphylaxis (SRS-A) when it was not yet known that the latter was composed mainly of LTs (2). Subsequently, the recognition of strict structural requirements for LT action (3, 4), their high potency (in the nanomolar range), and the development of a number of specific antagonists (5) made it clear that these lipid mediators elicit their biological actions through specific receptors.

The existence of different classes of Cys-LT receptors in human tissues is the subject of this article.

Cysteinyl-LTs have been recognized to play a key role in asthma (6), and, as a consequence, the large majority of studies aimed at the characterization of their receptors have been performed in the respiratory system. Asthma is a multifactorial disease, characterized by the presence of reversible airway obstruction, inflammation, and hyperresponsiveness (7). The most significant discovery in the more recent research on asthma pathophysiology has been the revelation that airway inflammation is the key component of this condition. Of the different mediators that are known to be involved in asthma, leukotrienes are considered to be among the most important because they participate both in bronchoconstriction and in the inflammatory component of this disease.

	THE CLASSIFICATION AND NOMENCLATURE OF LEUKOTRIENE RECEPTORS

TOP INTRODUCTION THE CLASSIFICATION AND... IDENTIFICATION OF CYSTEINYL-... NOTE ADDED IN PROOF REFERENCES

The classification and nomenclature of LT receptors have been proposed by an ad hoc committee appointed by the International Union of Pharmacology (IUPHAR), and has been officially approved by the latter (8). The receptors for LTB₄ have been termed BLT receptors (B refers to LTB), while the receptors for LTC₄, LTD₄, and LTE₄ have been named Cys-LT receptors (Table 1). As already mentioned, only the BLT receptor has been cloned so far (1), and a single class has been clearly identified.

On the other hand, it is recognized that Cys-LT receptors are heterogeneous. At present, firm evidence has been obtained only of two classes, namely, Cys-LT₁ and Cys-LT₂. The official classification of Cys-LT receptors has been performed on the basis of the sensitivity to the so-called "classic" antagonists (5, 9), the most widely used of which include SKF-104353, ICI-204,219, MK-571, MK-476, and ONO-1078. Thus, Cys-LT₁ receptors are defined as those sensitive to classic antagonists, while Cys-LT₂ receptors would be those that mediate effects not inhibited by the same antagonists (Table 1).

A new Cys-LT antagonist, i.e., BAY u9773 (10), has been reported to recognize the Cys-LT₂ receptor, as well as the Cys-LT₁ receptor. Although BAY u9773 is neither potent nor selective for the Cys-LT receptor classes, especially in human tissues, it is currently the only "dual" antagonist that clearly has activity at both the Cys-LT₁ and Cys-LT₂ receptor level and it might prove to be an interesting pharmacological tool to confirm the presence of Cys-LT₂ receptors in different tissues.

The available literature, as well as data obtained in our laboratory (11, 12), already indicate that additional classes of Cys-LT receptors might exist: not only of subclasses of both Cys-LT₁ and Cys-LT₂ (10, 13), but also of a third class of Cys-LT receptors (14). In fact, Cuthbert and coworkers (10) classified Cys-LT receptors in guinea pig (GP) trachea as Cys-LT₁ and as Cys-LT₂ according to IUPHAR criteria, but Cys-LT₁ receptors are predominantly activated by LTD₄/LTE₄, and Cys-LT₂ receptors are predominantly activated by LTC₄. Labat and co-workers (13) classified Cys-LT receptors in human lung as Cys-LT₁ and Cys-LT₂, again on the basis of IUPHAR criteria, but in this tissue both receptors are equally activated by LTD₄, LTE₄, and LTC₄. Thus, despite the problems that may be encountered when using agonists to define receptor subtypes (15), these data alone support our hypothesis of a subclassification of both Cys-LT₁ and Cys-LT₂, based on different potencies or affinities of the endogenous ligand (11).

	IDENTIFICATION OF CYSTEINYL-LEUKOTRIENE RECEPTORS IN AIRWAYS

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Functional Studies

Animal tissues. Despite the generally accepted assumption that cysteinyl-LTs exert their effects through specific receptors, none of the Cys-LT receptors has yet been purified or cloned. However, by means of pharmacological studies and radioligand-binding assays, Cys-LT receptors have been recognized in a number of cells, tissues, and organs in different species (16).

The first hint of the existence of distinct Cys-LT receptors in GP trachea had been given even as the first antagonist was developed, i.e., FPL-55712. In fact, LTD₄- and LTE₄-elicited contraction could be competitively antagonized by FPL-55712, while the effect of LTC₄ was insensitive to the same antagonist (17). Thus, GP trachea contains at least two different Cys-LT receptors, which, according to their sensitivity to the classic antagonists (IUPHAR nomenclature; see above), are termed Cys-LT₁ and Cys-LT₂. However, these receptors differ also in their selectivity to different agonists. In fact, in studies where LTC₄ conversion to LTD₄ was inhibited (14), LTC₄ was able to elicit relevant biological effects with a potency comparable to that of LTD₄, but independently from the latter.

Thus, both Cys-LT₁ and Cys-LT₂ receptors, officially defined on the basis of their antagonist selectivity, could actually show distinct preferences also toward different agonists.

The dual antagonist BAY u9773 is able to antagonize LTC₄- and LTD₄-induced contractions in a number of preparations, some of which contain a single population of Cys-LT₂ receptors, while others possess both Cys-LT₁ and Cys-LT₂ classes (18) [Table 2 (13, 14, 17)]. The existence of at least two distinct classes of Cys-LT receptors has been suggested also in rat lung (23) by ligand-binding assays (see below). In this latter tissue, however, functional data, which indicate the presence of homogeneous Cys-LT₁ receptors (25), are at variance with binding results.

Interestingly, GP lung parenchyma seems to behave differently from either the trachea or the ileum: Tudhope and co-workers (14) have found that the contractions induced by both LTC₄ and LTD₄ in GP lung strips are at least partially resistant not only to some of the classic antagonists (namely, ICI-198,615 and MK-571), but also to BAY u9773 itself (20), thus suggesting the existence of a third class of Cys-LT receptors (Cys-LT₃). However, these results are at variance with those of Snyder and coworkers (26), who reported that ICI-198,615 was potent in this tissue, provided the parenchyma was pretreated with the cycloxygenase inhibitor indomethacin (27).

Moreover, BAY u9773 is also able to contract GP lung strip in a concentration-dependent manner, thus behaving as a partial agonist. However, this contraction does not seem to be inhibited by the cycloxygenase inhibitor indomethacin (28), at variance with data previously reported (14). Interestingly, the thromboxane receptor (TP) antagonist BAY u3405 is able to inhibit the contraction induced by BAY u9773. Therefore, the mechanism of contraction evoked by BAY u9773 in GP lung parenchyma can pharmacologically be interpreted as Cys-LT₁ and TP receptor agonism. While the Cys-LT₁ receptor agonism was not unexpected on the basis of the similarity between BAY u9773 and LTE₄, the TP receptor agonism might be worth further investigation.

Human tissues. Despite the fact that GP is the animal of choice in airway studies, the situation in human intralobar airways seems to be quite different from that described in GP airways. In fact, in GP airways cysteinyl-LTs trigger the formation of thromboxane A₂ (TXA₂) (29), which contributes to the contractile response; this indirect response does not occur in humans. In addition, human bronchi (HB) apparently contain a homogeneous population of Cys-LT₁ receptors.

In fact, contractions elicited in HB by either LTC₄ or LTD₄ are sensitive to the classic antagonists (26, 30, 31). Furthermore, these receptors, at variance with those of GP trachea, seem to recognize LTC₄ and LTD₄ equally well. However, BAY u9773, which antagonizes LTD₄- and LTC₄-induced contractions, is far less potent in human airways as compared with GP trachea (13). This suggests that the Cys-LT population in human bronchi, although homogeneous, might have a profile different from that of the classic Cys-LT₁ receptor, perhaps reflecting species differences.

In addition, in human lung, Cys-LT₂ receptors have been demonstrated to be present also on pulmonary veins (13), which are contracted by both LTC₄ and LTD₄. Therefore, as in human bronchi, these receptors do not discriminate between LTD₄ or LTC₄. Moreover, endothelial cells of human pulmonary veins possess both a Cys-LT₁ receptor population, which mediates contraction of the veins, and a Cys-LT₂ population, mediating their relaxation (32).

As far as LTE₄ is concerned, its biological action is similar to that of the other cysteinyl-LTs, but it is usually less potent than its precursors. In addition, in many instances, such as in human pulmonary veins (13) and GP airways (33), it has been shown that LTE₄ has a lower efficacy than the other cysteinyl-LTs, thus behaving as a partial agonist; this does not apply to HB, where it is probably a full agonist.

In the great majority of investigated systems, LTE₄ elicits its effects by binding to the same receptor as LTD₄, although it has been suggested that in GP trachea LTE₄ might bind only to a subset of the Cys-LT₁ receptors (34).

Binding Studies

Binding studies with cysteinyl-LTs have proved difficult: the receptors seem to be present at a low density in most tissues; also, only tritiated ligands are commercially available so far, and their specific activity is much lower than that of iodinated analogs, thus decreasing the sensitivity of binding assays.

But the major problem in studying cysteinyl-LT binding is probably the fact that the predominant LTC₄-binding sites in cellular membranes from various tissues are not receptors but rather subunits of glutathione S-transferase in rat liver (35- 36). Furthermore, LTC₄ can bind to specific exporters (37), as well as to ATP-dependent carriers (38, 39).

However, more recently, by combining the use of an inhibitor of LTC₄ nonreceptor binding with sophisticated data analysis, we succeeded in identifying a high-affinity binding site for LTC₄ with receptor characteristics in human lung parenchyma (see below) (12).

Animal tissues. Ligand-binding studies have been performed under controlled metabolic conditions in various tissues such as GP lung (22, 40) and ileum (41) and rat lung (23). Binding data suggest that at least some of the Cys-LT receptors are coupled to GTP-binding proteins (G proteins) and therefore likely belong to the superfamily of the seven-transmembrane domain receptors. Indeed, the expected conversion of LTD₄-binding sites to lower affinity on GTP addition (42- 44) has been observed, for instance, in GP lung membranes and RBL-1 cells. Accordingly, the solubilized receptor is associated with a pertussis toxin-sensitive G protein (45).

The only antagonist that so far has been isotopically labeled and made commercially available for binding studies is [³H]ICI-198,615, which has been used to label the membranes from GP lung (46) and cardiac ventricle (47). Binding of [³H]ICI-198,615 could be inhibited by LTD₄, at least partially, indicating that most of the sites labeled by the antagonist represent the receptors for the endogenous agonists (48).

Photoaffinity labeling studies performed with [¹²⁵I]azido-LTD₄ allowed the evaluation of the size of the LTD₄ receptor (45 kD) in GP lung membranes (51); such a size is in agreement with that of most seven-transmembrane domain receptors, further supporting the idea that LTD₄ receptor is coupled to G proteins.

Human tissues. Specific binding sites for LTD₄ have been identified in human lung parenchyma (HLP) membranes (50). By means of more sophisticated binding protocols and analysis (12), we have demonstrated that LTD₄ binds to at least two different classes of sites: one with high affinity (K_d of 0.21 nM), and another endowed with lower affinity (K_d of 380 nM). We have also demonstrated that these two classes of sites actually represent the two affinity states of a G protein-coupled receptor, which can be interconverted into one another by GTP or its analogs (12).

Moreover, the binding of the antagonist [³H]ICI-198,615 in HLP membranes (51) occurs to at least two classes of sites, one of which has the characteristics of the LTD₄ receptor. However, as expected from the ternary-complex theory (58), an antagonist is not able to discriminate between the two affinity states of a G protein-coupled receptor and thus, this compound is not able to recognize the two distinct binding sites labeled by [³H]LTD₄ (12).

As far as [³H]LTC₄ binding is concerned, the identification of specific binding sites had been previously reported in membranes from both human lung (53, 54) and bronchi (55), but it was not clear which proportion of such sites might represent a receptor protein.

More recently (12), we have repeated the binding studies for [³H]LTC₄ in HLP in the absence and presence of S-decyl-glutathione, a high-affinity ligand for nonreceptor LTC₄-binding sites (56), devoid of either agonist or antagonist activities (41, 57). Because of these characteristics, S-decyl-glutathione abolishes binding to nonreceptor sites and therefore it has been able to unmask the possible LTC₄ receptor in human lung. In fact, computerized analysis of [³H]LTC₄ binding data revealed the presence of two different classes of binding sites in the presence of S-decyl-glutathione, while in its absence only one low-affinity class was detectable in most of the experiments. This confirms the hypothesis that the predominant binding of [³H]LTC₄ to nonreceptor sites could make receptor binding almost undetectable. At variance with [³H]LTD₄ binding, the high-affinity [³H]LTC₄ site was not affected by GTP, indicating that this site, although possessing most of the characteristics of a receptor, is not coupled to a G protein.

Computerized analysis of the binding curves for [³H]LTC₄ and [³H]LTD₄, as well as antagonist selectivity, demonstrated that the two high-affinity sites for these compounds are different from one another, despite some cross-reactivity. It is suggested, therefore, that in HLP, at variance with HB, LTC₄ and LTD₄ elicit some of their biological actions through different receptors. Figure 1 is a schematic representation of our current hypothesis on Cys-LT receptors in HLP.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Model for Cys-LT receptors in human lung parenchyma.

It is clear that the situation is far from being completely elucidated and that the IUPHAR classification of Cys-LT receptors is only a working hypothesis. Only the purification and cloning of the Cys-LT receptors will clarify definitively how many classes of receptors really exist in each particular tissue and whether the differences revealed by pharmacological studies really reflect different receptor proteins with different characteristics.

	NOTE ADDED IN PROOF

TOP INTRODUCTION THE CLASSIFICATION AND... IDENTIFICATION OF CYSTEINYL-... NOTE ADDED IN PROOF REFERENCES

During the review of this manuscript two groups published the cloning of the CysLT₁ receptor:

1. Lynch, K. R., G. P. Gary, P. O'Neill, Q. Qingyun Liu, D.-S. Im, N. Sawyer, K. M. Metters, N. Coulombe, M. Abramovitz, D. J. Figueroa, Z. Zeng, B. M. Connolly, C. Bai, C. P. Austin, A. Chaterauneuf, R. Stocco, G. M. Greig, S. Kargman, S. B. Hooks, E. Hosfield, D. L. Williams, Jr., A. W. Ford-Hutchinson, C. T. Caskey, and J. F. Evans. 1999. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 399:789-793.

2. Sarau, H. M., R. S. Ames, J. Chambers, C. Ellis, N. Elshourbagy, J. J. Foley, D. B. Schmidt, R. M. Muccitelli, O. Jenkins, P. R. Murdock, N. C. Herrity, W. Halsey, G. Sathe, A. I. Muir, P. Nuthulaganti, G. M. Dytko, P. T. Buckley, S. Wilson, D. J. Bergsma, and D. W. Hay. 1999. Identification, molecular cloning, expression, and characterization of a cysteinyl leukotriene receptor. Molecular Pharmacology 56:657-663.

	Footnotes

Correspondence and requests for reprints should be addressed to Simonetta Nicosia, Ph.D., Institute of Pharmacological Sciences, University of Milan, via Balzaretti 9, 20133 Milan, Italy.

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