Introduction

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It has long been accepted that cysteine-containing leukotrienes (cysteinyl-LTs) LTC₄, LTD₄, and LTE₄, play an important role in asthma, participating in both the bronchoconstriction and the chronic inflammatory component of the disease. CysLTs originate from the oxidative metabolism of arachidonic acid through a key enzyme, 5-lipoxygenase, in a number of inflammatory cells, including eosinophils, basophils, mast cells, and macrophages (Drazen and Austen, 1987; Hay et al., 1995).

CysLTs exert their actions through the activation of specific receptors, the first of which was recently cloned (Lynch et al., 1999). However, in human airways, all the interest has been focused on LTD₄, whereas LTC₄ has been considered either only a precursor or an equipotent/equieffective agonist (Buckner et al., 1986) and LTE₄ has been considered as a metabolite with partial agonist activity (Saussy et al., 1989). Moreover, it is generally believed that in human airways, LTC₄ acts on the same receptor as LTD₄, either CysLT₁ in bronchi (Buckner et al., 1986, 1990; Hay et al., 1987) or CysLT₂ in human pulmonary veins (Labat et al., 1992).

We recently pointed out that in human lung parenchyma (HLP) membranes, LTC₄ possesses a specific high-affinity binding site with characteristics distinct from those of LTD₄ (Capra et al., 1998). In particular, in this tissue, two of the classic CysLT₁ antagonists [i.e., pobilukast and ICI 198,615 (from which zafirlukast has been derived)] behaved differently against ³H-LTC₄ and ³H-LTD₄ at equilibrium, thus suggesting the idea that two different receptors might exist. However, all of the experiments have been performed in the presence of (S)-decyl-glutathione [(S)-decyl-GSH], a compound devoid of either agonist or antagonist activities, which, as it will be demonstrated, interferes with antagonist binding and prevents a complete pharmacological characterization. On the basis of these results, we avoided the use of (S)-decyl-GSH and characterized these two distinct binding sites with a series of antagonists (Fig. 1) in both kinetic binding studies in HLP membranes and contraction of HLP strips.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Chemical structures of LTC4, LTD4, and receptor antagonists.

	Experimental Procedures

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Materials. ³H-LTC₄ (164-173 Ci/mmol) and ³H-LTD₄ (164-173 Ci/mmol) were purchased from DuPont NEN (Boston, MA). LTC₄ and prostaglandin (PG)F₂ were purchased from Cayman Chemical Co. (Ann Arbor, MI). Pobilukast (SKF 104353) was kindly provided by SmithKline and Beecham Laboratories (King of Prussia, PA). LTD₄, zafirlukast (ICI 204,219), pranlukast (ONO 1078), iralukast (CGP 45715A), and CGP 57698 were a generous gift of Dr. A. von Sprecher (Novartis, Basel, Switzerland). Guanosine-5'-(,-imido) triphosphate [Gpp(NH)p], (S)-decyl-GSH, cysteine, glycine, boric acid, serine, indomethacin, Tyrode's salts, and HEPES were purchased from Sigma Chemical Co. (St Louis, MO). Filtercount was from Packard Instruments Co. (Meriden, CT). All the reagents used in HPLC analysis were of analytical grade and purchased from Carlo Erba (Milan, Italy), as were GF/C Whatman Fiberglas filters.

Preparation of HLP Membranes. Crude membranes were prepared from macroscopically normal specimens removed at thoracotomy for lung cancer as previously described (Rovati et al., 1985). Briefly, specimens were minced, homogenized at 4° in 10 mM HEPES buffer, pH 7.4 (1:24, w/v), and centrifuged at 770g for 10 min, and the supernatant was centrifuged at 27,000g for 20 min. The pellet was resuspended, centrifuged under the same condition, and finally resuspended in 5% of the homogenization volume. Membrane aliquots were frozen at 80° and stored for no longer than 3 months. Protein content was determined according to the Bradford dye-binding protein assay (Pierce Chemical Co., Rockford, IL). Before use, serine-borate complex (40 mM final concentration in the assay, prepared as an equimolar solution of serine and boric acid), cysteine (10 mM), and glycine (10 mM) were added to the membrane suspension to avoid cysteinyl-LT metabolism.

Reverse Phase HPLC. Labeled and unlabeled leukotriene purity was always assessed by reverse phase HPLC. Only leukotrienes with a purity grade greater than or equal to 90% were used. The Beckman HPLC system was equipped with a 110B Solvent Delivery Module, an ODS Ultrasphere C18 column, and a programmable detector module 166 set at 280 nm. Both labeled and unlabeled leukotrienes were eluted isocratically with a filtered and degassed mixture of CH₃OH:H₂O:CH₃COOH (65:35:0.02 v/v/v), adjusted at pH 5.8 with NH₄OH, at a flow rate of 1 ml/min. To check the purity of tritiated leukotrienes, fractions were collected every 30 s, and the radioactivity profile assessed by liquid scintillation counting (Ultima Gold; Packard).

Binding Studies. Equilibrium binding studies were performed at 25°C for 30 min with 0.5 nM ³H-LTD₄ or ³H-LTC₄ and unlabeled homologous or heterologous ligands at the indicated concentrations, in the absence and presence of 10 µM (S)-decyl-GSH.

Isolated HLP Strip Preparation. Strips of HLP (1.5-2 cm) were prepared from macroscopically normal human lung specimens placed in cold (4°C) saline solution and studied within 120 min from resection. The HLP strips were suspended in 5-ml organ baths containing Tyrode's solution (composed of 140 mM NaCl, 3 mM KCl, 1 mM CaCl₂, 0.05 mM MgCl₂, 0.5 mM NaH₂PO₄, 8.4 mM glucose, and 12 mM NaHCO₃), maintained at 37°C, and bubbled with 95% O₂, 5% CO₂, pH 7.4. Contractions were measured with a Basile 7004 isometric force transducer and recorded on a Basile Gemini 7070 polygraph. HLP strips were set at an initial tension of 1 g, washed with fresh buffer every 15 min over a 60-min equilibration period, and then treated with 40 mM serine-borate complex and 3 mM L-cysteine to inhibit LTC₄ and LTD₄ metabolism. For antagonist studies, after 15 min, cumulative concentration-response curves were obtained with an increasing concentration of LTC₄ or LTD₄ (0.1 nM to 1 µM). At 15 min later, either a concentration of 10 µM of each antagonist tested or the vehicle DMSO was added. Only one LTC₄ or LTD₄ concentration-response curve was obtained from each HLP strip. The contractile response to each concentration of LTC₄ or LTD₄ was expressed as percent of the maximal response to 300 µM PGF₂.

Computer Analysis. Analysis of binding data of association and homologous dissociation time-courses was performed using the program KINFIT II (Rovati et al., 1996) The computerized analysis of the data through KINFIT II has several advantages, as it allows 1) simultaneously analysis of association and dissociation time courses; 2) calculation of k_on, k_off, and B_max directly in the same analysis without any further approximation; 3) performance of association time courses using a mixture of labeled and unlabeled ligands; and 4) selective labeling of a high-affinity/low-capacity class of sites using a low-specific-activity compound. Binding is expressed as specific bound concentration versus time.

	Results

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Effect of (S)-Decyl-GSH on ³H-LTC₄ and ³H-LTD₄ Binding. The ability of a series of antagonists (10 µM) to compete for ³H-LTC₄ binding was assessed at equilibrium (Fig. 2A). In the presence of (S)-decyl-GSH, only pobilukast retained the ability to displace ³H-LTC₄ from its binding sites, whereas no appreciable effect was observed for agonist binding. The same experiment was repeated using ³H-LTD₄ in the absence and presence of 10 µM (S)-decyl-GSH. In the absence of (S)-decyl-GSH, all of the antagonists tested were able to inhibit ³H-LTD₄ binding, whereas in the presence of (S)-decyl-GSH, the profile of antagonism was identical to that obtained versus ³H-LTC₄ (Fig. 2B).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Effect of (S)-decyl-GSH on the ability of agonist and antagonist to dissociate 3H-LTC4 and 3H-LTD4 binding at equilibrium. A, effect of 10 µM (S)-decyl-GSH on displacement of 1 µM LTC4 and 10 µM concentration of the indicated antagonists versus 3H-LTC4. B, effect of 10 µM (S)-decyl-GSH on displacement of 1 µM LTD4 and 10 µM concentration of the indicated antagonists versus 3H-LTD4. Data are expressed as mean ± S.E. of three replicates from at least three experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effect of (S)-decyl-GSH on 3H-LTC4 and antagonist binding in kinetic studies. A, dissociation time courses of 3H-LTC4 in the absence () and presence () of 10 µM (S)-decyl-GSH. Inset, parameters C (amount bound, nM) and koff (s1) of the curves shown. B, heterologous dissociation time courses for 10 µM pranlukast versus 3H-LTC4 in the absence () and presence () of 10 µM (S)-decyl-GSH. Inset, parameters C (percent specific binding) and koff (s1) of the curves shown. Data are mean values of two replicates from a single experiment, representative of at least two other experiments.

³H-LTD₄ and ³H-LTC₄ Time Courses. ³H-LTD₄ and ³H-LTC₄ association time courses were performed at different concentrations of total ligand (see Experimental Procedures): 0.5 nM to prevalently label the high-affinity sites (Figs. 4 and 5, respectively, and Table 1) and 0.1 µM to also label the low-affinity sites (Table 1). Both dissociation curves are biphasic. Simultaneous computerized analysis of association and dissociation time courses performed at different total ligand concentrations confirmed the presence of two classes of binding sites for both LTC₄ and LTD₄ (P < .05). Parameters are reported in Table 1.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Association and dissociation time courses for 3H-LTD4. , 0.5 nM 3H-LTD4 in the association phase; dissociation was induced by 1 µM LTD4. , 10 nM total LTD4 (2 nM 3H-LTD4 plus 8 nM LTD4) in the association phase; dissociation was induced by 1 µM LTD4. Inset, 0.5 nM 3H-LTD4 in the association phase (data not shown); dissociation was induced by 1 µM LTD4 in the absence () and in the presence () of 30 µM Gpp(NH)p. Data are mean values of three replicates from a single experiment, representative of at least two other experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Association and dissociation time courses for 3H-LTC4. , 0.5 nM 3H-LTC4 in the association phase; dissociation was induced by 1 µM LTC4. Inset, 0.5 nM 3H-LTC4 in the association phase (data not shown); dissociation was induced by 1 µM LTC4 in the absence () and in the presence () of 30 µM Gpp(NH)p. Data are mean values of three replicates from a single experiment, representative of at least two other experiments.

View this table: [in this window] [in a new window]   TABLE 1 Kinetic parameters for 3H-LTD4 and 3H-LTC4 kon values are expressed in M1 · s1. koff values are expressed as s1. Kd values are the ratio of koff to kon and are expressed in nM. Bmax values are expressed as pmol/mg protein. Parameters are expressed as mean ± % CV.

Antagonist Binding Studies. Heterologous dissociation time courses were performed with a series of "classic" CysLT₁ antagonists (Brooks and Summers, 1996) and the dual antagonist BAY u9773 (Cuthbert et al., 1991).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Homologous and heterologous dissociation time courses. A, labeled ligand 0.5 nM 3H-LTD4; dissociation was induced by 1 µM LTD4 (), 10 µM pobilukast (), zafirlukast (), and BAY u9773 (). B, labeled ligand 0.5 nM 3H-LTC4; dissociation was induced by 1 µM LTC4 (), 10 µM pobilukast (), zafirlukast (), and BAY u9773 (). Data are mean values of three replicates from a single experiment, representative of at least two other experiments. S.E. values are not shown for the sake of clarity.

Isometric Contraction of Isolated HLP Strips. Figure 7 shows LTD₄ and LTC₄ cumulative concentration-response curves obtained from isometric contractions of HLP strips. The EC₅₀ values are 6.6 nM ±46% CV and 91 nM ±3.3% CV, and the maximal contractions (expressed as percent versus PGF₂) are 190 ± 9.5% CV and 111 ± 3.5% CV for LTD₄ and LTC₄, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Cumulative concentration-response curves of LTD4- () and LTC4- () induced contraction of isolated HLP strips. Data are mean values of four to six replicates ± S.E.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Effect of pranlukast (A) and iralukast (B) on cumulative concentration-response curve of LTC4 in isolated HLP strips. Data are from a single experiment, representative of at least two other experiments.

	Discussion

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It is well known that LTC₄ predominantly binds to a number of nonreceptor sites in cellular membranes (Keppler, 1992; Metters et al., 1994). As we previously demonstrated (Capra et al., 1998), to unmask a specific high-affinity binding site for LTC₄, (S)-decyl-GSH must be routinely included in the ³H-LTC₄ binding assay at equilibrium to inhibit the interaction with most of these lower-affinity nonreceptor sites. However, we observed that all of the antagonists tested, with the exception of pobilukast, were unable to compete for ³H-LTC₄ binding in the presence of (S)-decyl-GSH. To elucidate whether (S)-decyl-GSH might interfere with the antagonist binding, we selected one representative compound from each structural class of antagonists and tested them in HLP membranes, also against ³H-LTD₄ in the absence and presence of (S)-decyl-GSH. Surprisingly, in the presence of (S)-decyl-GSH, the antagonists were unable to also compete for ³H-LTD₄, indicating that this compound interferes with antagonist but not with agonist binding (Fig. 2). Interestingly, only the binding of pobilukast, the antagonist with the structure closest to that of cysteinyl-LTs, was not affected by (S)-decyl-GSH.

Because the presence of (S)-decyl-GSH prevents the pharmacological characterization of ³H-LTC₄ binding at equilibrium, to avoid the use of (S)-decyl-GSH, we have performed the pharmacological characterization of the ³H-LTC₄ high-affinity site by means of kinetic binding studies. This protocol is rarely used for this purpose, yet in this specific case, with K_d1 far apart from K_d2 (1600-fold difference), it is possible to choose a concentration of ³H-LTC₄ to saturate the high-affinity/low-capacity site without saturating the low-affinity/high-capacity site in the association phase (Rovati et al., 1996; Rovati, 1998). Clearly, a portion of the low-affinity sites, due to their abundance, is also labeled (dissociation time courses are always biphasic). However, with this approach, there is no longer a need to inhibit the binding to the lower-affinity sites by means of (S)-decyl-GSH. The same also applies, in part, to ³H-LTD₄, despite the difference between K_d1 and K_d2 being only 340-fold.

Thus, having primarily labeled the high-affinity binding sites, one can perturb the equilibrium with the antagonists to asses their ability to dissociate ³H-LTD₄ and ³H-LTC₄ from both sites. This protocol is indeed a heterologous dissociation time course, which allow a study of the interaction of unlabeled ligands (i.e., the antagonists) with ³H-LTD₄ and ³H-LTC₄. The only limitation of this type of protocol is that no dissociation constants for the antagonist can be calculated, but only their apparent potency order (Table 2).

We observed that (S)-decyl-GSH interferes with antagonist-induced ³H-LTC₄ dissociation from its high-affinity sites without interfering with the kinetic parameters of the agonist (Fig. 3), confirming the data obtained at equilibrium. A possible explanation for these findings could reside in a nontotal coincidence of agonist and antagonist sites on CysLT receptors and in the steric hindrance of (S)-decyl-GSH at the antagonist binding site.

The results obtained from the simultaneous computerized analysis of association and dissociation time courses for ³H-LTD₄ and ³H-LTC₄ confirmed the model and parameters (Table 1) for cysteinyl-LT binding sites in HLP (Capra et al., 1998), thus validating the kinetic approach in the absence of (S)-decyl-GSH. In fact, LTD₄ interacts with two interconvertible states of a G protein-coupled receptor, whereas LTC₄ displays a different kinetic profile, and both sites are GTP insensitive.

Heterologous dissociation time courses indicated that among all of the "classic" CysLT₁ antagonists we tested, only pobilukast, pranlukast, and CGP 57698 were able to dissociate both ³H-LTD₄ and ³H-LTC₄ from their high- and low-affinity binding sites. On the contrary, zafirlukast and iralukast were unable to interact with the ³H-LTC₄ high-affinity binding site (Fig. 6 and Table 2), whereas they retain the ability to dissociate the ligand from the nonreceptor sites (low-affinity component). Hence, ³H-LTC₄ high-affinity binding site has a unique pharmacological profile, suggesting the existence of a specific LTC₄ receptor different from that of LTD₄ (CysLT₁).

Among all of the cysteinyl-LT antagonists available, BAY u9773 is, until now, the only compound able to recognize both CysLT₁ and CysLT₂ receptors (Coleman et al., 1995). In HLP membranes, BAY u9773 is indeed able to dissociate ³H-LTD₄ from both of its sites but is unable to dissociate ³H-LTC₄ from its high-affinity sites, thus excluding that this LTC₄ specific site is a CysLT₂ receptor.

Taken together, these binding data confirm our hypothesis that HLP membranes contain two cysteinyl-LT high-affinity binding sites with different kinetic (sensitivity to GTP) and pharmacological profiles. LTD₄ binding sites can be classified as a CysLT₁ receptor (Lynch et al., 1999), whereas LTC₄ high-affinity binding site is neither a CysLT₁ nor a CysLT₂ receptor. Moreover, these results indicate that classic antagonists should no longer be considered a homogeneous class of compounds with respect to LTC₄ binding sites and that their specificity seems to be unrelated to the chemical structure, because antagonists of the same class (e.g., pobilukast, iralukast, and BAY u997) behave differently versus the two different receptors.

It is well known that in human airways, CysLT₁ receptors predominantly mediate the contraction of smooth muscle tissue, thus playing an important role in the acute phase of asthma. To evaluate whether LTD₄ and LTC₄ share the same effect and the same pharmacological profile in isolated HLP strips, all of the antagonists were also tested in a functional assay against LTD₄- and LTC₄-induced contractions. Despite the fact that LTD₄ and LTC₄ have different potencies (14-fold difference) and efficacies (1.7-fold difference; Fig. 7), all of the classic antagonists tested were able to reverse LTD₄- as well as LTC₄-induced contractions up to 85 to 100%. BAY u9773 showed a lower efficacy (60% inhibition of LTD₄- and LTC₄-induced contractions at the same time point), suggesting it could behave as a partial agonist, as already proposed both in this tissue (Wikstrom Jonsson et al., 1998) and in human pulmonary veins (Gardiner et al., 1994).

Thus, we can conclude that LTD₄ and LTC₄ contract isolated HLP strips through the same CysLT₁ receptor, as already suggested by Gardiner and Cuthbert (1988) on the basis of more limited data (only one antagonist, FPL 55712). The specific and characteristic LTC₄ high-affinity binding site cannot be classified among one of the officially recognized CysLT receptors, nor it is implicated in LTC₄-induced HLP strip contractions. The recent cloning of the CysLT₁ receptor (Lynch et al., 1999) will rapidly lead to the identification and characterization of the different classes and subclasses of CysLT receptors, but the LTC₄ specific binding site identified here is unlikely to be one of these. In fact, this binding site is not GTP sensitive (Fig. 5 and Capra et al., 1998) and thus should not belong to the superfamily of seven-transmembrane domain receptors.

It is tempting to speculate that this putative receptor is implicated in aspects of the asthmatic syndrome different from bronchoconstriction, such as smooth muscle hyperplasia and proliferation or mucus secretion. Indeed, there are data in the literature that indicate a proliferative role of cysteinyl-LTs in human airway epithelial (Leikauf et al., 1990) or smooth muscle (Panettieri et al., 1998) cells. These data not only suggest LTC₄ as a more potent mitogenic stimulus than LTD₄ (Leikauf et al., 1990) but also indicate LTD₄ to be a weak agonist with a different pharmacological profile compared with the classic contractile function mediated by the CysLT₁ receptor.

Although direct evidence to correlate proliferation with the putative LTC₄ receptor is still lacking, our findings might prompt a deeper investigation into the role of LTC₄, not only as a precursor of LTD₄/LTE₄ but also as an active independent agonist per se. This in turn might contribute to the discovery and development of new and more active drugs with a wider spectrum of action to be used in the treatment of an overlooked disease such as asthma.

Finally, our data also suggest that the homologous kinetic protocol is a valid alternative to classic equilibrium binding studies when supported by sophisticated data analysis. The intrinsic complexity of these experiments can be easily offset by the advantages that this type of protocol presents in particular biological systems where equilibrium studies might fail for theoretical or practical reasons (e.g., when one deals with a high-affinity ligand with a low specific activity; Rovati, 1998). Moreover, heterologous dissociation time courses, albeit with the limitation previously discussed, appear to be a powerful tool for the study of the kinetic characteristics of compounds not available in the labeled form.