Human CysLT Research Articles

A Selective Cysteinyl Leukotriene Receptor 2 Antagonist Blocks Myocardial Ischemia/Reperfusion Injury and Vascular Permeability in Mice

Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that predominantly exert their effects by binding to cysteinyl leukotriene receptors of the G protein-coupled receptor family. CysLT receptor 2 (CysLT(2)R), expressed in endothelial cells of some vascular beds, has been implicated in a variety of cardiovascular functions. Endothelium-specific overexpression of human CysLT(2)R in transgenic mice (hEC-CysLT(2)R) greatly increases myocardial infarction damage. Investigation of this receptor, however, has been hindered by the lack of selective pharmacological antagonists. Here, we describe the characterization of 3-(((3-carboxycyclohexyl)amino)carbonyl)-4-(3-(4-(4-phenoxybutoxy)phenyl)-propoxy)benzoic acid (BayCysLT(2)) and explore the selective effects of this compound in attenuating myocardial ischemia/reperfusion damage and vascular leakage. Using a recently developed β-galactosidase-β-arrestin complementation assay for CysLT(2)R activity (Mol Pharmacol 79:270-278, 2011), we determined BayCysLT(2) to be ∼20-fold more potent than the nonselective dual CysLT receptor 1 (CysLT(1)R)/CysLT(2)R antagonist 4-(((1R,2E,4E,6Z,9Z)-1-((1S)-4-carboxy-1-hydroxybutyl)-2,4,6,9-pentadecatetraen-1-yl)thio)benzoic acid (Bay-u9773) (IC(50) 274 nM versus 4.6 μM, respectively). Intracellular calcium mobilization in response to cysteinyl leukotriene administration showed that BayCysLT(2) was >500-fold more selective for CysLT(2)R compared with CysLT(1)R. Intraperitoneal injection of BayCysLT(2) in mice significantly attenuated leukotriene D(4)-induced Evans blue dye leakage in the murine ear vasculature. BayCysLT(2) administration either before or after ischemia/reperfusion attenuated the aforementioned increased myocardial infarction damage in hEC-CysLT(2)R mice. Finally, decreased neutrophil infiltration and leukocyte adhesion molecule mRNA expression were observed in mice treated with antagonist compared with untreated controls. In conclusion, we present the characterization of a potent and selective antagonist for CysLT(2)R that is useful for discerning biological activities of this receptor.

Construction of HEK293 cell lines expressing hCysLT₂ receptor and its application in screening of antagonists

To construct HEK293 cell lines stably expressing hCysLT(2) receptor, and to evaluate its application in screening of synthetic compounds with antagonist activity. The recombinant plasmid pcDNA3.1(+)-hCysLT(2) was transfected into HEK293 cells using Lipofectamin 2000. The transfected HEK293 cells were selected in 96 well plates by limiting dilution with 600 μg/ml C418 for 8 weeks. The expression of human CysLT(2) receptor was detected by RT-PCR and immunofluorescence staining. In HEK293 cells stably transfected with hCysLT(2), the agonist LTD(4)-induced elevation of intracellular calcium concentration ([Ca2(+)]i) was measured as the index for screening compounds with antagonist activity. After selection in 96 well plates by limiting dilution, 12 monoclones were obtained and 11 of them highly expressed hCysLT(2) receptor. The positive control ATP at 50 μmol/L and LTD(4) at 100 nmol/L elevated [Ca2(+)]i in hCysLT(2)-HEK293 cells. AP-2100984 inhibited LTD(4)-induced [Ca2(+)]i elevation, but selective CysLT(1) receptor antagonists did not exert such an effect. The newly synthesized compounds DXW2, DXW3, DXW4, DXW5, DXW9, DXW25, DXW26, DXW29 and DXW35 at 1 μmol/L significantly inhibited LTD(4)-induced [Ca2(+)]i elevation. The IC(50) values of DXW4 and DXW5 were 0.25 μmol/L and 7.5 μmol/L. HEK293 cell lines stably expressing hCysLT(2) receptor have been successfully constructed, and can be used to screen compounds with CysLT(2) receptor antagonist activity.

Cysteinyl leukotriene 2 receptor‐mediated vascular permeability via transendothelial vesicle transport

Cysteinyl leukotrienes (CysLTs) are potent mediators of inflammation synthesized by the concerted actions of 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), leukotriene C(4) synthase, and additional downstream enzymes, starting with arachidonic acid substrate. CysLTs produced by macrophages, eosinophils, mast cells, and other inflammatory cells activate 3 different high-affinity CysLT receptors: CysLT(1)R, CysLT(2)R, and GPR 17. We sought to investigate vascular sites of CysLT(2)R expression and the role and mechanism of this receptor in mediating vascular permeability events. Vascular expression of CysLT(2)R was investigated by reporter gene expression in a novel CysLT(2)R deficient-LacZ mouse model. CysLT(2)R was expressed in small, but not large, vessels in mouse brain, bladder, skin, and cremaster muscle. Intravital, in addition to confocal and electron, microscopy investigations using FITC-labeled albumin in cremaster postcapillary venule preparations indicated rapid CysLT-mediated permeability, which was blocked by application of BAY-u9773, a dual CysLT(1)R/CysLT(2)R antagonist or by CysLT(2)R deficiency. Endothelial human CysLT(2)R overexpression in mice exacerbated vascular leakage even in the absence of exogenous ligand. The enhanced vascular permeability mediated by CysLT(2)R takes place via a transendothelial vesicle transport mechanism as opposed to a paracellular route and is controlled via Ca(2+) signaling. Our results reveal that CysLT(2)R can mediate inflammatory reactions in a vascular bed-specific manner by altering transendothelial vesicle transport-based vascular permeability.

Transgenic smooth muscle expression of the human CysLT1 receptor induces enhanced responsiveness of murine airways to leukotriene D4.

Cysteinyl leukotrienes (CysLTs) exert potent proinflammatory actions and contribute to many of the symptoms of asthma. Using a model of allergic sensitization and airway challenge with Aspergillus fumigatus (Af), we have found that Th2-type inflammation and airway hyperresponsiveness (AHR) to methacholine (MCh) were associated with increased LTD(4) responsiveness in mice. To explore the importance of increased CysLT signaling in airway smooth muscle function, we generated transgenic mice that overexpress the human CysLT1 receptor (hCysLT(1)R) via the alpha-actin promoter. These receptors were expressed abundantly and induced intracellular calcium mobilization in airway smooth muscle cells from transgenic mice. Force generation in tracheal ring preparations ex vivo and airway reactivity in vivo in response to LTD(4) were greatly amplified in hCysLT(1)R-overexpressing mice, indicating that the enhanced signaling induces coordinated functional changes of the intact airway smooth muscle. The increase of AHR imposed by overexpression of the hCysLT(1)R was greater in transgenic BALB/c mice than in transgenic B6 x SJL mice. In addition, sensitization- and challenge-induced increases in airway responsiveness were significantly greater in transgenic mice than that of nontransgenic mice compared with their respective nonsensitized controls. The amplified AHR in sensitized transgenic mice was not due to an enhanced airway inflammation and was not associated with similar enhancement in MCh responsiveness. These results indicate that a selective hCysLT(1)R-induced contractile mechanism synergizes with allergic AHR. We speculate that hCysLT(1)R signaling contributes to a hypercontractile state of the airway smooth muscle.

Molecular and functional aspects of human cysteinyl leukotriene receptors

The cysteinyl leukotrienes (cys-LTs), i.e. LTC 4, LTD 4 and LTE 4, trigger contractile and inflammatory processes through the specific interaction with cell surface receptors belonging to the purine receptor cluster of the rhodopsin family of the G protein-coupled receptor (GPCR) genes. Cys-LTs have a clear role in pathophysiological conditions such as asthma, allergic rhinitis and other nasal allergies, and have been implicated in a number of inflammatory conditions including cardiovascular and gastrointestinal diseases. Pharmacological studies have identified two classes of cys-LT receptors (CysLT 1 and CysLT 2) based on their sensitivity to CysLT 1 selective antagonists, albeit there is evidence for additional subtypes. Molecular cloning of the human CysLT 1 and CysLT 2 receptors has confirmed both their structure as putative seven transmembrane domain G protein-coupled receptors and most of the previous pharmacological characterization. The rank order of potency of agonist activation for the CysLT 1 receptor is LTD 4>LTC 4>LTE 4 and for the CysLT 2 receptor is LTC 4=LTD 4>LTE 4. The CysLT 1 receptor is most highly expressed in spleen, peripheral blood leukocytes, interstitial lung macrophages and in airway smooth muscle. The CysLT 2 receptor is mostly expressed in heart, adrenals, placenta, spleen, peripheral blood leukocytes and less strongly in the brain. Gene cloning of CysLT 1 and CysLT 2 receptors has renewed the attention on the cys-LTs field and will, hopefully, encourage future studies on the regulation of CysLT receptors expression and the dissection of their signalling pathways. Furthermore, the peculiar pattern of expression of the two receptor subtypes will promote the discovery of new functions for cys-LTs in physiological and pathological conditions. Only CysLT 1 selective receptor antagonists have been described to date and are currently available for the treatment of asthma. Molecular cloning of different CysLT receptor subtypes will certainly foster the development of new selective antagonists based on molecular modelling studies.

Inverse agonist activity of selected ligands of the cysteinyl-leukotriene receptor 1.

Cysteinyl leukotrienes (CysLTs) are associated with several inflammatory processes, including asthma. Due to this association, considerable effort has been invested in the development of antagonists to the CysLT receptors (CysLT(1)R). Many of these molecules have been shown to specifically interact with CysLT(1)R, but little is known about their impact on the conformation of the receptor and its activity. We were especially interested in possible inverse agonist activity of the antagonists. Using a constitutively active mutant (N106A) of the human CysLT(1)R and the wild-type (WT) receptor coexpressed with the G(alphaq) subunit of the trimeric G protein, we were able to address this issue with ligands commonly used in therapy. We demonstrated that some of these molecules are inverse agonists, whereas others act as partial agonists. In cells expressing the CysLT(1)R mutant N106A exposed to Montelukast, Zafirlukast, or 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), the basal inositol phosphate production was reduced by 53 +/- 6, 44 +/- 3, and 54 +/- 4%, respectively. On the other hand, 6(R)-(4-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (BayU9773) and 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazole-5-YL)-butoxy]-phenyl ethanone] (LY171883) acted as partial agonists and alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV 5901) as a neutral antagonist. However, in cells expressing CysLT(1)R and G(alphaq), all antagonists used had inverse agonist activity. The decrease in basal inositol phosphate production by ligands with inverse agonist activity could be inhibited by a more neutral antagonist, confirming the specificity of the reaction. We demonstrate here that Montelukast, MK571, and Zafirlukast can act as inverse agonists on the human CysLT(1) receptor.

Cloning and characterization of human cysteinyl leukotriene receptors

The cysteinyl leukotrienes (CysLTs), namely LTC 4, LTD 4 and LTE 4, activate cellular signal transduction through their high affinity interaction with cell surface seven trans membrane G protein-coupled receptors (GPCRs). The identification and characterization of two human CysLT receptors are described here. The CysLT 1 receptor, a rhodopsin subfamily GPCR, was found during screening projects to identify endogenous ligands for orphan GPCRs. The CysLT 2 receptor has 38% identity to the CysLT 1 receptor and was identified through homology searching techniques. The agonist potencies on the CysLT 1 receptor are LTD 4>LTC 4>LTE 4 and for the CysLT 2 receptor are LTC 4=LTD 4>LTE 4. The CysLTs functionally couple to both receptors via calcium mobilization. The CysLT 1 receptor is most highly expressed in the spleen, myeloid tissues and on smooth muscle and interstitial macrophages in the lung. The CysLT 2 receptor is most highly expressed in the heart, adrenal medulla, myeloid cells and the placenta.

Pharmacological profile of MEN91507, a new CysLT 1 receptor antagonist

MEN91507 (8-[2-( E)-[4-[4-(4-fluorophenyl)butyloxy]phenyl]vinyl]-4-oxo-2-(5-1 H-tetrazolyl)-4 H-1-benzopyran sodium salt)) potently displaced [ 3H]leukotriene D 4 binding from guinea-pig lung and dimethylsulphoxide-differentiated U937 (dU937) cell membranes ( K i 0.50±0.16 and 0.65±0.29 nM, respectively). On the other hand, MEN91507 did not display significant binding affinity for a series of receptors or channels. In functional studies on dU937 cells, MEN91507 behaved as insurmountable antagonist of leukotriene D 4-induced calcium transients, with an apparent p K B of 10.25±0.15. In anaesthetized guinea-pigs, MEN91507 antagonized in a dose-dependent manner leukotriene D 4-induced bronchoconstriction following i.v. or oral administration: the ED 50s were 3.0±0.3 and 140±90 nmol/kg, respectively. The inhibition of leukotriene D 4-induced bronchoconstriction by MEN91507 was long-lasting, since a dose of 0.6 μmol/kg produced 74% reduction of the response after 8 h from administration. Likewise, leukotriene D 4-induced microvascular leakage was antagonized by MEN91507 either following i.v. or oral administration: a significant inhibitory effect was still evident at 16 h from oral administration of a dose of 6 μmol/kg. It is concluded that MEN91507 is a potent and selective antagonist of both guinea-pig and human CysLT 1 receptors; in addition, in vivo studies on guinea-pigs indicate that MEN91507 is an orally available and long-lasting antagonist of the bronchomotor and pro-inflammatory effects induced by leukotriene D 4 through the stimulation of CysLT 1 receptors.

Characterization of Mouse Cysteinyl Leukotriene Receptors mCysLT1 and mCysLT2: DIFFERENTIAL PHARMACOLOGICAL PROPERTIES AND TISSUE DISTRIBUTION

Cysteinyl leukotrienes (LTs) are important proinflammatory mediators. Their precise roles in mice need to be elucidated to interpret mouse models of inflammatory diseases. For this purpose, we cloned and characterized mouse receptors for cysteinyl LTs, mCysLT(1) and mCysLT(2). mCysLT(1) and mCysLT(2) were composed of 339 amino acids with 87.3% identity and 309 amino acids with 73.4% identity to human orthologues, respectively. A pharmacological difference was noted between mouse and human CysLT(2). Pranlukast, a specific inhibitor for human CysLT(1), antagonized mCysLT(2) responses as determined by Ca(2+) elevation and receptor-induced promoter activation. The mRNA expressions of both mCysLTs were higher in C57BL/6 mice than in 129 mice. mCysLT(1) mRNA was expressed mainly in skin, lung, and small intestine. mCysLT(2) was seen more ubiquitously with high expressions in spleen, lung, and small intestine. By in situ hybridization we demonstrated for the first time that mCysLT(1) and mCysLT(2) were expressed in subcutaneous fibroblasts. The different pharmacological characteristics of CysLT(2) between human and mouse and the different distributions of CysLTs between mouse strains suggest that careful choice and interpretation are necessary for a study of CysLTs using animal models.

Molecular cloning and functional characterization of murine cysteinyl-leukotriene 1 (CysLT 1) receptors

We sought to clone and characterize the murine cysteinyl-leukotriene D 4 receptor (mCysLT 1R) to complement our studies with leukotriene-deficient mice. A cDNA, cloned from trachea mRNA by reverse transcriptase-polymerase chain reaction, has two potential initiator ATG codons that would encode for polypeptides of 352 and 339 amino acids, respectively. These two potential forms, predicted to be seven transmembrane-spanning domain proteins, have 87% amino acid identity with the human CysLT 1 receptor (hCysLT 1R). Membrane fractions of Cos-7 cells transiently expressing the short mCysLT 1R demonstrated high affinity and specific binding for leukotriene D 4 (LTD 4, K d = 0.25 ± 0.04 nM). In competition binding experiments, LTD 4 was the most potent competitor ( K i = 0.8 ± 0.2 nM) followed by LTE 4 and LTC 4 ( K i = 86.6 ± 24.5 and 100.1 ± 17.1 nM, respectively) and LTB 4 ( K i > 1.5 μM). Binding of LTD 4 was competitively inhibited by the specific CysLT 1 receptor antagonists MK-571 [(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethylamino)-3-oxopropyl)thio)methyl)thio)propanoic acid], pranlukast (Onon™), and zafirlukast (Accolate™), while the CysLT 1/CysLT 2 receptor antagonist BAY-u9773 [6( R)-(4′-carboxyphenylthio)-5( S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetrenoic acid] was 1000 times less potent than LTD 4. In transiently transfected HEK293-T cells expressing either the long or short form of mCysLT 1R, LTD 4 induced an increase of intracellular calcium. In Xenopus laevis melanophores transiently expressing either isoform, LTD 4 induced the dispersion of pigment granules, consistent with the activation by LTD 4 of a G αq (calcium) pathway. Functional elucidation of mCysLT 1R properties as described here will enable further experiments to clarify the selective role of LTD 4 in murine models of inflammation and asthma.

Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing.

Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT(1)) and CysLT(2) receptors, recently have been characterized and cloned. Because the CysLT(1) receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT(1) receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT(1) receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT(1) receptor mRNA is expressed in lung and skin; and reverse transcription-PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT(1) receptor was mapped to band XD. Leukotriene (LT) D(4) induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT(1) receptor cDNA. This agonist effect of LTD(4) was fully inhibited by the CysLT(1) receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [(3)H]LTD(4); and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD(4) >> LTE(4) = LTC(4) >> LTB(4). Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.

Molecular cloning and characterization of a second human cysteinyl leukotriene receptor: discovery of a subtype selective agonist.

The cysteinyl leukotrienes (CysLTs) are potent biological mediators in the pathophysiology of inflammatory diseases, in particular of airway obstruction in asthma. Pharmacological studies have suggested the existence of at least two types of CysLT receptors, designated CysLT(1) and CysLT(2). The CysLT(1) receptor has been cloned recently. Here we report the molecular cloning, expression, localization, and functional characterization of a human G protein-coupled receptor that has the expected characteristics of a CysLT(2) receptor. This new receptor is selectively activated by nanomolar concentrations of CysLTs with a rank order potency of LTC(4) = LTD(4) >> LTE(4). The leukotriene analog BAY u9773, reported to be a dual CysLT(1)/CysLT(2) antagonist, was found to be an antagonist at CysLT(1) sites but acted as a partial agonist at this new receptor. The structurally different CysLT(1) receptor-selective antagonists zafirlukast, montelukast, and MK-571 did not inhibit the agonist-mediated calcium mobilization of CysLT(2) receptors at physiological concentrations. Localization studies indicate highest expression of CysLT(2) receptors in adrenal glands, heart, and placenta; moderate levels in spleen, peripheral blood leukocytes, and lymph nodes; and low levels in the central nervous system and pituitary. The human CysLT(2) receptor gene is located on chromosome 13q14.12-21.1. The new receptor exhibits all characteristics of the thus far poorly defined CysLT(2) receptor. Moreover, we have identified BAY u9773 as a CysLT(2) selective agonist, which could prove to be of immediate use in understanding the functional roles of the CysLT(2) receptor.

Characterization of the Human Cysteinyl Leukotriene 2 Receptor

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.