Cysteinyl-leukotriene receptors in pulmonary vessels.

Two categories of cysteinyl-leukotrienes have been proposed, namely, CysLT1 and CysLT2. These receptors are found not only on the vascular smooth muscle but also on the endothelium. Activation of the receptor(s) on vascular smooth muscle provokes contraction whereas activation of the receptors on the endothelium produces contraction and/or relaxation. These endothelium dependent effects are due to the release of both contractile and relaxant factors derived from the endothelium. While factors derived from either the cyclooxygenase or nitric oxide pathways are involved, in some vascular preparations other mediators such as endothelin may be involved. However, in isolated human pulmonary vascular preparations, this appears not to be the case and presently the nature and origin of the contractile factor remains to be established.     

The molecular characterization and tissue distribution of the human cysteinyl leukotriene CysLT(2) receptor

Cysteinyl leukotrienes (CysLTs), slow-reacting substances of anaphylaxis, are lipid mediators known to possess potent proinflammatory action. Pharmacological studies using CysLTs indicate that at least two classes of G protein-coupled receptors (GPCRs), named CysLT(1) and CysLT(2), exist; the former is sensitive and the latter is resistant to the CysLT(1) antagonists currently used to treat asthma. Although the CysLT(1) receptor gene has been recently cloned, the molecular identity of the CysLT(2) receptor has remained elusive. Here we show that the pharmacological profile of an orphan GPCR (PSEC0146) is consistent with that of the CysLT(2) receptor. In human embryonic kidney 293 cells that express the PSEC0146 cDNA, leukotriene C(4) (LTC(4)) and leukotriene D(4) (LTD(4)) induce equal increases in intracellular calcium mobilization; these increases are not affected by CysLT(1) antagonists. Additionally, [(3)H]LTC(4) specifically binds to membranes from COS-1 cells transiently transfected with PSEC0146. Large amounts of the PSEC0146 mRNA are found in human heart, placenta, spleen, and peripheral blood leukocytes but not in the lung and the trachea. Pharmacological feature and expression studies will eventually lead to a better understanding of the classification of CysLT receptors, possibly leading to a reconsideration of the pathological and physiological role of CysLTs.     

The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor

Nucleotides and cysteinyl-leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G-protein-coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi-coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist-response profile, as determined by [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.     

Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses

The cysteinyl leukotrienes (cys-LTs) are a family of potent bioactive lipids that act through two structurally divergent G protein-coupled receptors, termed the CysLT(1) and CysLT(2) receptors. The cloning and characterization of these two receptors has not only reconciled findings of previous pharmacologic profiling studies of contractile tissues, but also has uncovered their expression on a wide array of circulating and tissue-dwelling leukocytes. With the development of receptor-selective reagents, as well as mice lacking critical biosynthetic enzymes, transporter proteins, and the CysLT(1) receptor, diverse functions of cys-LTs and their receptors in immune and inflammatory responses have been identified. We review cys-LT biosynthesis; the molecular biology and distribution of the CysLT(1) and CysLT(2) receptors; the functions of cys-LTs and their receptors in the recruitment and activation of effector leukocytes and induction of adaptive immunity; and the development of fibrosis and airway remodeling in animal models of lung injury and allergic inflammation.     

Leukotriene D4 induces brain edema and enhances CysLT2 receptor-mediated aquaporin 4 expression

Cysteinyl leukotrienes (including LTC(4), LTD(4), and LTE(4)), potent inflammatory mediators, can induce brain-blood barrier (BBB) disruption and brain edema. These reactions are mediated by their receptors, CysLT(1) and CysLT(2) receptors. On the other hand, aquaporin 4 (AQP4) primarily modulates brain water homeostasis and edema after various injuries. Here, we aimed to determine whether AQP4 is involved in LTD(4)-induced brain edema. LTD(4) (1ng in 0.5mul PBS) microinjection into the cortex increased endogenous IgG exudation (BBB disruption) and water content (brain edema), and enhanced AQP4 expression in mouse brain. The selective CysLT(1) receptor antagonist pranlukast inhibited the IgG exudation, but not the increased water content and AQP4 expression induced by LTD(4). In the cultured rat astrocytes, LTD(4) (10(-9)-10(-7)M, for 24h) similarly enhanced AQP4 expression. The enhanced AQP4 expression was inhibited by Bay u9773, a non-selective CysLT(1)/CysLT(2) receptor antagonist, but not by pranlukast. LTD(4) (10(-9)-10(-7)M) also induced the mRNA expression of CysLT(2) (not CysLT(1)) receptor in astrocytes. These results indicate that LTD(4) modulates brain edema; CysLT(1) receptor mediates vasogenic edema while CysLT(2) receptor may mediate cytotoxic edema via up-regulating AQP4 expression.     

Distinct roles of CysLT1 and CysLT2 receptors in oxygen glucose deprivation-induced PC12 cell death

Cysteinyl leukotrienes are involved in ischemic brain injury, and their receptors (CysLT(1) and CysLT(2)) have been cloned. To clarify which subtype mediates the ischemic neuronal injury, we performed permanent transfection to increase CysLT(1) and CysLT(2) receptor expressions in PC12 cells. Oxygen glucose deprivation (OGD)-induced cell death was detected by Hoechst 33258 and propidium iodide fluorescent staining as well as by flow cytometry. OGD induced late phase apoptosis mainly and necrosis minimally. Over-expression of CysLT(1) receptor decreased and over-expression of CysLT(2) receptor increased OGD-induced cell death. An agonist LTD(4) (10(-7)M) also induced apoptosis, especially in CysLT(2) receptor over-expressing cells. A selective CysLT(1) receptor antagonist montelukast did not affect OGD-induced apoptosis; while non-selective CysLT receptor antagonist Bay u9773 inhibited OGD-induced apoptosis, especially in CysLT(2) receptor over-expressing cells. Thus, CysLT(1) and CysLT(2) receptors play distinct roles in OGD-induced PC12 cell death; CysLT(1) attenuates while CysLT(2) facilitates the cell death.     

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.     

Pharmacotherapy of diseases mediated by 5-lipoxygenase pathway eicosanoids

Inflammatory eicosanoids generated by the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism are now known to have at least 6 receptors: OXE, which recognizes 5-HETE and 5-oxo-ETE; a putative receptor recognizing a potent 5-oxo-ETE metabolite, FOG(7); the LTB(4) receptors, BLT1 and BLT2; the cysteinyl leukotriene receptors, CysLT(1) and CysLT(2), which recognize leukotrienes LTC(4), LTD(4), LTE(4) and LTF(4). The 5-LO pathway is activated in many diseases and invokes inflammatory responses not affected by glucocorticoids, but therapy with selective BLT1 or CysLT(1) antagonists in asthma has met with variable success. Studies show that 5-LO pathway eicosanoids are not primary mediators in all cases of asthma, but may be especially important in severe persistent asthma, aspirin- and exercise-induced asthma, allergic rhinitis, COPD, idiopathic pulmonary fibrosis, atherosclerosis, atopic dermatitis, acne and ischemia-related organ injury. These disorders appear to involve multiple 5-LO pathway eicosanoids and receptor subtypes, suggesting that inhibition of the pathway at the level of 5-LO may be necessary for maximal efficacy.     

G protein-coupled receptor crosstalk and signaling in hematopoietic stem and progenitor cells

A variety of G protein-coupled receptors (GPCRs) is expressed in hematopoietic stem and progenitor cells (HPCs), including the chemokine receptor CXCR4, the leukotriene receptor CysLT1, the sphingosine 1-phosphate receptor S1P1, the cannabinoid receptor CB2, and the complement receptor C3aR. While the role of CXCR4 in stem cell homing is largely established, the function of the other GPCRs expressed in HPCs is only partially understood. CXCR4 and CysLT1 inhibit their own activation after ligand binding (homologous desensitization). Stimulation of S1P1 or C3aR has been shown to activate CXCR4 in HPCs that may sensitize CXCR4-dependent stem cell homing. In contrast, activation of CXCR4 results in a loss of CysLT1 function, which is most likely mediated by protein kinase C (PKC) signaling (heterologous desensitization) and could explain the ineffectiveness of CysLT1 antagonists to mobilize HPCs in vivo. Further characterization of GPCR crosstalk will allow a better understanding of HPC trafficking.     

Involvement of prenylated proteins in calcium signaling induced by LTD4 in differentiated U937 cells

We investigated signal transduction pathways for LTD4 in the human promonocytic cell line U937 known, upon differentiation, to express CysLT1 receptors. We confirmed the presence of high-affinity binding sites for 3H-LTD4, which, in functional studies, displayed the features of CysLT1 receptor. In fact, three potent and selective CysLT1 receptor antagonists were able to completely inhibit LTD4-induced response. In turn, cytosolic Ca2+ ([Ca2+]i) increase (EC50 = 3.4 nM +/- 27% CV) was only partially sensitive to pertussis toxin (PTx) as well as to the prenylation inhibitor fluvastatin and to the specific geranylgeranylation and farnesylation inhibitors BAL 9504 and FPT II. Finally, Clostridium sordellii lethal toxin, inhibitor of the Ras family of GTPases, and FTS, a potent methyltransferase inhibitor, were both able to partially inhibit LTD4-induced [Ca2+] increase, suggesting a role for a Ras family member in [Ca2+]i regulation. In conclusion, in dU937 LTD4 signal transduction involves: (a) at least two pathways, one sensitive and one insensitive to PTx; (b) isoprenylated proteins, such as betagamma subunits and, possibly, a small G protein of the Ras family.     

Functional characterization of cysteinyl leukotriene CysLT(2) receptor on human coronary artery smooth muscle cells

Cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) are a class of biologically active lipids that exert potent effects on the heart. To assess their roles, we investigated the distribution of their receptors, CysLT(1) and CysLT(2), in the cardiovascular system. CysLT(2) mRNA was detected at high levels in the human atrium and ventricle and at intermediate levels in the coronary artery, whereas CysLT(1) mRNA was barely detected. Further analysis by in situ hybridization revealed that CysLT(2) mRNA was expressed in myocytes, fibroblasts, and vascular smooth muscle cells, but not in endothelial cells. When human coronary smooth muscle cells were stimulated with LTC(4), the intracellular calcium concentration increased in a dose-dependent manner, and this action was partially inhibited by nicardipine. Additionally, these cells showed chemotactic responses to LTC(4). This is the first report on the physiological role of CysLT(2), and the findings suggest that CysLT(2) has biological significance in the cardiovascular system.     

Human umbilical vein endothelial cells generate leukotriene C4 via microsomal glutathione S-transferase type 2 and express the CysLT(1) receptor.

Certain immunocompetent myeloid cells, such as eosinophils, basophils and mast cells, have a large capacity to synthesize the potent proinflammatory and spasmogenic mediator leukotriene (LT) C4 via a specific microsomal glutathione S-transferase (MGST) termed LTC4 synthase (LTC4S). Here, we report that MGST2, a distant homologue of LTC4S, is abundantly expressed in Human umbilical vein endothelial cells (HUVEC) and converts LTA4 into a single product, LTC4. Thus, using Northern blot, RT-PCR, Western blot, and enzyme activity assays, we show that MGST2 is the main, if not the only, enzyme that converts LTA4 into LTC4 in membrane preparations of HUVEC. In fact, we failed to detect any expression of LTC4S, MGST1 or MGST3 in these cells, indicating that MGST2 is a critical enzyme for transcellular LTC4 biosynthesis in the vascular wall. Unlike LTC4S, MGST2 prefers the naturally occurring free acid of LTA4 over the methyl ester as substrate and is also susceptible to product inhibition with an IC50 of about 1 microM for LTC4. Moreover, HUVEC were found to express the CysLT1 receptor in line with a paracrine and autocrine role for cysteinyl-leukotrienes in endothelial cell function.     

Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.     

Eosinophils and cysteinyl leukotrienes

Eosinophils are the main source of the cysteinyl leukotrienes, LTC(4)/D(4)/E(4), which are lipid mediators that play major roles in the pathogenesis of asthma and other forms of allergic inflammation. Here, we review the mechanisms governing eosinophil LTC(4) synthesis, focusing on the distinct intracellular domains that regulate eicosanoid formation and function within eosinophils. Cysteinyl leukotrienes exert their actions by engaging specific receptors. As recently shown, eosinophils express CysLT1 and CysLT2, the only cloned receptors for cysteinyl leukotrienes. Therefore, here we also present some of the new findings regarding the paracrine/autocrine activation of these CysLT receptors on eosinophils, and discuss some data on novel intracrine effects of LTC(4) triggered by a putative third CysLT receptor expressed intracellularly within eosinophils.     

Targeted gene disruption reveals the role of cysteinyl leukotriene 1 receptor in the enhanced vascular permeability of mice undergoing acute inflammatory responses

The cysteinyl leukotrienes (cysLTs), leukotriene (LT) C(4), LTD(4), and LTE(4), are proinflammatory lipid mediators generated in the mouse by hematopoietic cells such as macrophages and mast cells. There are two mouse receptors for the cysLTs, CysLT(1) receptor (CysLT(1)R) and CysLT(2)R, which are 38% homologous and are located on mouse chromosomes X and 14, respectively. To clarify the different roles of the CysLT(1)R and CysLT(2)R in inflammatory responses in vivo, we generated CysLT(1)R-deficient mice by targeted gene disruption. These mice developed normally and were fertile. In an intracellular calcium mobilization assay with fura-2 acetoxymethyl ester, peritoneal macrophages from wild-type littermates, which express both CysLT(1)R and CysLT(2)R, responded substantially to 1 x 10(-6) m LTD(4) and slightly to 1 x 10(-6) m LTC(4), whereas the macrophages from CysLT(1)R-deficient mice did not respond to either LTD(4) or LTC(4). Plasma protein extravasation, but not neutrophil infiltration, was significantly reduced in CysLT(1)R-deficient mice subjected to zymosan A-induced peritoneal inflammation. Plasma protein extravasation was also significantly diminished in CysLT(1)R-deficient mice undergoing IgE-mediated passive cutaneous anaphylaxis as compared with the wild-type mice. Thus, the cysLTs generated in vivo by either monocytes/macrophages or mast cells utilize CysLT(1)R for the response of the microvasculature in acute inflammation.     

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sj?gren's syndrome (SS).     

Leukotriene C4 synthase is a novel PPARγ target gene,and leukotriene C4 and D4 activate adipogenesis through cysteinyl LT1 receptors in adipocytes

Leukotriene (LT) C₄ synthase (LTC4S) catalyzes the conversion from LTA₄ to LTC_4, which is a proinflammatory lipid mediator in asthma and other inflammatory diseases. LTC₄ is metabolized to LTD₄ and LTE₄, all of which are known as cysteinyl (Cys) LTs and exert physiological functions through CysLT receptors. LTC4S is expressed in adipocytes. However, the function of CysLTs and the regulatory mechanism in adipocytes remain unclear. In this study, we investigated the expression of LTC4S and production of CysLTs in murine adipocyte 3T3-L1 cells and their underlying regulatory mechanisms. Expression of LTC4S and production of LTC₄ and CysLTs increased during adipogenesis, whereas siRNA-mediated suppression of LTC4S expression repressed adipogenesis by reducing adipogenic gene expression. The CysLT1 receptor, one of the two LTC₄ receptors, was expressed in adipocytes. LTC₄ and LTD₄ increased the intracellular triglyceride levels and adipogenic gene expression, and their enhancement was suppressed by co-treatment with pranlukast, a CysLT1 receptor antagonist. Moreover, the expression profiles of LTC4S gene/protein during adipogenesis resembled those of peroxisome proliferator-activated receptor (PPAR) γ. LTC4S expression was further upregulated by treatment with troglitazone, a PPARγ agonist. Promoter-luciferase and chromatin immunoprecipitation assays showed that PPARγ directly bound to the PPAR response element of the LTC4S gene promoter in adipocytes. These results indicate that the LTC4S gene expression was enhanced by PPARγ, and LTC₄ and LTD₄ activated adipogenesis through CysLT1 receptors in 3T3-L1 cells. Thus, LTC4S and CysLT1 receptors are novel potential targets for the treatment of obesity.     

IL-5 up-regulates cysteinyl leukotriene 1 receptor expression in HL-60 cells differentiated into eosinophils

The cysteinyl leukotrienes, leukotriene (LT) C(4), LTD(4), and LTE(4), are lipid mediators that have been implicated in the pathogenesis of several inflammatory processes, including asthma. The human LTD(4) receptor, CysLT(1)R, was recently cloned and characterized. We had previously shown that HL-60 cells differentiated toward the eosinophilic lineage (HL-60/eos) developed specific functional LTD(4) receptors. The present work was undertaken to study the potential modulation of CysLT(1)R expression in HL-60/eos by IL-5, an important regulator of eosinophil function. Here, we report that IL-5 rapidly up-regulates CysLT(1)R mRNA expression, with consequently enhanced CysLT(1)R protein expression and function in HL-60/eos. CysLT(1)R mRNA expression was augmented 2- to 15-fold following treatment with IL-5 (1-20 ng/ml). The effect was seen after 2 h, was maximal by 4 h, and maintained at 8 h. Although CysLT(1)R mRNA was constitutively expressed in undifferentiated HL-60 cells, its expression was not modulated by IL-5 in the absence of differentiation. Differentiated HL-60/eos cells pretreated with IL-5 (10 ng/ml) for 24 h showed enhanced CysLT(1)R expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-CysLT(1)R Ab. They also showed enhanced responsiveness to LTD(4), but not to LTB(4) or platelet-activating factor, in terms of Ca(2+) mobilization, and augmented the chemotactic response to LTD(4). Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil functions and particularly their responsiveness to LTD(4), and thus contribute to the pathogenesis of asthma and allergic diseases.