Control of NFAT Isoform Activation and NFAT-Dependent Gene Expression through Two Coincident and Spatially Segregated Intracellular Ca2+ Signals.

Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.

Do arachidonic acid metabolites affect apoptosis in bovine endometrial cells with silenced PPAR genes?

Peroxisome proliferator-activated receptors (PPARs) are expressed in bovine uterus, and their agonists are arachidonic acid (AA) metabolites. We hypothesised that silencing of PPAR genes in bovine endometrial stromal cells (ESC) would change the intracellular signalling through PPAR and affect apoptosis after cell treatment with different AA metabolites. The study's aims are detection of apoptosis and examining the influence of prostaglandins and leukotrienes on apoptosis occurring in physiological ESC and cells with silenced PPAR (α, δ, and γ) genes. Silencing the PPARα and PPARδ genes in cells resulted in increased DNA fragmentation and mRNA and protein expression of caspase (CASP) -3 and -8 (P < 0.05). Neither DNA fragmentation nor the mRNA and protein expression of CASP3 and -8 in cells with silenced PPARγ gene were changed compared to physiological cells (P > 0.05). Among PPARs, PPARα and PPARδ appear to inhibit apoptosis, and AA metabolites, as PPAR agonists, modify this process in bovine ESC.

Cysteinyl leukotriene type I receptor desensitization sustains Ca2+-dependent gene expression.

Receptor desensitization is a universal mechanism to turn off a biological response; in this process, the ability of a physiological trigger to activate a cell is lost despite the continued presence of the stimulus. Receptor desensitization of G-protein-coupled receptors involves uncoupling of the receptor from its G-protein or second-messenger pathway followed by receptor internalization. G-protein-coupled cysteinyl leukotriene type I (CysLT1) receptors regulate immune-cell function and CysLT1 receptors are an established therapeutic target for allergies, including asthma. Desensitization of CysLT1 receptors arises predominantly from protein-kinase-C-dependent phosphorylation of three serine residues in the receptor carboxy terminus. Physiological concentrations of the receptor agonist leukotriene C(4) (LTC(4)) evoke repetitive cytoplasmic Ca(2+) oscillations, reflecting regenerative Ca(2+) release from stores, which is sustained by Ca(2+) entry through store-operated calcium-release-activated calcium (CRAC) channels. CRAC channels are tightly linked to expression of the transcription factor c-fos, a regulator of numerous genes important to cell growth and development. Here we show that abolishing leukotriene receptor desensitization suppresses agonist-driven gene expression in a rat cell line. Mechanistically, stimulation of non-desensitizing receptors evoked prolonged inositol-trisphosphate-mediated Ca(2+) release, which led to accelerated Ca(2+)-dependent slow inactivation of CRAC channels and a subsequent loss of excitation-transcription coupling. Hence, rather than serving to turn off a biological response, reversible desensitization of a Ca(2+) mobilizing receptor acts as an 'on' switch, sustaining long-term signalling in the immune system.

Platelet-driven leukotriene C4-mediated airway inflammation in mice is aspirin-sensitive and depends on T prostanoid receptors.

Cysteinyl leukotrienes (cysLTs) are bronchoconstricting lipid mediators that amplify eosinophilic airway inflammation by incompletely understood mechanisms. We recently found that LTC4, the parent cysLT, potently activates platelets in vitro and induces airway eosinophilia in allergen-sensitized and -challenged mice by a platelet- and type 2 cysLT receptor-dependent pathway. We now demonstrate that this pathway requires production of thromboxane A2 and signaling through both hematopoietic and lung tissue-associated T prostanoid (TP) receptors. Intranasal administration of LTC4 to OVA-sensitized C57BL/6 mice markedly increased the numbers of eosinophils in the bronchoalveolar lavage fluid, while simultaneously decreasing the percentages of eosinophils in the blood by a TP receptor-dependent mechanism. LTC4 upregulated the expressions of ICAM-1 and VCAM-1 in an aspirin-sensitive and TP receptor-dependent manner. Both hematopoietic and nonhematopoietic TP receptors were essential for LTC4 to induce eosinophil recruitment. Thus, the autocrine and paracrine functions of thromboxane A2 act downstream of LTC4/type 2 cysLT receptor signaling on platelets to markedly amplify eosinophil recruitment through pulmonary vascular adhesion pathways. The findings suggest applications for TP receptor antagonists in cases of asthma with high levels of cysLT production.

The Responses of Pulmonary and Systemic Circulation and Airway to Allergic Mediators in Anesthetized Rats.

Lung allergic diseases sometimes accompany pulmonary vaso- and broncho-constriction. Rats are currently used for the experimental study of lung allergies. However, their hemodynamic mechanisms are not fully understood. Therefore the effects of allergic mediators were determined systematically in vivo in rats in terms of pulmonary vascular resistance (PVR), airway pressure (AWP) and total peripheral resistance (TPR). We directly measured pulmonary arterial pressure, left atrial pressure, systemic arterial pressure, central venous pressure and aortic blood flow to determine PVR and TPR, as well as AWP, following injections of platelet-activating factor (PAF), histamine, serotonin, leukotriene (LT) C4, and prostaglandin (PG) D2 in anesthetized open-chest artificially ventilated Sprague-Dawley (SD) rats. PVR was dose-dependently increased by consecutive administration of PAF, LTC4, and PGD2, with the maximal responsiveness being PAF>LTC4>PGD2. However, neither histamine nor serotonin changed PVR. TPR was decreased by all agents except LTC4 which actually increased it. PAF and serotonin, but not the other agents, increased AWP. In conclusion, allergic mediators exert non-uniform actions on pulmonary and systemic circulation and airways in anesthetized SD rats: PAF, LTC4 and PGD2, but not histamine or serotonin, caused substantial pulmonary vasoconstriction; LTC4 yielded systemic vasoconstriction, while the others caused systemic vasodilatation; only two mediators, PAF and serotonin, induce airway constriction.

Activation of calcium- and voltage-gated potassium channels of large conductance by leukotriene B4.

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory ß1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 µm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-ß2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK ß1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate ß1 subunit-containing channels when ß1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK ß1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of ß1-specific BK channel activators.

Autocrine activity of cysteinyl leukotrienes in human vascular endothelial cells: Signaling through the CysLT2 receptor.

We evaluated the autocrine activities of cysteinyl leukotrienes (cysteinyl-LTs) in HUVEC and studied the signaling and the pharmacological profile of the CysLT2 receptor (CysLT2R) expressed by ECs, finally assessing the role of the CysLT2R in permeability alterations in a model of isolated brain. Cysteinyl-LTs and their precursor LTA4 contracted HUVEC and increased permeability to macromolecules, increasing the formation of stress fibers through the phosphorylation of myosin light-chain (MLC) following Rho and PKC activation. Accordingly, in an organ model of cerebral vasculature with an intact intima, neutrophils challenge leaded to significant formation of cysteinyl-LTs and edema. Pretreatment with a selective CysLT2R antagonist prevented cytoskeleton rearrangement and HUVEC contraction, along with edema formation in the brain preparation, while leaving the synthesis of cysteinyl-LTs unaffected. We also demonstrate here that the CysLT1R antagonist zafirlukast, pranlukast, pobilukast and iralukast also possess CysLT2R antagonistic activity, which could help in reconsidering previous data on the role of cysteinyl-LTs in the cardiovascular system. The results obtained are further supporting a potential role for CysLT2R in cardiovascular disease.

Different agonists recruit different stromal interaction molecule proteins to support cytoplasmic Ca2+ oscillations and gene expression.

Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca(2+) oscillations. Although oscillations arise from regenerative Ca(2+) release, they are sustained by store-operated Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca(2+) oscillations, CRAC channel activity, and downstream Ca(2+)-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca(2+) oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca(2+) signals and downstream responses.

Leukotriene C4 induces migration of human monocyte-derived dendritic cells without loss of immunostimulatory function.

Generation of human monocyte-derived dendritic cells (DCs) for cancer vaccination involves ex vivo maturation in the presence of proinflammatory cytokines and prostaglandin E(2) (PGE(2)). Although the inclusion of PGE(2) during maturation is imperative for the induction of DC migration, PGE(2) has unfavorable effects on the immunostimulatory capacity of these cells. Like PGE(2), leukotrienes (LTs) are potent mediators of DC migration. We therefore sought to characterize the migratory and immunologic properties of DCs that matured in the presence of LTB(4), LTC(4), LTD(4), and PGE(2). Here, we demonstrate that DCs matured in the presence of LTC(4), but not LTB(4) or LTD(4), are superior to PGE(2)-matured DCs in stimulating CD4(+) T-cell responses and in inducing antigen-specific cytotoxic T lymphocytes (CTLs) in vitro without concomitant induction or recruitment of regulatory T cells (Tregs). LTC(4)-matured DCs migrate efficiently through layers of extracellular matrix and secrete higher levels of immunostimulatory IL-12p70 while producing reduced levels of immune-inhibitory IL-10, IL12p40, indoleamine-2,3-dioxidase, and TIMP-1 (tissue inhibitor of matrix metalloproteinases). Intracellular calcium mobilization and receptor antagonist studies reveal that, in contrast to LTD(4), LTC(4) did not signal through CysLTR(1) in DCs. Collectively, our data suggest that LTC(4) represents a promising candidate to replace PGE(2) in DC maturation protocols for cancer vaccination.

The function and three-dimensional structure of a thromboxane A2/cysteinyl leukotriene-binding protein from the saliva of a mosquito vector of the malaria parasite.

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the ω-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

Leukotriene C4 aggravates bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND AND OBJECTIVE: Synthesis of cysteinyl leukotrienes (cys-LT) is thought to cause inflammatory disorders such as bronchial asthma and allergic rhinitis. Recent reports have suggested that leukotriene C4 (LTC4 ) is an important regulator of pulmonary fibrosis. This study examined the effect of LTC4 in LTC4 synthase-overexpressed transgenic mice with bleomycin-induced pulmonary fibrosis. The function of lung-derived fibroblasts from transgenic mice was also investigated. METHODS: Bleomycin was administrated to transgenic mice and wild-type (WT) mice by intratracheal instillation. Concentrations of interleukin (IL)-4 and -13, interferon-γ, and transforming growth factor (TGF)-ß1 in bronchoalveolar lavage fluid were measured 1, 3, 7 and 14 days after the administration of bleomycin. Lung tissue was examined histopathologically on day 14. In addition, lung-derived fibroblasts from transgenic and WT mice were cultured for 7 days. Expression of TGF-ß1 mRNA was measured by real-time polymerase chain reaction. RESULTS: Both the pathological scores for pulmonary fibrosis (3.8 ± 0.4 vs 2.0 ± 0.1, P < 0.05) and the levels of IL-4 (12.1 ± 2.3 vs <7.8 pg/mL, P < 0.05), IL-13 (26.5 ± 5.2 vs <7.8 pg/mL, P < 0.01) and TGF-ß1 (211.1 ± 30.2 vs 21.3 ± 1.2 pg/mL, P < 0.01) on day 14 were significantly greater in transgenic than in WT mice. Furthermore, the reduction of LTC4 by pranlukast hydrate, a cys-LT1 receptor antagonist, in fibroblasts from transgenic significantly (P < 0.05) decreased the expression of TGF-ß1 mRNA (by ∼50%) compared with those from WT mice. CONCLUSIONS: Overexpression of LTC4 , amplifies bleomycin-induced pulmonary fibrosis in mice. Our findings suggest a role for LTC4 in lung fibrosis.

UDP-glucuronosyltransferase (UGT) 1A9-overexpressing HeLa cells is an appropriate tool to delineate the kinetic interplay between breast cancer resistance protein (BRCP) and UGT and to rapidly identify the glucuronide substrates of BCRP.

The interplay between phase II enzymes and efflux transporters leads to extensive metabolism and low bioavailability for flavonoids. To investigate the simplest interplay between one UDP-glucuronosyltransferase isoform and one efflux transporter in flavonoid disposition, engineered HeLa cells stably overexpressing UGT1A9 were developed, characterized, and further applied to investigate the metabolism of two model flavonoids (genistein and apigenin) and excretion of their glucuronides. The results indicated that the engineered HeLa cells overexpressing UGT1A9 rapidly excreted the glucuronides of genistein and apigenin. The kinetic characteristics of genistein or apigenin glucuronidation were similar with the use of UGT1A9 overexpressed in HeLa cells or the commercially available UGT1A9. Small interfering (siRNA)-mediated UGT1A9 silencing resulted in a substantial decrease in glucuronide excretion (>75%, p < 0.01). Furthermore, a potent inhibitor of breast cancer resistance protein (BCRP), 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), caused, in a dose-dependent manner, a substantial and marked reduction of the clearance (74-94%, p < 0.01), and a substantial increase in the intracellular glucuronide levels (4-8-fold, p < 0.01), resulting in a moderate decrease in glucuronide excretion (19-59%, p < 0.01). In addition, a significant, albeit moderate, reduction in the fraction of genistein metabolized (f(met)) in the presence of Ko143 was observed. In contrast, leukotriene C4 and siRNA against multidrug resistance protein (MRP) 2 and MRP3 did not affect excretion of flavonoid glucuronides. In conclusion, the engineered HeLa cells overexpressing UGT1A9 is an appropriate model to study the kinetic interplay between UGT1A9 and BCRP in the phase II disposition of flavonoids. This simple cell model should also be very useful to rapidly identify whether a phase II metabolite is the substrate of BCRP.

Coupling of UDP-glucuronosyltransferases and multidrug resistance-associated proteins is responsible for the intestinal disposition and poor bioavailability of emodin.

Emodin is a poorly bioavailable but promising plant-derived anticancer drug candidate. The low oral bioavailability of emodin is due to its extensive glucuronidation in the intestine and liver. Caco-2 cell culture model was used to investigate the interplay between UDP-glucuronosyltransferases (UGTs) and efflux transporters in the intestinal disposition of emodin. Bidirectional transport assays of emodin at different concentrations were performed in the Caco-2 monolayers with or without multidrug resistance-associated protein (MRP) and breast cancer resistance protein (BCRP) efflux transporter chemical inhibitors. The bidirectional permeability of emodin and its glucuronide in the Caco-2 monolayers was determined. Emodin was rapidly metabolized to emodin glucuronide in Caco-2 cells. LTC4, a potent inhibitor of MRP2, decreased the efflux of emodin glucuronide and also substantially increased the intracellular glucuronide level in the basolateral-to-apical (B-A) direction. MK-571, chemical inhibitor of MRP2, MRP3, and MRP4, significantly reduced the efflux of glucuronide in the apical-to-basolateral (A-B) and B-A directions in a dose-dependent manner. However, dipyridamole, a BCRP chemical inhibitor demonstrated no effect on formation and efflux of emodin glucuronide in Caco-2 cells. In conclusion, UGT is a main metabolic pathway for emodin in the intestine, and the MRP family is composed of major efflux transporters responsible for the excretion of emodin glucuronide in the intestine. The coupling of UGTs and MRP efflux transporters causes the extensive metabolism, excretion, and low bioavailability of emodin.

Montelukast inhibits leukotriene stimulation of human dendritic cells in vitro.

BACKGROUND: Leukotrienes are potent inflammatory mediators which modulate immune responses and induce bronchoconstriction in susceptible individuals. Montelukast (MK) is a leukotriene receptor (CysLT1) antagonist that has been shown to prevent exacerbation of asthma. Considering the plethora of potential cellular targets for MK, specific mechanisms for its therapeutic action are still not fully understood. In vitro, we determined whether human dendritic cell function could be affected by leukotriene C(4) (LTC(4)) treatment and whether MK had potential in modulating this response. We also studied the effect of LTC(4) in the context of response to an airway virus (respiratory syncytial virus, RSV). METHODS: Human monocyte-derived dendritic cells (moDCs) exposed to LTC(4), MK, or both, were cocultured with autologous T cells, with or without RSV. The effects of LTC(4) and MK on cell function were determined by ELISA and proliferation assays. RESULTS: Both moDCs and their precursors--monocytes--express LTC(4) receptor CysLT1, making them potential targets for MK. moDCs cultured with LTC(4) release the eosinophil chemoattractant RANTES (CCL5) and induce greater T cell proliferation. Both were blocked by the presence of MK. MK treatment, albeit anti-inflammatory, did not interfere with the moDC-dependent T cell-proliferative responses induced by RSV. CONCLUSIONS: LTC(4), chronically present in the airways of asthma patients, could induce an exaggerated inflammatory response to airway infection via dendritic cell activation, which would be prevented by MK. Our study provides additional insight into the mechanisms of action of this leukotriene receptor antagonist.

Non-genomic action of beclomethasone dipropionate on bronchoconstriction caused by leukotriene C4 in precision cut lung slices in the horse.

BACKGROUND: Glucocorticoids have been proven to be effective in the therapy of recurrent airway obstruction (RAO) in horses via systemic as well as local (inhalative) administration. Elective analysis of the effects of this drug on bronchoconstriction in viable lung tissue offers an insight into the mechanism of action of the inflammatory cascade occurring during RAO which is still unclear. The mechanism of action of steroids in treatment of RAO is thought to be induced through classical genomic pathways. We aimed at electively studying the effects of the glucocorticoid beclomethasone dipropionate on equine precision-cut lung slices (PCLS).PCLS were used to analyze ex-vivo effects of beclomethasone on inhibiting bronchoconstriction in the horse. The inhibiting effect was measured through instillation of a known mediator of inflammation and bronchoconstriction, leukotriene C4. For this, the accessory lobes of 13 horses subjected to euthanasia for reasons unrelated to the respiratory apparatus were used to obtain viable lung slices. RESULTS: After 30 minutes of PCLS incubation, beclomethasone showed to significantly inhibit the contraction of the bronchioles after instillation with leukotriene C4. The EC50 values of the two contraction curves (LTC4 with and without BDP) differed significantly from each other (p = 0.002). The possibility of a non-genomic rapid mechanism of action seems likely since transcriptional activities require a longer lag period. CONCLUSIONS: In human neuroendocrinology, high levels of glucocorticoids have been proven to function via a non-genomic mechanism of membrane receptors. The concentration of beclomethasone used on the lung slices in our study can be considered as high. This allows speculation about similar rapid non-genomic mechanisms of high-dosage inhaled glucocorticoids in the lower airways of horses. However, further assessment on a molecular basis is needed to confirm this.

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 LTC4, 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.

Inhibition of leukotriene C4 action reduces oxidative stress and apoptosis in cardiomyocytes and impedes remodeling after myocardial injury.

UNLABELLED: Tissue damage leads to release of pro-inflammatory mediators. Among these, leukotriene C(4) (LTC(4)) is a powerful, intracellularly induced mediator of inflammation, which requires inside-out transport of LTC(4). We investigated whether release of LTC(4)via the multidrug resistance related protein 1 (MRP1) induces apoptosis in cardiomyocytes in vitro and in vivo. METHODS AND RESULTS: Incubation of cultured embryonic cardiomyocytes (eCM) with recombined LTC(4) caused enhanced rates of reactive oxygen species (ROS) release measured via L012-luminescence method and apoptosis. Pharmacologic LTC(4) receptor blockade antagonized this effect in vitro. To evaluate the relevance of MRP1 mediated LTC(4) release after myocardial injury in vivo, MRP1(-/-) mice and FVB wildtype mice (WT) received cryoinjury of the left ventricle. Fourteen days after injury, left-ventricular ejection fraction (EF), end-diastolic volume (EDV), and akinetic myocardial mass (AMM) were quantified via echocardiography. MRP1(-/-) mice demonstrated increased EF (MRP1(-/-): 39 ± 3%, WT: 29 ± 4%) and reduced AMM (MRP1(-/-): 13 ± 2% WT: 16 ± 4%), indicating reduced post-infarction remodeling. Mechanistically, LTC(4) serum concentrations and levels of cellular apoptosis were increased in myocardial cryosections of FVB WT mice as compared to MRP1(-/-) mice. To identify key targets for pharmacological inhibition of LTC(4) actions, WT mice were treated with the specific Cys-LT1-receptor blocker Montelukast or the MRP1-Inhibitor MK571. Treatment of WT mice resulted in significant increase of EF (WT(Montelukast): 40 ± 5%, WT(MK571): 39 ± 3%, WT(vehicle): 33 ± 3% and decrease of AMM (WT(Montelukast): 12 ± 1%, WT(MK571): 10 ± 3%, WT(vehicle): 15 ± 5%) compared to untreated WT mice. CONCLUSION: Inhibition of leukotriene C(4) reduces levels of oxidative stress and apoptosis and demonstrates beneficial effects on myocardial remodeling after left ventricular injury.

Differential signaling of cysteinyl leukotrienes and a novel cysteinyl leukotriene receptor 2 (CysLT2) agonist, N-methyl-leukotriene C4, in calcium reporter and ß arrestin assays.

The cysteinyl leukotrienes (cysLTs) LTC4, LTD4, and LTE4 are lipid mediators with physiological and pathophysiological functions. They exert their effects through G protein-coupled receptors (GPCRs), most notably via CysLT1 and CysLT2 receptor. The roles of the CysLT2 receptor are beginning to emerge. Both LTC4 and LTD4 are potent agonists for the CysLT2 receptor; however, LTC4 is rapidly converted to LTD4, which is also the main endogenous ligand for the CysLT1 receptor. A selective and potent agonist at the CysLT2 receptor would facilitate studies to discern between receptor subtypes. We show here that N-methyl LTC4 (NMLTC4), a metabolically stable LTC4 mimetic, is a potent and selective CysLT2 receptor agonist. Two expression systems were used to evaluate the functional activity of NMLTC4 at human and/or mouse CysLT1 and CysLT2 receptors. Through the aequorin cell-based assay for calcium-coupled GPCRs, NMLTC4 was almost equipotent to LTC4 at CysLT2 receptors but was the least efficacious at CysLT2 receptors. In a ß-galactosidase-ß-arrestin complementation assay, the human (h) CysLT2 receptor can couple with ß-arrestin-2, and NMLTC4 is slightly more potent for eliciting ß-arrestin-2 binding compared with cysLTs. Furthermore, LTE4 is nearly inactive in this assay compared with its weak partial agonist activity in the aequorin system. In a vascular leakage assay, NMLTC4 is potent and active in mice overexpressing hCysLT2 receptor in endothelium, whereas the response is abrogated in CysLT2 receptor knockout mice. Therefore, NMLTC4 is a potent subtype selective agonist for the CysLT2 receptor in vitro and in vivo, and it will be useful to elucidate its biological roles.

Functional recognition of a distinct receptor preferential for leukotriene E4 in mice lacking the cysteinyl leukotriene 1 and 2 receptors.

The cysteinyl leukotrienes (cys-LTs) are a family of potent lipid mediators of inflammation derived from arachidonic acid. Activation of certain cell types results in the biosynthesis and export of leukotriene (LT) C(4), which then undergoes extracellular metabolism to LTD(4) and LTE(4). LTE(4), the most stable cys-LT, is only a weak agonist for the defined type 1 and type 2 cys-LT receptors (CysLT(1)R and CysLT(2)R, respectively). We had recognized a greater potency for LTE(4) than LTC(4) or LTD(4) in constricting guinea pig trachea in vitro and comparable activity in eliciting a cutaneous wheal and flare response in humans. Thus, we hypothesized that a vascular permeability response to LTE(4) in mice lacking both the CysLT(1)R and CysLT(2)R could establish the existence of a separate LTE(4) receptor. We now report that the intradermal injection of LTE(4) into the ear of mice deficient in both CysLT(1)R and CysLT(2)R elicits a vascular leak that exceeds the response to intradermal injection of LTC(4) or LTD(4), and that this response is inhibited by pretreatment of the mice with pertussis toxin or a Rho kinase inhibitor. LTE(4) is approximately 64-fold more potent in the CysLT(1)R/CysLT(2)R double-deficient mice than in sufficient mice. The administration of a CysLT(1)R antagonist augmented the permeability response of the CysLT(1)R/CysLT(2)R double-deficient mice to LTC(4), LTD(4), and LTE(4). Our findings establish the existence of a third receptor, CysLT(E)R, that responds preferentially to LTE(4), the most abundant cys-LT in biologic fluids, and thus reveal a new target for therapeutic intervention.

Expression of leukotriene receptors in the rat dorsal root ganglion and the effects on pain behaviors.

BACKGROUND: Leukotrienes (LTs) belong to the large family of lipid mediators implicated in various inflammatory conditions such as asthma and rheumatoid arthritis. Four distinct types (BLT1, BLT2, CysLT1 and CysLT2) of G-protein-coupled receptors for LTs have been identified. Several studies have reported that LTs are involved in inflammatory pain, but the mechanism and the expression of LT receptors in the nociceptive pathway are unknown. RESULTS: We investigated the precise expression of these four types of LT receptors in the adult rat dorsal root ganglion (DRG) using reverse transcription-polymerase reaction (RT-PCR) and radioisotope-labeled in situ hybridization histochemistry (ISHH). We detected mRNAs for BLT1 and CysLT2 in the DRG, but not for BLT2 and CysLT1. CysLT2 mRNA was preferentially expressed by small sized DRG neurons (about 36% of total neurons), whereas BLT1 mRNA was expressed by non-neuronal cells. Double labeling analysis of CysLT2 with NF-200, calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), transient receptor potential vanilloid subfamily 1 (TRPV1) and P2X3 receptor revealed that many CysLT2-labeled neurons were localized with unmyelinated and non-peptidergic neurons, and interestingly, CysLT2 mRNA heavily co-localized with TRPV1 and P2X3-positive neurons. Intraplantar injection of LTC4, a CysLT2 receptor agonist, itself did not induce the thermal hyperalgesia, spontaneous pain behaviors or swelling of hind paw. However, pretreatment of LTC4 remarkably enhanced the painful behaviors produced by alpha, beta-methylene adenosine 5'-triphosphate (αß-me-ATP), a P2X3 receptor agonist. CONCLUSIONS: These data suggests that CysLT2 expressed in DRG neurons may play a role as a modulator of P2X3, and contribute to a potentiation of the neuronal activity following peripheral inflammation.