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.     

Cutting edge: Interleukin 4-dependent mast cell proliferation requires autocrine/intracrine cysteinyl leukotriene-induced signaling

Reactive mastocytosis (RM) in epithelial surfaces is a consistent Th2-associated feature of allergic disease. RM fails to develop in mice lacking leukotriene (LT) C4 synthase (LTC4S), which is required for cysteinyl leukotriene (cys-LT) production. We now report that IL-4, which induces LTC4S expression by mast cells (MCs), requires cys-LTs, the cys-LT type 1 receptor (CysLT1), and Gi proteins to promote MC proliferation. LTD4 (10-1000 nM) enhanced proliferation of human MCs in a CysLT1-dependent, pertussis toxin-sensitive manner. LTD4-induced phosphorylation of ERK required transactivation of c-kit. IL-4-driven comitogenesis was likewise sensitive to pertussis toxin or a CysLT1-selective antagonist and was attenuated by treatment with leukotriene synthesis inhibitors. Mouse MCs lacking LTC4S or CysLT1 showed substantially diminished IL-4-induced comitogenesis. Thus, IL-4 induces proliferation in part by inducing LTC4S and cys-LT generation, which causes CysLT1 to transactivate c-kit in RM.     

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.     

Cysteinyl leukotriene-dependent [Ca2+]i responses to angiotensin II in cardiomyocytes

With the use of fura 2 measurements in multiple and single cells, we examined whether cysteinyl leukotrienes (CysLT) mediate angiotensin II (ANG II)-evoked increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in neonatal rat cardiomyocytes. ANG II-evoked CysLT release peaked at 1 min. The angiotensin type 1 (AT(1)) antagonist losartan, but not the AT(2) antagonist PD-123319, attenuated the elevations in [Ca(2+)](i) and CysLT levels evoked by ANG II. Vasopressin and endothelin-1 increased [Ca(2+)](i) but not CysLT levels. The 5-lipoxygenase (5-LO) inhibitor AA-861 and the CysLT(1)-selective antagonist MK-571 reduced the maximal [Ca(2+)](i) responses to ANG II but not to vasopressin and endothelin-1. While MK-571 reduced the responses to leukotriene D(4) (LTD(4)), the dual CysLT antagonist BAY-u9773 completely blocked the [Ca(2+)](i) elevation to both LTD(4) and LTC(4). These data confirm that ANG II-evoked increases, but not vasopressin- and endothelin-1-evoked increases, in [Ca(2+)](i) involve generation of the 5-lipoxygenase metabolite CysLT. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] antagonist 2-aminoethoxydiphenyl borate attenuated the [Ca(2+)](i) responses to ANG II and LTD(4). Thus AT(1) receptor activation by ANG II is linked to CysLT-mediated Ca(2+) release from Ins(1,4,5)P(3)-sensitive intracellular stores to augment direct ANG II-evoked Ca(2+) mobilization in rat cardiomyocytes.     

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.     

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.     

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.     

Leukotriene D4 mediates survival and proliferation via separate but parallel pathways in the human intestinal epithelial cell line Int 407

We demonstrated previously that leukotriene D4 (LTD4) regulates proliferation of intestinal epithelial cells through a CysLT receptor by protein kinase C (PKC)epsilon-dependent stimulation of the mitogen-activated protein kinase ERK1/2. Our current study provides the first evidence that LTD4 can activate 90-kDa ribosomal S6 kinase (p90RSK) and cAMP-responsive element-binding protein (CREB) via pertussis-toxin-sensitive Gi protein pathways. Transfection and inhibitor experiments revealed that activation of p90RSK, but not CREB, is a PKCepsilon/Raf-1/ERK1/2-dependent process. LTD4-mediated CREB activation was not affected by expression of kinase-dead p90RSK but was abolished by transfection with the regulatory domain of PKCalpha (a specific dominant-inhibitor of PKCalpha). Kinase-negative mutants of p90RSK and CREB (K-p90RSK and K-CREB) blocked the LTD4-induced increase in cell number and DNA synthesis (thymidine incorporation). Compatible with these results, flow cytometry showed that LTD4 caused transition from the G0/G1 to the S+G2/M cell cycle phase, indicating increased proliferation. Similar treatment of cells transfected with K-p90RSK resulted in cell cycle arrest in the G0/G1 phase, consistent with a role of p90RSK in LTD4-induced proliferation. On the other hand, expression of K-CREB caused a substantial buildup in the sub-G0/G1 phase, suggesting a role for CREB in mediating LTD4-mediated survival in intestinal epithelial cells. Our results show that LTD4 regulates proliferation and survival via distinct intracellular signaling pathways in intestinal epithelial cells.     

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.     

Conversion of leukotriene C4 to leukotriene D4 by a cell-surface enzyme of rat macrophages

Leukotriene (LT) C4-metabolizing enzyme was studied using rat leukocytes. Neutrophils and lymphocytes hardly metabolized LTC4, whereas macrophages rapidly converted LTC4 to LTD4. The LTC4-metabolizing enzyme of macrophages was present in the membrane fraction but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the LTC4-metabolizing activity of macrophages decreased significantly (greater than 90%). These findings suggest that rat macrophages possess the LTC4-metabolizing enzyme which converts LTC4 to LTD4, on the cell surface membrane.     

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.     

Mechanisms of leukotriene E4 partial agonist activity at leukotriene D4 receptors in differentiated U-937 cells

Leukotriene E4 (LTE4) is shown to be a partial agonist of leukotriene D4 (LTD4) in differentiated U-937 cells. The data that support this conclusion are: 1) LTE4 completely displaced [3H]LTD4 from its receptors in U-937 cell membranes. 2) LTE4 induced only 30 +/- 4% of the maximal Ca2+ transient induced by LTD4 in the presence of 1 mM extracellular Ca2+ and 60 +/- 4% of the maximal LTD4 response in the absence of extracellular Ca2+. 3) LTE4 induced only a fraction of the inositol phosphates metabolized by LTD4. Moreover, LTE4 resulted in essentially no production of the inositol 1,4,5-trisphosphate isomer, while LTD4 induced a rapid and substantial transient increase in this isomer. The generation of inositol phosphates by both agonists was unaffected by extracellular Ca2+. 4) The EC50 values for Ca2+ mobilization for LTD4 and LTE4 corresponded with their affinity (Kd values) for the LTD4 receptor. 5) A series of structurally diverse LTD4 receptor antagonists blocked the Ca2+ mobilization responses to LTD4 and LTE4 with identical rank orders of potency. 6) LTE4 acted as an antagonist of LTD4 of potency. 6) LTE4 acted as an antagonist of LTD4 effects when they were coadministered. 7) LTE4 and LTD4 acutely desensitized Ca2+ mobilization to each other. All of the effects of LTE4 are explained by its partial agonist activity at the LTD4 receptor as shown by the following data. 1) Neither LTD4 nor LTE4 had any effect on the agonist activity of fMet-Leu-Phe, LTB4, or platelet-activating factor. 2) None of the above agonists or antagonists to the above receptors affected any of the activities of LTD4 or LTE4. 3) Neither LTD4 nor LTE4 induced desensitization of Ca2+ mobilization to any of the non-LTD4 receptor agonists tested. 4) Under the conditions studied, we have not observed any evidence of multiple subclasses of LTD4 receptors in U-937 cells. LTE4 is a partial agonist of the LTD4 receptor, because it can only couple the LTD4 receptor to a portion of the signaling system available to the receptor when occupied by LTD4. Specifically, LTD4 caused the activation of receptor-operated calcium channels, mobilization of intracellular Ca2+, the activation of phosphatidylinositol-phospholipase C, and the liberation of an additional, as yet undefined, intracellular mediator. To do this, LTD4 receptors couple to at least two and perhaps more guanine nucleotide binding proteins. LTE4 is unable to activate the phosphatidylinositol-phospholipase C but can mimic the other effects of LTD4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Up-regulation of cysteinyl leukotriene 1 receptor by IL-13 enables human lung fibroblasts to respond to leukotriene C4 and produce eotaxin

Cysteinyl leukotrienes (CysLTs) play an important role in eosinophilic airway inflammation. In addition to their direct chemotactic effects on eosinophils, indirect effects have been reported. Eotaxin is a potent eosinophil-specific chemotactic factor produced mainly by fibroblasts. We investigated whether CysLTs augment eosinophilic inflammation via eotaxin production by fibroblasts. Leukotriene (LT)C(4) alone had no effect on eotaxin production by human fetal lung fibroblasts (HFL-1). However, LTC(4) stimulated eotaxin production by IL-13-treated fibroblasts, thereby indirectly inducing eosinophil sequestration. Unstimulated fibroblasts did not respond to LTC(4), but coincubation or preincubation of fibroblasts with IL-13 altered the response to LTC(4). To examine the mechanism(s) involved, the expression of CysLT1R in HFL-1 was investigated by quantitative real-time PCR and flow cytometry. Only low levels of CysLT1R mRNA and no CysLT1R protein were expressed in unstimulated HFL-1. In contrast, stimulation with IL-13 at a concentration of 10 ng/ml for 24 h significantly up-regulated both CysLT1R mRNA and protein expression in HFL-1. The synergistic effect of LTC(4) and IL-13 on eotaxin production was abolished by CysLT1R antagonists pranlukast and montelukast. These findings suggest that IL-13 up-regulates CysLT1R expression, which may contribute to the synergistic effect of LTC(4) and IL-13 on eotaxin production by lung fibroblasts. In the Th2 cytokine-rich milieu, such as that in bronchial asthma, CysLT1R expression on fibroblasts might be up-regulated, thereby allowing CysLTs to act effectively and increase eosinophilic inflammation.     

Conjunctival goblet cell secretion stimulated by leukotrienes is reduced by resolvins D1 and E1 to promote resolution of inflammation

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT(1) and CysLT(2,) were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB(4), LTC(4), LTD(4), and LTE(4), as well as PGD(2), stimulated goblet cell secretion in rat goblet cells. LTD(4) and LTE(4) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), and LTD(4) activated ERK1/2. The CysLT(1) receptor antagonist MK571 significantly decreased LTD(4)-stimulated rat goblet cell secretion and the increase in [Ca(2+)](i). Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD(4)-stimulated goblet cell secretion in cultured rat goblet cells. LTD(4)-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD(4)- and LTE(4)-stimulated increases in [Ca(2+)](i), as well as LTD(4) activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD(4) using the CysLT(1) receptor. Stimulated secretion is terminated by preventing the increase in [Ca(2+)](i) and activation of ERK1/2 by RvD1 and RvE1.     

Eosinophil peroxidase-mediated inactivation of leukotrienes B4, C4, and D4

The slow-reacting substance (SRS) bioactivity of leukotrienes C4 (LTC4) and D4 (LTD4) was rapidly decreased by incubation with eosinophil peroxidase (EPO), H2O2, and iodide, bromide, or to a lesser degree, chloride, LTB4 chemotactic activity was also decreased by the EPO-H2-H2-halide system, although at a slower rate. Myeloperoxidase could substitute for EPO in these reactions. Leukotriene inactivation was greatly decreased or abolished by deletion of any of the components of the system or by the addition of the hemeprotein inhibitors, azide, cyanide, or aminotriazole, indicating a requirement for peroxidase. The H2O2 concentration employed in the above studies was 10(-4) M. H2O2 at higher concentrations (5 x 10(-4) to 10(-2) M) inactivated LTC4 and LTD4 in the absence of EPO and a halide but had no effect on the chemotactic activity of LTB4. We have previously shown that horse eosinophils stimulated with the calcium ionophore A23187 generate SRS. In the present study, eosinophils stimulated in this way were found to release extracellularly both H2O2 and EPO. Incubation of eosinophils with azide that inhibits EPO, and catalase that degrades H2O2, significantly increased the amount of SRS activity detected in the extracellular medium after A23187 stimulation. These findings suggests eosinophils may play an important modulating role in hypersensitivity reactions both by the production of leukotrienes and by their inactivation through the release of H2O2 and EPO.     

Elucidation of signaling and functional activities of an orphan GPCR, GPR81

GPR81 is an orphan G protein-coupled receptor (GPCR) that has a high degree of homology to the nicotinic acid receptor GPR109A. GPR81 expression is highly enriched and specific in adipocytes. However, the function and signaling properties of GPR81 are unknown because of the lack of natural or synthetic ligands. Using chimeric G proteins that convert Gi-coupled receptors to Gq-mediated inositol phosphate (IP) accumulation, we show that GPR81 can constitutively increase IP accumulation in HEK293 cells and suggest that GPR81 couples to the Gi signaling pathway. We also constructed a chimeric receptor that expresses the extracellular domains of cysteinyl leukotriene 2 receptor (CysLT2R) and the intracellular domains of GPR81. We show that the CysLT2R ligand, leukotriene D(4) (LTD4), is able to activate this chimeric receptor through activation of the Gi pathway. In addition, LTD4 is able to inhibit lipolysis in adipocytes expressing this chimeric receptor. These results suggest that GPR81 couples to the Gi signaling pathway and that activation of the receptor may regulate adipocyte function and metabolism. Hence, targeting GPR81 may lead to the development of a novel and effective therapy for dyslipidemia and a better side effect profile than nicotinic acid.     

Differentiation of the mechanisms by which leukotrienes C4 and D4 elicit contraction of the guinea pig trachea

The contractions elicited by leukotriene (LT) C4 and D4 in isolated guinea pig trachea were characterized under conditions in which LTC4 to LTD4 metabolism was blocked by the presence of 45 mM l-serine-borate complex (SB). The presence of SB caused a shift of the LTC4-concentration-response curve to the left by 7.5-fold, and blocked the bioconversion of LTC4 to LTD4 by the trachea as estimated by HPLC analysis of the LTs present in the tissue bath fluid. The potency of FPL 55712 as an antagonist of the LTC4-induced contractions in the presence of SB was 15-30-fold less than its potency as an antagonist of the LTD4-induced contractions. In contrast, another LT antagonist, SK&F 101132, equally antagonized the contractions elicited by LTC4 and LTD4 in either the presence or absence of SB. The differential antagonism of LTC4 and LTD4 implies the existence of multiple pharmacologic receptors for the LTs. The calcium channel entry blockers, nifedipine and verapamil, at concentrations as high as 10 microM, suppressed the maximal LTC4-induced contraction by no more than 20%, whereas the purported intracellular calcium antagonist, TMB-8, completely suppressed the LTC4 concentration-response curve in the presence of SB, a profile identical to that previously reported for LTD4. Thus, if multiple LT receptors exist, they appear to mobilize calcium in a qualitatively similar fashion following LT stimulation.     

Leukotriene C4-induced phosphoinositide hydrolysis in rat basophilic leukemia cell.

Leukotriene C4 (LTC4), one of the major constituents of the slow reacting substance of anaphylaxis, induced a dose-dependent hydrolysis of phosphoinositides in [3H]inositol-prelabeled rat basophilic leukemia (RBL-1) cells. The EC50 for LTC4 to elicit the half maximum accumulation of [3H]inositol phosphates (IPs) was around 20 nM. The increase in the formation of [3H]inositol bisphosphate (IP2) and [3H]inositol trisphosphate (IP3) was detectable at 2 min after the stimulation and progressed up to 30 min. Accumulation of [3H]inositol monophosphate (IP1) was observed only during the late phase of 5-30 min in the presence of LiCl. When cells were stimulated with LTC4 and LTD4 together, there was no additive accumulation in [3H]IPs. Pretreatment of cells with either LTC4 or LTD4 resulted in a decrease in production of [3H]IPs on further stimulation with the same agonist. The desensitization appeared to be heterologous since pretreatment of cells with LTC4 attenuated the responsiveness to LTD4. Conversely, pretreatment with LTD4 also diminished the responsiveness to LTC4 markedly. These results suggest that both LTC4- and LTD4-induced hydrolysis of phosphoinositides are mediated through the same effector in RBL-1 cells.