SRS-A Research Articles

Peripheral Markers of Suicidal Behavior: Current Findings and Clinical Implications.

Biomarkers of suicidal behavior (SB), particularly peripheral biomarkers, may aid in the development of preventive and intervention strategies. The peripheral biomarkers of SB should be easily accessible, cost-effective, and minimally invasive. To identify peripheral biomarkers of SB, we summarized the current knowledge related to SB biomarkers with a focus on suicidal outcomes (suicidal ideation [SI], suicide risk [SR], suicide attempt [SA], and suicide death [SD]), measured site (center or periphery), and study design (cross-sectional or longitudinal). We also evaluated the central findings to validate the findings of peripheral biomarkers of SB. We found reduced peripheral interleukin (IL)-2 levels in individuals with a recent SA, higher cerebrospinal fluid (CSF) IL-6 levels in patients with a current SR and future SD, higher CSF tumor necrosis factor-α levels for current and future SRs, higher high-sensitivity C-reactive protein levels and lower peripheral total cholesterol levels for recent SAs, lower peripheral 5-HT levels for present SR, and a lower folate level for future SR and SA within 1 year. Previous studies have shown inconsistent associations of low peripheral leptin levels with SR and recent SA; therefore, further study is required. Given the multiple determinants of SB and weak associations with single biological markers, combinations of potential biological markers rather than single markers may improve the screening, diagnosis, and prediction of SB.

Lipid mass spectrometry: A path traveled for 50 years.

In the middle of the 1960s, I began graduate school and at the same time started on the path of using mass spectrometry to gain insight into various aspects of lipid biochemistry. This was not a straight path but one that went from organic geochemistry, to lunar sample analysis, to a pursuit of the structure of an elusive and very active, lipid mediator slow reacting substance of anaphylaxis (SRS-A). The discovery of the structure of SRS-A opened important questions about phospholipid biochemistry and the arachidonate cycle in cells. I have written this reflection to highlight the various advances in mass spectrometry that occurred during this time that had a great impact on our ability to study lipid biochemistry. I specifically applied these new advances to studies of leukotriene biosynthesis in vivo, leukotriene metabolism, and arachidonate-containing phospholipids that are essential in providing arachidonic acid for the 5-lipoxygenase pathway. Along the way, imaging mass spectrometry was shown to be a powerful tool to probe lipids as they exist in tissue slices. We found this as just one of the ways to use the emerging technology of lipidomics to study human pathophysiology. Our studies of neutral lipids and oxidized phospholipids were especially challenging due to the total number of molecular species that could be found in cells. Many challenges remain in using mass spectrometry for lipid studies, and a few are presented.

The Importance of CD19 Exon 2 for Surface Localization: Closing the Ig-like Loop

Background: CD19 is a near-universal surface marker of B-cell malignancies. Therefore, it is regarded as a target of choice for various immunotherapies, such as bi-specific T-cell engagers and chimeric antigen receptor-armed T-cells. Although the latter have proven remarkably effective in treating B-cell acute lymphoblastic leukemia (B-ALL), relapses caused by the loss of the CD19 surface epitopes are emerging as a major reason for treatment failure (Maude et al, 2014). However, the molecular mechanisms of epitope loss remain incompletely characterized. Recently, we have identified a novel alternatively spliced CD19 isoform lacking exon2 (Δex2 CD19) (Sotillo et al 2015). Although exon 2 encodes a single conserved N-linked glycosylation site (Asn-86) and two conserved cysteine residues (Cys-38 and Cys-97) responsible for the formation of a disulfide bond (Zhou et al 1991), the function and subcellular localization of the Δex2 isoform remained unknown. Our aim was to identify specific amino acids within exon 2 that are responsible for proper CD19 surface expression.Methods: CD19-null (ΔCD19) derivatives of Nalm6 and 697 B-ALL cell lines were generated using the CRISPR/Cas9 approach. We then reconstituted them with full-length and Δex2 CD19 isoforms, expressed either on their own or as CD19-GFP fusions. Additionally, CD19 N-terminus mutations (ΔSP [no signal peptide], N86A, C97A and N86A/C97A) were introduced in full-length CD19. Transduced cells were analyzed by flow cytometry, confocal microscopy, and western blotting. Glycosylation of the mutants was verified using treatment with swainsonine (Golgi glycosylation inhibitor) and an in vitro de-glycosylation assay.Results: As expected, deletion of the N-terminal signal peptide responsible for the endoplasmic reticulum translocation led to impaired surface expression. Surprisingly, deletion of exon 2 sequences had a similar effect: although up to 10% of Δex2 CD19 was found on the plasma membrane (where it enhanced pre-B-cell receptor signaling), the remainder was largely cytosolic. Furthermore, while the N86A substitution in full-length CD19 did not significantly affect its surface localization, substituting Cys-97 with Ala fully recapitulated the Δex2 cytosolic phenotype. In fact, the C97A mutant appeared to be stuck in the ER and never reach Golgi, since its electrophoretic mobility was not affected by swainsonine. In contrast, its glycosylation in ER was unperturbed, as evidenced by in vitro de-glycosylation assays.Conclusion: The immunoglobulin-like loop connecting Cys-38 and Cys-97 of CD19 is required for its plasma membrane localization. One possible mechanism is the N-termini-mediated interaction with CD81, which is known to be needed to transport CD19 to the cell surface (Matsumoto et al 1993).Maude SL, Frey, N, Shaw, PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, Shen A, Teachey DT, Levine BL, June CH, Porter DL, and Grupp SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014 371:1507-1517.Sotillo E, Barrett D, Bagashev A, Black K, Lanauze C, Oldridge D, Sussman R, Harrington C, Chung EY, Hofmann TJ, Maude SL, Martinez NM, Raman P, Ruella M, Allman D, Jacoby E, Fry T, Barash Y, Lynch KW, Mackall C, Maris J, Grupp SA, and Thomas-Tikhonenko A. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope andcontributes to treatment failure. 2015 AACR Annual Meeting, Philadelphia.Zhou LJ, Ord DC, Hughes AL and Tedder TF, Structure and domain organization of the CD19 antigen of human, mouse, and guinea pig B lymphocytes. Conservation of the extensive cytoplasmic domain. J Immunol. 1991 147(4):1424-32Matsumoto AK, Martin DR, Carter RH, Klickstein LB, Ahearn JM, and Fearon DT Functional dissection of the CD21/CD19/TAPA-1/Leu-13 complex of B lymphocytes. J Exp Med. 1993 178(4):1407-17. DisclosuresNo relevant conflicts of interest to declare.

Montelukast in Allergic Rhinitis: A Review

Leukotriene inhibitors are the first new class of medications for the treatment of persistent asthma that have been approved by the U.S food and drug Administration in more than two decades. They also have been approved for the treatment of Allergic Rhinitis. Leukotrienes are inflammatory mediators that are known as the slow-reacting substance of anaphylaxis. Synthesis of these mediators results from the cleavage of arachidonic acid in cell membranes, exert their biologic effect by binding and activating specific adaptors. This occurs in a series of events that lead to their important role in the Allergic Rhinitis. Because agents lead to the production of symptoms, the use of leukotriene inhibitors, particularly montelukast seems appropriate. These classes of drug can block the binding of leukotrienes to CysLT1 receptors and can also result in the prevention of leukotriene activity. Montelukast is the most commonly used inhibitor of the leukotriene pathway because of its ease of use , good safety profile, and once-daily regimen. We conduct a systematic review of studies that have evaluated montelikast in the treatment of Allergic Rhinitis. This review focuses on the pathophysiology and effect of montelukast in Allergic Rhinitis. The purpose of this paper is to provide an overview of the accumulation data concerning these agents in treating Allergic Rhinitis. Montelukast provides an effective and well tolerated oral treatment for Allergic Rhinitis.DOI: http://dx.doi.org/10.3329/medtoday.v24i2.15012 Medicine Today 2012 Vol.24(2): 75-78

Release of histamine and slow reacting substance of anaphylaxis (SRS-A) from the sensitized guinea-pig lungs. 1. Experimental studies

Release of histamine and slow reacting substance of anaphylaxis (SRS-A) from the sensitized guinea-pig lungs. 1. Experimental studies

構造生物学研究の新展開 ２．基礎研究から応用展開に向けて（ｃ）応用志向型研究 システインロイコトリエン研究の新知見

Cysteinyl leukotriene has been known to be a major component of SRS-A (Slow Reacting Substance of Anaphylaxis). Inhibitors affecting on its biosynthesis and antagonists of its G-protein coupled receptor are therapeutic agents for acute inflammatory diseases, such as bronchial asthma and rhinitis. Latest findings in pathobiology of asthma suggest that cysteinyl leukotriene would take a key role in not only acute but also chronic inflammation of asthma. The structure basis of biosynthesis of cysteinyl leukotriene was revealed by crystallographic analysis of human membrane protein leukotriene C4 synthase.

Leukotrienes as mediators of skin inflammation.

Leukotrienes (LTs) derived from the 5-lipoxygenase pathways of arachidonic acid metabolism, are a new group of biologically active mediators. LTC4, LTD4 and LTE4 are considered collectively to account for the activity of slow reacting substance of anaphylaxis (SRS-A). They have potent smooth muscle contracting activity (Drazen et al., 1980; Holme et al., 1980) and cause vascular permeability changes in guinea-pig skin (Peck, Piper & Williams, 1981). LTB4 has been shown to be a potent chemotactic and chemokinetic agent for polymorphonuclear leucocytes (PMNs) (Ford-Hutchinson et al., 1980; Smith, Ford-Hutchinson & Bray, 1980) and to cause vascular permeability changes in rabbit, rat and guinea-pig skin (Bray et al., 1981a). In human skin LTB4 has been shown to be a chemotactic agent for neutrophils (Bray, Ford-Hutchinson & Smith, 1981b; Camp et al., 1982). LTC4 and LTD4 have pronounced inflammatory actions in human skin causing weal and flare responses and increases in blood flow at low concentrations (Camp et al., 1982; Bisgard, Kristensen & Sondergaard, 1982). The present studies were designed to investigate a variety of leukotrienes on permeability responses in rabbit and guinea-pig skin.

Roflumilast Inhibition of Pulmonary Leukotriene Production and Bronchoconstriction in Ovalbumin-Sensitized and -Challenged Guinea Pigs

This study investigated the effects of roflumilast, a PDE4 inhibitor, on slow-reacting substance of anaphylaxis (SRS-A)-mediated bronchoconstriction and pulmonary leukotriene (LT) release in ovalbumin (OVA)-sensitized and -challenged guinea pigs. Animals were treated with roflumilast orally (0.04, 0.12, 0.4, or 4 mg/kg) or placebo 1 hour before OVA challenge. Bronchoconstriction was quantified by measuring airway conductance (Gaw) and dynamic lung compliance (Cdyn). Roflumilast significantly attenuated the decrease in Gaw (50% inhibitory dose [ID50] = 0.33 mg/kg) and Cdyn (ID50 = 0.25 mg/kg) in a dose-dependent manner and significantly inhibited Cys-LT (ID50 = 0.06 mg/kg) and LTB4 (ID50 = 0.05 mg/kg) release versus placebo-treated animals. Roflumilast did not affect LTD4-induced bronchoconstriction. These findings support the role of roflumilast as an anti-inflammatory treatment for asthma.

A review of montelukast in the treatment of asthma and allergic rhinitis

Montelukast sodium (Singulair®, Merck) is a selective and orally-active leukotriene-receptor antagonist (LTRA) that inhibits the cysteinyl leukotriene 1 (CysLT1) receptor. Montelukast is an effective and well-tolerated preventative treatment for asthma and allergic rhinitis in adults and children. The upper and lower airway show similar inflammatory responses to allergen challenge. Leukotrienes are inflammatory mediators that are known as the slow-reacting substance of anaphylaxis produced by a number of cell types including mast cells, eosinophils, basophils, macrophages and monocytes. Synthesis of these mediators results from the cleavage of arachidonic acid in cell membranes and they exert their biological effects by binding and activating specific receptors. This occurs in a series of events that lead to contraction of the human airway smooth muscle, chemotaxis and increased vascular permeability. These effects have led to their important role in the diseases of asthma and allergic rhinitis. As these agents lead to the production of symptoms in patients that are asthmatic or allergic, the use of LTRAs, particularly montelukast, may seem appropriate. Clinical trials have shown that montelukast is effective and safe in the treatment of patients with asthma, allergic rhinitis or both diseases.

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The first drugs affecting the leukotriene-lipoxygenase pathway, which have been introduced in clinical application, inhibit effects of slow reacting substance of anaphylaxis (SRS-A). Although, a 5-lipoxygenase inhibitor was first used in clinical practice as an anti-asthma drug, cysteinyl-leukotriene type 1 receptor (cysLT(1)R) antagonists are preferred as anti-asthma and anti-rhinitis drugs because they are almost as effective as the 5-lipoxygenase inhibitors but have fewer side effects. The cloning of genes related to lipoxygenase-leukotriene metabolism prompted us to try to elucidate the role of leukotrienes in various inflammations. There are at least two types of cysLTRs known: cysLT(1)R and cysLT(2)R. CysLT(1)R plays an important role in the pathophysiology of asthma; however, the role of the cysLT(2)R remains unknown. The abundant distribution of cysLT(2)R in heart and brain tissues suggests that cysLTs play an important role in the pathophysiology of ischemic heart diseases or arrhythmias and through this receptor (cysLT(2)R), psychoneurological disorders. The use of a selective cysLT(2)R antagonist may clarify these questions. Since the 5-lipoxygenase pathway is abundantly expressed in atherosclerotic lesions, and 12/15-lipoxygenase is able to oxygenate polyunsaturated fatty acid esterified in the membranous phospholipids, 5-lipoxygenase or 12/15-lipoxygenase inhibitors may prevent progression of atherosclerosis. In addition, it has been reported that 15-lipoxygenase participates in suppression of prostate cancer. In conclusion, the leukotriene-lipoxygenase metabolism may be involved in the pathophysiology of acute inflammatory to chronic progressive disorders. We think that more drugs modifying leukotriene-lipoxygenase metabolism will be introduced into clinical practice in the future.

Leukotrienes in asthma.

One of the major reasons for pursuing the chemical structure of the biological material known as slow-reacting substance of anaphylaxis (SRS-A) was that this material was known to be a potent bronchoconstrictor substance in guinea pigs [1] and in isolated human airways [2]. Thirty years ago, the simple concept was that SRS-A was released from sensitized cells following antigen sensitization and challenge, and that the released material transduced a signal at an as yet to be identified receptor leading to smooth muscle constriction and therefore the manifestations of human asthmA. It has been almost 25 years since the elucidation of the structure of SRS-A as a mixture of the cysteinyl leukotrienes (LT) [3] and just over 5 years since agents that act on leukotriene pathway have been available as asthma treatments [4,5]. What have we learned about asthma from the use of these agents and what is the role of these agents in the treatment of asthma?

Leukotrienes and the pulmonary vascular bed.

The antigenic stimulation of respiratory tissues has always been associated with the release of slow reacting substance of anaphylaxis (SRS-A), an entity which is now known to be a composite of metabolites of the 5-lipoxygenase enzymatic pathway, namely, the cysteinyl-leukotrienes (LTC4, LTD4 and LTE4). Evidence that these mediators may play a role in asthma was based on the clinical observations that several cell types that produce cysteinyl-leukotrienes are present in increased numbers in the lung of asthmatic patients. Furthermore, these metabolites were detected in biological fluids derived from asthmatics following allergen inhalation. Such clinical data implicated the cysteinyl-leukotrienes in not only the allergic reactions to inhaled allergens but also supported the notion that these mediators may play a role in asthma.

Leukotrienes in respiratory disease.

Arachidonic acid metabolism via 5-lipoxygenase gives rise to a group of biologically active lipids known as leukotrienes: leukotriene B(4), which is a potent activator of leukocyte chemotaxis, and cysteinyl leukotrienes (leukotriene C(4), D(4)and E(4)) which account for the spasmogenic activity previously described as slow-reacting substance of anaphylaxis. The biological actions of leukotrienes and the observations that leukotrienes are synthesised in the lung following antigen provocation and are elevated in asthma, stimulated considerable activity in the pharmaceutical industry to find drugs that modulate the synthesis or actions of leukotrienes. Three cysteinyl leukotriene antagonists (zafirlukast [Accolate], montelukast [Singulair] and pranlukast) and one 5-lipoxygenase inhibitor (zileuton) have received regulatory approval for the treatment of asthma. The clinical data obtained from using these drugs are generally consistent and complimentary. As a class the leukotriene modulators produce a rapid improvement in lung function after the first oral dose. Lung function improvements are maintained on chronic administration and are associated with reductions in a variety of asthma symptom scores. All of the available data are consistent with the hypothesis that all the leukotriene modulators exert their clinical benefit primarily through interference with cysteinyl leukotrienes. There are no compelling clinical data for an additional contribution by leukotriene B(4)in human asthma. In other respiratory conditions such as COPD, which are characterised by pronounced neutrophil infiltration, it may be that the chemotactic properties of leukotriene B(4)are more important and therefore evaluation of 5-lipoxygenase inhibitors in this condition is warranted. The introduction of the leukotriene modulators into clinical practice is the culmination of over 60 years of research since the initial discovery of the slow-reacting substances. The leukotriene modulators, and in particular the cysteinyl leukotriene antagonists, provide respiratory physicians with an oral therapeutic option and have set an efficacy standard which new oral anti-inflammatory approaches will have to beat.

Discovery of leukotrienes and development of antileukotriene agents

This article presents information on the origin of leukotrienes (LTs) and the development of antileukotriene (anti-LT) agents. After reading this article, readers should have an understanding of the chemical mediators involved in the pathogenesis of asthma, the structural features of LTs, and the role of anti-LTs in the management of asthma symptoms. Studies considered relevant and appropriately controlled were used. Only literature in the English language was reviewed. Material was taken from academic/scholarly journals and abstracts. One of the important chemical mediators implicated in the pathogenesis of asthma is the slow-reacting substance of anaphylaxis, which was subsequently found to comprise LTs C4, D4, and E4. 5-lipoxygenase products from arachidonic acid metabolism, LTs are released from the lung tissue of asthmatic patients and purified human lung mast cells by antigens. The LTs directly induce contraction of bronchial smooth muscle. The use of anti-LT agents, particularly the receptor antagonists zafirlukast and montelukast and the biosynthesis inhibitor zileuton, reverses the bronchoconstrictive effects of LTs and significantly improve asthma symptoms. Extensive in vitro and in vivo evidence supports the role of LTs in the pathogenesis of asthma. Their discovery has had a significant impact on treatment strategies, including the use of anti-LT agents, for the management of asthma.

The 30th anniversary of the American Board of Allergy and Immunology: Then and now

Thirty years ago the Allergy Subspecialty Boards of the American Board of Pediatrics (ABP) and the American Board of Internal Medicine (ABIM) merged to form the American Board of Allergy and Immunology (ABAI). The ABAI mission was to: establish qualifications and examine physician candidates for certification as specialists in allergy and immunology; serve the public, physicians, hospitals, and medical schools by providing the names of physicians certified by the Board; assist educational and professional organizations to improve the quality of care and availability of allergists to deliver such care, to establish and improve standards for the teaching of allergy and immunology, to establish standards for training programs, and to encourage development of increased opportunities for training of physicians interested in allergy and immunology. This mission statement has guided the activities of the Board ever since by providing a strong focus on the 2 major responsibilities: examining and certifying candidates in a fair objective way, and setting standards for the content and conduct of training programs.

Outpatient Chemotherapy: Hypersensitivity and Anaphylaxis

Outpatient Chemotherapy: Hypersensitivity and Anaphylaxis

The leukotriene C(4) synthase gene and asthma.

The cysteinyl leukotrienes (LT), LTC 4 , LTD 4 , and LTE 4 , are oxygenated metabolites of arachidonic acid derived from the 5-lipoxygenase (5-LO) pathway (Figure 1) (1). The term leukotriene was originally proposed by Samuelsson as a trivial name to describe these compounds that are derived from leukocytes and are conjugated trienes (2). 5-LO is an iron-containing enzyme most often found free in the cytosol of cells of myeloid origin (3). With cellular activation, this enzyme translocates to the nuclear envelope (4). Once translocated, it is responsible for the first committed steps of the 5-LO pathway in association with the 5-LO–activating protein (FLAP). The precise role of FLAP remains obscure; however, it is required for appreciable 5-LO activity in intact inflammatory cells (5). 5-LO inserts molecular oxygen at the carbon 5 position of arachidonic acid forming the hydroperoxy-intermediate, 5-hydroperoxyeicosatetraenoic acid. This intermediate is further metabolized by 5-LO via a dehydration reaction to form the epoxy intermediate, LTA 4 . LTA 4 can be further metabolized to LTB 4 by the enzyme LTA 4 hydrolase, an epoxide hydrolase enzyme with a ubiquitous distribution (6). The role of LTB 4 in asthma remains to be clarified. LTA 4 can also be metabolized to LTC 4 by the enzyme LTC 4 synthase (7). This enzyme is a microsomal glutathioneS -transferase that belongs to a larger family of membrane-bound proteins involved in eicosanoid and glutathione metabolism (8). LTC 4 synthase appears to have a more restricted distribution than 5-LO and appears to be limited to eosinophils, mast cells, basophils, mononuclear phagocytes, and some leukemic cells lines such as THP-1 (9) and KG-1 (10). The concept that lipid soluble mediators may play a role in asthma is not new. Van Leeuwen and Zeydner demonstrated that alcohol extracts of blood obtained from subjects with asthma, but not healthy individuals, contained a substance that caused contraction of smooth muscle (11). Moreover, Harkavy reported in 1930 that alcohol extracts of sputum obtained from asthmatics during exacerbation also contained a substance that could induce sustained smooth muscle contraction, which he termed “spasm-producing substance” (12). In studies performed by Feldberg and Kellaway (13, 14), and subsequently by Kellaway and Trethewie (15), a substance that could induced sustained smooth muscle contraction was present in the blood of antigen-challenged guinea pigs. These authors coined the term “slow-reacting smooth-muscle stimulating substance in anaphylaxis,” which ultimately was refined to “slow-reacting substance of anaphylaxis” (SRS-A) by Brocklehurst (16). The subsequent forty years of research and major efforts of many investigators culminated in the characterization of SRS-A as the cysteinyl leukotrienes C 4 , D 4 , and E 4 (17). These compounds mediate sustained smooth muscle contraction, mucus hypersecretion, airway edema (18), and eosinophil recruitment (19). Since 1979 a substantial body of evidence has accumulated demonstrating that the cysteinyl LTs are increased in the sputum (20), lung lining fluid (21), blood, and urine (22) of asthmatics when compared with healthy subjects. Aspirin-intolerant asthma (AIA) is a syndrome of aspirin sensitivity, asthma, and nasal polyposis (23). Individuals with this syndrome often have profound systemic reactions when challenged with aspirin. Reactions to aspirin may include cutaneous and gastrointestinal manifestations, in addition to potentially life-threatening acute airway obstruction. The onset of AIA is usually beyond the third decade, and these individuals commonly have severe asthma, which may require systemic corticosteroids. We now recognize that this is not an allergically mediated phenomenon, but is instead a disorder that can occur with any nonsteroidal anti-inflammatory drug that acts via inhibition of cyclo-oxygenase. However, the molecular mechanism(s) involved in the pathogenesis of AIA remains unclear (23). The initial hypothesis suggested that AIA was due to a shunt of arachidonic acid from the cyclo-oxygenase pathway to the lipoxygenase pathway (24). We now understand that the pathogenesis of AIA is more complex. Utilization of arachidonic acid within the cell is a highly ordered process with multiple regulatory steps, and simple shunting of substrate does not appear to play a major role. Additional hypotheses have been put forth to explain AIA. For example, studies suggest that a failure of cyclooxygenase-2 expression in the nasal mucosa of patients with AIA may be a feature (25). The largest body of evidence, however, supports a central role for cysteinyl LT synthesis and an enzyme responsible for this synthesis, LTC 4 synthase, in AIA. AIA patients have markedly elevated cysteinyl LT levels at baseline that increase dramatically with aspirin challenge (26). Moreover, studies have demonstrated that LTC 4 synthase is strikingly increased in eosinophils within the airways of aspirin-intolerant asth( Received in original form July 10, 2000 )

The Molecular Characterization and Tissue Distribution of the Human Cysteinyl Leukotriene CysLT2 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 CysLT1 and CysLT2, exist; the former is sensitive and the latter is resistant to the CysLT1 antagonists currently used to treat asthma. Although the CysLT1 receptor gene has been recently cloned, the molecular identity of the CysLT2 receptor has remained elusive. Here we show that the pharmacological profile of an orphan GPCR (PSEC0146) is consistent with that of the CysLT2 receptor. In human embryonic kidney 293 cells that express the PSEC0146 cDNA, leukotriene C4 (LTC4) and leukotriene D4 (LTD4) induce equal increases in intracellular calcium mobilization; these increases are not affected by CysLT1 antagonists. Additionally, [3H]LTC4 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.

Why does airway inflammation persist? Is it leukotrienes?

The leukotrienes are eicosanoids, derived from a cell membrane phospholipid constituent arachidonic acid, which is selectively cleaved by phospholipase A 2 from cell membranes. Arachidonic acid is converted sequentially to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and then to leukotriene A 4 (LTA 4 ) by a catalytic complex consisting of 5-lipoxygenase (5LO) and the 5-lipoxygenase-activating protein (FLAP). In the presence of leukotriene-C 4 synthase, glutathione is adducted at the C-6 position of LTA 4 to yield the molecule known as LTC 4 , which is exported from the cytosol to the extracellular microenvironment, where the glutamic acid moiety is cleaved to form LTD 4 . Cleavage of the glycine moiety from LTD 4 results in the formation of LTE 4 , which is the stable excretory product. LTC 4 , LTD 4 , and LTE 4 , are known as the cysteinyl leukotrienes; together these molecules constitute the material formerly known as the slow-reacting substance of anaphylaxis (SRS-A). All three cysteinyl leukotrienes have the same range of biological effects; however, LTE 4 is much less potent than its precursor molecules. Among the cells in the lung that possess the enzymatic activities to produce the cysteinyl leukotrienes are mast cells, eosinophils, and alveolar macrophages. There is, now, compelling information to implicate the cysteinyl leukotrienes as mediators of asthmatic bronchoconstriction. Increased production of the cysteinyl leukotrienes, as measured by increases in urinary excretion of LTE 4 , has been demonstrated after allergen inhalation (1, 2) in subjects with atopic asthma and during episodes of acute severe asthma (2). Also, subjects with aspirin-induced asthma have higher baseline levels of urinary LTE 4 when compared with other subjects with asthma (3); this level increases after aspirin ingestion, and is associated with an overexpression of LTC 4 synthase in airway biopsies (4). Treatment with drugs that inhibit the synthesis of the cysteinyl leukotrienes or block the CysLT 1 receptor, which mediates the action of the cysteinyl leukotrienes in the airways, markedly attenuates allergen-induced, aspirin-induced, and exercise-induced bronchoconstriction (5–7) and allergen-induced airway hyperresponsiveness (8), as well as improving spontaneous bronchoconstriction (9, 10), and other indices of asthma control (11). Taken together, these studies indicate an important role for the cysteinyl leukotrienes in causing bronchoconstriction.

From bench to bedside. The hurdles of discovering a new leukotriene receptor antagonist.

The discovery of leukotrienes dates back to the period between 1938 and 1940. During this period Kellaway, Feldberg, Trethewie, and Holden published the results of a series of experiments on snake venoms that led them to conclude that there is formed from tissue constituents by enzymic action of the venoms a 'slow-reaction smooth muscle-stimulating substance' which may largely determine the nature of the responses (1, 2). It was, however, in their seminal paper in 1940, using guinea pigs sensitized to egg albumin, that Kellaway and Trethewie (3) coined the term SRS and concluded that SRS was formed by the action of antigen upon sensitized tissue. It was not until some 20 yr later, when Brocklehurst was performing elegant experiments on perfused guinea pig lungs, that he confirmed the release of SRS-like activity on anaphylactic challenge of sensitized tissue (4). In his article Brocklehurst named this biological material slow-reacting substance of anaphylaxis (SRS-A) to differentiate it from other material that could induce slow-reacting contractions. Thus, the term SRS-A was born. Despite intensive research efforts by a wide variety of scientists it was not until 1979 that the chemical constituents of SRS-A were identified, by B. Samuelsson and colleagues at the Karolinska Institute in Stockholm, as the cysteinyl leukotrienes (LTs), namely LTC 4 , LTD 4 , and LTE 4 (5, 6). In the period up until the chemical elucidation of SRS-A it had become progressively more widely accepted that SRS-A played a pivotal role in the bronchoconstriction that is one of the hallmarks of acute asthma attacks. Thus, the seminal discovery that the cysteinyl leukotrienes were the active principle of SRS-A and the elucidation of their biosynthetic pathway from arachidonic acid were key events that provided a rational basis for the design of molecules designed to interfere with these events. Not surprisingly, several pharmaceutical companies (e.g., Merck, ICI [now Zeneca], Abbott, Smith Kline & French [now SmithKline Beecham], Bayer, Lilly, Ono, Ciba Geigy [now Novartis]) mounted research programs that were designed to discover molecules that either inhibited leukotriene biosynthesis or blocked the receptors via which the leukotrienes exerted their effects. The purpose of this article is to review some of the hurdles that are an inevitable component of the drug discovery process. In the interests of brevity, focus is directed particularly at the discovery of drugs by Merck designed to antagonize the cysteinyl leukotriene receptors. This focus, however, in no way diminishes or dismisses the significance of the complementary approach of inhibiting leukotriene biosynthesis, far from it. Indeed, 5-lipoxygenase-activating protein (FLAP) inhibitors have been developed by several companies including Merck (7, 8), and a direct 5-lipoxygenase inhibitor, zileuton, by Abbott (9).