Montelukast Use Decreases Cardiovascular Events in Asthmatics.

Cysteinyl leukotrienes are proinflammatory mediators with a clinically established role in asthma and a human genetic and preclinical role in cardiovascular pathology. Given that cardiovascular disease has a critical inflammatory component, the aim of this work was to conduct an observational study to verify whether the use of a cysteinyl leukotriene antagonist, namely, montelukast, may protect asthmatic patients from a major cardiovascular event and, therefore, represent an innovative adjunct therapy to target an inflammatory component in cardiovascular disease. We performed an observational retrospective 3-year study on eight hundred adult asthmatic patients 18 years or older in Albania, equally distributed into two cohorts, exposed or nonexposed to montelukast usage, matched by age and gender according to information reported in the data collection. Patients with a previous history of myocardial infarction or ischemic stroke were excluded. In summary, 37 (4.6%) of the asthmatic patients, 32 nonexposed, and five exposed to montelukast suffered a major cardiovascular event during the 3-year observation period. All the cardiovascular events, in either group, occurred among patients with an increased cardiovascular risk. Our analyses demonstrate that, independent from gender, exposure to montelukast remained a significant protective factor for incident ischemic events (78% or 76% risk reduction depending on type of analysis). The event-free Kaplan-Meier survival curves confirmed the lower cardiovascular event incidence in patients exposed to montelukast. Our data suggest that there is a potential preventative role of montelukast for incident cardiac ischemic events in the older asthmatic population, indicating a comorbidity benefit of montelukast usage in asthmatics by targeting cysteinyl leukotriene-driven cardiac disease inflammation.

Cysteinyl Leukotrienes Pathway Genes, Atopic Asthma and Drug Response: From Population Isolates to Large Genome-Wide Association Studies.

Genetic variants associated with asthma pathogenesis and altered response to drug therapy are discussed. Many studies implicate polymorphisms in genes encoding the enzymes responsible for leukotriene synthesis and intracellular signaling through activation of seven transmembrane domain receptors, such as the cysteinyl leukotriene 1 (CYSLTR1) and 2 (CYSLTR2) receptors. The leukotrienes are polyunsaturated lipoxygenated eicosatetraenoic acids that exhibit a wide range of pharmacological and physiological actions. Of the three enzymes involved in the formation of the leukotrienes, arachidonate 5 lipoxygenase 5 (ALOX5), leukotriene C4 synthase (LTC4S), and leukotriene hydrolase (LTA4H) are all polymorphic. These polymorphisms often result in variable production of the CysLTs (LTC4, LTD4, and LTE4) and LTB4. Variable number tandem repeat sequences located in the Sp1-binding motif within the promotor region of the ALOX5 gene are associated with leukotriene burden and bronchoconstriction independent of asthma risk. A 444A > C SNP polymorphism in the LTC4S gene, encoding an enzyme required for the formation of a glutathione adduct at the C-6 position of the arachidonic acid backbone, is associated with severe asthma and altered response to the CYSLTR1 receptor antagonist zafirlukast. Genetic variability in the CysLT pathway may contribute additively or synergistically to altered drug responses. The 601 A > G variant of the CYSLTR2 gene, encoding the Met201Val CYSLTR2 receptor variant, is associated with atopic asthma in the general European population, where it is present at a frequency of ∼2.6%. The variant was originally found in the founder population of Tristan da Cunha, a remote island in the South Atlantic, in which the prevalence of atopy is approximately 45% and the prevalence of asthma is 36%. In vitro work showed that the atopy-associated Met201Val variant was inactivating with respect to ligand binding, Ca2+ flux and inositol phosphate generation. In addition, the CYSLTR1 gene, located at Xq13-21.1, has been associated with atopic asthma. The activating Gly300Ser CYSLTR1 variant is discussed. In addition to genetic loci, risk for asthma may be influenced by environmental factors such as smoking. The contribution of CysLT pathway gene sequence variants to atopic asthma is discussed in the context of other genes and environmental influences known to influence asthma.

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.

Pharmacogenetics of the G protein-coupled receptors.

Pharmacogenetics investigates the influence of genetic variants on physiological phenotypes related to drug response and disease, while pharmacogenomics takes a genome-wide approach to advancing this knowledge. Both play an important role in identifying responders and nonresponders to medication, avoiding adverse drug reactions, and optimizing drug dose for the individual. G protein-coupled receptors (GPCRs) are the primary target of therapeutic drugs and have been the focus of these studies. With the advance of genomic technologies, there has been a substantial increase in the inventory of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms and insertion or deletions that have potential to alter GPCR expression of function. In vivo and in vitro studies have determined functional roles for many GPCR variants, but genetic association studies that define the physiological impact of the majority of these common variants are still limited. Despite the breadth of pharmacogenetic data available, GPCR variants have not been included in drug labeling and are only occasionally considered in optimizing clinical use of GPCR-targeted agents. In this chapter, pharmacogenetic and genomic studies on GPCR variants are reviewed with respect to a subset of GPCR systems, including the adrenergic, calcium sensing, cysteinyl leukotriene, cannabinoid CB1 and CB2 receptors, and the de-orphanized receptors such as GPR55. The nature of the disruption to receptor function is discussed with respect to regulation of gene expression, expression on the cell surface (affected by receptor trafficking, dimerization, desensitization/downregulation), or perturbation of receptor function (altered ligand binding, G protein coupling, constitutive activity). The large body of experimental data generated on structure and function relationships and receptor-ligand interactions are being harnessed for the in silico functional prediction of naturally occurring GPCR variants. We provide information on online resources dedicated to GPCRs and present applications of publically available computational tools for pharmacogenetic studies of GPCRs. As the breadth of GPCR pharmacogenomic data becomes clearer, the opportunity for routine assessment of GPCR variants to predict disease risk, drug response, and potential adverse drug effects will become possible.

Rosuvastatin inhibits human airway smooth muscle cells mitogenic response to eicosanoid contractile agents.

BACKGROUND: The concept of permanent narrowing of the airways resulting from chronic inflammation and fibrosis is called remodeling and is a common feature of asthma and chronic obstructive pulmonary disease (COPD). The eicosanoid contractile agents thromboxane A(2) (TxA(2)) and cysteinyl-leukotriene D(4) (LTD(4)) are among the recognized mitogens for human airway smooth muscle (ASM) cells. Statins are known to possess anti-inflammatory and immunomodulatory properties that are independent on their cholesterol-lowering effects and may result in clinical lung benefits. Rosuvastatin is the last agent of the lipid-lowering drugs to be introduced and experimental evidence indicates that it possess favorable pleiotropic effects in the cardiovascular and nervous systems. Yet, no data is available in the literature regarding its effects on human airway remodeling. The present study was aimed at examining the effect of rosuvastatin and the involvement of prenylated proteins in the response of human ASM cells to serum, epidermal growth factor (EGF) and eicosanoid contractile mitogens that activate TxA(2) prostanoid and LTD(4) receptors. METHODS: Cell growth was assessed by nuclear incorporation of [(3)H]thymidine in human ASM cells serum-starved and then stimulated for 48 h in MEM plus 0.1% BSA containing mitogens in the absence and presence of modulators of the mevalonate and prenylation pathways. RESULTS: We found that rosuvastatin dose-dependently inhibited serum-, EGF-, the TxA(2) stable analog U46619-, and LTD(4)-induced human ASM cells growth. All these effects were prevented by pretreatment with mevalonate. Addition of the prenylation substrates farnesol and geranylgeraniol reversed the effect of rosuvastatin on EGF and U46619, respectively. Interestingly, only mevalonate showed restoration of cell growth following rosuvastatin treatment in LTD(4) and LTD(4) plus EGF treated cells, suggesting a possible involvement of both farnesylated and geranylgeranylated proteins in the cysteinyl-LT-induced cell growth. CONCLUSIONS: The hydrophilic statin rosuvastatin exerts direct effects on human ASM cells mitogenic response in vitro by inhibiting prenylation of signaling proteins, likely small G proteins. These findings are consistent with previous observed involvement of small GTPase signaling in EGF- and U46619-induced human airway proliferation and corroborate the recent interest in the potential clinical benefits of statins in asthma/COPD.

Synthesis of cysteinyl leukotrienes in human endothelial cells: subcellular localization and autocrine signaling through the CysLT2 receptor.

The purpose of this study was to characterize enzyme, receptor, and signaling involved in the synthesis and the activity of cysteinyl leukotrienes (cys-LTs) in human umbilical vein endothelial cells (HUVECs). We used primary cultures of HUVECs and evaluated the formation of cys-LTs by RP-HPLC. Suicide inactivation and subcellular localization of the enzyme responsible for the conversion of leukotriene (LT) A(4) into LTC(4) were studied by repeated incubations with LTA(4) and immunogold electron microscopy. The CysLT(2) receptor in HUVECs was characterized by equilibrium binding studies, Western blot analysis, and immunohistochemistry. Concentration-response curves in HUVECs and in transfected COS-7 cells were used to characterize a novel specific CysLT(2) receptor antagonist (pA(2) of 8.33 and 6.79 against CysLT(2) and CysLT(1) receptors, respectively). The results obtained provide evidence that the mGST-II synthesizing LTC(4) in HUVECs is pharmacologically distinguishable from the LTC(4)-synthase (IC(50) of MK886 <5 µM for LTC(4)-synthase and >30 µM for mGST-II), is not suicide-inactivated and is strategically located on endothelial transport vesicles. The CysLT(2) receptor is responsible for the increase in intracellular Ca(2+) following exposure of HUVECs to cys-LTs and is coupled to a pertussis toxin-insensitive G(q) protein. The synthesis of cys-LTs from LTA(4) by endothelial cells is directly associated with the activation of the CysLT(2) receptor (EC(50) 0.64 µM) in a typical autocrine fashion.

Leukotrienes and atherosclerosis.

Evidence from experimental and genetic studies suggest the existence of a potential link between the leukotrienes (LT) signalling cascade, and the pathogenesis/progression of atherosclerosis and its serious consequences such as acute myocardial infarction (AMI), stroke, aortic aneurysms, and intimal hyperplasia. LT biosynthetic enzymes are expressed within atherosclerotic lesion, leading to production of cysteinyl-leukotrienes (Cys-LTs) and LTB4 that exert potent pro-inflammatory action through interaction with CysLT and BLT receptor subtypes expressed on inflammatory and structural cells within the vascular wall. Genetic variants in the genes of the 5-LO pathway have been associated with the risk of developing AMI and stroke. As a result, anti-LT have recently received renewed interest for the treatment of atherosclerosis and its ischemic complications. The aim of this short review is to summarize our current understanding of the role of LTs and their receptors in the genesis and progression of atherosclerosis and review the recent developments on the use of antagonists.

A role for inflammatory mediators in heterologous desensitization of CysLT1 receptor in human monocytes.

Cysteinyl-leukotrienes (cysteinyl-LT) are rapidly generated at sites of inflammation and, in addition to their role in asthma, rhinitis, and other immune disorders, are increasingly regarded as significant inflammatory factors in cancer, gastrointestinal, cardiovascular diseases. We recently demonstrated that in monocyte/macrophage-like U937 cells, extracellular nucleotides heterologously desensitize CysLT(1) receptor (CysLT(1)R)-induced Ca(2+) transients. Given that monocytes express a number of other inflammatory and chemoattractant receptors, this study was aimed at characterizing transregulation between these different stimuli. We demonstrate that in U937 cells and in primary human monocytes, a series of inflammatory mediators activating G(i)-coupled receptor (FPR1, BLT(1)) desensitize CysLT(1)R-induced Ca(2+) response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to G(q), cross-desensitizes CysLT(1)R without the apparent involvement of any kinase. Interestingly, G(s)-coupled receptors (beta(2)AR, H(1/2)R, EP(2/4)R) are also able to desensitize CysLT(1)R response through activation of PKA. Heterologous desensitization seems to affect mostly the G(i)-mediated signaling of the CysLT(1)R. The hierarchy of desensitization among agonists may be important for leukocyte signal processing at the site of inflammation. Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and inflammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating this complex network of integrated signals.

Leukotriene modifiers in the treatment of cardiovascular diseases.

Cysteinyl-leukotrienes (Cys-LTs) and LTB4 are potent proinflammatory mediators derived from arachidonic acid through the 5-lipoxygenase (5-LO) pathway, which exerts important pharmacological effects through their interaction with specific receptors: Cys-LT receptors (CysLT1 and CysLT2) and LTB4 receptors (BLT1 and BLT2). Published evidence justifies a broader role for LT receptor antagonists (LTRAs), in particular, montelukast, in the treatment of bronchial asthma, allergic rhinitis, and recently, in cardiocerebrovascular disease. The actions of Cys-LTs on the cardiovascular (CV) system are well-documented and include a broad array of activities with promising therapeutic targets in animal models exploring the use of selective 5-LO (or 5-LO-activating protein) inhibitors or dual LO-cycloxygenase-blocking agents in experimentally induced acute myocardial infarction. The picture that emerges from studies with LTRAs is more controversial at the moment, and some findings suggest a role for Cys-LTs in the extension of ischemic damage and in cardiac dysfunction during reperfusion; others do not. The aim of this short review is to summarize the state of present research about LT modifier treatment in CV disease.

Cysteinyl-leukotriene receptors and cellular signals.

Cysteinyl-leukotrienes (cysteinyl-LTs) exert a range of proinflammatory effects, such as constriction of airways and vascular smooth muscle, increase of endothelial cell permeability leading to plasma exudation and edema, and enhanced mucus secretion. They have proved to be important mediators in asthma, allergic rhinitis, and other inflammatory conditions, including cardiovascular diseases, cancer, atopic dermatitis, and urticaria. The classification into subtypes of the cysteinyl-LT receptors (CysLTRs) was based initially on binding and functional data, obtained using the natural agonists and a wide range of antagonists. CysLTRs have proved remarkably resistant to cloning. However, in 1999 and 2000, the CysLT1R and CysLT2R were successfully cloned and both shown to be members of the G-protein coupled receptors (GPCRs) superfamily. Molecular cloning has confirmed most of the previous pharmacological characterization and identified distinct expression patterns only partially overlapping. Recombinant CysLTRs couple to the Gq/11 pathway that modulates inositol phospholipids hydrolysis and calcium mobilization, whereas in native systems, they often activate a pertussis toxin-insensitive Gi/o-protein, or are coupled promiscuously to both G-proteins. Interestingly, recent data provide evidence for the existence of an additional receptor subtype that seems to respond to both cysteinyl-LTs and uracil nucleosides, and of an intracellular pool of CysLTRs that may have roles different from those of plasma membrane receptors. Finally, a cross-talk between the cysteinyl-LT and the purine systems is being delineated. This review will summarize recent data derived from studies on the molecular and cellular pharmacology of CysLTRs.

A functional G300S variant of the cysteinyl leukotriene 1 receptor is associated with atopy in a Tristan da Cunha isolate.

Atopy is a well-defined immune phenotype that is reported to be a risk factor for asthma. Among the many loci that contribute to a genetic predisposition to asthma, the cysteinyl leukotriene receptor genes and their variants have been important subjects of study because they are functionally and pharmacologically implicated in the atopy phenotype affecting many asthma subjects. Moreover, the product of cysteinyl-leukotriene 1 receptor gene (CysLT1), located at Xq13.2, is targeted by LT receptor antagonists. In our earlier association study, the M201V variant of the cysteinyl-leukotriene 2 receptor gene (CysLT2), located at 13q14, was implicated in atopic asthma. Here we report the screening of the coding region of the CysLT1, gene in the highly asthmatic Tristan da Cunha population. In this population, we discovered a CysLT1 G300S variant that is carried with a significantly higher frequency in atopics and asthmatics from the Tristan da Cunha population. Furthermore, we report the asthma independent association of the CysLT1 G300S variant with atopy. Subsequently, we compared the changes conferred by each SNP on CysLT function. The CysLT1 300S receptor interacts with LTD4 with significantly greater potency. For the 300S variant, a statistically significant decrease in the effector concentration for half-maximum response (EC50) for intracellular Ca flux and total InsP generation is observed. Other aspects of the receptor function and activity, such as desensitization, pharmacologic profile in response to montelukast, and cellular localization, are unchanged. These in vitro analyses provide evidence that the 300S CysLT1 variant, found more commonly in atopics in the Tristan da Cunha population, encodes a functionally more sensitive variant.

Cysteinyl-leukotrienes and their receptors in asthma and other inflammatory diseases: critical update and emerging trends.

Cysteinyl-leukotrienes (cysteinyl-LTs), that is, LTC4, LTD4, and LTE4, trigger contractile and inflammatory responses through the specific interaction with G protein-coupled receptors (GPCRs) belonging to the purine receptor cluster of the rhodopsin family, and identified as CysLT receptors (CysLTRs). Cysteinyl-LTs have a clear role in pathophysiological conditions such as asthma and allergic rhinitis (AR), and have been implicated in other inflammatory conditions including cardiovascular diseases, cancer, atopic dermatitis, and urticaria. Molecular cloning of human CysLT1R and CysLT2R subtypes has confirmed most of the previous pharmacological characterization and identified distinct expression patterns only partially overlapping. Interestingly, recent data provide evidence for the immunomodulation of CysLTR expression, the existence of additional receptor subtypes, and of an intracellular pool of CysLTRs that may have roles different from those of plasma membrane receptors. Furthermore, genetic variants have been identified for the CysLTRs that may interact to confer risk for atopy. Finally, a crosstalk between the cysteinyl-LT and the purine systems is being delineated. This review will summarize and attempt to integrate recent data derived from studies on the molecular pharmacology and pharmacogenetics of CysLTRs, and will consider the therapeutic opportunities arising from the new roles suggested for cysteinyl-LTs and their receptors.

The highly conserved DRY motif of class A G protein-coupled receptors: beyond the ground state.

Despite extensive study of heptahelical G protein-coupled receptors (GPCRs), the precise mechanism of G protein activation is unknown. The role of one highly conserved stretch of residues, the amino acids glutamic acid/aspartic acid-arginine-tyrosine (i.e., the E/DRY motif), has received considerable attention with respect to regulating GPCR conformational states. In the consensus view, glutamic acid/aspartic acid maintains the receptor in its ground state, because mutations frequently induce constitutive activity (CA). This hypothesis has been confirmed by the rhodopsin ground-state crystal structure and by computational modeling approaches. However, some class A GPCRs are resistant to CA, suggesting alternative roles for the glutamic acid/aspartic acid residue and the E/DRY motif. Here, we propose two different subgroups of receptors within class A GPCRs that make different use of the E/DRY motif, independent of the G protein type (G(s), G(i), or G(q)) to which the receptor couples. In phenotype 1 receptors, nonconservative mutations of the glutamic acid/aspartic acid-arginine residues, besides inducing CA, increase affinity for agonist binding, retain G protein coupling, and retain an agonist-induced response. In contrast, in second phenotype receptors, the E/DRY motif is more directly involved in governing receptor conformation and G protein coupling/recognition. Hence, mutations of the glutamic acid/aspartic acid residues do not induce CA. Conversely, nonconservative mutations of the arginine of the E/DRY motif always impair agonist-induced receptor responses and, generally, reduce agonist binding affinity. Thus, it is essential to look beyond the rhodopsin ground-state model of conformational activation to clarify the role of this highly conserved triplet in GPCR activation and function.

The role of the antioxidant vitamin supplementation in the prevention of cardiovascular diseases.

Industrial and technological revolutions have resulted in dramatic shifts in the diseases that are responsible for illness and death. In particular, cardiovascular disease (CVD) has emerged as the dominant chronic disease in many parts of the world. Diet, tobacco smoking, physical inactivity, obesity, lipid levels, hypertension and diabetes mellitus have contributed to their wide diffusion. Oxidative damage and the production of free radicals in the endothelium are two of the main factors involved in the pathogenesis of the atherosclerotic process that causes CVD. One of the more important results of basic research on dietetic regimes has shown that people who consume large amounts of fruits and vegetables have a lower incidence of CVD, stroke and tumours, but the specific mechanisms of these foods (which have an apparently protective effect) are still not completely clear. Possible reasons could include a greater consumption of fruit and vegetables, and an increased consumption of dietetic fibres. Recently, it been proposed that micronourishments with an antioxidant activity could be responsible for the reduction of chronic diseases. Research supplies a hypothetical mechanism by which antioxidant substances may be reducing the risk of atherosclerosis through the inhibition of oxidative damage. Appropriate nutritional practices are of central importance in managing risk and treatment of CVD; in fact, many current guidelines for a healthy general population contain nutritional recommendations to reduce the risk of these diseases. A large number of descriptive and case-control studies suggests that the consumption of many antioxidant vitamins (A, C and E) reduces the risk of CVD. Such data raises the following problem of whether supplementation of vitamins A, C and E emerges as being useful in the primary prevention of CVD. Many important studies involving a great number of participants have not confirmed this hypothesis and the results are often contradictory. This review examines the studies published in the literature that document the effect of supplementation with antioxidant vitamins (A, C and E) in the primary and secondary prevention of CVD due to an atherosclerosis process.

G-protein-coupled receptors and asthma endophenotypes: the cysteinyl leukotriene system in perspective.

Genetic variation in specific G-protein coupled receptors (GPCRs) is associated with a spectrum of respiratory disease predispositions and drug response phenotypes. Although certain GPCR gene variants can be disease-causing through the expression of inactive, overactive, or constitutively active receptor proteins, many more GPCR gene variants confer risk for potentially deleterious endophenotypes. Endophenotypes are traits, such as bronchiole hyperactivity, atopy, and aspirin intolerant asthma, which have a strong genetic component and are risk factors for a variety of more complex outcomes that may include disease states. GPCR genes implicated in asthma endophenotypes include variants of the cysteinyl leukotriene receptors (CYSLTR1 and CYSLTR2), and prostaglandin D2 receptors (PTGDR and CRTH2), thromboxane A2 receptor (TBXA2R), beta2-adrenergic receptor (ADRB2), chemokine receptor 5 (CCR5), and the G protein-coupled receptor associated with asthma (GPRA). This review of the contribution of variability in these genes places the contribution of the cysteinyl leukotriene system to respiratory endophenotypes in perspective. The genetic variant(s) of receptors that are associated with endophenotypes are discussed in the context of the extent to which they contribute to a disease phenotype or altered drug efficacy.

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

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

CysLT1 receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation.

BACKGROUND: Cysteine-containing leukotrienes (cysteinyl-LTs) are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC) proliferation. We used human ASMC (HASMC) to identify the signal transduction pathway(s) of the leukotriene D4 (LTD4)-induced DNA synthesis. METHODS: Proliferation of primary HASMC was measured by [3H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R) and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS) was estimated by measuring dichlorodihydrofluorescein (DCF) oxidation. RESULTS: We demonstrate that in HASMC LTD4-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT1-R. Accordingly, we found that LTD4 stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX) and phosphoinositide 3-kinase (PI3K) inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD4-induced DNA synthesis following Src inhibition. More interestingly, CysLT1-R stimulation increased the production of ROS and N-acetylcysteine (NAC) abolished LTD4-induced EGF-R phosphorylation and thymidine incorporation. CONCLUSION: Collectively, our data demonstrate that in HASMC LTD4 stimulation of a Gi/o coupled CysLT1-R triggers the transactivation of the EGF-R through the intervention of PI3K and ROS. While PI3K and ROS involvement is an early event, the activation of Src occurs downstream of EGF-R activation and is followed by the classical Ras-ERK1/2 signaling pathway to control G1 progression and cell proliferation.

CysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation.

Both cysteinyl-leukotrienes and extracellular nucleotides mediate inflammatory responses via specific G-protein-coupled receptors, the CysLT and the P2Y receptors, respectively. Since these mediators accumulate at sites of inflammation, and inflammatory cells express both classes of receptors, their responses are likely to be crossregulated. We investigated the molecular basis of desensitization and trafficking of the CysLT1 receptor constitutively and transiently expressed in the human monocyte/macrophage-like U937 or COS-7 cells in response to LTD4 or nucleotides. Exposure to agonist induced a rapid homologous desensitization of the CysLT1 receptor [as measured by the reduction in the maximal agonist-induced intracellular cytosolic Ca2+ ([Ca2+]i) transient], followed by receptor internalization (as assessed by equilibrium binding and confocal microscopy). Activation of P2Y receptors with ATP or UDP induced heterologous desensitization of the CysLT1 receptor. Conversely, LTD4-induced CysLT1 receptor activation had no effect on P2Y receptor responses, which suggests that the latter have a hierarchy in producing desensitizing signals. Furthermore, ATP/UDP-induced CysLT1 receptor desensitization was unable to cause receptor internalization, induced a faster recovery of CysLT1 functionality and was dependent upon protein kinase C. By contrast, homologous desensitization, which is probably dependent upon G-protein-receptor kinase 2 activation, induced a fast receptor downregulation and, accordingly, a slower recovery of CysLT1 functionality. Hence, CysLT1 receptor desensitization and trafficking are differentially regulated by the CysLT1 cognate ligand or by extracellular nucleotides. This crosstalk may have a profound physiological implication in the regulation of responses at sites of inflammation, and may represent just an example of a feedback mechanism used by cells to fine-tune their responses.

CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors.

Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y(1,2,4,6,11,12,13,14) receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 microM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 microM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y(1,2,4,6) receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were <1 microM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 microM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions.

Molecular and functional aspects of human cysteinyl leukotriene receptors.

The cysteinyl leukotrienes (cys-LTs), i.e. LTC(4), LTD(4) and LTE(4), trigger contractile and inflammatory processes through the specific interaction with cell surface receptors belonging to the purine receptor cluster of the rhodopsin family of the G protein-coupled receptor (GPCR) genes. Cys-LTs have a clear role in pathophysiological conditions such as asthma, allergic rhinitis and other nasal allergies, and have been implicated in a number of inflammatory conditions including cardiovascular and gastrointestinal diseases. Pharmacological studies have identified two classes of cys-LT receptors (CysLT(1) and CysLT(2)) based on their sensitivity to CysLT(1) selective antagonists, albeit there is evidence for additional subtypes. Molecular cloning of the human CysLT(1) and CysLT(2) receptors has confirmed both their structure as putative seven transmembrane domain G protein-coupled receptors and most of the previous pharmacological characterization. The rank order of potency of agonist activation for the CysLT(1) receptor is LTD4 > LTC4 > LTE4 and for the CysLT(2) receptor is LTC4 = LTD4 > LTE4. The CysLT(1) receptor is most highly expressed in spleen, peripheral blood leukocytes, interstitial lung macrophages and in airway smooth muscle. The CysLT(2) receptor is mostly expressed in heart, adrenals, placenta, spleen, peripheral blood leukocytes and less strongly in the brain. Gene cloning of CysLT(1) and CysLT(2) receptors has renewed the attention on the cys-LTs field and will, hopefully, encourage future studies on the regulation of CysLT receptors expression and the dissection of their signalling pathways. Furthermore, the peculiar pattern of expression of the two receptor subtypes will promote the discovery of new functions for cys-LTs in physiological and pathological conditions. Only CysLT(1) selective receptor antagonists have been described to date and are currently available for the treatment of asthma. Molecular cloning of different CysLT receptor subtypes will certainly foster the development of new selective antagonists based on molecular modelling studies.