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.

Montelukast inhibits tumour necrosis factor-alpha-mediated interleukin-8 expression through inhibition of nuclear factor-kappaB p65-associated histone acetyltransferase activity.

BACKGROUND: Montelukast is a potent cysteinyl leukotriene-1 receptor antagonist possessing some anti-inflammatory effects although the molecular mechanism of these anti-inflammatory effects is unknown. In this study, we aimed to investigate the effect of montelukast on nuclear factor (NF)-kappaB-associated histone acetylation activity in phorbol myristate acetate (PMA)-differentiated U937 cells. METHODS: We examined the inhibitory effects of montelukast on TNF-alpha-induced IL-8 production in PMA-differentiated U-937 cells. U-937 cells were exposed to PMA (50 ng/mL) for 48 h to allow differentiation to macrophages. Macrophages were then exposed to TNF-alpha (10 ng/mL) in the presence or absence of montelukast (0.01-10 microm) for 24 h. After this time, the concentration of IL-8 in the culture supernatant was measured by sandwich-type ELISA kit. The effect of signalling pathways on TNF-alpha-induced IL-8 release was examined pharmacologically using selective NF-kappaB/IKK2 (AS602868, 3 microm), (PD98059, 10 microm) and p38 mitogen activated protein kinase (MAPK) (SB203580, 1 microm) inhibitors. NF-kappaB DNA binding activity was measured by a DNA-binding ELISA-based assay. NF-kappaB-p65-associated histone acetyltransferase (HAT) activity was measured by immunoprecipitation linked to commercial fluorescent HAT. RESULTS: TNF-alpha-induced IL-8 release was suppressed by an NF-kappaB inhibitor but not by MEK or p38 MAPK inhibitors. Montelukast induced a concentration-dependent inhibition of TNF-alpha-induced IL-8 release and mRNA expression that reached a plateau at 0.1 microm without affecting cell viability. Montelukast did not affect NF-kappaB p65 activation as measured by DNA binding but suppressed NF-kappaB p65-associated HAT activity. CONCLUSION: Montelukast inhibits TNF-alpha-stimulated IL-8 expression through changes in NF-kappaB p65-associated HAT activity. Drugs targeting these enzymes may enhance the anti-inflammatory actions of montelukast.

Antagonism of thromboxane receptors by diclofenac and lumiracoxib.

BACKGROUND AND PURPOSE: Non-steroidal anti-inflammatory drugs (NSAIDs) are analgesic and anti-inflammatory by virtue of inhibition of the cyclooxygenase (COX) reaction that initiates biosynthesis of prostaglandins. Findings in a pulmonary pharmacology project gave rise to the hypothesis that certain members of the NSAID class might also be antagonists of the thromboxane (TP) receptor. EXPERIMENTAL APPROACH: Functional responses due to activation of the TP receptor were studied in isolated airway and vascular smooth muscle preparations from guinea pigs and rats as well as in human platelets. Receptor binding and activation of the TP receptor was studied in HEK293 cells. KEY RESULTS: Diclofenac concentration-dependently and selectively inhibited the contraction responses to TP receptor agonists such as prostaglandin D2 and U-46619 in the tested smooth muscle preparations and the aggregation of human platelets. The competitive antagonism of the TP receptor was confirmed by binding studies and at the level of signal transduction. The selective COX-2 inhibitor lumiracoxib shared this activity profile, whereas a number of standard NSAIDs and other selective COX-2 inhibitors did not. CONCLUSIONS AND IMPLICATIONS: Diclofenac and lumiracoxib, in addition to being COX unselective and highly COX-2 selective inhibitors, respectively, displayed a previously unknown pharmacological activity, namely TP receptor antagonism. Development of COX-2 selective inhibitors with dual activity as potent TP antagonists may lead to coxibs with improved cardiovascular safety, as the TP receptor mediates cardiovascular effects of thromboxane A2 and isoprostanes.

Cysteinyl-leukotriene receptor antagonists: present situation and future opportunities.

Cysteinlyl-leukotriene receptor antagonists (LTRAs) were introduced as oral preventative anti-asthma medications in the late 1990s and, very recently, montelukast has been approved also for the relief of symptoms of perennial and seasonal allergic rhinitis. Although clinical trials and clinical practice showed LTRAs to be effective in the treatment of asthma patients with a wide range of disease severity, their exact role in the therapy of asthma is not well defined and possibly under-appreciated. As for other anti-asthma drugs, clinical trials with LTRAs uncovered a range of patient responses, so that an understanding of the variability mechanisms (e.g. acquired or genetic factors, etc.) is needed to maximize the probability of a beneficial response. Since the molecular cloning of CysLT receptors (CysLTRs) has been achieved, new roles for cysteinyl-LTs in pathophysiological conditions have been suggested or established from the observed distribution in cells and tissues other than the lung. Cysteinyl-LTs and CysLTRs have been implicated in the pathophysiology of other inflammatory conditions including cancer, atopic dermatitis, idiopathic chronic urticaria, and cardiovascular diseases. As a result, LTRAs might be worth assessing for a therapeutic role in some of these pathologies. This review summarizes and attempts to integrate recent data on the therapeutic efficacy, effectiveness and safety of LTRAs in asthma and allergic rhinitis, and speculates on other therapeutic opportunities.

Developmental expression of heteromeric nicotinic receptor subtypes in chick retina.

Acting through nicotinic acetylcholine receptors (nAChRs), acetylcholine plays an important role in retinal development and the formation of retinal connections to target tissues, but very little is known about the nAChR subtypes expressed in vertebrate retina during neuronal development. We used immunoprecipitation and [3H]epibatidine binding to study the expression of chick retina alpha-bungarotoxin-insensitive heteromeric nAChRs during development and adulthood, and found that it is strictly developmentally regulated, reaching a peak on postnatal day 1. The increase in [3H]epibatidine receptors is caused mainly by an increase in the receptors containing the alpha2, alpha6, beta3, and beta4 subunits. The contribution of beta subunits to [3H]epibatidine receptors significantly changes during development: the beta2 subunit is contained in the majority (84%) of receptors on embryonic day (E) 7 but in only 32% on postnatal day (P) 1, whereas the beta4-containing receptors increase from 22% to 78% during the same period. Using a sequential immunodepletion procedure, we purified the beta2- and beta4-containing subtypes and found that they coassemble with alpha4 and/or alpha3 on E11, and also with the alpha2, alpha6, and beta3 on P1. After the immunodepletion of alpha6-containing receptors, the beta2- and beta4-containing receptors have a very similar pharmacological profile on P1. Parallel immunoprecipitation experiments in other brain areas showed that the developmentally regulated receptors in optic lobe are those containing the alpha2, alpha5, and beta2 subunits and those containing the alpha4 and beta2 subunits, whereas the receptors in forebrain-cerebellum contain the alpha4 and beta2 subunits with or without the alpha5 subunit. These results indicate that there is an increase in receptor heterogeneity and complexity in chick retina during development that is also maintained in adulthood.

Leukotriene D4-induced activation of smooth-muscle cells from human bronchi is partly Ca2+-independent.

Cysteine-containing leukotrienes (cysteinyl-LTs) are potent bronchoconstrictors and play a key role in asthma. We found that histamine and LTD4 markedly constrict strips of human bronchi (HB) with similar efficacy. However, in human airway smooth-muscle (HASM) cells, LTD4, at variance with histamine, elicited only a small, transient change in intracellular calcium ion concentration. HASM cells express both Ca2+-dependent and -independent isoforms of protein kinase C (PKC) (i.e., PKC-alpha and PKC-alpha ). Western blot analysis showed that PKC-alpha is activated by histamine and, to a lesser extent, by LTD4, whereas only LTD4 translocates PKC-alpha. This translocation was specifically inhibited by the LTD4 antagonist pobilukast. Phorbol-dibutyrate ester (PDBu) (a PKC activator) contracted HB strips to the same extent in the presence as in the absence of extra- and intracellular Ca2+. In the absence of Ca2+, LTD4 contracted HB strips to the same extent as did PDBu, suggesting the involvement of a Ca2+-independent PKC in LTD4-mediated signal transduction. PDBu-induced desensitization and the PKC inhibitor H7 abolished the slow and sustained LTD4-triggered contraction of HB strips in the absence of Ca2+, although H7 did not greatly affect the response in the presence of the ion. Thus, in human airways, we identified a novel LTD4 transduction mechanism linked to bronchial smooth-muscle contraction, which is partly independent of Ca2+ and involves the activation of PKC-alpha.

Leukotrienes as mediators of asthma.

This review describes the aspects of leukotriene (LT) pharmacology and biology that are relevant to their important role in asthma. The biosynthesis and metabolism, including transcellular metabolism, of LTB4 and the cysteinyl-LTs (i.e. LTC4, LTD4 and LTE4) are described, and their transport is briefly outlined. The existence, distribution and pharmacological characterization of the receptors (BLT, CysLT1, CysLT2), as well as the transduction mechanisms triggered, are discussed in detail. We also describe their effects on airway smooth muscle tone, hyperresponsiveness and proliferation, on vascular tone and permeability, on mucus secretion, on neural fibers and inflammatory cell functions. Finally, the evidence supporting their role as asthma mediators is reviewed, including the effects of anti LT drugs (both biosynthesis inhibitors and receptor antagonists) in experimental and clinical asthma.

A kinetic binding study to evaluate the pharmacological profile of a specific leukotriene C(4) binding site not coupled to contraction in human lung parenchyma.

We report the identification of a novel pharmacological profile for the leukotriene (LT)C(4) binding site we previously identified in human lung parenchyma (HLP). We used a series of classic cysteinyl-LT (CysLT)(1) receptor antagonists belonging to different chemical classes and the dual CysLT(1)-CysLT(2) antagonist BAY u9773 for both binding and functional studies. Because the presence of (S)-decyl-glutathione interfered with cysteinyl-LT binding, with a kinetic protocol we avoided the use of this compound. By means of heterologous dissociation time courses, we demonstrated that zafirlukast, iralukast, and BAY u9773 selectively competed only for (3)H-LTD(4) binding sites, whereas pobilukast, pranlukast, and CGP 57698 dissociated both (3)H-LTC(4) and (3)H-LTD(4) from their binding sites. Thus, with binding studies, we have been able to identify a pharmacological profile for LTC(4) distinct from that of LTD(4) receptor (CysLT(1)) in HLP. On the contrary, in functional studies, all of the classic antagonists tested were able to revert both LTC(4)- and LTD(4)-induced contractions of isolated HLP strips. Thus, LTD(4) and LTC(4) contract isolated HLP strips through the same CysLT(1) receptor. The results of kinetic binding studies, coupled to a sophisticated data analysis, confirm our hypothesis that HLP membranes contain two cysteinyl-LT high-affinity binding sites with different pharmacological profiles. In functional studies, however, LTD(4)- and LTC(4)-induced contractions are mediated by the same CysLT(1) receptor. In conclusion, the specific LTC(4) high-affinity binding site cannot be classified as one of the officially recognized CysLT receptors, and it is not implicated in LTC(4)-induced HLP strip contractions.

Expression of prostacyclin receptors in luteinizing hormone-releasing hormone immortalized neurons: role in the control of hormone secretion.

PGs of the E series are involved in the control of LHRH secretion. The present experiments were conducted to clarify whether PGI2 (prostacyclin) might be also involved in such a control, using multiple methodological approaches on immortalized LHRH-secreting neurons. A RT-PCR procedure to detect mouse PGI2 receptor (IP) messenger RNA was first applied, and the results obtained showed the presence of a specific transcript in two cell lines of immortalized LHRH neurons (GT1-1 and GN11 cell lines). Receptor binding assays on membrane preparations from GT1-1 cells showed the presence of a single specific and saturable class of binding sites (Kd = 4.6 nM; 10,000 sites/cell) for [3H]iloprost, a stable analog of PGI2. Competition experiments showed that the binding sites labeled by [3H]iloprost possess the pharmacological characteristics of IP receptors. In functional studies, PGI2 and its analogs, iloprost and cicaprost, were able to stimulate LHRH release from the GT1-1 cells with elevated potencies (EC50 = 0.6-4.3 nM); PGE1 was only slightly less active (EC50 = 28.5 nM), whereas PGE2, considered the major PG involved in LHRH secretion, was poorly effective (EC50 = 921 nM). The relative potencies (EC50) of these compounds in stimulating the intracellular accumulation of cAMP were in line with their LHRH-releasing activities. In conclusion, these results indicate that immortalized LHRH-secreting neurons express IP receptors through which PGI2 may exert relevant effects on LHRH release.

Ligand-binding studies: old beliefs and new strategies.

Ligand-binding studies remain a very popular technique among many experimentalists. As far as equilibrium experiments are concerned, saturation and displacement curves are commonly performed for simplicity, convenience or for the sake of tradition. However, alternative protocols, such as 'mixed'-type protocols or multiligand experiments, are also possible. Indeed, there are cases where kinetic experiments, usually considered a 'second-choice' experiment, might have a superior resolving power compared to equilibrium ones. A combination of equilibrium and kinetic experiments might be a powerful solution to overcome limits and shortcomings of each specific technique and is discussed in this issue by G. Enrico Rovati. Thus, a careful choice of the design, a protocol optimization and a computerized analysis of the data can yield a dramatic improvement in the precision of the parameter estimation over more conventional approaches.

4-Oxystilbene compounds are selective ligands for neuronal nicotinic alphaBungarotoxin receptors.

1. Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes. 2. MG 624, F3, F3A and F3B inhibited of 125I-alphaBungarotoxin (alphaBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited 125I-alphaBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations. 3. We immobilized the alpha7, beta2 and beta4 containing chick neuronal nicotinic AChR subtypes using anti-subunit specific antibodies. MG 624, F3, F3A and F3B inhibited 125I-alphaBgtx binding to the alpha7-containing receptors with nM affinity, but inhibited 3H-Epi binding to beta2-containing receptors only at very high concentrations (more than 35 microM); their affinity for the beta4-containing receptors was ten times more than for the beta2-containing subtype. 4. Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte-expressed chick alpha7 receptors with an IC50 of respectively 94 and 119 nM. 5. High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve-stomach preparations although at different IC50s (49.4 vs 166.2 microM) The effect of MG624 on rat nerve-hemidiaphragm preparations was 33 times less potent than that of F3 (IC50 486 vs 14.5 microM). 6. In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic alphaBgtx receptors containing the alpha7 subunit.

Identification and characterization of two cysteinyl-leukotriene high affinity binding sites with receptor characteristics in human lung parenchyma.

We report the characterization of two distinct binding sites with receptor characteristics for leukotriene (LT)D4 and LTC4 in membranes from human lung parenchyma. The use of S-decyl-glutathione allowed us to characterize a previously unidentified high affinity binding site for LTC4. Computerized analysis of binding data revealed that each leukotriene interacts with two distinct classes of binding sites (Kd = 0.015 and 105 nM for LTC4 and 0.023 and 230 nM for LTD4) and that despite cross-reactivity, the two high affinity sites are different entities. LTD4 binding sites displayed features of G protein-coupled receptors, whereas LTC4 binding sites did not show any significant modulation by guanosine-5'-(beta, gamma-imido)triphosphate or stimulation of GTPase activity. The antagonists ICI 198,615 and SKF 104353 were unselective for the high and low affinity states of LTD4 receptor, whereas only SKF 104353 was able to recognize the two [3H]LTC4 binding sites although with different affinities. These data indicate that in human lung parenchyma, LTD4 and LTC4 recognize two different binding sites; these binding sites are different entities; and for LTD4, the two binding sites represent the interconvertible affinity states of a G protein-coupled receptor, whereas for LTC4, the high affinity site is likely to be a specific LTC4 receptor.

Pharmacological characterization of the cysteinyl-leukotriene antagonists CGP 45715A (iralukast) and CGP 57698 in human airways in vitro.

1. Cysteinyl-leukotrienes (cysteinyl-LTs) are important mediators in the pathogenesis of asthma. They cause bronchoconstriction, mucus hypersecretion, increase in microvascular permeability, plasma extravasation and eosinophil recruitment. 2. We investigated the pharmacological profile of the cysteinyl-LT antagonists CGP 45715A (iralukast), a structural analogue of LTD4 and CGP 57698, a quinoline type antagonist, in human airways in vitro, by performing binding studies on human lung parenchyma membranes and functional studies on human isolated bronchial strips. 3. Competition curves vs [3H]-LTD4 on human lung parenchyma membranes demonstrated that: (a) both antagonists were able to compete for the two sites labelled by [3H]-LTD4; (b) as in all the G-protein coupled receptors, iralukast and CGP 57698 did not discriminate between the high and the low affinity states of the CysLT receptor labelled by LTD4 (Ki1=Ki2= 16.6 nM+/-36% CV and Ki1= Ki2 = 5.7 nM+/-19% CV, respectively); (c) iralukast, but not CGP 57698, displayed a slow binding kinetic, because preincubation (15 min) increased its antagonist potency. 4. In functional studies: (a) iralukast and CGP 57698 antagonized LTD4-induced contraction of human bronchi, with pA2 values of 7.77+/-4.3% CV and 8.51+/-1.6% CV, respectively, and slopes not significantly different from unity; (b) the maximal LTD4 response in the presence of CGP 57698 was actually increased, thus clearly deviating from apparent simple competition. 5. Both antagonists significantly inhibited antigen-induced contraction of human isolated bronchial strips in a concentration-dependent manner, lowering the upper plateau of the anti-IgE curves. 6. In conclusion, the results of the present in vitro investigation indicate that iralukast and CGP 57698 are potent antagonists of LTD4 in human airways, with affinities in the nanomolar range, similar to those obtained for ICI 204,219 and ONO 1078, two of the most clinically advanced CysLT receptor antagonists. Thus, these compounds might be useful drugs for the therapy of asthma and other allergic diseases.