The cysteinyl leukotrienes (CysLTs) mimic many of the features of asthma and are implicated in its pathophysiology. Little, however, is known about the effects of the CysLTs on airways remodeling. In this study the effects of leukotriene D4 (LTD4) on human airway smooth muscle (HASM) cell proliferation and expression of extracellular matrix proteins were investigated. LTD4 (0.1-10 microM) alone had no effect on DNA synthesis in HASM. LTD4, however, markedly augmented proliferation induced by the mitogen, epidermal growth factor (EGF, 1 ng/ml). The potentiating effect of LTD4 (1 microM) on EGF-induced DNA synthesis was abolished by pranlukast (1 microM) or pobilukast (30 microM), but unaffected by zafirlukast (1 microM). In contrast, pranlukast (pKB = 6.9), pobilukast (pKB = 7.0), and zafirlukast (pKB = 6.5) had equivalent potencies for inhibition of LTD4-induced contraction in human bronchus. LTD4 (0.1 or 10 microM) did not increase the total messenger RNA expression of the extracellular matrix proteins (pro-alpha[I] type I or alpha1[IV] type IV collagen), elastin, biglycan, decorin, and fibronectin, and did not influence tumor growth factor-beta (10 ng/ml)-induced effects on the expression of these proteins in HASM cells. These data indicate that LTD4 augments growth factor-induced HASM proliferation but does not alter the expression of various extracellular matrix components. The observed differences in sensitivity to the antagonists suggests that the former phenomenon may be mediated by a CysLT receptor distinct from that which mediates LTD4-induced HASM contraction. Collectively, these results provide preliminary evidence that CysLTs may play a role in airways remodeling in asthma.
1 In this study the endothelin (ET) receptor subtypes mediating contractions produced by ET-1 in human and guinea-pig pulmonary tissues were investigated. In addition the receptor responsible for ET-1-induced prostanoid release in human bronchus was determined.2 In human bronchus and human pulmonary artery ET-l (0.1 nM-0.3 JIM) was a potent and effective contractile agent (pD2 = 7.58 + 0.15, n = 6, and 8.48 + 0.11, n = 7, respectively). BQ-123 (1-10 JiM), a potent and selective ETA receptor antagonist, potently antagonized ET-1-induced contraction in human pulmonary artery (pKB = 6.8 with 1 JIM BQ-123, n = 7) but had no effect in human bronchus (n = 6). 3 Sarafotoxin S6c (0.1 nM-0. IliM), the ETB-selective agonist, did not contract human pulmonary artery (n = 5), but potently and effectively contracted human bronchus: pD2 = 8.41 ± 0.17, maximum response =74.4 ± 3.1% of 1O JM carbachol; n = 5. BQ-123 (1-1 I1M) did not antagonize sarafotoxin S6c-induced contraction in human bronchus (n = 5). 4 ET-1 potently contracted guinea-pig trachea, bronchus, pulmonary artery and aorta (pD2 = 8.15 + 0.14, 7.72 + 0.12, 8.52 + 0.12, and 8.18 ± 0.12, respectively, n = 6-14). BQ-123 (0.1-101M) antagonized ET-1-induced contractions in guinea-pig pulmonary artery (pKB = 6.7 with 1 JIM BQ-123, n = 6), aorta (pKB = 7.1 with 1 JIM BQ-123, n = 6) and trachea (pKB = 6.2 with 1 AIM BQ-123, n = 6) but was without marked effect in bronchus (n = 4). In contrast, sarafotoxin S6c (0.1 nM-0.l JIM) did not contract guinea-pig aorta (n = 4) or guinea-pig pulmonary artery (n = 6) but potently and effectively contracted guinea-pig bronchus: pD2= 8.55 + 0. 1; maximum contraction = 63.6 ± 3.1% of 1O JIM carbachol, n = 4. Sarafotoxin S6c (0.1 nM-0. 1 JIM) was a much less effective agonist in guinea-pig trachea: maximum contraction = 13.9 ± 2.5% of 10 JM carbachol, n = 4; P < 0.0001, compared to bronchus. Contractions produced by sarafotoxin S6c in guinea-pig bronchus or trachea were unaffected by (IO IM, n=4). 5 Significant differences were observed in the efficacy, relative to carbachol, but not the potency of sarafotoxin S6c in guinea-pig airways, with a much greater maximum contractile response in bronchus (69.6 ± 2.4% of 1O JM carbachol, n = 6) or lower region of the trachea (48.5 ± 5.9% of 1OJIM carbachol, n = 6) than in the middle region of the trachea (14.4 ± 4.0% of 10 JiM carbachol, n = 6) or the upper region of the trachea (19.3 ± 2.7% of 1O JM carbachol, n = 6). There were minimal regional differences in either ET-1-induced contraction or the potency of BQ-123 (3 JIM) for inhibition of responses to ET-1 in guinea-pig airways. 6 Release of various prostanoids in human bronchus induced by ET-1 (0.3 JiM) was essentially abolished with 10 IM These data provide evidence that distinct ET receptors mediate ET-1-induced contraction in human pulmonary artery, guinea-pig pulmonary artery and guinea-pig aorta (ETA subtype) compared with human bronchus and guinea-pig bronchus (non-ETA, perhaps ETB subtype). Contractions to ET-1 in guinea-pig trache...
The potent bronchoconstrictor and mitogenic actions of the peptide endothelin-1 (ET-1) on airway smooth muscle may contribute significantly to the bronchial obstruction observed in asthma. However, the status of the receptor-effector systems that mediate these actions of ET-1 in asthmatic airways is currently unknown. Thus, we have used quantitative autoradiographic and isometric-tension recording techniques to evaluate the density, distribution, and function of the specific receptors that mediate the actions of ET-1 in both asthmatic and nonasthmatic airways. Here, we report that similar numbers of specific binding sites for [125I]-ET-1 exist in asthmatic and nonasthmatic airways, with the greatest densities located in airway smooth muscle in both tissue types. The ETB-receptor subtype constituted approximately 82% and 88% of these receptors for ET-1 in asthmatic and nonasthmatic human bronchial smooth muscle, respectively, and mediated contraction in response to this peptide. In addition, a component of ET-1-induced contraction appeared to be mediated by a non-ETB, BQ-123-resistant mechanism. Furthermore, a small population of ETA sites was identified that did not mediate contraction, but which may have a role in ET-1-induced prostanoid release and airway smooth-muscle proliferation. Interestingly, bronchial smooth muscle from asthmatic lung was significantly less sensitive to the contractile effects of ETB receptor activation, consistent with desensitization of this receptor subtype in response to the increased production and release of ET-1 that occurs in this disease.
A novel class of potent and selective non-peptide neurokinin-3 (NK-3) receptor antagonists, featuring the 4-quinolinecarboxamide framework, has been designed based upon chemically diverse NK-1 receptor antagonists. The novel compounds 33-76, prompted by chemical modifications of the prototype 4, have been characterized by binding analysis using a membrane preparation of chinese hamster ovary (CHO) cells expressing the human neurokinin-3 receptors (hNK-3-CHO), and clear structure-activity relationships (SARs) have been established. From SARs, (R)-N-[alpha-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (65, SB 218795, hNK-3-CHO binding Ki = 13 nM) emerged as one of the most potent compounds of this novel class. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 65 is about 90-fold selective for hNK-3 versus hNK-2 receptors (hNK-2-CHO binding Ki = 1221 nM) and over 7000-fold selective versus hNK-1 receptors (hNK-1-CHO binding Ki = > 100 microM). In vitro functional studies in rabbit isolated iris sphincter muscle preparation demonstrated that 65 is a competitive antagonist of the contractile response induced by the potent and selective NK-3 receptor agonist senktide with a Kb = 43 nM. Overall, the data indicate that 65 is a potent and selective hNK-3 receptor antagonist and a useful lead for further chemical optimization.
Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl) bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA 2 5 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED 50 value was 0.02 mg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 mg elicited 50% bronchoprotection for .24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.
Optimization of the previously reported 2-phenyl-4-quinolinecarboxamide NK-3 receptor antagonist 14, with regard to potential metabolic instability of the ester moiety and affinity and selectivity for the human neurokinin-3 (hNK-3) receptor, is described. The ester functionality could be successfully replaced by the ketone (31) or by lower alkyl groups (Et, 21, or n-Pr, 24). Investigation of the substitution pattern of the quinoline ring resulted in the identification of position 3 as a key position to enhance hNK-3 binding affinity and selectivity for the hNK-3 versus the hNK-2 receptor. All of the chemical groups introduced at this position, with the exception of halogens, increased the hNK-3 binding affinity, and compounds 53 (3-OH, SB 223412, hNK-3-CHO binding Ki = 1.4 nM) and 55 (3-NH2, hNK-3-CHO binding Ki = 1.2 nM) were the most potent compounds of this series. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 53 is about 100-fold selective for the hNK-3 versus hNK-2 receptor, with no affinity for the hNK-1 at concentrations up to 100 microM. In vitro studies demonstrated that 53 is a potent functional antagonist of the hNK-3 receptor (reversal of senktide-induced contractions in rabbit isolated iris sphincter muscles and reversal of NKB-induced Ca2+ mobilization in CHO cells stably expressing the hNK-3 receptor), while in vivo this compound showed oral and intravenous activity in NK-3 receptor-driven models (senktide-induced behavioral responses in mice and senktide-induced miosis in rabbits). Overall, the biological data indicate that (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (53, SB 223412) may serve as a pharmacological tool in animal models of disease to assess the functional and pathophysiological role of the NK-3 receptor and to establish therapeutic indications for non-peptide NK-3 receptor antagonists.
In this report the in vitro and in vivo pharmacological and pharmacokinetic profile of (Ϫ)-(S)-N-(␣-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB 235375), a low central nervous system (CNS)-penetrant, human neurokinin-3 (NK-3) receptor (hNK-3R) antagonist, is described. SB 235375 inhibited 125 I- [MePhe 7 ]-neurokinin B (NKB) binding to membranes of Chinese hamster ovary (CHO) cells expressing the hNK-3R (CHO-hNK-3R) with a K i ϭ 2.2 nM and antagonized competitively NKB-induced Ca 2ϩ mobilization in human embryonic kidney (HEK) 293 cells expressing the hNK-3R (HEK 293-hNK-3R) with a K b ϭ 12 nM. SB 235375 antagonized senktide (NK-3R)-induced contractions in rabbit isolated iris sphincter (pA 2 ϭ 8.1) and guinea pig ileal circular smooth muscles (pA 2 ϭ 8.3). SB 235375 was selective for the hNK-3R compared with hNK-1 (K i Ͼ 100,000 nM) and hNK-2 receptors (K i ϭ 209 nM), and was without effect, at 1 M, in 68 other receptor, enzyme, and ion channel assays. Intravenous SB 235375 produced a dose-related inhibition of miosis induced by i.v. senktide in the rabbit (ED 50 of 0.56 mg/kg). Intraperitoneal SB 235375 (10 -30 mg/kg) inhibited citric acid-induced cough and airways hyper-reactivity in guinea pigs. In mice oral SB 235375 (3-30 mg/kg) was without significant effect on the behavioral responses induced by intracerebral ventricular administration of senktide. Pharmacokinetic evaluation in the mouse and rat revealed that oral SB 235375 was well absorbed systemically but did not effectively cross the blood-brain barrier. The preclinical profile of SB 235375, encompassing high affinity, selectivity, oral activity, and low CNS penetration, suggests that it is an appropriate tool compound to define the pathophysiological roles of the NK-3Rs in the peripheral nervous system.
Prior studies have demonstrated that the ion channel transient receptor potential vanilloid 4 (TRPV4) is functionally expressed in airway smooth muscle cells and that TRPV4 single nucleotide polymorphisms are associated with airflow obstruction in patients with chronic obstructive pulmonary disease. We sought to use isometric tension measurements in ex vivo airways to determine whether short-term pharmacological activation of TRPV4 with thewould constrict human bronchial tissue. As predicted, transient receptor potential vanilloid 4 activation in the human airway produces contractions that are blocked by the nonselective transient receptor potential channel blocker ruthenium red. Moreover, the novel TRPV4-selective blocker GSK2334775 [(R)-6-(methylsulfonyl)-3-((4-(pyrrolidin-1-yl)piperindin-1-yl)methyl)-N-(2,2,2,-trifluoro-1-phenylethyl)-2-(3-(trifluoromethyl)phenyl)quinoline-4-carboxamide] inhibited these contractions over a concentration range consistent with its in vitro potency against recombinant and native TRPV4-containing channels. Surprisingly, TRPV4-dependent contractions were also blocked by a 5-lipoxygenase inhibitor and two structurally distinct cysteinyl leukotriene 1 receptor antagonists. In aggregate, our results fail to support the hypothesis that TRPV4 in airway smooth muscle cells regulates airway contractility short term. Rather, we provide pharmacological evidence that TRPV4 activation causes human airway constriction that is entirely dependent upon the production of cysteinyl leukotrienes. Together, these data identify a novel mechanism by which TRPV4 activation may contribute to pathologic remodeling and inflammation, in addition to airflow obstruction, in the diseased human respiratory tract.
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