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.