Douglas W. P. Hay

Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14

Urotensin-II (U-II) is a vasoactive ‘somatostatin-like’ cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 (refs 4, 5) and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

Cysteinyl leukotrienes in asthma: old mediators up to new tricks

The cysteinyl leukotrienes have long been suspected to play a role in the pathogenesis of asthma. This speculation was based largely on their release in human lung following antigen challenge as well as their potent bronchoconstrictor activity. However, there is increasing evidence that the cysteinyl leukotrienes also produce several pro-inflammatory effects and alter the activity of neuronal pathways in the airways. Douglas Hay, Theodore Torphy and Bradley Undem review these recent data and discuss the therapeutic possibilities of cysteinyl leukotriene receptor antagonists and 5-lipoxygenase inhibitors.

A Potent and Selective Nonpeptide Antagonist of CXCR2 Inhibits Acute and Chronic Models of Arthritis in the Rabbit

Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits 125I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC50 = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC50 = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-α, IL-8, PGE2, leukotriene B4, and leukotriene C4 levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-α and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC50 = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.

Endothelin‐1‐induced potentiation of human airway smooth muscle proliferation: an ETA receptor‐mediated phenomenon

1 . In this study the mitogenic effects in human cultured tracheal smooth muscle cells of endothelin‐1 (ET‐1), ET‐3, and sarafotoxin S6c (S6c), the ETB receptor‐selective agonist, were explored either alone or in combination with the potent mitogen, epidermal growth factor (EGF). 2 . In confluent, growth‐arrested human airway smooth, neither ET‐1 (0.01 nM‐1μm) nor ET‐3 (0.001 nM‐1 μm) or S6c (0.01 nM‐1 μm) induced cell proliferation, as assessed by [3H]‐thymidine incorporation. In contrast, EGF (1.6 pM − 16 nM) produced concentration‐dependent stimulation of DNA synthesis (EC50 of about 0.06 nM). The maximum increase of about 60 fold above control, elicited by 16nM EGF, was similar to that obtained with 10% foetal bovine serum (FBS). EGF (0.16‐16 nM) also produced a concentration‐dependent increase in cell counts, whereas ET‐1 (1 − 100 nM) was without effect on this index of mitogenesis. 3 . ET‐1 (1–100 nM) potentiated EGF‐induced proliferation of human tracheal smooth muscle cells. For example, ET‐1 (100 nM), which alone was without significant effect, increased by 3.0 to 3.5 fold the mitogenic influence of EGF (0.16 nM). The potentiating effect of ET‐1 on EGF‐induced proliferation was antagonized by BQ‐123 (3 μm), the ETA receptor antagonist, but was unaffected by the ETB receptor antagonist BQ‐788 (10 μm). 4 . Neither ET‐3 (1 − 100 nM) nor S6c (1 − 100 nM) influenced the mitogenic effects of EGF (0.16‐1.6 nM). 5 . [125I]‐ET‐1 binding studies revealed that on average the ratio of ETA to ETB receptors in human cultured tracheal smooth muscle cells was 35:65 (±3; n=4), confirming the predominance of the ETB receptor subtype in human airway smooth muscle. 6 . These data indicate that ET‐1 alone does not induce significant human airway smooth muscle cell proliferation. However, it potently potentiated mitogenesis induced by EGF, apparently via an ETA receptor‐mediated mechanism. These findings suggest that ET‐1, a mediator detected in increased amounts in patients with acute asthma, may potentiate the proliferative effects of mitogens and contribute to the airway smooth muscle hyperplasia associated with chronic severe asthma.

Endothelin receptor subtypes in human and guinea‐pig pulmonary tissues

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‐1 (0.1 nm–0.3 μm) 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 μm), a potent and selective ETA receptor antagonist, potently antagonized ET‐1‐induced contraction in human pulmonary artery (pKB = 6.8 with 1 μm BQ‐123, n = 7) but had no effect in human bronchus (n = 6). 3 Sarafotoxin S6c (0.1 nm–0.1 μm), 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 10 μm carbachol; n = 5. BQ‐123 (1–10 μm) 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 (0.1–10 μm) antagonized ET‐1‐induced contractions in guinea‐pig pulmonary artery (pKB = 6.7 with 1 μm BQ‐123, n = 6), aorta (pKB = 7.1 with 1 μm BQ‐123, n = 6) and trachea (pKB = 6.2 with 1 μm BQ‐123, n = 6) but was without marked effect in bronchus (n = 4). In contrast, sarafotoxin S6c (0.1 nm–0.1 μm) 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 10 μm carbachol, n = 4. Sarafotoxin S6c (0.1 nm–0.1 μm) was a much less effective agonist in guinea‐pig trachea: maximum contraction = 13.9 ± 2.5% of 10 μm carbachol, n = 4; P < 0.0001, compared to bronchus. Contractions produced by sarafotoxin S6c in guinea‐pig bronchus or trachea were unaffected by BQ‐123 (10 μm, 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 10 μm carbachol, n = 6) or lower region of the trachea (48.5 ± 5.9% of 10 μm carbachol, n = 6) than in the middle region of the trachea (14.4 ± 4.0% of 10 μm carbachol, n = 6) or the upper region of the trachea (19.3 ± 2.7% of 10 μm carbachol, n = 6). There were minimal regional differences in either ET‐1‐induced contraction or the potency of BQ‐123 (3 μm) 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 μm) was essentially abolished with 10 μm BQ‐123. 7 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 trachea appear to involve both ETA and non‐ETA (ETB?) receptor subtypes. Furthermore, regional differences appear to exist in the relative distribution of ET receptor subtypes in guinea‐pig airways. In human bronchus ET‐1‐induced prostanoid release, unlike the contractile response, appears to be mediated via ETA receptor activation.

Endothelin and the respiratory system

Pharmacological research involving the endothelin peptides has emphasized their activities in vascular systems, from both physiological and pathophysiological perspectives. However, the endothelins are known also to be synthesized and released from respiratory epithelial cells and to have potent effects in nonvascular components of the respiratory tract. Douglas Hay, Peter Henry and Roy Goldie summarize present understanding of the pharmacology of the endothelins in the respiratory system and assess the potential pathophysiological role in asthma.

Involvement of cysteinyl leukotrienes in airway smooth muscle cell DNA synthesis after repeated allergen exposure in sensitized Brown Norway rats

Airway smooth muscle thickening is a characteristic feature of airway wall remodelling in chronic asthma. We have investigated the role of the leukotrienes in airway smooth muscle (ASM) and epithelial cell DNA synthesis and ASM thickening following repeated allergen exposure in Brown Norway rats sensitized to ovalbumin. There was a 3 fold increase in ASM cell DNA synthesis, as measured by percentage bromodeoxyuridine (BrdU) incorporation, in repeatedly ovalbumin‐exposed (4.1%, 3.6–4.6; mean, 95% c.i.) compared to chronically saline‐exposed rats (1.3%, 0.6–2.1; P<0.001). Treatment with a 5‐lipoxygenase enzyme inhibitor (SB 210661, 10 mg kg−1, p.o.) and a specific cysteinyl leukotriene (CysLT1) receptor antagonist, pranlukast (SB 205312, 30 mg kg−1, p.o.), both attenuated ASM cell DNA synthesis. Treatment with a specific leukotriene B4 (BLT) receptor antagonist (SB 201146, 15 mg kg−1, p.o.) had no effect. There was also a significant, 2 fold increase in the number of epithelial cells incorporating BrdU per unit length of basement membrane after repeated allergen exposure. This response was not inhibited by treatment with SB 210661, pranlukast or SB 201146. A significant increase in ASM thickness was identified following repeated allergen exposure and this response was attenuated significantly by SB 210661, pranlukast and SB 201146. Rats exposed to chronic allergen exhibited bronchial hyperresponsiveness to acetylcholine and had significant eosinophil recruitment into the lungs. Treatment with SB 210661, pranlukast or SB 201146 significantly attenuated eosinophil recruitment into the lungs, whilst having no significant effect on airway hyperresponsiveness. These data indicate that the cysteinyl leukotrienes are important mediators in allergen‐induced ASM cell DNA synthesis in rats, while both LTB4 and cysteinyl leukotrienes contribute to ASM thickening and eosinophil recruitment following repeated allergen exposure.

Airway epithelium-derived inhibitory factor

Various bronchoactive agents can induce the release from the airway epithelium of an inhibitory substance that is able to relax certain tissues including rat aorta and possibly also airway smooth muscle. This substance, whose existence has recently been confirmed using a new bioassay system, is distinct from nitric oxide (EDRF) and is also known to be non-prostanoid in nature. Roy Goldie and colleagues describe the properties of this factor, and its potential clinical significance.

Receptors for endothelin‐1 in asthmatic human peripheral lung

[125I]‐endothelin‐1 ([125I]‐ET‐1) binding was assessed by autoradiography in peripheral airway smooth muscle and alveolar wall tissue in human non‐asthmatic and asthmatic peripheral lung. Levels of specific binding to these structures were similar in both non‐asthmatic and asthmatic lung. The use of the receptor subtype‐selective ligands, BQ‐123 (ETA) and sarafotoxin S6c (ETB), demonstrated the existence of both ETA and ETB sites in airway smooth muscle and in alveoli. In airway smooth muscle from both sources, the great majority of sites were of the ETB subtype. Quantitative analyses of asthmatic and non‐asthmatic alveolar wall tissue demonstrated that 29–32% of specific [125I]‐ET‐1 binding was to ETA sites and 68–71% was to ETB sites. Thus, asthma was not associated with any significant alteration in the densities of ETA and ETB receptors in peripheral human lung.

Endothelin‐induced contraction and mediator release in human bronchus

1 To elucidate the role of acetylcholine and various autacoids in endothelin‐1 (ET‐1)‐induced contraction in human bronchus, the effects of various receptor antagonists were examined. In addition, the ability of ET‐1 to stimulate the release of histamine, peptidoleukotrienes and prostanoids was determined. 2 ET‐1 was a potent and effective contractile agonist in human bronchus, possessing similar potency and efficacy to leukotriene D4 (LTD4); EC50 (−log m): ET‐1 = 7.76 ± 0.09, n = 7; LTD4 = 8.46 ± 0.53, n = 7; P > 0.2; maximum response (% 10 μm pre‐carbachol): ET‐1 = 103.8 ± 17.4, n = 7; LTD4 = 95.5 ± 9.3, n = 7; P > 0.6. 3 The cyclo‐oxygenase inhibitor, sodium meclofenamate (1 μm) or the potent and selective thromboxane receptor antagonist, SQ 29,548 (1 μm) were without significant effect on ET‐1 concentration‐response curves. 4 In the presence of sodium meclofenamate (1 μm), the muscarinic receptor antagonist, atropine (1 μm), the platelet activating factor (PAF) receptor antagonist, WEB 2086 (1 μm) or the combination of the H1‐histamine receptor antagonist, mepyramine (10 μm) and the leukotriene receptor antagonist, SK&F 104353 (10 μm), were without marked effect on ET‐1 concentration‐response curves. In addition, the combination of all four receptor antagonists did not antagonize ET‐1‐induced contraction. 5 ET‐1 (0.3 μm) did not stimulate the release of histamine or immunoreactive leukotrienes from human bronchus. 6 ET‐1 (0.3 μm) significantly stimulated the release of prostaglandin D2 (PGD2), 9α, 11β PGF2 (PGD2 metabolite), PGE2, 6‐keto PGF1α (PGI2 metabolite), PGF2α and thromboxane B2 (TxB2) a lower concentration, 10 nm, was without effect on prostanoid release. The production of PGD2 was increased 7.5 fold, whereas the release of the other prostanoids was stimulated only about 1.6 to 2.7 fold. 7 These data provide evidence that ET‐1 elicits contraction of human isolated bronchus predominantly via a direct mechanism with no significant involvement of the release of acetylcholine, leukotrienes, histamine or PAF. Although ET‐1 increased the release of several prostanoids they did not have a significant modulatory effect on the smooth muscle contraction.