Yasushi Fujitani

Pronounced eosinophilic lung inflammation and Th2 cytokine release in human lipocalin-type prostaglandin D synthase transgenic mice

PGD2 is a major lipid mediator released from mast cells, but little is known about its role in the development of allergic reactions. We used transgenic (TG) mice overexpressing human lipocalin-type PGD synthase to examine the effect of overproduction of PGD2 in an OVA-induced murine asthma model. The sensitization of wild-type (WT) and TG mice was similar as judged by the content of OVA-specific IgE. After OVA challenge, PGD2, but not PGE2, substantially increased in the lungs of WT and TG mice with greater PGD2 increment in TG mice compared with WT mice. The numbers of eosinophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid were significantly greater in TG mice than in WT mice on days 1 and 3 post-OVA challenge, whereas the numbers of macrophages and neutrophils were the same in both WT and TG mice. The levels of IL-4, IL-5, and eotaxin in BAL fluid were also significantly higher in TG mice than in WT mice, although the level of IFN-γ in the BAL fluid of TG mice was decreased compared with that in WT mice. Furthermore, lymphocytes isolated from the lungs of TG mice secreted less IFN-γ than those from WT mice, whereas IL-4 production was unchanged between WT and TG mice. Thus, overproduction of PGD2 caused an increase in the levels of Th2 cytokines and a chemokine, accompanied by the enhanced accumulation of eosinophils and lymphocytes in the lung. These results indicate that PGD2 plays an important role in late phase allergic reactions in the pathophysiology of bronchial asthma.

A potent and specific agonist, Suc-[Glu9, Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.

Inducible Nitric Oxide Synthase Inhibitors Suppress Airway Inflammation in Mice Through Down-Regulation of Chemokine Expression

Growing evidence demonstrates that inducible NO synthase (iNOS) is induced in the airways of asthmatic patients. However, the precise role of NO in the lung inflammation is unknown. This study investigated the effect of both selective and nonselective iNOS inhibitors in an allergen-driven murine lung inflammation model. OVA challenge resulted in an accumulation of eosinophils and neutrophils in the airways. Expression of iNOS immunostaining in lung sections together with an increase in calcium-independent NOS activity in lung homogenates was also observed after OVA challenge. Treatment with iNOS inhibitors from the day of challenge to the day of sacrifice resulted in an inhibition of the inflammatory cell influx together with a down-regulation of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 production. In contrast, eosinophilic and neutrophilic inhibition was not observed with treatment during the sensitization. Both treatments induced an increased production of Th2-type cytokines (IL-4 and IL-5) with a concomitant decrease in production of Th1-type cytokine (IFN-γ). In vitro exposure of primary cultures of murine lung fibroblasts to a NO donor, hydroxylamine, induced a dose-dependent release of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1. Our results suggest that lung inflammation after allergen challenge in mice is partially dependent on NO produced mainly by iNOS. NO appears to increase lung chemokine expression and, thereby, to facilitate influx of inflammatory cells into the airways.

An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11–21)

In the inhibition of specific binding or [125]endothelins (ETs) to membranes from various tissues of rats, guinea pigs, pigs and humans, [Cys11‐Cys15]‐ET‐1(11–21), IRL 1038, has a much higher affinity for ETB receptors (K i = 6–11 nM) than for ETA receptors (K i = 0.4–0.7 μM). In contraction assays, with ET‐3 as a stimulant, 3 μM IRL 1038 antagonized the ETB receptor‐mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor‐mediated contraction of rat aortic smooth muscle. IRL 1038 is, therefore, considered to be the first antagonist selective to the ETB receptor.

IL‐5 deficiency abolishes aspects of airway remodelling in a murine model of lung inflammation

Background and objectives Lung remodelling is a recognized feature of chronic asthma. In the present study, we have used IL‐5‐deficient mice to evaluate the role of this cytokine and eosinophilic inflammation in the initial stages of the structural changes occurring in the lung after antigen challenge.

Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein

Human umbilical vein endothelial cells (HUVECs) in primary culture produced and secreted endothelin I (ET‐1) actively. Specific binding of [125I]ET‐1 to these cells was not detectable because of the saturation of ET receptors with endogenously produced ET‐1. However, addition of phosphoramidon, an inhibitor of ET‐converting enzyme, to the medium reduced the production of ET‐1 and thus the receptors on HUVECs were made available for exogenously added [125I]ET‐1. Binding studies using phosphoramidon‐treated HUVECs indicated the existence of a non‐isopeptide‐selective type (ETB) or ET receptor with a K d of 17 pM. This receptor is thought to be involved in ET‐induced vasodilation in autocrine manner in vivo.

Endothelin stimulates both cAMP formation and phosphatidylinositol hydrolysis in cultured embryonic bovine tracheal cells

Embryonic bovine tracheal (EBTr) cells were found to possess receptors for endothelin (ET) of ET‐1‐selective (ETA)subtype with a K d for ET‐1 of 114 pM and a B max of 12.9 fmol/105 cells. Stimulation of EBTr cells with 100 pM to 100 nM ET‐1 increased the contents of both inositol phosphates and cAMP in a concentration‐dependent manner, indicating that the receptors are coupled to both phosphatidylinositol hydrolysis and cAMP formation in EBTr cells.

Retraction concerning an endothelin B receptor-selective antagonist: Urade, Y., Fujitani, Y., Oda, K., Watakabe, T., Umemura, L., Takai, M., Okada, T., Sakata, K. and Karaki, H. (1992) FEBS Lett. 311, 12-16 : An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11-21)

In the inhibition of specific binding of [125I]endothelins (ETs) to membrane from various tissues of rats, guinea pigs, pigs and humans, [Cys11-Cys15]-ET-1(11-21), IRL 1038, has a much higher affinity for ETB receptors (Ki = 6-11 nM) than for ETA receptors (Ki = 0.4-0.7 microM). In contraction assays, with ET-3 as a stimulant, 3 microM IRL 1038 antagonized the ETB receptor-mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor-mediated contraction of rat aortic smooth muscle. IRL 1038 is therefore, considered to be the first antagonist selective to the ETB receptor.The article describes the synthetic peptide IRL 1038 as having a much higher affinity to ETB receptors (Ki = 6 11 nM) than to ET, receptors (Ki = 4OC700 nM) in various tissue membranes. It was also shown that this peptide antagonized the ET,-mediated contraction of guinea pig ileal and tracheal smooth muscles. Since the publication of the data, we have synthesized more than 20 different batches of IRL 1038, some of which showed potencies and selectivities almost equivalent to the published data. However, the other batches, including batches synthesized by external suppliers, exhibited significantly higher Ki of 5&400 nM for the binding to ET, receptors in porcine lung membranes. We have very carefully investigated the physico-chemical properties of IRL 1038 in aqueous solutions at different pH (pH 7-10) and peptide concentrations, using fluorescence and proton NMR spectroscopy. At high pH (pH lo), the peptide does not aggregate and two kinds of non-aggregated states were observed by NMR spectroscopy. There was, however, no evident correlation between the two non-aggregated states and binding af-

Modulation of ET-1-induced contraction of human bronchi by airway epithelium-dependent nitric oxide release via ETA receptor activation

The purpose of this work was to investigate whether endothelin‐1 (ET‐1) was able to induce the release of an inhibitory factor from the airway epithelium in isolated human bronchi and to identify this mediator as well as the endothelin receptor involved in this phenomenon. In intact bronchi, ET‐1 induced a concentration‐dependent contraction (−logEC50=7.92±0.09, n=18) which was potentiated by epithelium removal (−logEC50=8.65±0.11, n=17). BQ‐123, an ETA receptor antagonist, induced a significant leftward shift of the ET‐1 concentration‐response curve (CRC). This leftward shift was abolished after epithelium removal. L‐NAME (3×10−3 M), an inhibitor of nitric oxide (NO) synthase, induced a significant leftward shift of the ET‐1 CRC, and abolished the potentiation by BQ‐123 (10−8 M) of ET‐1‐induced contraction. In intact preparations, the ETB receptor antagonist BQ‐788 induced only at 10−5 M a slight rightward shift of the ET‐1 CRC. In contrast, in epithelium‐denuded bronchi or in intact preparations in the presence of L‐NAME, BQ‐788 displayed a non‐competitive antagonism toward ET‐1‐induced contraction. IRL 1620, a selective ETB receptor agonist, induced a contraction of the isolated bronchus (−logEC50=7.94±0.11, n=19). This effect was not modified by epithelium removal or by BQ‐123. BQ‐788 exerted a competitive antagonism against IRL 1620 which was similar in the presence or absence of epithelium. These results show that ET‐1 exerts two opposite effects on the human airway smooth muscle. One is contractile via ETB‐receptor activation, the other is inhibitory and responsible of NO release which counteracts via ETA‐receptor activation the contraction.

Effect of a novel bifunctional endothelin receptor antagonist, IRL 3630A, on guinea pig respiratory mechanics.

This study characterized the in vitro pharmacological properties of a newly developed endothelin receptor antagonist, N-butanesulfonyl-[N-(3, 5-dimethylbenzoyl)-N-methyl-3-[4-(5-isoxazolyl)-phenyl]-(D)- alanyl]-( L)-valineamide sodium salt (IRL 3630A), and its in vivo effects on respiratory mechanics were determined. IRL 3630A showed highly balanced affinities to human endothelin ET(A) and ET(B) receptors, giving apparent K(i) values of 1.5 and 1.2 nM, respectively. This compound also potently antagonized the endothelin-1-induced intracellular Ca(2+) increases in both embryonic bovine tracheal (EBTr) cells expressing endothelin ET(A) receptors and human Girardi heart (hGH) cells expressing endothelin ET(B) receptors. In guinea pig isolated tracheas having both endothelin ET(A) and ET(B) receptors, IRL 3630A greatly inhibited endothelin-1-induced contraction (pA(2)=7.1), which was partially or scarcely suppressed by the endothelin ET(A) receptor antagonist cyclo[-(D)-Trp-(D)-Asp-(L)-Pro-(D)-Val-(L)-Leu-] (BQ-123) or the endothelin ET(B) receptor antagonist N-(3, 5-dimethylbenzoyl)-N-methyl-3-(4-phenyl)-(D)-phenylalanyl-(L)-t ryptop han (IRL 2500), respectively. Bolus i.v. injections of IRL 3630A administered into anaesthetized guinea pigs at 10 and 30 microg/kg inhibited endothelin-1 (1.3 microg/kg)-induced changes in respiratory resistance and compliance in a dose dependent manner, whereas both sodium 2-benzo[1, 3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4, 5-trimethoxy-benzyl)-but-2-enoate (an endothelin ET(A) receptor antagonist: PD 156707) and IRL 2500 at doses of up to 30 microg/kg did not affect endothelin-1-induced changes in respiratory mechanics, reflecting the in vitro results. IRL 3630A is thus an effective bifunctional endothelin receptor antagonist, and will be useful in clarifying the role of endothelin in pulmonary diseases such as bronchial asthma.