Azithromycin inhibits mucin secretion, mucous metaplasia, airway inflammation, and airways hyperresponsiveness in mice exposed to house dust mite extract.

Excessive production, secretion, and retention of abnormal mucus is a pathological feature of many obstructive airways diseases including asthma. Azithromycin is an antibiotic that also possesses immunomodulatory and mucoregulatory activities, which may contribute to the clinical effectiveness of azithromycin in asthma. The current study investigated these nonantibiotic activities of azithromycin in mice exposed daily to intranasal house dust mite (HDM) extract for 10 days. HDM-exposed mice exhibited airways hyperresponsiveness to aerosolized methacholine, a pronounced mixed eosinophilic and neutrophilic inflammatory response, increased airway smooth muscle (ASM) thickness, and elevated levels of epithelial mucin staining. Azithromycin (50 mg/kg sc, 2 h before each HDM exposure) attenuated HDM-induced airways hyperresponsiveness to methacholine, airways inflammation (bronchoalveolar lavage eosinophil and neutrophils numbers, and IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and RANTES levels), and epithelial mucin staining (mucous metaplasia) by at least 50% (compared with HDM-exposed mice, P < 0.05). Isolated tracheal segments of HDM-exposed mice secreted Muc5ac and Muc5b (above baseline levels) in response to exogenous ATP. Moreover, ATP-induced secretion of mucins was attenuated in segments obtained from azithromycin-treated, HDM-exposed mice (P < 0.05). In additional ex vivo studies, ATP-induced secretion of Muc5ac (but not muc5b) from HDM-exposed tracheal segments was inhibited by in vitro exposure to azithromycin. In vitro azithromycin also inhibited ATP-induced secretion of Muc5ac and Muc5b in tracheal segments from IL-13-exposed mice. In summary, azithromycin inhibited ATP-induced mucin secretion and airways inflammation in HDM-exposed mice, both of which are likely to contribute to suppression of airways hyperresponsiveness.

Pharmacological ablation of the airway smooth muscle layer-Mathematical predictions of functional improvement in asthma.

Airway smooth muscle (ASM) plays a major role in acute airway narrowing and reducing ASM thickness is expected to attenuate airway hyper-responsiveness and disease burden. There are two therapeutic approaches to reduce ASM thickness: (a) a direct approach, targeting specific airways, best exemplified by bronchial thermoplasty (BT), which delivers radiofrequency energy to the airway via bronchoscope; and (b) a pharmacological approach, targeting airways more broadly. An example of the less well-established pharmacological approach is the calcium-channel blocker gallopamil which in a clinical trial effectively reduced ASM thickness; other agents may act similarly. In view of established anti-proliferative properties of the macrolide antibiotic azithromycin, we examined its effects in naive mice and report a reduction in ASM thickness of 29% (p < .01). We further considered the potential functional implications of this finding, if it were to extend to humans, by way of a mathematical model of lung function in asthmatic patients which has previously been used to understand the mechanistic action of BT. Predictions show that pharmacological reduction of ASM in all airways of this magnitude would reduce ventilation heterogeneity in asthma, and produce a therapeutic benefit similar to BT. Moreover there are differences in the expected response depending on disease severity, with the pharmacological approach exceeding the benefits provided by BT in more severe disease. Findings provide further proof of concept that pharmacological targeting of ASM thickness will be beneficial and may be facilitated by azithromycin, revealing a new mode of action of an existing agent in respiratory medicine.

Functional characterisation and application of an ex vivo perfusion-superfusion system in murine airways.

INTRODUCTION: The aim of this study was to develop two dynamic ex vivo airway explant systems, a perfusion-superfusion system and a ventilation-superfusion system, for the study of toxic airborne substances, such as the prevalent smoke constituent acrolein. METHODS: Mouse isolated tracheal segments were perfused with physiological media or ventilated with humidified air at 37°C to mimic dynamic flow conditions, and superfused with media over the exterior surface. At selected time points, the histological and functional integrity of segments was evaluated. The perfusion-superfusion system was subsequently used to examine mucin secretory responses elicited by acrolein in airways in which mucous metaplasia had been induced with lipopolysaccharide (LPS; 1µgml-1) prior to 24h of media perfusion, followed by stimulation with acrolein or ATP for 15min. Epithelial mucin levels were determined by quantitative analysis of periodic acid-Schiff's reagent (PAS)-stained sections. RESULTS: Epithelial morphology was successfully preserved in the perfusion-superfusion and ventilation-superfusion systems for at least 24h and up to 18h, respectively. At these time points, the contractile and relaxation responses of perfused and ventilated tracheal segments to carbachol, the neuropeptide substance P, and the prostanoid PGE2 were also preserved. Using the perfusion-superfusion system, acute exposure to acrolein caused a dose-dependent reduction in the levels of PAS-positive mucin stores induced by LPS, consistent with mucin secretion. DISCUSSION: Both the perfusion-superfusion and ventilation-superfusion systems successfully preserved the viability of mouse isolated tracheal segments on a histological and functional level, and the perfusion-superfusion system was used to characterise the mucin secretory responses elicited by acrolein. Thus, this system may be a useful model through which to conduct further toxicological studies in mammalian airways.

Investigating the role of MRGPRC11 and capsaicin-sensitive afferent nerves in the anti-influenza effects exerted by SLIGRL-amide in murine airways.

BACKGROUND: The hexapeptide SLIGRL-amide activates protease-activated receptor-2 (PAR-2) and mas-related G protein-coupled receptor C11 (MRGPRC11), both of which are known to be expressed on populations of sensory nerves. SLIGRL-amide has recently been reported to inhibit influenza A (IAV) infection in mice independently of PAR-2 activation, however the explicit roles of MRGPRC11 and sensory nerves in this process are unknown. Thus, the principal aim of this study was to determine whether SLIGRL-amide-induced inhibition of influenza infection is mediated by MRGPRC11 and/or by capsaicin-sensitive sensory nerves. METHODS: The inhibitory effect of SLIGRL-amide on IAV infection observed in control mice in vivo was compared to effects produced in mice that did not express MRGPRC11 (mrgpr-cluster∆ (-/-) mice) or had impaired sensory nerve function (induced by chronic pre-treatment with capsaicin). Complementary mechanistic studies using both in vivo and ex vivo approaches investigated whether the anti-IAV activity of SLIGRL-amide was (1) mimicked by either activators of MRGPRC11 (BAM8-22) or by activators (acute capsaicin) or selected mediators (substance P, CGRP) of sensory nerve function, or (2) suppressed by inhibitors of sensory nerve function (e.g. NK1 receptor antagonists). RESULTS: SLIGRL-amide and BAM8-22 dose-dependently inhibited IAV infection in mrgpr-cluster∆ (-/-) mice that do not express MRGPRC11. In addition, SLIGRL-amide and BAM8-22 each inhibited IAV infection in capsaicin-pre-treated mice that lack functional sensory nerves. Furthermore, the anti-IAV activity of SLIGRL-amide was not mimicked by the sensory neuropeptides substance P or CGRP, nor blocked by either NK1 (L-703,606, RP67580) and CGRP receptor (CGRP8-37) antagonists. Direct stimulation of airway sensory nerves through acute exposure to the TRPV1 activator capsaicin also failed to mimic SLIGRL-amide-induced inhibition of IAV infectivity. The anti-IAV activity of SLIGRL-amide was mimicked by the purinoceptor agonist ATP, a direct activator of mucus secretion from airway epithelial cells. Additionally, both SLIGRL-amide and ATP stimulated mucus secretion and inhibited IAV infectivity in mouse isolated tracheal segments. CONCLUSIONS: SLIGRL-amide inhibits IAV infection independently of MRGPRC11 and independently of capsaicin-sensitive, neuropeptide-releasing sensory nerves, and its secretory action on epithelial cells warrants further investigation.

Influenza A infection attenuates relaxation responses of mouse tracheal smooth muscle evoked by acrolein.

The airway epithelium is an important source of relaxant mediators, and damage to the epithelium caused by respiratory tract viruses may contribute to airway hyperreactivity. The aim of this study was to determine whether influenza A-induced epithelial damage would modulate relaxation responses evoked by acrolein, a toxic and prevalent component of smoke. Male BALB/c mice were inoculated intranasally with influenza A/PR-8/34 (VIRUS-infected) or allantoic fluid (SHAM-infected). On day 4 post-inoculation, isometric tension recording studies were conducted on carbachol pre-contracted tracheal segments isolated from VIRUS and SHAM mice. Relaxant responses to acrolein (30 µM) were markedly smaller in VIRUS segments compared to SHAM segments (2 ± 1% relaxation vs. 28 ± 5%, n=14, p<0.01). Similarly, relaxation responses of VIRUS segments to the neuropeptide substance P (SP) were greatly attenuated (1 ± 1% vs. 47 ± 6% evoked by 1 nM SP, n=14, p<0.001). Consistent with epithelial damage, PGE2 release in response to both acrolein and SP were reduced in VIRUS segments (>35% reduction, n=6, p<0.01), as determined using ELISA. In contrast, exogenous PGE2 was 2.8-fold more potent in VIRUS relative to SHAM segments (-log EC50 7.82 ± 0.14 vs. 7.38 ± 0.05, n=7, p<0.01) whilst responses of VIRUS segments to the ß-adrenoceptor agonist isoprenaline were similar to SHAM segments. In conclusion, relaxation responses evoked by acrolein were profoundly diminished in tracheal segments isolated from influenza A-infected mice. The mechanism through which influenza A infection attenuates this response appears to involve reduced production of PGE2 in response to SP due to epithelial cell loss, and may provide insight into the airway hyperreactivity observed with influenza A infection.

Airborne acrolein induces keratin-8 (Ser-73) hyperphosphorylation and intermediate filament ubiquitination in bronchiolar lung cell monolayers.

The combustion product acrolein is a key mediator of pulmonary edema in victims of smoke inhalation injury. Since studying acrolein toxicity in conventional in vitro systems is complicated by reactivity with nucleophilic culture media constituents, we explored an exposure system which delivers airborne acrolein directly to lung cell monolayers at the air-liquid interface. Calu-3 lung adenocarcinoma cells were maintained on membrane inserts such that the basal surface was bathed in nucleophile-free media while the upper surface remained in contact with acrolein-containing air. Cells were exposed to airborne acrolein for 30 min before they were allowed to recover in fresh media, with cell sampling at defined time points to allow evaluation of toxicity and protein damage. After prior exposure to acrolein, cell ATP levels remained close to controls for 4h but decreased in an exposure-dependent manner by 24h. A loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-labeled dextran preceded ATP loss. Use of antibody arrays to monitor protein expression in exposed monolayers identified strong upregulation of phospho-keratin-8 (Ser(73)) as an early consequence of acrolein exposure. These changes were accompanied by chemical damage to keratin-8 and other intermediate filament family members, while acrolein exposure also resulted in controlled ubiquitination of high mass proteins within the intermediate filament extracts. These findings confirm the usefulness of systems allowing delivery of airborne smoke constituents to lung cell monolayers during studies of the molecular basis for acute smoke intoxication injury.

Acrolein relaxes mouse isolated tracheal smooth muscle via a TRPA1-dependent mechanism.

Airway sensory C-fibres express TRPA1 channels which have recently been identified as a key chemosensory receptor for acrolein, a toxic and highly prevalent component of smoke. TRPA1 likely plays an intermediary role in eliciting a range of effects induced by acrolein including cough and neurogenic inflammation. Currently, it is not known whether acrolein-induced activation of TRPA1 produces other airway effects including relaxation of mouse airway smooth muscle. The aims of this study were to examine the effects of acrolein on airway smooth muscle tone in mouse isolated trachea, and to characterise the cellular and molecular mechanisms underpinning the effects of acrolein. Isometric tension recording studies were conducted on mouse isolated tracheal segments to characterise acrolein-induced relaxation responses. Release of the relaxant PGE2 was measured by EIA to examine its role in the response. Use of selective antagonists/inhibitors permitted pharmacological characterisation of the molecular and cellular mechanisms underlying this relaxation response. Acrolein induced dose-dependent relaxation responses in mouse isolated tracheal segments. Importantly, these relaxation responses were significantly inhibited by the TRPA1 antagonists AP-18 and HC-030031, an NK1 receptor antagonist RP-67580, and the EP2 receptor antagonist PF-04418948, whilst completely abolished by the non-selective COX inhibitor indomethacin. Acrolein also caused rapid PGE2 release which was suppressed by HC-030031. In summary, acrolein induced a novel bronchodilator response in mouse airways. Pharmacologic studies indicate that acrolein-induced relaxation likely involves interplay between TRPA1-expressing airway sensory C-fibres, NK1 receptor-expressing epithelial cells, and EP2-receptor expressing airway smooth muscle cells.

Early induction of uncoupling protein-2 in pulmonary macrophages in hyperoxia-associated lung injury.

CONTEXT: High concentrations of inspired oxygen contribute to the pathogenesis of neonatal bronchopulmonary dysplasia and adult acute respiratory distress syndrome. Animal models of hyperoxia-associated lung injury (HALI) are characterized by enhanced generation of reactive oxygen species (ROS) and an adaptive antioxidant response. ROS contribute to pathogenesis, partly through enhancing pro-inflammatory activity in macrophages. Uncoupling protein-2 (UCP2) is an inner mitochondrial membrane protein whose expression lowers mitochondrial superoxide (O2ⁱ⁻) production. UCP2, therefore, has potential to contribute to antioxidant response. It is inducible in macrophages. OBJECTIVES AND METHODS: We hypothesized that induction of UCP2 occurred in response to pulmonary hyperoxia in vivo and that expression localized to pulmonary macrophages. We then investigated mechanisms of UCP2 regulation in hyperoxia-exposed macrophages in vitro and correlated changing UCP2 expression with mitochondrial membrane potential (Δψm) and O2ⁱ⁻ production. RESULTS: UCP2 is induced in lungs of mice within 1 h of hyperoxia exposure. Induction occurs in pulmonary alveolar macrophages in vivo, and can be replicated in vitro in isolated macrophages. UCP2 mRNA does not change. UCP2 increases quickly after the first hyperoxia-induced burst of mitochondrial O2ⁱ⁻ generation. Suppression of Δψm and mitochondrial O2ⁱ⁻ production follow and persist while UCP2 is elevated. DISCUSSION AND CONCLUSIONS: Induction of UCP2 is an early response to hyperoxia in pulmonary macrophages. The mechanism is post-transcriptional. UCP2 induction follows a transient rise in mitochondrial ROS generation. The subsequent falls in Δψm and mitochondrial O2ⁱ⁻ support the notion that regulable UCP2 expression in macrophages acts to contain mitochondrial ROS generation. That, in turn, may limit inappropriate pro-inflammatory activation in HALI.

Inhibitory influence of the hexapeptidic sequence SLIGRL on influenza A virus infection in mice.

Proteinase-activated receptor 2 (PAR(2)) is widely expressed in the respiratory tract and is an integral component of the host antimicrobial defense system. The principal aim of this study was to investigate the influence of a PAR(2)-activating peptide, SLIGRL, on influenza A virus (IAV)-induced pathogenesis in mice. Intranasal inoculation of BALB/c mice with influenza A/PR/8/34 virus caused time-dependent increases in the number of pulmonary leukocytes (recovered from bronchoalveolar lavage fluid), marked airway histopathology characterized by extensive epithelial cell damage, airway hyper-responsiveness to the bronchoconstrictor methacholine, and elevated levels of inflammatory chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein 2) and cytokines (interferon-γ). It is noteworthy that these IAV-induced effects were dose-dependently attenuated in mice treated with a PAR(2)-activating peptide, SLIGRL, at the time of IAV inoculation. However, SLIGRL also inhibited IAV-induced increases in pulmonary leukocytes in PAR(2)-deficient mice, indicating these antiviral actions were not mediated by PAR(2). The potency order obtained for a series of structural analogs of SLIGRL for anti-IAV activity (IGRL > SLIGRL > LSIGRL >2-furoyl-LIGRL) was also inconsistent with a PAR(2)-mediated effect. In further mechanistic studies, SLIGRL inhibited IAV-induced propagation in ex vivo perfused segments of trachea from wild-type or PAR(2)(-/-) mice, but did not inhibit viral attachment or replication in Madin-Darby canine kidney cells and chorioallantoic membrane cells, which are established hosts for IAV. In summary, SLIGRL protected mice from IAV infection independently of PAR(2) and independently of direct inhibition of IAV attachment or replication, potentially through the activation of endogenous antiviral pathways within the mouse respiratory tract.

Prior exposure to acrolein accelerates pulmonary inflammation in influenza A-infected mice.

The combustion product acrolein contributes to several smoke-related health disorders, but whether this immunomodulatory toxicant alters pulmonary susceptibility to viruses has received little attention. To study the effects of prior acrolein dosing on the severity of influenza A viral infection, male BALB/c mice received acrolein (1mg/kg) or saline (control) via oropharyngeal aspiration either 4- or 7-days prior to intranasal inoculation with either influenza A/PR/8/34 virus or vehicle. At 0, 2, 4 and 7 days post-inoculation, lung samples were assessed for histological changes while pulmonary inflammation was monitored by estimating immune cell numbers and cytokine levels in bronchoalveolar lavage fluid (BALF). After viral challenge, animals that were exposed to acrolein 4 days previously experienced greater weight loss and exhibited an accelerated inflammatory response at 2 days after viral inoculation. Thus compared to saline-pretreated, virus-challenged controls, BALF recovered from these mice contained higher numbers of macrophages and neutrophils in addition to increased levels of several inflammatory cytokines, including IL-1α, IL-1ß, IL-6, TNF, IFN-γ, KC, and MCP-1. The acrolein-induced increase in viral susceptibility was suppressed by the carbonyl scavenger bisulphite. These findings suggest acute acrolein intoxication "primes" the lung to mount an accelerated immune response to inhaled viruses.

Influence of influenza A infection on capsaicin-induced responses in murine airways.

The principal aim of the study was to determine the influence of influenza A virus infection on capsaicin-induced relaxation responses in mouse isolated tracheal segments and clarify the underlying mechanisms. Anesthetized mice were intranasally inoculated with influenza A/PR-8/34 virus (VIRUS) or vehicle (SHAM), and 4 days later tracheal segments were harvested for isometric tension recording and biochemical and histologic analyses. Capsaicin induced dose-dependent relaxation responses in carbachol-contracted SHAM trachea (e.g., 10 µM capsaicin produced 66 ± 4% relaxation; n = 11), which were significantly inhibited by capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist], (2S,3S)-3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine hydrochloride (L-733,060) [neurokinin 1 (NK1) receptor antagonist], indomethacin [cyclooxygenase (COX) inhibitor], and the combination of 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) and 7-[5α-([1S,1α(Z)-biphenyl]-4-ylmethoxy)-2ß-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid, calcium salt, hydrate (AH23848) [E-prostanoid (EP)2 and EP4 receptor antagonists, respectively], indicating that capsaicin-induced relaxation involved the TRPV1-mediated release of substance P (SP), activation of epithelial NK1 receptors, and production of COX products capable of activating relaxant EP2/EP4 receptors. Consistent with this postulate, capsaicin-induced relaxation was associated with the significant release of SP and prostaglandin E2 (PGE2) from mouse tracheal segments. As expected, influenza A virus infection was associated with widespread disruption of the tracheal epithelium. Tracheal segments from VIRUS mice responded weakly to capsaicin (7 ± 3% relaxation) and were 25-fold less responsive to SP than tracheas from SHAM mice. In contrast, relaxation responses to exogenous PGE2 and the ß-adrenoceptor agonist isoprenaline were not inhibited in VIRUS trachea. Virus infection was associated with impaired capsaicin-induced release of PGE2, but the release of SP was not affected. In summary, influenza A virus infection profoundly inhibits capsaicin- and SP-induced relaxation responses, most likely by inhibiting the production of PGE2.

Inhibitory influence of protease-activated receptor 2 and E-prostanoid receptor stimulants in lipopolysaccharide models of acute airway inflammation.

Protease-activated receptors (PARs) are widely expressed throughout the respiratory tract, and PAR(2) has been investigated as a potential drug target for inflammatory airway diseases. The primary focus of this study was to determine the extent to which PAR(2)-activating peptides modulate lipopolysaccharide (LPS)-induced airway neutrophilia in mice and establish the underlying mechanisms. Intranasal administration of LPS induced dose- and time-dependent increases in the number of neutrophils recovered from bronchoalveolar lavage (BAL) fluid of mice. Coadministration of the PAR(2)-activating peptide f-LIGRL inhibited LPS-induced neutrophilia at 3 and 6 h after inoculation. PAR(2)-mediated inhibition of LPS-induced neutrophilia was mimicked by prostaglandin E(2) (PGE(2)) and butaprost [selective E-prostanoid (EP(2)) receptor agonist], and blocked by parecoxib (cyclooxygenase 2 inhibitor) and 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) (EP(1)/EP(2) receptor antagonist). PAR(2)-activating peptides also blunted early increases in the levels of the key neutrophil chemoattractants keratinocyte-derived chemokine and macrophage inflammatory protein 2 (MIP-2) in the BAL of LPS-exposed mice. However, neither PAR(2)-activating peptides nor PGE(2) inhibited LPS-induced generation of MIP-2 in cultures of primary murine alveolar macrophages In summary, PAR(2)-activating peptides and PGE(2) suppressed LPS-induced neutrophilia in murine airways, independently of an inhibitory action on MIP-2 generation by alveolar macrophages.

Protease-activated receptors and prostaglandins in inflammatory lung disease.

Protease-activated receptors (PARs) are a novel family of G protein-coupled receptors. Signalling through PARs typically involves the cleavage of an extracellular region of the receptor by endogenous or exogenous proteases, which reveals a tethered ligand sequence capable of auto-activating the receptor. A considerable body of evidence has emerged over the past 20 years supporting a prominent role for PARs in a variety of human physiological and pathophysiological processes, and thus substantial attention has been directed towards developing drug-like molecules that activate or block PARs via non-proteolytic pathways. PARs are widely expressed within the respiratory tract, and their activation appears to exert significant modulatory influences on the level of bronchomotor tone, as well as on the inflammatory processes associated with a range of respiratory tract disorders. Nevertheless, there is debate as to whether the principal response to PAR activation is an augmentation or attenuation of airways inflammation. In this context, an important action of PAR activators may be to promote the generation and release of prostanoids, such as prostglandin E(2), which have well-established anti-inflammatory effects in the lung. In this review, we primarily focus on the relationship between PARs, prostaglandins and inflammatory processes in the lung, and highlight their potential role in selected respiratory tract disorders, including pulmonary fibrosis, asthma and chronic obstructive pulmonary disease.

Influence of dexamethasone on protease-activated receptor 2-mediated responses in the airways.

Stimulants of protease-activated receptor (PAR)(2) promote the generation of the bronchoprotective prostanoid prostaglandin (PG) E(2) by airway epithelial cells. In contrast, glucocorticoids reduce the levels of PGE(2) in airway epithelial cell cultures by concomitantly inhibiting pathways required for PGE(2) synthesis and facilitating pathways involved in PGE(2) inactivation. The aim of this study was to determine whether glucocorticoids inhibited PAR(2)-mediated, PGE(2)-dependent responses in epithelial cell cultures, in intact airway preparations, and in whole animals. In cultures of A549 cells, a PAR(2)-activating peptide SLI-GRL-NH(2) produced concentration and time-dependent increases in PGE(2) levels, which were significantly enhanced after exposure to lipopolysaccharide (LPS). However, SLIGRL-NH(2)-induced increases in PGE(2) levels were abolished by pretreatment of cells with the glucocorticoid, dexamethasone. In mouse isolated tracheal preparations, SLIGRL-NH(2) and PGE(2) induced concentration-dependent relaxation responses that were unaffected by dexamethasone, irrespective of whether dexamethasone exposure occurred in vitro or in vivo. Intranasal administration of LPS produced a pronounced increase in the numbers of neutrophils recovered from the bronchoalveolar lavage fluid of BALB/c mice. Numbers of recovered neutrophils were 40 to 60% lower in mice that received f-LIGRL-NH(2) (PAR(2)-activating peptide, 30 microg intranasally), PGE(2) (10 mugintranasally), or dexamethasone (1 mg/kg i.p.). In the combined presence of dexamethasone and f-LIGRL-NH(2) or dexamethasone and PGE(2), the number of neutrophils was suppressed further (80-83% lower). Thus, although dexamethasone abolished PAR(2)-mediated generation of PGE(2) in A549 cells, neither the smooth muscle relaxant nor the anti-inflammatory effects of PAR(2)-activating peptides (and PGE(2)) were diminished by in vitro or in vivo exposure to dexamethasone.

Rho kinase as a therapeutic target in the treatment of asthma and chronic obstructive pulmonary disease.

Asthma is a complex inflammatory disease of the airways involving reversible bronchoconstriction. Chronic obstructive pulmonary disease is typified by inflammation and airflow limitation that has an irreversible component. There is now substantial evidence that Rho kinase is involved in many of the pathways that contribute to the pathologies associated with these respiratory diseases including bronchoconstriction, airway inflammation, airway remodelling, neuromodulation and exacerbations due to respiratory tract viral infection. Indeed the Rho kinase inhibitor Y-27632 causes bronchodilatation and reduces pulmonary eosinophilia trafficking and airways hyperresponsiveness. Furthermore, accumulating evidence suggests that inhibition of Rho kinase could have a major beneficial impact on symptoms and disease progression in asthma and COPD by modulating several other systems and processes. Thus, the Rho kinase pathway may indeed be a worthwhile therapeutic target in the treatment of asthma and chronic obstructive pulmonary disease.

The protease-activated receptor2 (PAR2)-prostaglandin E2-prostanoid EP receptor axis: a potential bronchoprotective unit in the respiratory tract?

Protease-activated receptor2 (PAR2) is a subtype of G protein-coupled receptor that is widely expressed within the respiratory tract. Stimulation of PAR2 by proteases such as trypsin and tryptase, or by small peptidic activators induces a complex array of effects within the airways. One such PAR2-mediated effect by basal airway epithelial cells is the generation of prostaglandin E2. Prostaglandin E2 produces a raft of anti-inflammatory effects within the airways, principally through the activation of the prostanoid EP2 and EP3 receptor subtypes. This article reviews the PAR2-prostaglandin E2-prostanoid EP receptor axis and discusses approaches through which its activation may provide beneficial effects in respiratory disease.

Toxins not neutralized by brown snake antivenom.

The Australian snakes of the genus Pseudonaja (dugite, gwardar and common brown) account for the majority of snake bite related deaths in Australia. Without antivenom treatment, the risk of mortality is significant. There is an accumulating body of evidence to suggest that the efficacy of the antivenom is limited. The current study investigates the protein constituents recognized by the antivenom using 2-DE, immuno-blot techniques and rat tracheal organ bath assays. The 2-DE profiles for all three snake venoms were similar, with major species visualized at 78-132 kDa, 32-45 kDa and 6-15 kDa. Proteins characterized by LC-MS/MS revealed a coagulant toxin ( approximately 42 kDa) and coagulant peptide ( approximately 6 kDa), as well as two PLA(2) ( approximately 14 kDa). Peptides isolated from approximately 78 kDa and 15-32 kDa protein components showed no similarity to known protein sequences. Protein recognition by the antivenom occurred predominantly for the higher molecular weight components with little recognition of 6-32 kDa MW species. The ability of antivenom to neutralize venom activity was also investigated using rat tracheal organ bath assays. The venoms of Pseudonaja affinis affinis and Pseudonaja nuchalis incited a sustained, significant contraction of the trachea. These contractions were attributed to PLA(2) enzymatic activity as pre-treatment with the PLA(2) inhibitor 4-BPB attenuated the venom-induced contractions. The venom of Pseudonaja textilis incited tracheal contractility through a non-PLA(2) enzymatic activity. Neither activity was attenuated by the antivenom treatment. These results represent the first proteomic investigation of the venoms from the snakes of the genus Pseudonaja, revealing a possible limitation of the brown snake antivenom in binding to the low MW protein components.

Inhibitors of prostaglandin transport and metabolism augment protease-activated receptor-2-mediated increases in prostaglandin E2 levels and smooth muscle relaxation in mouse isolated trachea.

Stimulants of protease-activated receptor-2 (PAR(2)), such as Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), cause airway smooth muscle relaxation via the release of the bronchodilatory prostanoid prostaglandin E(2) (PGE(2)). The principal aim of the current study was to determine whether compounds that inhibit PGE(2) reuptake by the prostaglandin transporter [bromocresol green and U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF2alpha) and PGE(2) metabolism by 15-hydroxyprostaglandin dehydrogenase (thiazolidenedione compounds rosiglitazone and ciglitazone) significantly enhanced the capacity of SLIGRL to elevate PGE(2) levels and produce relaxation in isolated segments of upper and lower mouse trachea. SLIGRL produced concentration-dependent increases in PGE(2) levels and smooth muscle relaxation, although both effects were significantly greater in lower tracheal segments than in upper tracheal segments. SLIGRL-induced increases in PGE(2) levels were significantly enhanced in the presence of ciglitazone and rosiglitazone, and these effects were not inhibited by GW9662 (2-chloro-5-nitrobenzanilide), a peroxisome proliferator-activated receptor-gamma antagonist. SLI-GRL-induced relaxation responses were also significantly enhanced by ciglitazone and rosiglitazone, whereas responses to isoprenaline, a PGE(2)-independent smooth muscle relaxant, were unaltered. Ciglitazone and rosiglitazone alone produced concentration-dependent increases in PGE(2) levels and smooth muscle relaxation, and these responses were inhibited by indomethacin, a cyclooxygenase inhibitor. Bromocresol green, an inhibitor of prostaglandin transport, significantly enhanced SLIGRL-induced increases in PGE(2) levels and relaxation. Immunohistochemical staining for 15-hydroxyprostaglandin dehydrogenase was relatively intense over airway smooth muscle, as was staining for the prostaglandin transporter over both airway smooth muscle and epithelium. In summary, inhibitors of PGE(2) reuptake and metabolism significantly potentiate PAR(2)-mediated increases in PGE(2) levels and smooth muscle relaxation in murine-isolated airways.

Stimulation of protease-activated receptor-2 inhibits airway eosinophilia, hyperresponsiveness and bronchoconstriction in a murine model of allergic inflammation.

1. An emerging body of evidence indicates that PGE(2) has a privileged anti-inflammatory role within the airways. Stimulants of protease-activated receptor-2 (PAR(2)) inhibit airway smooth muscle tone in vitro and in vivo predominantly via cyclooxygenase (COX)-dependent generation of prostaglandin E(2) (PGE(2)). Thus, the current study tested the hypothesis that PAR(2)-induced generation of PGE(2) inhibits the development of allergic airways inflammation and hyperresponsiveness. 2. Bronchoalveolar lavage (BAL) fluid recovered from ovalbumin (OVA)-sensitised and -challenged (allergic) mice contained elevated numbers of eosinophils, which peaked at 48 h postchallenge. Intranasal (i.n.) administration of a PAR(2)-activating peptide (PAR(2)-AP) SLIGRL (25 mg kg(-1), at the time of OVA challenge) caused a 70% reduction in the numbers of BAL eosinophils (compared to the scrambled peptide LSIGRL, 25 mg kg(-1)). 3. Pretreatment of allergic mice with either indomethacin (1 mg kg(-1), dual COX inhibitor) or nimesulide (3 mg kg(-1), COX-2-selective inhibitor) blocked SLIGRL-induced reductions in BAL eosinophils. 4. I.n. SLIGRL, but not LSIGRL, inhibited the development of antigen-induced airways hyperresponsiveness. The inhibitory effect of SLIGRL was blocked by indomethacin. 5. Exposure of isolated tracheal preparations from allergic mice to 100 microM SLIGRL was associated with a 5.0-fold increase in PGE(2) levels (P<0.05, compared to 100 microM LSIGRL). SLIGRL induced similar increases in PGE(2) levels in control mice (OVA-sensitised, saline-challenged). 6. I.n. administration of PGE(2) (0.15 mg kg(-1)) to allergic mice significantly inhibited eosinophilia and airways hyperresponsiveness to methacholine. 7. In anaesthetised, ventilated allergic mice, SLIGRL (5 mg kg(-1), i.v.) inhibited methacholine-induced increases in airways resistance. Consistent with this bronchodilator effect, SLIGRL induced pronounced relaxation responses in isolated tracheal preparations obtained from allergic mice. LSIGRL did not inhibit bronchomotor tone in either of these in vivo or in vitro experiments. 8. In summary, a PAR(2)-AP SLIGRL inhibited the development of airway eosinophilia and hyperresponsiveness in allergic mice through a COX-dependent pathway involving COX-2-mediated generation of the anti-inflammatory mediator PGE(2). SLIGRL also displayed bronchodilator activity in allergic mice. These studies support the concept that PAR(2) exerts predominantly bronchoprotective actions within allergic murine airways.