Bronchoconstriction in nonhuman primates: a species comparison

Bronchoconstriction is a characteristic symptom of various chronic obstructive respiratory diseases such as chronic obstructive pulmonary disease and asthma. Precision-cut lung slices (PCLS) are a suitable ex vivo model to study physiological mechanisms of bronchoconstriction in different species. In the present study, we established an ex vivo model of bronchoconstriction in nonhuman primates (NHPs). PCLS prepared from common marmosets, cynomolgus macaques, rhesus macaques, and anubis baboons were stimulated with increasing concentrations of representative bronchoconstrictors: methacholine, histamine, serotonin, leukotriene D? (LTD?), U46619, and endothelin-1. Alterations in the airway caliber were measured and compared with previously published data from rodents, guinea pigs, and humans. Methacholine induced maximal airway constriction, varying between 74 and 88% in all NHP species, whereas serotonin was ineffective. Histamine induced maximal bronchoconstriction of 77 to 90% in rhesus macaques, cynomolgus macaques, and baboons and a lesser constriction of 53% in marmosets. LTD? was ineffective in marmosets and rhesus macaques but induced a maximum constriction of 44 to 49% in cynomolgus macaques and baboons. U46619 and endothelin-1 caused airway constriction in all NHP species, with maximum constrictions of 65 to 91% and 70 to 81%, respectively. In conclusion, PCLS from NHPs represent a valuable ex vivo model for studying bronchoconstriction. All NHPs respond to mediators relevant to human airway disorders such as methacholine, histamine, U46619, and endothelin-1 and are insensitive to the rodent mast cell product serotonin. Only PCLS from cynomolgus macaques and baboons, however, responded also to leukotrienes, suggesting that among all compared species, these two NHPs resemble the human airway mechanisms best.     

Comparison of Airway Responses in Sheep of Different Age in Precision-Cut Lung Slices (PCLS)

Background

Animal models should display important characteristics of the human disease. Sheep have been considered particularly useful to study allergic airway responses to common natural antigens causing human asthma. A rationale of this study was to establish a model of ovine precision-cut lung slices (PCLS) for the in vitro measurement of airway responses in newborn and adult animals. We hypothesized that differences in airway reactivity in sheep are present at different ages.

Methods

Lambs were delivered spontaneously at term (147d) and adult sheep lived till 18 months. Viability of PCLS was confirmed by the MTT-test. To study airway provocations cumulative concentration-response curves were performed with different allergic response mediators and biogenic amines. In addition, electric field stimulation, passive sensitization with house dust mite (HDM) and mast cells staining were evaluated.

Results

PCLS from sheep were viable for at least three days. PCLS of newborn and adult sheep responded equally strong to methacholine and endothelin-1. The responses to serotonin, leukotriene D₄ and U46619 differed with age. No airway contraction was evoked by histamine, except after cimetidine pretreatment. In response to EFS, airways in PCLS from adult and newborn sheep strongly contracted and these contractions were atropine sensitive. Passive sensitization with HDM evoked a weak early allergic response in PCLS from adult and newborn sheep, which notably was prolonged in airways from adult sheep. Only few mast cells were found in the lungs of non-sensitized sheep at both ages.

Conclusion

PCLS from sheep lungs represent a useful tool to study pharmacological airway responses for at least three days. Sheep seem well suited to study mechanisms of cholinergic airway contraction. The notable differences between newborn and adult sheep demonstrate the importance of age in such studies.     

Neurally Mediated Airway Constriction in Human and Other Species: A Comparative Study Using Precision-Cut Lung Slices (PCLS)

The peripheral airway innervation of the lower respiratory tract of mammals is not completely functionally characterized. Recently, we have shown in rats that precision-cut lung slices (PCLS) respond to electric field stimulation (EFS) and provide a useful model to study neural airway responses in distal airways. Since airway responses are known to exhibit considerable species differences, here we examined the neural responses of PCLS prepared from mice, rats, guinea pigs, sheep, marmosets and humans. Peripheral neurons were activated either by EFS or by capsaicin. Bronchoconstriction in response to identical EFS conditions varied between species in magnitude. Frequency response curves did reveal further species-dependent differences of nerve activation in PCLS. Atropine antagonized the EFS-induced bronchoconstriction in human, guinea pig, sheep, rat and marmoset PCLS, showing cholinergic responses. Capsaicin (10 µM) caused bronchoconstriction in human (4 from 7) and guinea pig lungs only, indicating excitatory non-adrenergic non-cholinergic responses (eNANC). However, this effect was notably smaller in human responder (30±7.1%) than in guinea pig (79±5.1%) PCLS. The transient receptor potential (TRP) channel blockers {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 and ruthenium red antagonized airway contractions after exposure to EFS or capsaicin in guinea pigs. In conclusion, the different species show distinct patterns of nerve-mediated bronchoconstriction. In the most common experimental animals, i.e. in mice and rats, these responses differ considerably from those in humans. On the other hand, guinea pig and marmoset monkey mimic human responses well and may thus serve as clinically relevant models to study neural airway responses.     

Comparative effects of platelet activating factor, leukotriene D4 and histamine on guinea pig trachea, bronchus and lung parenchyma

The myotropic effect of platelet activating factor (PAF), leukotriene D4 (LTD4) and histamine were compared on guinea pig pulmonary tissues. The initial administration of PAF induced a contraction of strips of trachea, bronchus and lung parenchyma. However subsequent injections were characterized by relaxation of trachea and bronchus and a highly reduced (if any) contraction of the parenchyma. The three tissues of the guinea pig respiratory system contracted strongly to leukotriene D4 and histamine. Indomethacin blocked PAF-induced relaxation of the trachea and bronchus and reduced the contraction of the lung parenchyma. The injection of PAF in the pulmonary circulation stimulated the release of substance(s) causing the contraction of the trachea, bronchus and parenchyma. This study suggests that PAF is not a direct agonist of bronchoconstriction.     

Early onset airway obstruction in response to organic dust in the horse.

Equine recurrent airway obstruction (RAO) has been used as a naturally occurring model of human asthma. However, it is unknown whether there is an early-phase response in RAO. The aim of this study was to determine whether exposure to organic dust induces immediate changes in lung function in RAO-affected horses, which could be mediated by airway mast cells. Six RAO-affected horses in remission and six control horses were challenged with hay-straw dust suspension by nebulization. Total respiratory resistance at 1 Hz, measured by forced oscillation, was increased from 0.62 +/- 0.09 cmH(2)O.l(-1).s (mean +/- SE) to 1.23 +/- 0.20 cmH(2)O.l(-1).s 15 min after nebulization in control horses (P = 0.023) but did not change significantly in the RAO group. Total respiratory reactance at 1 Hz (P = 0.005) was significantly lower in the control horses (-0.77 +/- 0.07 cmH(2)O.l(-1).s) than in the RAO group (-0.49 +/- 0.04 cmH(2)O.l(-1).s) 15 min after nebulization. Bronchoalveolar lavage fluid (BALF) histamine concentration was significantly elevated 10 and 20 min postnebulization in control horses but not in RAO horses. Minimum reactance at 1 Hz in the early postnebulization period significantly correlated with both prechallenge BALF mast cell numbers (r = -0.65, P = 0.02) and peak BALF histamine concentration postnebulization (r = -0.61, P = 0.04). In conclusion, RAO horses, unlike human asthmatic patients, do not exhibit an early-phase response. However, healthy control horses do demonstrate a mild but significant early (<20 min) phase response to inhaled organic dust. This response may serve to decrease the subsequent dose of dust inhaled and as such provide a protective mechanism, which may be compromised in RAO horses.     

Comparative effects of platelet activating factor, leukotriene D4 and histamine on guinea pig trachea, bronchus and lung parenchyma

The myotropic effect of platelet activating factor (PAF), leukotriene D₄ (LTD₄) and histamine were compared on guinea pig pulmonary tissues. The initial administration of PAF induced a contraction of strips of trachea, bronchus and lung parenchyma. However subsequent injections were characterized by relaxation of trachea and bronchus and a highly reduced (if any) contraction of the parenchyma. The three tissues of the guinea pig respiratory system contracted strongly to leukotriene D₄ and histamine. Indomethacin blocked PAF-induced relaxation of the trachea and bronchus and reduced the contraction of the lung parenchyma. The injection of PAF in the pulmonary circulation stimulated the release of substance(s) causing the contraction of the trachea, bronchus and parenchyma. This study suggests that PAF is not a direct agonist of bronchoconstriction.     

Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production,Phagocytosis and Extracellular Trap Formation

Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.     

Propranolol potentiates leukotriene D4-induced bronchoconstriction and enhances antagonism by FPL 55712

Aerosol LTD4-induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs was potentiated by either pretreatment with propranolol or bilateral adrenalectomy, whereas bilateral vagotomy did not affect the LTD4 response. The dose-response curve describing LTD4-induced changes in dynamic lung compliance (CDYN) and pulmonary resistance (RL) [as reflective indices of bronchoconstriction] was shifted to the left by approximately 20-fold by propranolol. Against an equal degree of LTD4-induced bronchoconstriction, the leukotriene antagonist, FPL 55712, had an apparent 20-fold greater potency in propranolol-pretreated animals vis a vis saline-treated controls. The duration of action of aerosol FPL 55712 was similar in both propranolol-treated and saline-treated animals. These results demonstrate that aerosol LTD4-induced bronchoconstriction is modulated by an adrenergic compensatory bronchodilator mechanism that is apparently dependent upon the adrenals and independent of vagal influences. Inhibition of the effect of this reflex with propranolol also enhances the apparent potency of an aerosol leukotriene antagonist, FPL 55712, presumably reflecting a constant LTD4 to antagonist ratio in the saline-treated and propranolol-pretreated guinea pigs.     

LPS-induced lung inflammation in marmoset monkeys - an acute model for anti-inflammatory drug testing

Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1β) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50)). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.     

Electric field stimulation of precision-cut lung slices

The precision-cut lung slice (PCLS) technique is widely used to examine airway responses in different species. We developed a method to study nerve-dependent bronchoconstriction by the application of electric field stimulation (EFS) to PCLS. PCLS prepared from Wistar rats were placed between two platinum electrodes to apply serial rectangular impulses (5-100 Hz), and bronchoconstriction was studied by videomicroscopy. The extent of airway contractions increased with higher frequencies. Stable repeated airway contractions were obtained at a frequency of 50 Hz, a width of 1 ms, and an output of 200 mA for 2.5 s each minute. Larger airways showed stronger responses. The EFS-triggered contractions were increased by the acetylcholine esterase inhibitor neostigmine (10 μM) and reversed by the muscarinic antagonist atropine (10 μM), whereas the thromboxane protanoid receptor antagonist SQ29548 (10 μM) had no effect. Magnesium ions (10 mM) antagonized airway contractions induced by EFS, but not by methacholine, indicating that nerve endings remain intact in PCLS. Our data further show that the electrically evoked airway contractions in PCLS are mediated by cholinergic nerves, independent of thromboxane and more prominent in larger airways. Taken together these findings show that nerve endings remain intact in PCLS, and they suggest that the present method is useful to study neurogenic responses in airways of different size.     

Differential Gene Expression Profiles and Selected Cytokine Protein Analysis of Mediastinal Lymph Nodes of Horses with Chronic Recurrent Airway Obstruction (RAO) Support an Interleukin-17 Immune Response

Recurrent airway obstruction (RAO) is a pulmonary inflammatory condition that afflicts certain mature horses exposed to organic dust particulates in hay. Its clinical and pathological features, manifested by reversible bronchoconstriction, excessive mucus production and airway neutrophilia, resemble the pulmonary alterations that occur in agricultural workers with occupational asthma. The immunological basis of RAO remains uncertain although its chronicity, its localization to a mucosal surface and its domination by a neutrophilic, non-septic inflammatory response, suggest involvement of Interleukin-17 (IL-17). We examined global gene expression profiles in mediastinal (pulmonary-draining) lymph nodes isolated from RAO-affected and control horses. Differential expression of > 200 genes, coupled with network analysis, supports an IL-17 response centered about NF-κB. Immunohistochemical analysis of mediastinal lymph node sections demonstrated increased IL-17 staining intensity in diseased horses. This result, along with the finding of increased IL-17 concentrations in lymph node homogenates from RAO-affected horses (P = 0.1) and a down-regulation of IL-4 gene and protein expression, provides additional evidence of the involvement of IL-17 in the chronic stages of RAO. Additional investigations are needed to ascertain the cellular source of IL-17 in this equine model of occupational asthma. Understanding the immunopathogenesis of this disorder likely will enhance the development of therapeutic interventions beneficial to human and animal pulmonary health.     

Universality in kinetic models of circadian rhythms in $$Arabidopsis\,\,thaliana$$

Journal of Mathematical Biology - Precision-cut lung-slices (PCLS), in which viable airways embedded within lung parenchyma are stretched or induced to contract, are a widely used ex vivo assay to...     

Increased hypoxia-inducible factor 1alpha expression in lung cells of horses with recurrent airway obstruction

ABSTRACT: BACKGROUND: Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition caused by exposure of susceptible horses to organic dusts in hay. The immunological processes responsible for the development and the persistence of airway inflammation are still largely unknown. Hypoxia-inducible factor (Hif) is mainly known as a major regulator of energy homeostasis and cellular adaptation to hypoxia. More recently however, Hif also emerged as an essential regulator of innate immune responses. Here, we aimed at investigating the potential involvement of Hif1-alpha in myeloid cells in horse with recurrent airway obstruction. RESULTS: In vitro, we observed that Hif is expressed in equine myeloid cells after hay dust stimulation and regulates genes such as tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A). We further showed in vivo that airway challenge with hay dust upregulated Hif1-alpha mRNA expression in myeloid cells from the bronchoalveolar lavage fluid (BALF) of healthy and RAO-affected horses, with a more pronounced effect in cells from RAO-affected horses. Finally, Hif1-alpha mRNA expression in BALF cells from challenged horses correlated positively with lung dysfunction. CONCLUSION: Taken together, our results suggest an important role for Hif1-alpha in myeloid cells during hay dust-induced inflammation in horses with RAO. We therefore propose that future research aiming at functional inactivation of Hif1 in lung myeloid cells could open new therapeutic perspectives for RAO.     

Persistent mucin glycoprotein alterations in equine recurrent airway obstruction

Horses with the episodic asthmalike condition of recurrent airway obstruction (RAO) have bouts of inflammation and bronchoconstriction associated with indoor housing. To assess the potential differences in airway secretions between RAO-affected and control horses, methods to quantify mucus secretions were developed and applied to bronchoalveolar lavage fluid. The relative difference in the amount of mucin glycoproteins between control and RAO-affected horses was assessed with a carbohydrate side chain-specific monoclonal antibody (4E4) in an enzyme-linked immunosorbent assay and by carbohydrate-specific enzyme-linked lectin assays. Significantly increased levels of 4E4-immunoreactive glycoprotein and the mucin-associated carbohydrates fucose (alpha-1,2 linkage) and N-acetylglucosamine were detected in RAO-affected horses in acute disease. RAO-affected horses in remission maintained significantly elevated levels of alpha-1,2-fucose and N-acetylglucosamine, whereas the 4E4-immunoreactive glycoprotein levels displayed a trend toward an increase over control levels. These results indicated that persistent changes in the quantity and/or quality of mucus glycoproteins occurred in the RAO-affected horses.     

Energetic cost of breathing, body composition, and pulmonary function in horses with recurrent airway obstruction.

This study was conducted to determine whether horses with naturally occurring, severe chronic recurrent airway obstruction (RAO) 1). have a greater resting energy expenditure (REE) than control horses, 2). suffer body mass depletion, and 3). have significantly decreased REE after bronchodilation and, therefore, also 4). whether increased work of breathing contributes to the cachexia seen in some horses with RAO. Six RAO horses and six control horses underwent indirect calorimetric measures of REE and pulmonary function testing using the esophageal balloon-pneumotachograph method before and after treatment with ipratropium bromide, a parasympatholytic bronchodilator agent, at 4-h intervals for a 24-h period. Body condition scoring was performed, and an estimate of fat mass was determined via B-mode ultrasonography. O(2) and CO(2) fractions, respiratory airflow, respiratory rate, and pleural pressure changes were recorded, and O(2) consumption, CO(2) production, REE, pulmonary resistance, dynamic elastance, and tidal volume were calculated. In addition, we performed lung function testing and calorimetry both before and after sedation in two control horses. RAO horses had significantly lower body condition scores (2.8 +/- 1.0 vs. 6.4 +/- 1.2) and significantly greater O(2) consumption than controls (4.93 +/- 1.30 vs. 2.93 +/- 0.70 ml.kg(-1).min(-1)). After bronchodilation, there was no significant difference in O(2) consumption between RAO horses and controls, although there remained evidence of residual airway obstruction. There was a strong correlation between O(2) consumption and indexes of airway obstruction. Xylazine sedation was not associated with changes in pulmonary function but did result in markedly decreased REE in controls.     

Chlamydophila spp. infection in horses with recurrent airway obstruction: similarities to human chronic obstructive disease

Background

Recurrent airway obstruction (RAO) in horses is a naturally occurring dust-induced disease mainly characterized by bronchiolitis which shows histological and pathophysiological similarities to human chronic obstructive pulmonary disease (COPD). In human COPD previous investigations indicated an association with Chlamydophila psittaci infection. The present study was designed (1) to clarify a possible role of this infectious agent in RAO and (2) to investigate the suitability of this equine disorder as a model for human COPD.

Methods

Clinico-pathological parameters of a total of 45 horses (25 horses with clinical signs of RAO and 20 clinically healthy controls) were compared to histological findings in lung tissue samples and infection by Chlamydiaceae using light microscopy, immunohistochemistry, and PCR.

Results

Horses with clinical signs of RAO vs. controls revealed more inflammatory changes in histology (p = 0.01), and a higher detection rate of Chlamydia psittaci antigens in all cells (p < 0.001) and bronchiolar epithelial cells alone (p < 0.001) by immunohistochemistry. The abundance of chlamydial inclusions increased with the severity of disease. PCR was positive in 60% of horses with RAO vs. 45% of the controls (p = 0.316). OmpA sequencing identified Chlamydophila psittaci (n = 9) and Chlamydophila abortus (n = 13) in both groups with no significant differences. Within the group of clinically healthy horses subgroups with no changes (n = 15) and slight inflammation of the small airways (n = 5) were identified. Also in the group of animals with RAO subgroups with slight (n = 16) and severe (n = 9) bronchiolitis could be formed. These four subgroups can be separated in parts by the number of cells positive for Chlamydia psittaci antigens.

Conclusion

Chlamydophila psittaci or abortus were present in the lung of both clinically healthy horses and those with RAO. Immunohistochemistry revealed acute chlamydial infections with inflammation in RAO horses, whereas in clinically healthy animals mostly persistent chlamydial infection and no inflammatory reactions were seen. Stable dust as the known fundamental abiotic factor in RAO is comparable to smoking in human disease. These results show that RAO can be used as a model for human COPD.     

糖皮质激素在孤束核内去甲肾上腺素／神经肽Y引起的心血管效应的影响及其机

本研究观察了糖皮质激素自身在孤束核ＮＴＳ内的心血管效应,以及它在ＮＴＳ内对ＮＡ／ＮＰＹ诱导的心血管活动变化的影响及机制。结果发现,大剂量地塞米松在大鼠ＮＴＳ的内能很快导致血压下降,血清中ＮＯ浓度升高。小剂量Ｄｅｘ在ＮＴＳ内能很快抑制ＮＡ／ＮＰＹ在ＮＴＳ内诱导的心血管效应,并维持较长时间。表明Ｄｅｘ对ＮＡ／ＮＰＹ在ＮＴＳ诱导的心血管效应,并维持较长时间。表明Ｄｅｘ对ＮＡ／ＮＰＹ在ＮＴＳ诱导的心血管效     

Leukotriene A4-induced bronchoconstriction in the guinea pig in vivo

Administration of leukotriene A4 (0.03-0.3 microgram kg-1 i.v.) to anesthetized spontaneously breathing guinea pigs produced pronounced changes in pulmonary resistance, dynamic compliance and blood pressure. The pulmonary responses were unaffected either by pretreatment with indomethacin or following desensitization to leukotriene B4 but were significantly attenuated by the leukotriene D4 receptor antagonist, FPL-55712. Following administration of leukotriene A4 increased levels of leukotriene C4-immunoreactive material were determined in the plasma and neutrophil accumulation was observed in the lung. It was concluded that leukotriene A4 induced bronchoconstriction in the guinea pig either by acting directly on the leukotriene D4 receptor site or more probably through efficient metabolism in the lung to peptido-lipid leukotrienes which in turn exerted direct bronchoconstrictor actions.     

Does thromboxane mediate the indirect contractile action of leukotriene D4 in guinea-pig isolated lung parenchyma under equilibrium conditions?

The role that thromboxane A2 plays in contractions induced by leukotriene D4 in guinea-pig isolated lung parenchyma was investigated under equilibrium conditions. Lung tissue generated thromboxane A2 and prostacyclin spontaneously as evidenced by the slow accumulation of their biologically inactive metabolites, thromboxane B2 and 6-keto-prostaglandin F1 alpha, in the bathing buffer. Challenge of guinea-pig lung parenchyma with a high concentration (EC90 for tension generation) of leukotriene D4 (200 nM) produced a biphasic contraction of the tissue that consisted of an initial rapid increase in isometric tension followed by a slowly developing, well-sustained contracture. In addition, leukotriene D4 (200 nM) effected a transient increase (over basal) in the generation of thromboxane A2 and prostacyclin that lagged significantly behind the tension response. Kinetic analysis of the mechanical and eicosanoid-generating effect of leukotriene D4 revealed that tension development could be suitably fitted to a biexponential function, whilst the release of both eicosanoids from the lung occurred monoexponentially. Pretreatment of the lung with the cyclooxygenase inhibitor, flurbiprofen, which effectively abolished both the spontaneous and the leukotriene D4-stimulated eicosanoid biosynthesis, significantly reduced both the first order rate coefficient of the first exponent and the maximum amplitude of this function with respect to control. This change in the kinetics describing leukotriene D4-induced contractions was explained by the fact that the initial rate of tension development was markedly reduced following pretreatment of the lung with flurbiprofen. Neither the inhibitor of thromboxane synthetase, dazmegrel, which selectively inhibited (by 95%) leukotriene D4-stimulated thromboxane A2 formation, nor blockade of 11 alpha,9 alpha-epoxymethano-prostaglandin H2 (U-46619)-sensitive (thromboxane A2) receptors with either AH 23848 or EP 092 affected the profile of leukotriene D4-induced tension development in guinea-pig lung. It is concluded that a high concentration of leukotriene D4 (200 nM) contracts guinea-pig lung by both a direct and indirect mechanism. Initially, a rapid short-lived contraction of the lung is manifest which is dependent, in part, upon the synthesis and release of cyclooxygenase product(s) other than thromboxane A2. This initial response occurs coincidently with, and is subsequently followed by, the development of a tonic well-sustained contracture that is the result of a direct action of leukotriene D4 on the contractile cells that comprise the lung.