Lead acetate induced contraction in rat tracheal smooth muscle is independent of epithelium

Airways are the primary target of lead exposure from atmospheric pollution, its effect on airway smooth muscle and their responsiveness to bronchoactive agents is not clearly understood. In the present investigation the effect of lead on the isolated airway smooth muscle activity was studied in organ bath set-up. Further the involvement of airway epithelium was examined and the responsiveness of airway smooth muscle to adenosine, acetylcholine (bronchoconstrictors) and isoproterenol (bronchodilator) was also investigated. Lead in concentration of 10(-12) M to 10(-4) M produced concentration-dependant contractile response in rat tracheal rings. Acetylcholine and adenosine induced concentration-dependent contractile response was slightly inhibited after lead exposure. The relaxant response to isoproterenol was also inhibited in lead exposed tissues. Epithelium removal did not significantly change the contractile response to lead suggesting that the lead induced contraction of airway smooth muscle is epithelium independent.     

Release of prostaglandin E2 and leukotriene C4/D4 from airway segments isolated from rats after exposure to ozone for 20 months

Metabolites of arachidonic acid have been implicated as mediators of some of the pulmonary effects observed after acute exposure to ozone. Accordingly, recent studies have focused on the effects of acute ozone exposure on the arachidonic acid cascade, however, whether eicosanoid metabolism is altered after chronic exposure to ozone is unknown. To begin to address this issue, we examined the effects of near-lifetime exposure to ozone on release of prostaglandin E₂ (PGE₂) and leukotriene C₄/D₄ from airway segments isolated from exposed Fischer-344 rats. Airway segments representing approximately eighth to tenth generation airways were isolated from rats of both genders that had been exposed for 6 h per day, 5 days per week for 20 months to filtered air or 0.12, 0.5 or 1.0 parts per million (ppm) ozone. Basal and stimulated release of eicosanoids were measured in the medium surrounding airway segments using enzymoimmunoassay. Basal release of PGE₂ was detected in the medium surrounding airway segments and this release was unaffected by ozone exposure. Incubation of the segments with the calcium ionophore, A23187, increased the release of the prostaglandin; the A23187-induced release of PGE₂ was significantly enhanced in airway segments isolated from rats in the 1.0 ppm exposure group. Basal release of leukotriene C₄/D₄ was not detected in the medium surrounding airway segments regardless of ozone exposure. Measurable amounts of the leukotriene were released during incubation with A23187, however, ozone was without affect on these levels. The results suggest that the cyclooxygenase pathway of the arachidonic acid cascade appears to be affected by ozone exposure. Which of the processes of prostaglandin production and release are affected by chronic ozone exposure remains to be determined.     

Racemic salbutamol administration to guinea-pigs selectively augments airway smooth muscle responsiveness to cholinoceptor agonists

1. An aim of this study was to investigate whether continuous in vivo administration of a low dose of salbutamol to guinea-pigs alters the responsiveness of airway smooth muscle in vitro. 2. Osmotic minipumps containing a solution of racemic salbutamol were implanted subcutaneously in guinea-pigs. The drug was infused at a dose of 0.2 mg kg(-1) day(-1) for 10 days and, at the end of that time, the trachea was isolated and concentration-response relationships to several contractile agonists were examined. 3. This treatment resulted in significant increases in the maximum tension developed by tracheal preparations in response to cholinoceptor agonists, carbachol and methacholine. 4. Cumulative concentration-response curves for histamine, leukotriene D4, and KCl were similar in tracheal segments from saline-control and salbutamol-infused animals. 5. Time course experiments showed that augmented airway contractile responsiveness to cholinoceptor agonists was reversible within 3 days after cessation of the 10 day salbutamol infusion. 6. Our findings support the hypothesis that beta2-adrenoceptor agonist drugs, administered over time in vivo, induce a transient hyperresponsiveness of airway smooth muscle to cholinergic bronchoconstrictor stimuli.     

Aliphatic chlorinated hydrocarbons alter the contractile responses of tracheal smooth muscle in piglet

Aliphatic chlorinated hydrocarbons (ACHs) are widely used in several industrial processes and are also found in many commercial household products. They are classified as hazardous air pollutants, since ACHs exposure induces respiratory complications including airway hyperactivity. However, the contribution of airway smooth muscle tone to ACH-induced respiratory dysfunction has not been elucidated. Thus, the effects of ACHs such as dichloromethane (DCM), dichloroethane (DCE), and trichloromethane (TCM), on the basal and stimulant-induced contractile responses in piglet tracheal smooth muscle were investigated. ACHs at 100-1,000 ppm were found to evoke the basal contraction of tracheal smooth muscle strips. Although DCM, DCE, and TCM enhanced the muscle tone precontracted by KCl, they exerted differential effects on acetylcholine- or histamine-induced muscle contraction. DCE did not alter the muscle tone activated by acetylcholine and histamine. DCM at 1,000 ppm enhanced the muscle tension precontracted by acetylcholine but not by histamine. TCM at 30-1,000 ppm increased the histamine-induced muscle contraction, but at 1,000 ppm relaxed the muscle precontracted by acetylcholine. DCE and TCM at the highest concentration (1,000 ppm) provoked a biphasic response with an initial increase in KCI-induced muscle tension followed by a decrease. Furthermore, pretreatment with DCE potentiated the acetylcholine-, histamine-, and KCl-induced muscle contractile responses. Pretreatment with TCM potentiated the histamine-, and KCl-induced response, but DCM only potentiated the KCl-induced response. The results suggest that ACH exposure altering the basal and spasmogen-induced contractile responses might participate in airway impairment with hyperresponsiveness.     

Ozone effects on airway responsiveness, lung injury, and inflammation. Comparative rat strain and in vivo/in vitro investigations

Asthmatic individuals appear to be particularly sensitive to the effects of certain air pollutants-including ozone (O(3)), an oxidant ambient air pollutant-for reasons that are poorly understood. The general purpose of these studies, therefore, was to expand and improve upon toxicologic methods for assessing ozone-induced effects on the airways of the rat by (1) developing an in vivo testing procedure that allows detection of airway responsiveness changes in rats exposed to ozone; (2) identifying a strain of rat that may be inherently more sensitive to the effects of ozone; and (3) validation of an in vitro epithelial culture system to more directly assess airway cellular/subcellular effects of ozone. Using methacholine inhalation challenges, we detected increased airway responsiveness in senescent F344 rats acutely after ozone exposure (2 ppm x 2 h). We also determined that acutely after ozone exposure (0.5 ppm x 8 h), Wistar rats developed significantly greater lung injury, neutrophilic inflammation, and bronchoalveolar lavage (BAL) fluid concentrations of IL-6 than either Sprague-Dawley (SD) or F344 rats. SD rats had greater BAL fluid concentrations of prostaglandin E(2) (PGE(2)), while F344 rats consistently exhibited the least effect. Wistar rat-derived tracheal epithelial (RTE) cultures were exposed in vitro to air or ozone (0.1-1.0 ppm x 1 h), and examined for analogous effects. In a concentration-dependent manner, ozone exposure resulted in acute but minor cytotoxicity. RT polymerase chain reaction (PCR) analysis of RNA isolated from ozone-exposed cells demonstrated variable increases in steady-state gene expression of IL-6 at 4 h postexposure, while at 24 h cellular fibronectin expression (EIIIA domain) was decreased. Exposure was without effect on macrophage inflammatory protein 2 (MIP-2) or gamma-glutamyl cysteine synthetase expression. At 6 h postexposure, IL-6 synthesis and apical release appeared increased in ozone-exposed cells (1 ppm x 1 h). MIP-2 release was not significantly increased in ozone-exposed cells. At 2 h postexposure, ozone exposure resulted in minor increases in apical fibronectin, but exposure was without effect on basolateral accumulation of fibronectin. Exposure to 1.0, but not 0.1 ppm (x 1 h), increased production of cyclooxygenase (i.e., PGE(2)) and noncyclooxygenase products of arachidonic acid. Results demonstrate that multiple inflammatory mediator pathways are affected by ozone exposure. Such effects could exacerbate morbidity in individuals with preexisting airway inflammation such as asthmatics.     

Structural and functional localization of airway effects from episodic exposure of infant monkeys to allergen and/or ozone

Both allergen and ozone exposure increase asthma symptoms and airway responsiveness in children. Little is known about how these inhalants may differentially modify airway responsiveness in large proximal as compared to small distal airways. We evaluated whether bronchi and respiratory bronchioles from infant monkeys exposed episodically to allergen and/or ozone differentially develop intrinsic hyperresponsiveness to methacholine and whether eosinophils and/or pulmonary neuroendocrine cells play a role. Infant monkeys were exposed episodically for 5 months to: (1) filtered air, (2) aerosolized house dust mite allergen, (3) ozone 0.5 ppm, or (4) house dust mite allergen + ozone. Studying the function/structure relationship of the same lung slices, we evaluated methacholine airway responsiveness and histology of bronchi and respiratory bronchioles. In bronchi, intrinsic responsiveness was increased by allergen exposure, an effect reduced by bombesin antagonist. In respiratory bronchioles, intrinsic airway responsiveness was increased by allergen + ozone exposure. Eosinophils were increased by allergen and allergen + ozone exposure in bronchi and by allergen exposure in respiratory bronchioles. In both airways, exposure to allergen + ozone resulted in fewer tissue eosinophils than did allergen exposure alone. In bronchi, but not in respiratory bronchioles, the number of eosinophils and neuroendocrine cells correlated with airway responsiveness. We conclude that episodically exposing infant monkeys to house dust mite allergen with or without ozone increased intrinsic airway responsiveness to methacholine in bronchi differently than in respiratory bronchioles. In bronchi, eosinophils and neuroendocrine cells may play a role in the development of airway hyperresponsiveness.     

Racemic salbutamol administration to guinea‐pigs selectively augments airway smooth muscle responsiveness to cholinoceptor agonists

1 An aim of this study was to investigate whether continuous in vivo administration of a low dose of salbutamol to guinea‐pigs alters the responsiveness of airway smooth muscle in vitro. 2 Osmotic minipumps containing a solution of racemic salbutamol were implanted subcutaneouly in guinea‐pigs. The drug was infused at a dose of 0.2 mg kg^?1 day^?1 for 10 days and, at the end of that time, the trachea was isolated and concentration–response relationships to several contractile agonists were examined. 3 This treatment resulted in significant increases in the maximum tension developed by tracheal preparations in response to cholinoceptor agonists, carbachol and methacholine. 4 Cumulative concentration–response curves for histamine, leukotriene D4, and KCl were similar in tracheal segments from saline‐control and salbutamol‐infused animals. 5 Time course experiments showed that augmented airway contractile responsiveness to cholinoceptor agonists was reversible within 3 days after cessation of the 10 day salbutamol infusion. 6 Our findings support the hypothesis that β₂‐adrenoceptor agonist drugs, administered over time in vivo, induce a transient hyperresponsiveness of airway smooth muscle to cholinergic bronchoconstrictor stimuli.     

The effects of inhaled corticosteroids on intrinsic responsiveness and histology of airways from infant monkeys exposed to house dust mite allergen and ozone

Inhaled corticosteroids (ICS) are recommended to treat infants with asthma, some with intermittent asthma. We previously showed that exposing infant monkeys to allergen/ozone resulted in asthma-like characteristics of their airways. We evaluated the effects of ICS on histology and intrinsic responsiveness of allergen/ozone-exposed and normal infant primate airways. Infant monkeys were exposed by inhalation to (1) filtered air and saline, (2) house dust mite allergen (HDMA)+ozone and saline, (3) filtered air and ICS (budesonide) or (4) HDMA+ozone and ICS. Allergen/ozone exposures started at 1 month and ICS at 3 months of age. At 6 months of age, methacholine-induced changes in luminal area of airways in proximal and distal lung slices were determined using videomicrometry, followed by histology of the same slices. Proximal airway responsiveness was increased by allergen/ozone and by ICS. Eosinophil profiles were increased by allergen/ozone in both proximal and distal airways, an effect that was decreased by ICS in distal airways. In both allergen/ozone- and air-exposed monkeys, ICS increased the number of alveolar attachments in distal airways, decreased mucin in proximal airways and decreased epithelial volume in both airways. ICS increased smooth muscle in air-exposed animals while decreasing it in allergen/ozone-exposed animals in both airways. In proximal airways, there was a small but significant positive correlation between smooth muscle and airway responsiveness, as well as between alveolar attachments and responsiveness. ICS change morphology and function in normal airways as well as allergen/ozone-exposed airways, suggesting that they should be reserved for infants with active symptoms.     

Ovalbumin-Induced Airway Inflammation and Fibrosis in Mice Also Exposed to Ozone

A murine model of allergen-induced airway inflammation was used to examine the effects of exposure to ozone on airway inflammation and remodeling. Sensitized BALB/c mice were exposed to ovalbumin aerosol for 4 wk before and after 2 wk of exposure to either 0.2 ppm or 0.5 ppm ozone. Other groups of mice were exposed to ovalbumin aerosol for 6 wk with continuous concurrent exposure to ozone during wk 1–6, or during intermittent concurrent exposure to ozone. Lung inflammation was measured by quantitative differential evaluation of lung lavage cells and by histological evaluation of stained lung sections. Alterations in lung structure (airway fibrosis) were evaluated by quantitative biochemical analysis of microdissected airways. The same total number of cells was observed in lavage fluid from animals exposed for 4 wk to ovalbumin alone or to ovalbumin for 4 wk immediately before or after exposure to 2 wk of 0.2 or 0.5 ppm ozone. Mice exposed to ovalbumin for 6 wk with concurrent exposure to either 0.2 ppm or 0.5 ppm ozone during wk 3–6 had a significant decrease in the total number of cells recovered by lavage. Values as low as 7% of the cell number found in mice exposed to ovalbumin aerosol alone were observed in the mice exposed to ovalbumin plus 0.2 ppm ozone during wk 3–6. There were significant differences in the cell differential counts in the lavage fluid from mice exposed to ovalbumin alone as compared with values from mice exposed to ovalbumin and ozone under all of the protocols studied. When ozone was given for 2 wk prior to ovalbumin exposure (Experiment 1), there were a high percentage of macrophages and low percentages of lymphocytes and eosinophils in the lung lavage. When ozone was given for 2 wk after ovalbumin exposure (Experiment 2), there were a moderate percentage of macrophages, a low percentage of eosinophils, and a high percentage of lymphocytes in the lung lavage. When ozone and ovalbumin were given simultaneously (Experiments 3 and 4), there were a high percentage of macrophages in the lavage with 0.2 ppm ozone and a high percentage of eosinophils. Ozone appears to antagonize the specific inflammatory effects of ovalbumin exposure, especially when given before or during exposure to ovalbumin. Airway remodeling was examined by two different quantitative methods. None of the groups exposed concurrently to ovalbumin and ozone had a significant increase in airway collagen content as compared to the matched groups of mice exposed to ovalbumin alone. The findings were consistent with an additive response of mice to simultaneous exposure to ovalbumin and ozone. Ozone exposure alone for 6 wk did not affect the number of goblet cells in the airways, while mice exposed to ovalbumin aerosol alone for 6 wk had about 25% goblet cells in their conducting airways. Concurrent exposure to ovalbumin and 0.2 ppm ozone caused significant increases in goblet cells (to 43% of total cells) in the conducting airways of the exposed mice. We conclude that when mice with allergen-induced airway inflammation induced by ovalbumin are also exposed to ozone, the lung inflammatory response may be modified, but that this altered response is dependent on the sequence of exposure and the concentration of ozone to which they are exposed. At the concentrations of ozone tested, we did not see changes in airway fibrosis. However, goblet-cell hyperplasia appeared to be increased in mice exposed concurrently to ovalbumin and 0.2 ppm ozone.     

Ovalbumin-induced airway inflammation and fibrosis in mice also exposed to ozone

A murine model of allergen-induced airway inflammation was used to examine the effects of exposure to ozone on airway inflammation and remodeling. Sensitized BALB/c mice were exposed to ovalbumin aerosol for 4 wk before and after 2 wk of exposure to either 0.2 ppm or 0.5 ppm ozone. Other groups of mice were exposed to ovalbumin aerosol for 6 wk with continuous concurrent exposure to ozone during wk 1-6, or during intermittent concurrent exposure to ozone. Lung inflammation was measured by quantitative differential evaluation of lung lavage cells and by histological evaluation of stained lung sections. Alterations in lung structure (airway fibrosis) were evaluated by quantitative biochemical analysis of microdissected airways. The same total number of cells was observed in lavage fluid from animals exposed for 4 wk to ovalbumin alone or to ovalbumin for 4 wk immediately before or after exposure to 2 wk of 0.2 or 0.5 ppm ozone. Mice exposed to ovalbumin for 6 wk with concurrent exposure to either 0.2 ppm or 0.5 ppm ozone during wk 3-6 had a significant decrease in the total number of cells recovered by lavage. Values as low as 7% of the cell number found in mice exposed to ovalbumin aerosol alone were observed in the mice exposed to ovalbumin plus 0.2 ppm ozone during wk 3-6. There were significant differences in the cell differential counts in the lavage fluid from mice exposed to ovalbumin alone as compared with values from mice exposed to ovalbumin and ozone under all of the protocols studied. When ozone was given for 2 wk prior to ovalbumin exposure (Experiment 1), there were a high percentage of macrophages and low percentages of lymphocytes and eosinophils in the lung lavage. When ozone was given for 2 wk after ovalbumin exposure (Experiment 2), there were a moderate percentage of macrophages, a low percentage of eosinophils, and a high percentage of lymphocytes in the lung lavage. When ozone and ovalbumin were given simultaneously (Experiments 3 and 4), there were a high percentage of macrophages in the lavage with 0.2 ppm ozone and a high percentage of eosinophils. Ozone appears to antagonize the specific inflammatory effects of ovalbumin exposure, especially when given before or during exposure to ovalbumin. Airway remodeling was examined by two different quantitative methods. None of the groups exposed concurrently to ovalbumin and ozone had a significant increase in airway collagen content as compared to the matched groups of mice exposed to ovalbumin alone. The findings were consistent with an additive response of mice to simultaneous exposure to ovalbumin and ozone. Ozone exposure alone for 6 wk did not affect the number of goblet cells in the airways, while mice exposed to ovalbumin aerosol alone for 6 wk had about 25% goblet cells in their conducting airways. Concurrent exposure to ovalbumin and 0.2 ppm ozone caused significant increases in goblet cells (to 43% of total cells) in the conducting airways of the exposed mice. We conclude that when mice with allergen-induced airway inflammation induced by ovalbumin are also exposed to ozone, the lung inflammatory response may be modified, but that this altered response is dependent on the sequence of exposure and the concentration of ozone to which they are exposed. At the concentrations of ozone tested, we did not see changes in airway fibrosis. However, goblet-cell hyperplasia appeared to be increased in mice exposed concurrently to ovalbumin and 0.2 ppm ozone.     

Disruption of tracheobronchial airway growth following postnatal exposure to ozone and ultrafine particles

This study examined airway structure changes in adult rats after a long recovery period due to sub-chronic juvenile exposure to ozone and ultrafine particles that have a high organic fraction. Neonatal male Sprague-Dawley rats were exposed during lung development to 3 cycles of 0.5?ppm ozone from postnatal day 7 through 25. Two different exposure patterns were used: 5-day exposure per week (Ozone52) or 2-day exposure per week (Ozone25) with or without co-exposure to ultrafine particles (OPFP5252, OPFP5225). Airway architecture was evaluated at 81 days of age, after 56 days of continued development beyond the exposure period in filtered air (FA). By analyzing CT images from lung airway casts, we determined airway diameter, length, branching angle, and rotation angle for most conducting airways. Compared with the FA control group, the Ozone52 group showed significant decreases in airway diameter in generations larger than 10 especially in the right diaphragmatic lobe and in airway length in distal generations, while changes in airway structure due to the Ozone25 exposure were not appreciable. Interaction effects of ozone and ultrafine particle exposures were not significant. These results suggest that airway alterations due to postnatal ozone exposure are not limited to the distal region but occur extensively from the middle to distal conducting airways. Further, alterations due to early ozone exposure do not recover nearly 2 months after exposure has ceased demonstrating a persistent airway structural change following an early life exposure to ozone.     

Disruption of tracheobronchial airway growth following postnatal exposure to ozone and ultrafine particles

This study examined airway structure changes in adult rats after a long recovery period due to sub-chronic juvenile exposure to ozone and ultrafine particles that have a high organic fraction. Neonatal male Sprague-Dawley rats were exposed during lung development to 3 cycles of 0.5 ppm ozone from postnatal day 7 through 25. Two different exposure patterns were used: 5-day exposure per week (Ozone52) or 2-day exposure per week (Ozone25) with or without co-exposure to ultrafine particles (OPFP5252, OPFP5225). Airway architecture was evaluated at 81 days of age, after 56 days of continued development beyond the exposure period in filtered air (FA). By analyzing CT images from lung airway casts, we determined airway diameter, length, branching angle, and rotation angle for most conducting airways. Compared with the FA control group, the Ozone52 group showed significant decreases in airway diameter in generations larger than 10 especially in the right diaphragmatic lobe and in airway length in distal generations, while changes in airway structure due to the Ozone25 exposure were not appreciable. Interaction effects of ozone and ultrafine particle exposures were not significant. These results suggest that airway alterations due to postnatal ozone exposure are not limited to the distal region but occur extensively from the middle to distal conducting airways. Further, alterations due to early ozone exposure do not recover nearly 2 months after exposure has ceased demonstrating a persistent airway structural change following an early life exposure to ozone.     

Comparison of acute ozone-induced nasal and pulmonary inflammatory responses in rats

The centriacinar pulmonary lesion induced by ozone has been extensively characterized, but little is known about the effects of this oxidant gas in the upper airways. The present study was designed to compare the effects of acute ozone exposure in the nose and lungs of rats. We examined the cellular inflammatory responses in the nasal cavity and lower respiratory tract by means of nasal and bronchoalveolar lavage and morphometric quantitation of neutrophils within the nasal mucosa and pulmonary terminal bronchioloalveolar duct regions (i.e., centriacinar). Rats were exposed to 0.0, 0.12, 0.8, or 1.5 ppm ozone for 6 hr and were sacrificed immediately or 3, 18, 42, or 66 hr following exposure. Eighteen hours after exposure, increased numbers of neutrophils, as compared to controls, were recovered from nasal lavage fluid (NLF) of rats exposed to 0.12 ppm ozone. There was no change in the number of neutrophils recovered from bronchoalveolar lavage fluid (BALF) at any time after exposure. Rats exposed to 0.8 ppm ozone had more neutrophils in NLF than controls immediately after exposure, but no concomitant increase in BALF neutrophils at that time. However, as the number of neutrophils in BALF increased (maximum at 42 hr), the number of neutrophils recovered from NLF decreased (minimum at 42 hr). Rats exposed to 1.5 ppm ozone had no significant increases in nasal neutrophils in NLF at any time after exposure but had greatly increased numbers of neutrophils in BALF 3, 18, and 42 hr after exposure. The number of neutrophils recovered by nasal and bronchoalveolar lavage accurately reflected the tissue neutrophil response at sites within the nasal cavity and lung that were injured by acute ozone exposure. Our results suggest that at high ozone concentrations (0.8 and 1.5 ppm), the acute nasal inflammatory response is attenuated by a simultaneous, competing, inflammatory response within the centriacinar region of the lung. Analysis of nasal lavage fluid for changes in cellular composition may be a useful indicator of acute exposure to ambient levels of ozone, but at higher ozone levels, the nasal cellular inflammatory response may underestimate the effects of ozone on nasal and pulmonary epithelia.     

TIME COURSE OF RESPONSE TO OZONE EXPOSURE IN HEALTHY ADULT FEMALES

Ozone exposure causes acute decrements in pulmonary function, increases airway responsiveness, and changes the breathing pattern. We examined these responses in 19 ozone-responsive (D FEV1 > 5%) young females exposed to both air and 0.35 ppm ozone. The randomized 75-min exposures included two 30-min exercise periods at V E 40 L/min. Responses were measured before, during, and after exposure and at 18 and 42 h postexposure. FVC, FEV1, and FIV0.5 decreased (p < .01) immediately postexposure by 13.2%, 19.9%, and 20.8%, respectively, and the airway responsiveness was significantly increased. Raw increased (p < .05), while TGV remained essentially unchanged. At 18 h postexposure, the airways were still hyperresponsive and FEV1 and FIV0.5 were still 5% below the preexposure levels. There were no residual effects in any of the variables at 42 h postexposure. During exercise in ozone the tidal volume was decreased (-14%) and respiratory frequency increased (+15%). The changes in airway responsiveness were not related to changes in spirometric measurements. We found no significant differences between postair and postozone mouth occlusion pressure (Pm0.1) and the hypercapnic response to CO2 rebreathing. We conclude that ozone induced typical acute changes in airway responsiveness and that ventilatory (exercise), spirometric (inspiratory and expiratory), and plethysmographic pulmonary function may show some residual effects for up to 18 h after exposure. The ozoneinduced alteration in breathing pattern during exercise does not appear to be related to a change in ventilatory drive.     

Bronchopulmonary inflammation and airway smooth muscle hyperresponsiveness induced by nitrogen dioxide in guinea pigs.

We investigated whether acute exposure to nitrogen dioxide (NO2) causes major inflammatory responses (inflammatory cell recruitment, oedema and smooth muscle hyperresponsiveness) in guinea pig airways. Anaesthetised guinea pigs were exposed to 18 ppm NO2 or air for 4 h through a tracheal cannula. Bronchoalveolar lavage was performed and airway microvascular permeability and in vitro bronchial smooth muscle responsiveness were measured. Exposure to NO2 induced a significant increase in eosinophils and neutrophils in bronchoalveolar lavage fluid, microvascular leakage in the trachea and main bronchi (but not in peripheral airways), and a significant in vitro hyperresponsiveness to acetylcholine, electrical field stimulation, and neurokinin A, but not to histamine. Thus, this study shows that in vivo exposure to high concentrations of NO2 induces major inflammatory responses in guinea pig airways that mimic acute bronchitis induced by exposure to irritant gases in man.     

Time course of response to ozone exposure in healthy adult females

Ozone exposure causes acute decrements in pulmonary function, increases airway responsiveness, and changes the breathing pattern. We examined these responses in 19 ozone-responsive (DeltaFEV(1) > 5%) young females exposed to both air and 0.35 ppm ozone. The randomized 75-min exposures included two 30-min exercise periods at V(E) approximately 40 L/min. Responses were measured before, during, and after exposure and at 18 and 42 h postexposure. FVC, FEV(1), and FIV(0.5) decreased (p <.01) immediately postexposure by 13.2%, 19.9%, and 20.8%, respectively, and the airway responsiveness was significantly increased. Raw increased (p <.05), while TGV remained essentially unchanged. At 18 h postexposure, the airways were still hyperresponsive and FEV(1) and FIV(0.5) were still 5% below the preexposure levels. There were no residual effects in any of the variables at 42 h postexposure. During exercise in ozone the tidal volume was decreased (-14%) and respiratory frequency increased (+15%). The changes in airway responsiveness were not related to changes in spirometric measurements. We found no significant differences between postair and postozone mouth occlusion pressure (Pm(0.1)) and the hypercapnic response to CO(2) rebreathing. We conclude that ozone induced typical acute changes in airway responsiveness and that ventilatory (exercise), spirometric (inspiratory and expiratory), and plethysmographic pulmonary function may show some residual effects for up to 18 h after exposure. The ozone-induced alteration in breathing pattern during exercise does not appear to be related to a change in ventilatory drive.     

Dose-response relationship of ozone-induced airway hyperresponsiveness in unanesthetized guinea pigs

The effect of ozone dose (the product of ozone concentration and exposure time) on airway responsiveness was examined in unanesthetized, spontaneously breathing guinea pigs. Airway responsiveness was assessed by measuring specific airway resistance (sRaw) as a function of increasing concentration of inhaled methacholine (Mch) aerosol (the concentration of Mch required in order to double the baseline sRaw: PC200Mch). The airway responsiveness was measured before and at 5 min, 5 h, and 24 h after exposure. A 30-min exposure to 1 ppm ozone (dose 30 ppm.min) did not change PC200Mch at any time after exposure. Both a 90-min exposure to 1 ppm ozone and a 30-min exposure to 3 ppm ozone, which are identical in terms of ozone dose (90 ppm.min), decreased PC200Mch to a similar degree. A 120-min exposure to 3 ppm ozone (360 ppm.min) produced a much greater decrease of PC200Mch at 5 min and 5 h after exposure, compared with low-dose exposure. There was a significant correlation between ozone dose and the change in airway responsiveness. In all groups, the baseline sRaw was increased by approximately 50% at 5 min after exposure, but there was no correlation between the changes in PC200Mch and the baseline sRaw. This study suggests that ozone-induced airway hyperresponsiveness in guinea pigs is closely related to ozone dose.     

Regional differences in reactivity and in the influence of the epithelium on canine intrapulmonary bronchial smooth muscle responsiveness

Differences in the reactivity and in the influence of the epithelium on responsiveness of canine 2nd and 3rd generation airway smooth muscle were examined. Epithelium-containing 3rd generation airways produced a greater maximum contraction and were more sensitive to methacholine and histamine, but not to KCl, than corresponding 2nd generation airways. Mechanical removal of the epithelium increased the sensitivity to methacholine and histamine in 2nd generation airways; there was also an increase in the maximum response elicited by histamine, but not by methacholine, in epithelium-free preparations. In contrast, there was no significant difference in the sensitivity to or the maximum response elicited by histamine or methacholine in epithelium-containing and epithelium-free 3rd generation airways. Epithelium removal had no effect on KCl-induced responses in either airway region. The inhibitory effects of verapamil (1 microM) against KCl- and methacholine-induced responses were identical in preparations containing and lacking the epithelium. The results support the postulate of an epithelium-derived inhibitory factor modulating airway smooth muscle reactivity. Furthermore, the influence of the epithelium exhibits regional differences, being greater in larger airways.     

Changes in neuroreceptor function of tracheal smooth muscle following acute ozone exposure of guinea pigs

We studied the effect of in vivo ozone inhalation (3 ppm, 2 h) on neuroreceptor function in guinea pig tracheal smooth muscle in vitro and the role of the epithelial layer in this process. Changes in smooth muscle tension after stimulation of the muscarinic- and β-adrenergic receptor were recorded isometrically and stained tracheal tissue sections were histologically evaluated for changes in the epithelial and smooth muscle layer. Ozone exposure resulted in an increase in maximal contraction following stimulation of the muscarinic receptor, whereas pD₂ values remained unchanged. After stimulation of the β-adrenergic receptor no increase in maximal relaxation but only an increase in pD₂ value was observed after correction for differences in precontraction level in control- and ozone-exposed situations. Mechanical removal of the epithelial layer resulted in a slight increase of the maximal contraction level after stimulation with methacholine in the control situation, whereas exposure to ozone resulted in a strong decrease of the maximal contraction level under these conditions. Histological stainings showed a slight and focal influx of neutrophilic granulocytes in the epithelial layer, submucosal layer and airway lumen after exposure to ozone. These data support the idea that ozone is able to increase the maximal degree of airway narrowing upon muscarinergic stimulation i.e. a hyperreactivity response. The results also suggest that functionally altered epithelium plays an important role in the process of ozone-induced hyperreactivity, possibly linked with an early inflammatory response.     

Acetylcholine-induced smooth muscle contraction of intrapulmonary small bronchi is augmented in antigen-induced airway hyperresponsive rats

Smooth muscle responsiveness of intrapulmonary small bronchi obtained from repeatedly antigen-challenged rats was compared with that from control animals to determine whether smooth muscle contractility of peripheral airways is augmented by such repeated challenge. In intact (non-permeabilized) smooth muscles of intrapulmonary bronchi, the acetylcholine (ACh)-induced contractile response was significantly augmented in the repeated challenge group, although 60-mM K+-induced contraction was within the normal level. In beta-escin-permeabilized muscles, no significant difference between groups was observed in the Ca2+-induced contractile responses. Thus, augmented ACh-induced contraction of intact intrapulmonary small bronchial smooth muscle might be, at least in part, due to an enhanced ACh-mediated Ca2+-sensitizing signal.