Exposing guinea pigs to ozone for 1wk enhances responsiveness of rapidly adapting receptors

Acute exposure to ozone causes changes inbreathing pattern and lung function which may be caused in part bystimulation of rapidly adapting receptors (RARs). The consequences ofrepeated daily ozone exposure on RAR responsiveness are unknown,although ozone-induced changes in pulmonary function diminish withrepeated exposure. Accordingly, we investigated whether repeated daily ozone exposure diminishes the general responsiveness of RARs. Guineapigs (n = 30) were exposed to 0.5 parts/million ozone or filtered air (8 h/day for 7 days). The animalswere then anesthetized, and RAR impulse activity, dynamic compliance(Cdyn), and lung resistance were recorded at baseline and in responseto four stimuli: substance P, methacholine, hyperinflation, and removalof positive end-expiratory pressure. Repeated daily ozone exposureexaggerated RAR responses to substance P, methacholine, andhyperinflation without causing physiologically relevant effects onbaseline or substance P- and methacholine-induced changes in Cdyn andlung resistance. Because agonist-evoked changes in RAR activitypreceded Cdyn changes, the data suggest that repeated daily ozoneexposure enhances RAR responsiveness via a mechanism other than changes in Cdyn.

     

Enhanced airway responses to substance P after repeated challenge in guinea pigs

We performed three consecutive dose-response curves to rapid intravenous infusions of substance P (SP) in anesthetized, mechanically ventilated guinea pigs. The dose of SP required to decrease pulmonary conductance to 50% of its base-line value (ED50GL) decreased 2.8-fold (P less than 0.002) and 3.3-fold (P less than 0.001) on the second and third dose-response curves, respectively, compared with the first. SP did not alter airway responses to intravenous histamine but did cause a significant (3.7-fold) decrease in ED50GL for dose-response curves to intravenous capsaicin, an agent that causes bronchoconstriction by release of endogenous tachykinins. The neutral metalloendopeptidase inhibitor thiorphan (0.5 mg) and the angiotensin-converting enzyme inhibitor captopril (1.7 mg) both caused a marked enhancement of airway responses to SP observed on the first dose-response curve but did not alter the enhancement of SP-induced airway responses produced by repeated SP challenge. The anticholinergic atropine (5 mg/kg iv), the antihistamine mepyramine (8 mg/kg iv), and the cyclooxygenase inhibitor indomethacin (30 mg/kg ip) had no effect on the first SP dose-response curve. Atropine and mepyramine did not prevent the enhancement of SP responses observed with repeated challenge, but after pretreatment with either indomethacin or acetylsalicylic acid, dose-response curves to SP were reproducible. Our results indicate that airway responses to intravenous SP are enhanced with repeated SP challenge and suggest that cyclooxygenase products of arachidonic acid metabolism are involved in the mediation of this phenomenon.     

Chronic exposure to sidestream tobacco smoke augments lung C-fiber responsiveness in young guinea pigs.

Children chronically exposed to environmental tobacco smoke (ETS) have more coughs, wheezes, and airway obstruction, which may result in part from stimulation of lung C fibers. We examined the effect of chronic exposure to sidestream tobacco smoke (SS, a surrogate for ETS) on lung C-fiber responsiveness in guinea pigs, in which dynamic compliance (Cdyn), lung resistance, tracheal pressure, arterial blood pressure, and heart rate were also monitored. Guinea pigs were exposed to SS (1 mg/mm(3) total suspended particulates) or filtered air 5 days/wk from 1 to 6 wk of age. They were then anesthetized, and lung C fibers (n = 55), identified by a conduction velocity of <2.0 m/s, were tested for responsiveness to chemical and mechanical stimuli. SS exposure doubled C-fiber responsiveness to left atrial capsaicin (P = 0.02) and lung hyperinflation (P = 0.03) but had no effect on responsiveness to inhaled capsaicin or bradykinin or on baseline activity. The data indicate that chronically exposing young guinea pigs to SS enhances C-fiber sensitivity to certain stimuli and may help explain respiratory symptoms in children exposed to ETS.     

Tobacco smoke exposure and bombesin-like peptides in guinea pigs

Bombesin-like peptides (BLPs) are associated with tobacco smoke (TS)-induced diseases. We sought to determine if acute TS exposure releases BLPs into the pulmonary circulation. Sensitized and non-sensitized guinea pigs were chronically exposed to TS or compressed air. Thereafter, the lungs were acutely challenged with TS while perfused. Perfusates were analyzed for BLPs. TS increased BLPs in non-sensitized guinea pigs. A separate study determined daily bombesin exposure increased lung cell counts but not airway hyperresponsivensess. TS exposure releases BLPs into the pulmonary circulation but can be modified by host factors and bombesin itself does not induce airway hyperresponsiveness.     

Cigarette smoke exposure potentiates bleomycin-induced lung fibrosis in guinea pigs

The role of tobacco smoking in the development and outcome of pulmonary fibrosis is uncertain. To approach the effects of cigarette smoke on bleomycin-induced lung fibrosis, we studied five groups of guinea pigs: 1) controls, 2) instilled with bleomycin (B), 3) exposed to tobacco smoke for 6 wk (TS), 4) bleomycin instillation plus tobacco smoke exposure for 6 wk (B+TS), and 5) tobacco smoke exposure for 6 wk and bleomycin after smoking (TS/B). Guinea pigs receiving bleomycin and tobacco smoke exposure exhibited higher fibrotic lesions including a significant increase in the number of positive alpha-smooth muscle actin cells compared with bleomycin alone (B+TS, 3.4 +/- 1.2%; TS/B, 3.7 +/- 1.5%; B, 2.3 +/- 1.5%; P < 0.01). However, only the TS/B group reached a significant increase in lung collagen compared with the bleomycin group (TS/B, 3.5 +/- 0.7; B +/- TS, 2.9 +/- 0.4; B, 2.4 +/- 0.2 mg hydroxyproline/lung; P < 0.01). Bronchoalveolar lavage (BAL) from TS/B showed an increased number of eosinophils and higher levels of IL-4 and tissue inhibitor of metalloproteinase-2 (P < 0.01 for all comparisons) and induced a significant increase in fibroblast proliferation (P < 0.05). Importantly, smoke exposure alone induced an increase in BAL neutrophils, matrix metalloproteinase-9, and fibroblast proliferation compared with controls, suggesting that tobacco smoke creates a profibrotic milieu that may contribute to the increased bleomycin-induced fibrosis.     

Respiratory epithelial permeability after cigarette smoke exposure in guinea pigs

The purpose of this study was to determine the pathology of cigarette smoke-increased permeability at the bronchioalveolar junction of the guinea pig. After exposure to either smoke or room air, guinea pigs were anesthetized and fluorescein isothiocyanate-dextran (FITC-D, mol wt 10,000) was aerosolized into their lungs. Blood samples taken through a carotid arterial cannula were analyzed by gel chromatography and spectrofluorometry for the presence of FITC-D. The results confirmed that, after smoke exposure, increased amounts of intact FITC-D molecules with a reported Einstein-Stokes radius of 22.2 A crossed the respiratory epithelium into the vascular space. Transmission electron-microscopic studies showed that the FITC-D diffused across damaged type I pneumocyte membranes and cytoplasm to reach the basal lamina and entered the alveolar capillaries through endothelial tight junctions. Damage to the alveolar epithelium was more frequent for the smoke-exposed animals than the room air-exposed animals (P less than 0.05). We conclude that smoke exposure damages type I cells and that inhaled FITC-D crosses the epithelial barrier at damaged type I cells of the bronchioloalveolar junctions.     

Airway hyperresponsiveness to bronchoconstrictor challenge after wood smoke exposure in guinea pigs.

Prior airway exposure to wood smoke induces an increase in airway responsiveness to subsequent smoke inhalation in guinea pigs (Life Sci. 63: 1513, 1998; 66: 971, 2000). To further characterize this airway hyperreactivity, we investigated and compared the airway responsiveness to bronchoconstrictor challenge before and 30 min after sham air exposure or wood smoke exposure in anesthetized and artificially ventilated guinea pigs. Various doses of substance P (0.8-6.4 microg/kg), capsaicin (0.2-3.2 microg/kg), prostaglandin F2alpha (30-3000 microg/kg), histamine (1-8 microg/kg), or acetylcholine (5-20 microg/kg) were intravenously injected at 2-min intervals in successively increasing doses to obtain the dose required to provoke a 200% increase in baseline total lung resistance (ED200). Wood smoke exposure significantly lowered the ED200 of substance P, capsaicin, and prostaglandin F2alpha whereas sham air exposure failed to do so. Furthermore, wood smoke exposure did not significantly alter the ED200 of histamine or acetylcholine. Pretreatment with phosphoramidon (2 mg/kg), an inhibitor of the neutral endopeptidase (the major degradation enzyme of substance P), before smoke exposure did not significantly affect the smoke-induced reduction in ED200 of substance P. Sectioning both cervical vagi before smoke exposure did not significantly alter the smoke-induced reduction in ED200 of capsaicin or prostaglandin F2alpha. These results suggest that airway exposure to wood smoke acutely produces airway hyperresponsiveness to substance P, capsaicin, and prostaglandin F2alpha, but not to histamine or acetylcholine. Since the combination of phosphoramidon and wood smoke exposure did not result in an additive potentiation of smoke-induced airway hyperresponsiveness to substance P, it is suggested that an inhibition of the degradation enzyme of substance P may contribute to this increase in airway reactivity. Furthermore, vagally-mediated bronchoconstriction does not play a vital role in enhanced airway responsiveness to capsaicin or prostaglandin F2alpha.     

Effect of the NEP inhibitor SCH32615 on airway responses to intravenous substance P in guinea pigs.

We examined the effects of the selective neutral endopeptidase (NEP) inhibitor SCH32615 on airway responses to rapid intravenous infusions of substance P (SP) and neurokinin A (NKA) and on recovery of administered tachykinins from arterial blood in anesthetized mechanically ventilated guinea pigs. SCH32615, in doses that cause a marked increase in the magnitude of bronchoconstriction induced by infused NKA, had little effect on the changes in pulmonary conductance (GL) or dynamic compliance induced by SP. In animals in which SCH32615 (1 mg/kg) was administered in combination with the angiotensin-converting enzyme (ACE) inhibitor captopril (5.7 mg/kg), the dose of SP required to decrease GL by 50% was fourfold less than in animals that received captopril alone (P < 0.005). SP measured in arterial blood withdrawn within 45 s of intravenous administration of this tachykinin was not different in control and SCH32615-treated animals, whereas captopril caused an approximately threefold increase in SP concentrations (P < 0.005). When SCH32615 and captopril were administered together, significantly more SP was recovered than when captopril or SCH32615 was administered alone (P < 0.0005). Our results are consistent with the hypothesis that both NEP and ACE contribute to the degradation of intravenously infused SP. ACE degradation of SP is sufficient to limit SP-induced bronchoconstriction even in the presence of specific NEP inhibition.     

Chronic tobacco smoke exposure increases airway sensitivity to capsaicin in awake guinea pigs.

Tobacco smoke (TS) exposure induces airway hyperreactivity, particularly in sensitive individuals with asthma. However, the mechanism of this airway hyperreactivity is not well understood. To investigate the relative susceptibility of atopic and nonatopic individuals to TS-induced airway hyperreactivity, we exposed ovalbumin (OA)-sensitized and nonsensitized guinea pigs to TS exposure (5 mg/l air, 30-min exposure, 7 days/wk for 120-156 days). Two similar groups exposed to compressed air served as controls. Airway reactivity was assessed as an increase in enhanced pause (Penh) units using a plethysmograph that allowed free movement of the animals. After 90 days of exposure, airway reactivity increased in OA-TS guinea pigs challenged with capsaicin, bradykinin, and neurokinin A fragment 4--10 aerosols. In addition, substance P content increased in lung perfusate of OA-TS guinea pigs in response to acute TS challenge compared with that of the other groups. Airway hyperirritability was not enhanced by phosphoramidon but was attenuated by a cocktail of neurokinin antagonists, nor was airway hyperreactivity observed after either methacholine or histamine challenge in OA-TS guinea pigs. Chronic TS exposure enhanced neither airway reactivity to histamine or methacholine nor contractility of isolated tracheal rings. In conclusion, chronic TS exposure increased airway reactivity to capsaicin and bradykinin aerosol challenge, and OA-TS guinea pigs were most susceptible to airway dysfunction as the result of exposure to TS compared with the other groups. Increased airway reactivity to capsaicin suggests a mechanism involving neurogenic inflammation, such as increased activation of lung C fibers.     

Prostaglandin involvement in lung C-fiber activation by substance P in guinea pigs.

Airway hyperresponsiveness is a cardinal feature of asthma. Lung C-fiber activation induces central and local defense reflexes that may contribute to airway hyperresponsiveness. Initial studies show that substance P (SP) activates C fibers even though it is produced and released by these same C fibers. SP may induce release of other endogenous mediators. Bradykinin (BK) is an endogenous mediator that activates C fibers. The hypothesis was tested that SP activates C fibers via BK release. Guinea pigs were anesthetized, and C-fiber activity (FA), pulmonary insufflation pressure (PIP), heart rate, and arterial blood pressure were monitored before and after intravenous injection of capsaicin (Cap), SP, and BK. Identical agonist challenges were repeated after infusion of an antagonist cocktail of des-Arg9-[Leu8]-BK (10(-3) M, B1 antagonist), and HOE-140 (10(-4) M, B2 antagonist). After antagonist administration, BK increased neither PIP nor FA. Increases in neither PIP nor FA were attenuated after Cap or SP challenge. In a second series of experiments, Cap and SP were injected before and after infusion of indomethacin (1 mg/kg iv) to determine whether either agent activates C fibers through release of arachidonic acid metabolites. Indomethacin administration decreased the effect of SP challenge on FA but not PIP. The effect of Cap on FA or PIP was not altered by indomethacin. In subsequent experiments, C fibers were activated by prostaglandin E2 and F2alpha. Therefore, exogenously applied SP stimulates an indomethacin-sensitive pathway leading to C-fiber activation.     

Airway hyperresponsiveness induced by chronic exposure to cigarette smoke in guinea pigs: role of tachykinins.

This study was carried out to determine whether tachykinins released from lung C-fiber afferents play a part in the bronchial hyperreactivity induced in guinea pigs by chronic exposure to cigarette smoke (CS). Two matching groups of young guinea pigs were exposed to either mainstream CS (CS group) or air (control group) for 20 min twice daily for 14-17 days. There was no difference in the baseline total pulmonary resistance (RL) between the two groups, but the baseline dynamic lung compliance was reduced ( approximately 19%) in CS animals. The responses of RL to intravenous injections of ACh, neurokinin (NK) A, and capsaicin were all markedly increased in CS animals; for example, ACh at the same dose of 5.06 microg/kg increased RL by 207% in the control group and by 697% (n = 8; P < 0. 001) in the CS group. The increased responsiveness was accompanied by significant increases in the numbers of neutrophils, eosinophils, and macrophages in the bronchoalveolar lavage fluid in CS animals. Pretreatment with SR-48968 and CP-99994, antagonists of NK(1) and NK(2) receptors, respectively, did not alter the response of RL to ACh in control animals, but it abolished the elevated bronchoconstrictive response in the CS animals. Furthermore, the immunoreactivities of substance P and calcitonin gene-related peptide in the bronchoalveolar lavage fluid collected after capsaicin challenge were significantly increased in CS animals. These results show that chronic exposure to CS induced airway mucosal inflammation accompanied by bronchial hyperreactivity in guinea pigs and that the tachykininergic mechanism plays an important role in this augmented responsiveness.     

Allergic inflammation-induced neuropeptide production in rapidly adapting afferent nerves in guinea pig airways

In the vagal-sensory system, neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) are synthesized nearly exclusively in small-diameter nociceptive type C-fiber neurons. By definition, these neurons are designed to respond to noxious or tissue-damaging stimuli. A common feature of visceral inflammation is the elevation in production of sensory neuropeptides. Little is known, however, about the physiological characteristics of vagal sensory neurons induced by inflammation to produce substance P. In the present study, we show that allergic inflammation of guinea pig airways leads to the induction of substance P and CGRP production in large-diameter vagal sensory neurons. Electrophysiological and anatomical evidence reveals that the peripheral terminals of these neurons are low-threshold Adelta mechanosensors that are insensitive to nociceptive stimuli such as capsaicin and bradykinin. Thus inflammation causes a qualitative change in chemical coding of vagal primary afferent neurons. The results support the hypothesis that during an inflammatory reaction, sensory neuropeptide release from primary afferent nerve endings in the periphery and central nervous system does not require noxious or nociceptive stimuli but may also occur simply as a result of stimulation of low-threshold mechanosensors. This may contribute to the heightened reflex physiology and pain that often accompany inflammatory diseases.     

Lung glutathione adaptive responses to cigarette smoke exposure

Background

Smoking tobacco is a leading cause of chronic obstructive pulmonary disease (COPD), but although the majority of COPD cases can be directly related to smoking, only a quarter of smokers actually develop the disease. A potential reason for the disparity between smoking and COPD may involve an individual''s ability to mount a protective adaptive response to cigarette smoke (CS). Glutathione (GSH) is highly concentrated in the lung epithelial lining fluid (ELF) and protects against many inhaled oxidants. The changes in GSH that occur with CS are not well investigated; therefore the GSH adaptive response that occurs with a commonly utilized CS exposure was examined in mice.

Methods

Mice were exposed to CS for 5 h after which they were rested in filtered air for up to 16 h. GSH levels were measured in the ELF, bronchoalveolar lavage cells, plasma, and tissues. GSH synthesis was assessed by measuring γ-glutamylcysteine ligase (GCL) activity in lung and liver tissue.

Results

GSH levels in the ELF, plasma, and liver were decreased by as much as 50% during the 5 h CS exposure period whereas the lung GSH levels were unchanged. Next, the time course of rebound in GSH levels after the CS exposure was examined. CS exposure initially decreased ELF GSH levels by 50% but within 2 h GSH levels rebound to about 3 times basal levels and peaked at 16 h with a 6-fold increase and over repeat exposures were maintained at a 3-fold elevation for up to 2 months. Similar changes were observed in tissue GCL activity which is the rate limiting step in GSH synthesis. Furthermore, elevation in ELF GSH levels was not arbitrary since the CS induced GSH adaptive response after a 3d exposure period prevented GSH levels from dropping below basal levels.

Conclusions

CS exposures evoke a powerful GSH adaptive response in the lung and systemically. These data suggests there may be a sensor that sets the ELF GSH adaptive response to prevent GSH levels from dipping below basal levels. Factors that disrupt GSH adaptive responses may contribute to the pathophysiology of COPD.     

Lysophosphatidic acid enhances airway response to acetylcholine in guinea pigs.

Inhalation of lysophosphatidic acid (LPA, 1-100 microg/ml) for 2 min enhanced the airway response induced by intravenous injection of ACh in guinea pigs. At 30 min after inhalation of LPA, the airway response to ACh was two fold higher than that before inhalation. This enhancement of airway response to ACh was partially inhibited by capsaicin desensitization or bilateral vagotomy. These results suggested that the enhancement of airway response to ACh induced by LPA may be due to the activation of capsaicin-sensitive fibers. It can be also contribute to bronchial asthma or other types of pulmonary disease such as cough variant asthma and atopic cough.     

Bronchial reactivities in guinea pigs to acetylcholine or histamine exposure

The bronchial reactivities in Hartley guinea pigs to acetylcholine (ACh) or histamine (Hist) were investigated, and the following results obtained; 1. Eight-week-old animals were exposed to ACh and Hist. A significant relationship was observed between the concentrations of the chemicals and the time needed to produce falling down (TNPFD) due to the asthmatic reaction to ACh and Hist. 2. Eight-week-old animals were exposed to 0.1% ACh and 0.05% Hist, for which the mean TNPFD +/- standard error were 377 +/- 33 sec and 122 +/- 5 sec respectivity. However, no difference in reactivity between male and female animals was noted. 3. Eight- and 9-week-old animals were exposed to 0.01% ACh and 0.025% Hist. A positive correlation was observed (r = 0.736, p less than 0.01) between the TNPFD for ACh and that for Hist. 4. Growing animals from 2 weeks to 20 weeks old were exposed to 0.08% ACh and 0.025% Hist. After inhalation of both chemicals, 6-week-old animals showed the greatest prolongation of mean TNPFD (lowest sensitivity). 5. Eight-week-old animals were exposed to 0.08% ACh and 0.025% Hist. With both of these chemicals, a positive correlation was observed between TNPFD and dose threshold (ACh r = 0.886, p less than 0.001; Hist r = 0.891, p less than 0.001).