INTRODUCTION

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The acute inhalation of ozone, a ubiquitous component of photochemical air pollution, has been shown to result in numerous pathophysiologic responses. The acute physiologic responses induced by the inhalation of ambient levels of ozone in healthy human subjects include a decreased inspiratory capacity, a mild bronchoconstriction, a rapid shallow breathing pattern during exercise, and subjective symptoms of throat tickle and/or irritation, cough, shortness of breath, and pain on deep inspiration in the absence of a significant change in residual volume (1). The decrease in inspiratory capacity results in a decrease in FVC and, in combination with mild bronchoconstriction, contributes to a decrease in FEV₁.

Bates and colleagues (2) and Hazucha and colleagues (3) observed that the inhalation of ozone by human subjects resulted in a reduction in transpulmonary pressure at maximal inspiratory volume without a concomitant decrease in lung compliance, demonstrating that the ozone-induced decrease in inspiratory capacity is not the result of an alteration in lung mechanics. These investigators suggest that ozone inhalation results in the stimulation of nerve endings located in the tracheobronchial tree and that these airway afferents initiate a reflex inhibition of maximal inspiratory effort. Studies in animals have demonstrated that acute ozone inhalation excites bronchial C-fibers and rapidly adapting pulmonary stretch receptors (RARs) (4), sensitizes pulmonary C-fibers to lung inflation (5), and induces rapid shallow breathing via a reflex primarily initiated by lung C-fibers (6, 7).

Hazbun and colleagues (8) found a significant increase in substance P, a neuropeptide released by C-fiber afferent endings, in airway washings of healthy human subjects after acute ozone inhalation. This finding, in combination with the observations of Krishna and colleagues (9) that acute ozone inhalation in healthy subjects results in a marked reduction in substance P immunoreactivity in endobronchial biopsies, suggests that ozone stimulates bronchial C-fibers in humans. Passannante and colleagues (10), have shown that injection of the potent opioid analgesic, sufentanil, significantly attenuates ozone-induced symptoms of breathing discomfort and lung volume decrements. These observations support the notion that bronchial C-fibers serve a nociceptor role in the airway, contributing to ozone-induced symptoms of respiratory discomfort, as well as the inhibition of the ability to inspire maximally. There is, therefore, strong evidence that ozone-induced decreases in inspiratory capacity, rapid shallow breathing, and subjective symptoms are all mediated by neural mechanisms (2). However, the generally weak association among these responses from individual to individual (11, 12) would be consistent with several neural pathways interacting to produce a response profile that is unique for each individual.

In this study, we sought to explore the role of neural afferents located in the large conducting airways in the responses induced by acute ozone inhalation in human subjects. To accomplish this goal we designed an experiment in which our subjects inhaled an approximately 3.5-µm aerosol of the local anesthetic, tetracaine, part way through an ozone inhalation protocol. The preferential delivery of a higher dose (mg/cm²) of this size aerosol to the large conducting airway of the tracheobronchial tree (13) allowed us to block the excitation of afferent nerves located primarily in the large conducting airways during ozone inhalation and to examine the effect of this block on ozone-induced rapid shallow breathing, pulmonary function impairment, and subjective symptoms of breathing discomfort.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

A total of 45 subjects participated in this study, including nine from a previous study and 36 who underwent screening for ozone sensitivity (described below). All subjects were nonsmoking young adults 18 to 30 yr of age, who had not lived during the previous 6 mo where the State of California air quality standard for ozone (0.09 ppm) was exceeded. Subjects were solicited volunteers from the University of California, Davis, or the surrounding community. They were screened for absence of asthma or allergic rhinitis requiring medication and had normal baseline pulmonary function. All subjects were engaged in a regular program of aerobic training to ensure their ability to complete the exercise protocols. Prospective participants read and signed an Institutional Review Board informed consent form. They were shown the equipment to be used in the study, and any questions were answered before they signed the consent form.

Subject Orientation

Each subject participated in a 1-h orientation session, during which he/she first performed at least three maximum forced expiratory maneuvers. Each then pedaled an electronically braked cycle ergometer (model 845; Quinton Instrument Co., Bothell, WA) at 3 or 4 work rates for at least 3 min each until they reached steady-state minute ventilation (E) values of > 30 l/min/m² of body surface area (BSA). During this ergometer exercise, E determinations were made with the subject wearing a translucent silicone rubber face mask (Hans Rudolph, Kansas City, MO) with the inside surface lined with a Teflon overlay "coating", to which a two-way non-rebreathing nylon plastic valve was attached (16). Inspired filtered air (FA) was provided via the ozone delivery system described hereafter. Heart rate (HR) was monitored via an electrocardiograph R-wave detector. Each orientation session was completed with the performance of at least two maximum forced expiratory maneuvers.

Evaluation of Ozone Sensitivity

Nine subjects (four women and five men) had been subjects in a recent study, in which they completed a 1-h 0.30-ppm ozone exposure with exercise E similar to that used in the present study. Each had demonstrated at least a 13% FEV₁ decrement immediately after this exposure, thus obviating the need for them to complete the following ozone sensitivity exposure in this study. A total of 36 subjects (17 women and 19 men) completed the ozone sensitivity exposure, which consisted of 1 h of continuous exercise at a cycle ergometer work load resulting in a mean E of approximately 30 l/min/m² of BSA while exposed to 0.30 ppm ozone. This ozone sensitivity test resulted in seven women and six men being found to show significant subjective symptoms of breathing discomfort and an FEV₁ decrement of > 13%. Thus, a total of 22 subjects, 11 women and 11 men, completed the protocols described below.

Experimental Design and Protocols

Each of 22 subjects completed four 80-min protocols: (1) FA with saline; (2) FA with tetracaine; (3) 0.30 ppm ozone with saline; (4) 0.30 ppm ozone with tetracaine. After initial pulmonary function measurement, each subject exercised at a E of ~ 30 l/min/m² of BSA for 50 min, followed by a 15-min rest period. The rest period consisted of a pulmonary function test and then seated rest the remainder of the first 5 min followed by tetracaine (or isotonic saline) aerosol inhalation for 5 min, and then seated rest during the last 5 min. The subject then completed an additional 15 min of exercise at the same work load (i.e., mean E of ~ 30 l/min/m² of BSA), followed by after exposure pulmonary function. The protocols were conducted in single blind fashion and completed by each subject in near random order, with a minimum of 4 d intervening between each subject's individual protocol (17).

Pulmonary Function Measurements

Subjects performed two to four forced maximal expiratory maneuvers immediately before and immediately after the first 50 min of exercise. In addition, after the second period of exercise (i.e., 80 min), each subject again performed two to four forced maximal expiratory maneuvers. On-line measurements of FVC and FEV₁ were made with a data collection system consisting of a Collins 10-L spirometer (Model 3000; Warren E. Collins, Braintree, MA). The output of the spirometer was analyzed using a modified Lab View software package (LabView Inc, Austin, TX). If the sums of the FVC and FEV₁ values for the first two maneuvers were within 200 ml of each other, the mean for each was used in all statistical analyses. If not, then additional maneuvers were performed until this criteria was met for any two maneuvers.

Exercise Measurements

Minute-by-minute ventilatory data, including E, breathing frequency (f), and tidal volume (VT), and respiratory metabolism data were obtained every 15 s using a turbotachometer ventilation measurement module (VMM-2; Interface Assoc., Aliso Viejo, CA), an LB-2 carbon dioxide analyzer (Beckman Instruments, Fullerton, CA), an S-3A oxygen analyzer (Applied Electrochemistry, Pasadena, CA), an electrocardiograph R-wave detector, and a temperature thermistor located in the expired gas line, interfaced to a modified Lab View data acquisition package (Lab View).

Subjective symptoms of breathing discomfort were monitored from the 8th to 9th min and the 49th to the 50th min of the first exercise period. During the second exercise period of each protocol, subjective symptoms were evaluated from the 8th to 9th min and the 14th to the 15th min. In each case, subjects were asked to rate the severity of each of four symptomsthroat tickle and/or irritation (TTI), cough, shortness of breath (SOB), and pain on deep inspiration (PDI)by pointing to a visual display. Each symptom was rated according to a severity scale ranging from 0 (not present) to 40 (incapacitating) as previously described (18). Total symptom severity (TSS) was calculated as the sum of the severity ratings for the four individual symptoms.

Ozone Generation, Delivery, and Monitoring

All air inhaled by the subjects during the exposure protocols was first filtered via a Barneby-Cheney (Columbus, OH) activated charcoal filter. In those protocols involving ozone inhalation, ozone was introduced and mixed into the FA supply, as described in detail elsewhere (19). The ozone was generated by passing purified O₂ through a Sander Ozonizer (Modell II; Erwin Elektroapparatebau G.mbH, Uetze-Eltze, Germany). The air mixture was delivered to the subject via the silicone rubber face mask described above. The subject's expired gas was directed through a 5-L stainless steel mixing and sampling chamber to the turbotachometer ventilation measurement module (Interface Assoc.). The subject's expired air was combined with the pollutant mixture not inspired by the subject and passed through a QDF multistage filter assembly (Barneby-Cheney) and then to the laboratory ventilation exhaust outlet.

Appropriate levels of ozone were maintained by continuous sampling from the inspiratory side of the Hans Rudolph valve and face mask assembly, through 0.64-cm inner diameter Teflon tubing, connected to an ozone analyzer (Model 1003-AH; Dasibi Environmental Corp, Glendale, CA). Continuous measurement of ozone was accomplished by an on-line data acquisition system with minute-by-minute averages obtained from the voltage output generated by the Dasibi analyzer. The Dasibi analyzer was calibrated before and after the study, using the ultraviolet (UV) absorption photometric method, at the University of California, Davis, Primate Research Center.

Airway Anesthesia

Airway anesthesia was accomplished by inhaling tetracaine aerosol (35B ultrasonic nebulizer; DeVilbiss Health Care, Somerset, PA) at a maximal dose of 20 mg dissolved in 20 ml of isotonic saline, during a 5-min administration. At this dose, tetracaine gave approximately 30 min of effective airway anesthesia as indicated by the inhibition of citrate-induced cough (unpublished pilot study using 40 mg/ml citrate). Thus, tetracaine (or isotonic saline) was administered 5 min after completion of the first 50 min of each experimental exposure. After tetracaine (or isotonic saline) administration, the subject was allowed to become accustomed to the sensations of airway anesthesia for 5 min before commencing the second exercise period.

Subject Characterization

After completion of all experimental exposures, subjects were characterized via body composition analysis and maximal aerobic power assessment. Body composition was assessed via hydrostatic weighing. Each subject's maximal oxygen uptake (O₂max) was determined using a progressive graded exercise test to voluntary exhaustion, which occurred when the subject ceased pedaling (20). A summary of each subject's anthropometry, O₂max, and pulmonary function is given in Table 1. The subjects' mean FVC was 98.5% of predicted, whereas that for FEV₁ was 100.5%.

Statistical Procedures

All spirometric volumes and flows were measured as earlier described and expressed as mean values for analysis. The treatment effect was determined as percent change from the preexposure value. Absolute changes for TSS ratings for all reported symptoms, as well as individual symptoms of TTI, cough, SOB, and PDI, were calculated by subtracting the "initial" 5th min exposure values (almost always zero) from those obtained during the last minute of exercise. The treatment effect for f and VT was analyzed as the percent change from the "initial" values for those obtained in the next to last minute of both exercise periods. These data were analyzed for statistical significance (p < 0.05) using a two-way analysis of variance (ANOVA) with repeated measures (Super ANOVA), which tested for gas concentration effects and airway treatment effects. Upon obtaining a significant F ratio for main effects, because of ozone concentration or airway treatment, multiple mean contrasts were performed to determine the source of significance (Super ANOVA).

Correlation matrices were calculated to examine the extent of the linear relationship between the magnitude of the ozone-induced effect on FEV₁, f, SOB, cough, and PDI, as well as the reversal of each of these responses produced by tetracaine aerosol inhalation (Statview; SAS Institute, Cary, NC). Reversal was calculated as the fraction of the difference between the end of ozone exposure value minus the end of exposure/tetracaine value divided by the absolute change induced by ozone for each response. Fisher's r to z transformation was done to determine if a correlation coefficient was significantly different from zero (Statview; SAS).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Tetracaine inhalation produced a discordant effect on ozone-induced lung volume decrements, rapid shallow breathing, and respiratory symptoms of discomfort. This discordant effect is illustrated in Figure 1, which compares the FEV₁, f, and TSS responses for all four protocols.

View larger version (17K): [in this window] [in a new window]   Figure 1.   Illustration of the differential effect of tetracaine aerosol inhalation on ozone-induced FEV1, F, and TSS responses in the four protocols (FA, saline; FA, tetracaine; O3, saline; O3, tetracaine). TSS represents total symptom score.

Pulmonary Function Responses

Group mean data for preexposure, after the first exercise period, and after the treatment period for FVC and FEV₁ are given in Table 2. There were no significant differences between any of the preexposure FVC or FEV₁ values. The inhalation of FA during the first 50-min exercise period did not significantly change FVC or FEV₁. The inhalation of 0.30 ppm ozone during the first 50-min exercise period in either ozone inhalation protocol resulted in significant reductions in FVC and FEV₁ when compared with either of the FA inhalation protocols. There were no significant differences between the two ozone inhalation protocols for either FVC or FEV₁.

The inhalation of either saline or tetracaine aerosol after the inhalation of FA in the first exercise period did not significantly affect FVC or FEV₁ measured at the end of these protocols (Table 2). The inhalation of either saline or tetracaine aerosol after the inhalation of 0.30 ppm ozone in the first exercise period produced similar small nonsignificant attenuations in the ozone-induced decrements in FVC and FEV₁ (Table 2)

Breathing Pattern Responses

Group mean values for f and VT for the initial period (7 to 10 min), end of the first exercise period (47 to 50 min), and the last 3 min of the second exercise period are given in Table 2. There were no significant differences in initial f or VT values between the four exposures. For the two FA exposures, an exercise-induced rapid shallow breathing pattern (i.e., ~ 10% increase in f and ~ 10% decrease in VT) developed by the end of the first 50 min and persisted at the end of the second (15-min) exercise period regardless of whether the second exercise period was preceded by the inhalation of saline or tetracaine aerosols. In contrast, ozone inhalation during the first exercise period induced a much greater rapid shallow breathing pattern, which peaked by the end of the first exercise period and was not significantly changed by the inhalation of either saline or tetracaine aerosol. The E values for all time periods and all protocols were not significantly different from each other.

Subjective Symptoms Responses

Group mean subjective symptoms score responses are given in Table 3. The inhalation of FA during the first exercise period did not significantly result in the reporting of any of the subjective symptoms. The inhalation of either saline or tetracaine aerosol after the inhalation of FA did not result in a significant change in any of the subjective symptoms (i.e., cough, SOB, PDI, or TTI). Total, as well as individual, symptom responses were significantly elevated at the end of the first exercise period in the both ozone inhalation protocols. After saline aerosol inhalation there was a significant attenuation of TSS, cough, and PDI at the end of the second exercise period after ozone inhalation. There was a similar nonsignificant trend toward attenuation of TTI and SOB. The inhalation of tetracaine aerosol elicited a significant marked attenuation of TSS, as well as the individual symptoms of TTI, cough, SOB, and PDI after ozone inhalation.

Correlation Analysis

Correlation analysis revealed significant linear relationships between ozone-induced alterations in FEV₁ and the subjective symptoms of cough and SOB (Table 4). Similarly, a near significant (p < 0.10) relationship was found between ozone-induced tachypnea and cough and SOB. In contrast, correlation analysis revealed no relationship between the tetracaine-induced recovery in FEV₁ or f, and cough, SOB, and PDI (Table 4). As an example of the dissociation of ozone-induced functional and subjective symptoms responses produced by tetracaine aerosol inhalation in this study, two scattergrams that relate the ozone-induced FEV₁ decrements and SOB, and the tetracaine-induced recovery in these end-points, are shown in Figure 2.

View larger version (12K): [in this window] [in a new window]   Figure 2.   Relationships between the ozone- induced FEV1 decrements and shortness of breath SOB (A), and the tetracaine-induced recovery in these end-points (B). Both illustrate the dissociation of ozone-induced functional and subjective symptom responses produced by tetracaine aerosol inhalation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study, inhalation of tetracaine aerosol resulted in marked reductions in ozone-induced subjective symptoms of cough, TTI, SOB, and PDI. Tetracaine inhalation was so effective in some subjects that it virtually abolished all symptoms of breathing discomfort. In contrast, inhalation of tetracaine resulted in only minor and inconsistent rectification of pulmonary function impairment and no alleviation of rapid shallow breathing. This differential effect of tetracaine treatment on ozone-induced responses was further demonstrated using correlation analysis. This analysis showed that tetracaine inhalation resulted in a dissociation of ozone-induced FEV₁ and f responses from ozone-induced subjective symptoms responses. This finding is the strongest evidence to date that ozone-induced inhibition of maximal inspiratory effort is reflex in origin and not dependent on conscious sensations of inspiratory discomfort.

The differential effect of inhaled tetracaine aerosol on ozone-induced subjective symptoms, ozone-induced rapid shallow breathing, and pulmonary function decrements is consistent with two possible interpretations. The first is that the inhalation of tetracaine blocked all the afferent nerve endings contained in the airways and parenchyma. If true, then the ozone- induced rapid shallow breathing and pulmonary function decrements are of a non-neurogenic origin, possibly involving some degree of small airways dysfunction. The other interpretation is that the inhalation of tetracaine only partially blocked the afferent nerves contained within the airways and parenchyma. If this is correct, then the ozone-induced rapid shallow breathing and pulmonary function decrements are of neurogenic origin involving receptors that are only minimally effected, if at all, by the inhalation of tetracaine as delivered in this study. The weight of evidence derived from human and animal studies would tend to support the latter of these two interpretations. The setting of breathing pattern during rest and exercise has been shown to be determined by multiple neural inputs to the respiratory centers in the brainstem including inputs from intrapulmonary and extrapulmonary mechanoreceptors and chemoreceptors. Furthermore, studies in dogs (4, 6) and rats (5, 7) support the notion that ozone-induced rapid shallow breathing is mediated by afferent nerve endings located in the lower airways. In addition, a significant contribution to the alteration in small airways mechanics contributing to ozone-induced pulmonary function decrements is excluded by the results of numerous human exposure studies. The primary pulmonary function decrements induced by the acute inhalation of ozone in concentrations used in this experiment are a decrease in inspiratory capacity and a mild increase in airway resistance. These pulmonary function decrements occur despite the observation of only small and variable effects of acute ozone inhalation on residual volume (19) and lung compliance (2, 3). These observations support the notion that ozone-induced decreases in FVC are the result of a decrease in inspiratory capacity that is not related to a restrictive limitation on the ability to inspire or to significant gas trapping caused by collapse of small airways. Furthermore, the observation of Beckett and colleagues (21) that atropine treatment abolishes ozone-induced increases in airway resistance and decreases in the FEV₁/FVC ratio demonstrates the reflex nature of ozone-induced bronchoconstriction. In addition, atropine treatment did not effect ozone-induced decreases in inspiratory capacity, demonstrating the independence of this response on changes in lung and/or airway mechanics. Further, the observation of Passannante and colleagues (10) that sufentanil, a µ-receptor agonist, markedly attenuates ozone-induced decreases in FVC clearly demonstrates the neurogenic origin of the inspiratory capacity response.

Tetracaine is a highly water-soluble, rapidly acting local anesthetic that stabilizes neural membranes, preventing the initiation and transmission of nerve impulses. We used tetracaine in the present study because of these properties and because of its relatively prolonged duration of action (2 to 3 h). Tetracaine was administered as an aerosol using an ultrasonic nebulizer that has been shown previously to produce an aerosol with a mass median aerodynamic diameter (MMAD) of 3.52 µm and a geometric standard deviation (GSD) of 1.92. On the basis of previously published data, we would expect that less than 20% of the tetracaine loaded into the nebulizer in these experiments would be deposited in the subjects upper and lower airways (22). Kim and colleagues (14) examined the regional deposition of three monodispersed aerosols with MMADs of 5, 3, and 1 µm within the lower respiratory tract of human subjects. Results from this study showed that when examined as dose delivered per surface area of airway (mg/cm²), a greater dose was delivered to the larger conducting airways for all aerosol sizes studied regardless of inspiratory flow rate used (150, 250, or 500 ml/s). This differential regional dose distribution was greatest for the 5- and 3-µm aerosols and least for the 1-µm aerosol. On the basis of the aerosol deposition data of Kim and colleagues (14) and of that of other investigators (13, 15), we would expect that the polydispersed aerosol used in the present experiment would deposit the greatest dose of tetracaine (mg/cm²) to large conducting airways and that neural receptors within these airways would be more effectively anesthetized. Supporting this contention is the observation that delivery of a polydispersed aerosol to the lower respiratory tract of cats (23) was 100% effective in anesthetizing airway receptors known (24) to be located primarily in the large conducting airways (low threshold slowly adapting stretch receptors) and only partially effective in anesthetizing receptors known to be distributed in more distal conducting airways (high threshold slowly adapting stretch receptors). In addition, a polydispersed aerosol delivered to the lower airway of rabbits (25) was shown to be ineffective in blocking the pulmonary C-fiber mediated pulmonary chemoreflex. On the basis of these physiologic and aerosol delivery/deposition studies, our observation of a preferential effect of tetracaine on ozone-induced subjective symptoms suggests that neural receptors located in the larger conducting airways of the lower respiratory tract are mainly responsible for mediating these responses. Furthermore, our findings suggest that neural afferents located in more distal airways mediate ozone-induced lung volume decrements and rapid shallow breathing in human subjects.

Our findings differ from the results of the exploratory study conducted by Hazucha and colleagues (3). These investigators found that approximately 1-h after a 2-h ozone inhalation protocol (0.5 ppm with intermittent exercise) the inhalation of an aerosol of 20% lidocaine partially reversed ozone-induced altered lung volumes, resting breathing pattern, and subjective symptoms. The reversal in all these ozone-induced responses were of similar magnitude. However, the considerable time (approximately 55 min) separating the pulmonary function tests done immediately after their ozone inhalation protocol and after lidocaine inhalation, combined with the lack of a vehicle control, limited the interpretation of their data. In order to avoid similar limitations in the interpretation of our data we designed our experimental protocol to allow for the evaluation of the effectiveness of tetracaine and saline aerosol during and immediately after ozone inhalation. We found that the inhalation of saline aerosol alone produced a small improvement in ozone-induced alterations in lung volumes and exercise breathing pattern that was similar in magnitude to that produced by tetracaine. Similarly, inhalation of saline aerosol resulted in mild improvements in ozone-induced subjective symptoms of breathing discomfort, whereas the inhalation of tetracaine aerosol markedly attenuated and, in some cases, abolished them.

It is uncertain which specific airway afferents initiated the ozone-induced subjective symptom responses blocked in the present study, but they most likely include C-fibers and RARs. A comparison of our findings with those of Passannante and colleagues (10) provides considerable insight into what airway afferents were or were not blocked in the current study and what ozone-induced responses they induce. Passannante and colleagues (10) examined the role of airway nociception, most likely mediated by airway C-fibers, in ozone-induced responses. They found that the intravenous injection of the potent opioid analgesic sufentanil, after ozone inhalation, significantly attenuated ozone-induced symptoms of cough and PDI and decrements in FEV₁. Sufentanil treatment did not significantly affect the symptoms of TTI and SOB. These observations support the notion that lung C-fibers serve a nociceptor role in the airway, contributing to ozone-induced symptoms of cough and PDI as well as the inhibition of the ability to inspire maximally. When combined, the results of the present study and those of Passannante and colleagues (10) suggest that the symptoms of cough and PDI are in part mediated by airway C-fibers located in proximal conducting airways, whereas the symptoms of TTI and SOB are mediated primarily by RARs, also located in proximal airways. The observation of Passannante and colleagues (10) observation that sufentanil injection markedly attenuated ozone-induced decrements in FEV₁, whereas our observation that tetracaine inhalation did not, suggests that the reflex inhibition of the ability to inspire is mediated by bronchial C-fibers located in distal airways and/ or pulmonary C-fibers. The possible role of pulmonary C-fibers in ozone-induced FEV₁ decrements and rapid shallow breathing is supported by the observation in rats that acute ozone inhalation sensitizes pulmonary C-fibers to lung inflation (5).

In conclusion, our observation that ozone-induced decrements in FVC and FEV₁ survive tetracaine inhalation while ozone-induced symptoms of breathing discomfort were markedly attenuated, further supports the notion that ozone-induced inhibition of maximal inspiratory effort is reflex in origin and not the result of breathing discomfort. In addition, our results are consistent with neural afferent endings located within the large conducting airways of the tracheobronchial tree being primarily responsible for ozone-induced subjective symptoms and only partially responsible for pulmonary function decrements. Furthermore, it appears that afferent endings located in more distal airways and possibly the alveoli mediate the majority of the ozone-induced reductions in inspiratory capacity and rapid shallow breathing. Further studies need to be conducted to determine the airway level at which this transition from receptors primarily mediating ozone-induced subjective symptoms to those primarily mediating ozone-induced reductions in inspiratory capacity and rapid shallow breathing is located.