Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 85: 442-450, 1998;
8750-7587/98 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takebayashi, T.
Right arrow Articles by Shore, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takebayashi, T.
Right arrow Articles by Shore, S. A.
Vol. 85, Issue 2, 442-450, August 1998

Role of tachykinins in airway responses to ozone in rats

Toru Takebayashi1, Joseph Abraham1, G. G. Krishna Murthy1, Craig Lilly2, Ian Rodger3, and Stephanie A. Shore1

1 Physiology Program, Harvard School of Public Health, and 2 Pulmonary Division, Brigham and Women's Hospital, Boston, Massachusetts 02115; and 3 Merck Frosst Center for Therapeutic Research, Kirkland, Quebec, Canada H9H 3L1

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Previous studies that used neonatal capsaicin (Cap) treatment to ablate C fibers indicate that C fibers act to inhibit lung damage and airway hyperresponsiveness after ozone (O3) exposure in rats. The purpose of this study was to determine 1) the role of tachykinins in these protective effects and 2) whether differences in minute ventilation (VE) during O3 exposure might account for the effect of Cap. In the first study, male Sprague-Dawley rats were exposed to 1 part/million O3 or air for 3 h. Four hours later, a bronchoalveolar lavage (BAL) was performed or airway responsiveness was measured. Rats were treated with CP-99994 and SR-48968, selective neurokinin-1- and -2-receptor antagonists, respectively, or with vehicle (Veh). O3 caused an increase in the number of neutrophils recovered from BAL fluid in both the Veh-treated and tachykinin-receptor antagonist (TKRA)-treated rats, but the number of neutrophils was approximately twofold greater in the TKRA-treated rats. In contrast, TKRA treatment had no effect on baseline pulmonary mechanics or airway responsiveness. After O3 exposure, the number of neutrophils in BAL fluid was also greater in Cap- than in Veh-treated rats. O3 reduced VE in both Veh- and Cap-treated rats, but the response was greater (reduction of 44.7 ± 3.7 vs. 27.8 ± 6.8%) and occurred earlier (10 vs. 70 min) in Cap- than in Veh-treated rats (P < 0.02). These results suggest that tachykinins mediate protective effects of C fibers against O3-induced lung inflammation. The results also indicate that the more pronounced effect of O3 on BAL neutrophils in Cap-treated rats is not the result of a greater inhaled dose of O3 resulting from greater VE.

airway responsiveness; bronchoconstriction; methacholine; C fibers; ventilation; CP-99994; SR-48968; bronchoalveolar lavage; tidal volume; breathing frequency

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

C FIBERS, a class of unmyelinated sensory neurons, innervate the lungs and airways of many species, including humans. The terminal ramifications of these neurons are found underneath and between epithelial cells, around blood vessels, and near airway smooth muscle and mucous glands (2, 24). In response to stimulation, the tachykinins, substance P (SP) and neurokinin A (NKA), are released from the peripheral endings of these neurons. The biological effects of these peptides in the lung and airways are diverse. Tachykinins can induce bronchoconstriction, increase microcirculation, cause mucous secretion, and increase vascular permeability (19). Stimulation of C fibers also causes apnea, followed by rapid, shallow breathing (33). C fibers are stimulated by various inhaled irritants including ozone (O3) (4) as well as by many endogenous inflammatory mediators (19). In addition, SP is released into the airway lavage fluid of humans exposed to O3 (12).

Neonatal capsaicin (Cap) pretreatment causes degeneration of C fibers and decreases neuropeptide content in the lungs and airways (14, 27). Our laboratory (15) has previously reported that, in Sprague-Dawley rats treated neonatally with Cap, airway hyperresponsiveness to inhaled methacholine is induced by exposure to concentrations of O3, which do not affect responsiveness in control rats. Neonatal Cap treatment also results in more airway epithelial injury and more inflammation after O3 exposure, as determined by pathology and bronchoalveolar lavage (BAL) in Wistar rats (37). Taken together, these results suggest that C fibers protect the airways during exposure to O3, minimizing airway injury and inflammation and inhibiting the development of airway hyperresponsiveness. C fibers have also been shown to play a protective role in the responses to other inhaled irritants such as sulfur dioxide (22, 23), hydrogen sulfide (30), and acrolein (41).

One aim of this study was to determine whether tachykinins were important in mediating these protective effects of C fibers against O3-induced airway inflammation and airway hyperresponsiveness in rats. To this end, we treated rats with tachykinin-receptor antagonists (TKRAs) or the vehicle (Veh) used to dissolve them, exposed them to O3 or air, and then either performed a BAL or instrumented them for the measurement of pulmonary mechanics and responsiveness to inhaled aerosolized methacholine. The TKRAs, CP-99994 and SR-48968, which possess high potency and selectivity in blocking the neurokinin-1 (NK1) and -2 (NK2) receptors, respectively (8, 26), were employed. Because our experiments were performed in Sprague-Dawley rats, whereas those of Sterner-Kock et al. (37) were performed in Wistar rats, and because differences in responses to tachykinins and to O3 have been observed among strains of rat (9, 16, 39, 42), we also examined the effect of neonatal Cap treatment on O3-induced alterations in BAL cells and protein and confirmed the results of Sterner-Kock et al. (37) indicating that ablation of C fibers causes more lung injury and inflammation after O3 inhalation.

One explanation for the greater inflammatory response of the Cap-treated rats is that a larger dose of O3 is delivered to the lungs of these rats despite a similar O3 concentration. The dose of O3 delivered to the lungs is a function of both O3 concentration and minute ventilation (VE) (47). During O3 exposure, rats decrease their VE (1, 25). Because O3 stimulates C fibers and stimulation of C fibers alters VE (4, 33), it is possible that C fibers are the sensory arm of O3-induced changes in VE. If so, one might expect VE to be reduced during O3 exposure in normal rats, but not in Cap-treated rats lacking C fibers, leading to an overall greater dose of O3 in the Cap-treated rats. To test this hypothesis, we measured VE and its components, tidal volume (VT) and breathing frequency (f), during exposure of awake Veh- and Cap-treated rats to O3 or air.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Animals. This study protocol was approved by the Harvard Medical Area Standing Committee on Animals. For the studies with TKRAs, pathogen-free Sprague-Dawley rats were obtained from Charles River Breeding Laboratories (Wilmington, MA) and were housed in group cages. For the study with neonatal Cap treatment, pregnant female Sprague-Dawley rats were purchased from Charles River and were housed individually. In each litter, rats were treated either with 50 mg/kg of Cap, dissolved in a mixture of 10% Tween 80 and 10% ethanol in saline, or with an equivalent amount of Veh subcutaneously 2 or 3 days after their birth. During Cap treatment, rats were anesthetized with halothane. The rats were weaned at 1 mo of age, and males and females were separated.

Verification of Cap desensitization. Our laboratory has previously reported that neonatal Cap pretreatment performed in this manner decreases tachykinins in lung and other tissues (15, 23). To confirm a decrease in tachykinins in these rats, six Veh- and six Cap-pretreated rats from the same cohort used for measurements of ventilatory responses to O3 were killed with an overdose of pentobarbital sodium, and the lungs were excised. The lungs were immediately frozen in liquid nitrogen and stored at -70°C until extraction and assay for SP content by an enzyme-linked immunosorbent assay. SP was extracted from the lungs as previously described (18). The enzyme-linked immunosorbent assay for SP was performed by using methods described by Folkesson et al. (10). The antibody for SP was purchased from Peninsula Laboratories (Belmont, CA). The lower limit of detection of this assay was 3 fmol of SP, and the assay has <0.01% cross-reactivity with NKA (18). Cap pretreatment significantly reduced the SP content of the lungs from 2.83 ± 0.5 to 1.75 ± 0.1 pmol/g tissue (P < 0.002).

O3 exposure. Exposure to O3 or filtered room air was conducted in a stainless steel chamber with a Plexiglas door on the front (~145 liters in volume). O3 was generated by passing dry 100% O2 through ultraviolet light and mixing it with filtered room air in the chamber. Chamber atmosphere was drawn continuously via a sampling port, and O3 concentration was measured by an O3 chemiluminescent analyzer (model 49, Thermoelectron Instruments, Hopkinton, MA), which was calibrated by an ultraviolet photometric O3 calibrator (model 49PS, Thermoelectron Instruments).

Experimental protocol. Two different studies were performed. The purpose of the first study (TKRA) was to determine whether tachykinins act to limit airway injury and inflammation induced by O3 and thereby limit the degree of airway hyperresponsiveness induced by O3. In this study, rats were treated with CP-99994 (0.75 mg/kg ip) and SR-48968 (0.75 mg/kg ip) or with Veh 15 min before exposure to filtered air or O3. The rats were then exposed to air or O3 [1 part/million (ppm), for 3 h] and were retreated with the same doses of CP-99994 and SR-48968 or the Veh immediately after the exposure to ensure continued receptor blockade. These doses of TKRAs were chosen based on reports that indicated their effectiveness in inhibiting responses to SP and NKA in rats and guinea pigs (8, 13, 26, 36). Four hours after the air or O3 exposure had ended, rats were killed, and BAL was performed. Alternatively, 2 h after exposure, rats were anesthetized for the measurement of airway responsiveness to inhaled methacholine, followed by administration of intravenous SP to confirm the efficacy of the antagonists. The time required to anesthetize the animals, perform the necessary surgery, instrument them, and perform the measurements of responsiveness was such that the measurements of the steep part of the dose-response curve coincided in time with BAL performed in the other animals.

The purpose of the second study was to confirm, in Sprague-Dawley rats, reports about Wistar rats indicating that Cap-treated rats that lack C fibers have increased airway injury and airway inflammation after O3 exposure (37). We also sought to determine whether differences in VE during O3 exposure might contribute to these enhanced responses. Two cohorts of rats were used in this study (Cap). In the first cohort, adult rats with or without neonatal Cap treatment were exposed either to 1 ppm O3 or to filtered air for 3 h. To evaluate inflammatory changes induced by O3, BAL was performed 4 h after the end of O3 exposure. In the second cohort, adult rats treated neonatally with either Cap or Veh were placed in head-out restraining tubes that served as body plethysmographs. Once the animal was in the tube, the cranial end of the tube was inserted into a port in the door of the O3-exposure chamber, so that animals breathed air from within the chamber. We then monitored VE for 45 min before O3 was introduced into the chamber. O3 exposures (2 ppm) were for 90 min followed by a 45- to 90-min recovery period during which we continued to monitor VE. For these experiments, the washin of O3 was such that 2 ppm was reached within 5 min of initiating the exposure. The washout was such that O3 had decreased to <0.02 ppm within 5 min after the O3 flow to the chamber was stopped. Other Cap- and Veh-treated rats were treated identically but were exposed to filtered air only.

Measurements of pulmonary mechanics and airway responsiveness to inhaled aerosolized methacholine were performed as previously described (15). Briefly, rats were anesthetized with pentobarbital sodium (64.8 mg/kg ip), tracheostomized with a tubing adapter (14 gauge, 1.65-mm ID), and mechanically ventilated with a rodent ventilator (Harvard Apparatus, Natick, MA) at 8 ml/kg and 60 breaths/min. End-expiratory pressure was maintained at 3.5 cmH2O throughout the experiment. The left carotid and right jugular vein were catheterized for blood pressure measurement and drug administration, respectively. The chest wall was opened widely to expose the lungs to atmospheric pressure, and the rat was then placed in a body plethysmograph. Plethysmograph pressure relative to a reference bottle of a similar size was measured with a differential pressure transducer (model LCVR, Celesco, Canoga Park, CA) and used as a volume signal. Flow was calculated by electrical differentiation of the volume signal. Transpulmonary pressure was obtained with a second pressure transducer (Celesco) placed between a side tap in a tracheal cannula and the inside of the plethysmograph. All three signals were recorded on a strip chart (model RS65P, General Scanning, Watertown, MA) and fed to a computer for calculation of dynamic compliance (Cdyn) and pulmonary conductance (GL), according to the method of Von Neergard and Wirz (44, 45).

Airway responsiveness was measured as follows. A breath equal to three times VT was given to provide a constant volume history. Baseline measurements of GL and Cdyn were obtained 1 min later. Animals were then given 20 tidal breaths of aerosolized saline generated in an acorn nebulizer. A flow rate of 4.5 l/min was used to generate the aerosol. GL and Cdyn were then measured repeatedly over the next 3 min, and the peak response was recorded. The procedure was then repeated by using methacholine solutions in the nebulizer, beginning with 0.1 mg/ml and increasing in one-half log units up to 300 mg/ml. The time interval between doses was sufficient for GL to return to within 10% of its original value or the time interval was 10 min, whichever came first. GL and Cdyn values measured before the methacholine challenge are reported as baseline values. The methacholine doses required to produce a 50% decrease of GL and Cdyn were determined by log-linear interpolation between the two doses bounding the point at which GL or Cdyn was exactly 50% of its baseline value.

At the end of the methacholine challenge, changes in blood pressure caused by intravenous infusions of increasing concentrations of SP were measured to confirm the efficacy of the TKRAs. The doses of SP ranged from 10-12 to 10-10 mol/kg. SP was injected over 20 s, and each administration was separated by 5 min. This time interval was sufficient for blood pressure to return to the baseline level.

BAL. Animals were given a lethal dose of pentobarbital sodium (64.8 mg in 1.0 ml) intravenously. The chest wall was opened, and the lungs were then lavaged twice with 5 ml of saline. For the lavages, saline was injected and withdrawn gently over 30 s. The recovered BAL fluid was centrifuged at 1,200 rpm at 4°C for 10 min. Cell pellets were resuspended in 5 ml of saline, and the total number of cells was counted by a hemocytometer. Aliquots of cells were also centrifuged onto glass slides at 800 rpm for 5 min (Cytospin2, Shandon, Sewickley, PA), air-dried, and stained with Wright-Giemsa (LeukoStat, Fisher Scientific, Pittsburgh, PA). Cell differentiation was determined by the counting of 1,200 cells under ×400 magnification. Total protein concentration in the BAL supernatant was determined by the Bradford technique.

Monitoring of VE. Rats were placed in a Plexiglas restraining tube, which served as a head-out flow plethysmograph. The tube was fitted with a silicone rubber gasket designed to fit snugly around the animal's neck and seal the head from the rest of the body. Once the animal was in the tube, a large piston was moved into placed behind the animal. The piston served to prevent the animal from moving and to seal the body chamber from the outside air. Air, displaced at the body surface as the animal breathed, passed across a pneumotachograph (8-mm diameter fitted with a screen filter), designed and fabricated in-house and attached to a differential pressure transducer (model 163PC01D75, Omega Engineering). The resulting flow signal was analyzed by a computer program (BUXCO, Troy, NY) that computed VE, VT, f, and inspiratory (TI) and expiratory time (TE) on a breath-by-breath basis and reported the average of each of these values every minute.

Chemicals. Methacholine chloride and SP were purchased from Sigma Chemical (St. Louis, MO) and were dissolved in saline. CP-99994 and SR-48968 were synthesized at Merck Frosst and were dissolved in a 1:1 solution of saline and polyethylene glycol (average mol wt 200; Sigma Chemical). Cap was obtained from Spectrum Chemical Manufacturing.

Statistical analysis. Results are expressed as means ± SE. For baseline values of pulmonary mechanics and log 50% effective dose data, analysis of variance was employed to detect differences across all groups in the TKRA study. The effect of TKRAs on changes in blood pressure induced by SP was examined by using Student's t-test. For BAL data, analysis of variance was first employed to detect differences across all groups. If a statistically significant result was observed, follow-up t-tests were used to determine the significance of differences across groups. The Bonferroni correction was employed to correct for multiple comparisons. Because there were no significant differences in any BAL parameter between the two air-exposed groups, the air-exposed animals were combined for these comparisons. For ventilatory parameters, mean values over the 20 min immediately preceding O3 exposure were determined for each animal, and data are reported as percent changes from those values. In each animal, 5-min averages around the 10-, 30-, 50-, 70-, and 90-min time points for O3 exposure were computed. The effect of Cap treatment on these parameters was then assessed by repeated-measures analysis of variance. A P value of <0.05 was considered statistically significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

TKRA study. To determine whether tachykinins released from C fibers act to limit O3-induced airway injury and inflammation, we examined the effect of TKRAs on BAL cells and protein in O3-exposed animals. The profile of cells recovered from BAL was affected by both O3 exposure and TKRA treatment (Table 1). Analysis of variance indicated significant differences across the groups in the number of BAL neutrophils and ciliated epithelial cells and the BAL protein concentration, whereas total cell numbers, lymphocytes, and macrophages were unchanged. Treatment with TKRAs did not affect either the BAL cell profile or the BAL protein concentration in air-exposed rats. O3 exposure increased the number of neutrophils in both the Veh- and the TKRA-treated rats, compared with air-exposed controls. However, the number of neutrophils recovered from the TKRA-treated rats exposed to O3 was significantly greater than that from the Veh-treated rats exposed to O3. O3 exposure also increased BAL epithelial cells and BAL protein concentration in both the Veh- and the TKRA-treated rats, compared with air-exposed controls (Table 1). However, in contrast to the effect of TKRA on BAL neutrophils, there was no difference in BAL epithelial cells or BAL protein concentration between the TKRA-treated rats exposed to O3 and the Veh-treated rats exposed to O3.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Effects of O3 and inhibition of tachykinin receptors on no. of cells and total protein concentration in bronchoalveolar lavage fluid

We also measured pulmonary mechanics and airway responsiveness to inhaled aerosolized methacholine in TKRA- and Veh-treated rats exposed to air or O3. Exposure to O3 did not alter baseline airway mechanics or induce airway hyperresponsiveness to inhaled methacholine in either the Veh- or TKRA-treated rats (Table 2).

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Effects of O3 and inhibition of tachykinin receptors on airway responsiveness to inhaled methacholine

To confirm the efficacy of the TKRA treatment, after the measurements of airway responsiveness to methacholine were taken, changes in blood pressure induced by SP were assessed (Table 3). Infusion of SP caused a triphasic blood pressure change as reported by others (35): an immediate fall phase, recovery within 1 min, and, at higher doses, an extended secondary decrease. The lowest mean blood pressure observed in the first phase is reported. Intravenous injection of 10-12 mol/kg of SP did not alter blood pressure in either the Veh- or the TKRA-treated groups. In the Veh-treated animals, 10-11 mol/kg SP caused a significant decrease in blood pressure. In the TKRA-treated groups, the same dose of SP had no effect (P < 0.05 compared with the Veh group). At 10-10 mol/kg, blood pressure fell in both groups, but the decrease was greater in the Veh-treated than in the TKRA-treated group.

                              
View this table:
[in this window]
[in a new window]
  		Table 3.   Effects of intravenous substance P infusion on mean arterial blood pressure (mmHg)

Cap study. To verify a protective role for C fibers in the airway inflammation and injury induced by O3 exposure in Sprague-Dawley rats, as had previously been demonstrated in Wistar rats (37), adult rats treated neonatally with Cap or the Veh used to dissolve Cap were exposed to air or O3, and a BAL was performed. The results are summarized in Table 4. Analysis of variance indicated a statistically significant difference across groups in the numbers of neutrophils, lymphocytes, and ciliated epithelial cells and in total protein. Follow-up analysis indicated that, compared with air-exposed controls, the numbers of neutrophils and ciliated epithelial cells were increased significantly in both the O3-exposed, Cap-treated group and the O3-exposed, Veh-treated group. As was observed with TKRA treatment, the number of neutrophils in BAL fluid recovered from Cap-treated animals exposed to O3 was significantly greater than was the number of neutrophils recovered from BAL of Veh-treated animals exposed to O3. Compared with air-exposed animals, the total protein concentration was elevated in the O3-exposed, Cap-treated group but not in the Veh-treated group.

                              
View this table:
[in this window]
[in a new window]
  		Table 4.   Effects of O3 and C-fiber ablation on no. of cells and total protein concentration in bronchoalveolar lavage fluid

To determine whether the effective dose of O3 delivered to the lungs might be different in Cap- and Veh-treated rats, we measured VE and examined the pattern of breathing during air or O3 exposure. Table 5 shows mean baseline ventilatory parameters measured during the 20 min immediately before O3 (or air) exposure (after the animals were allowed 25 min to settle in the restraining tubes). No significant differences between Cap- and Veh-treated rats were observed.

                              
View this table:
[in this window]
[in a new window]
  		Table 5.   Effect of capsaicin on baseline ventilatory parameters

In Cap- and Veh-treated rats exposed to filtered air, there was substantial minute-to-minute variability in VE, but no significant trend over time in either group and no significant effect of Cap treatment (Fig. 1). Similarly, no significant changes in VT, f, TI, or TE were observed with time in air-exposed animals (data not shown).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1.   Effects of exposure to filtered air on minute ventilation (VE). Results are means ± SE of data from 3 vehicle (Veh)-treated (open circle ) and 3 capsaicin (Cap)-treated () rats and are expressed as %changes from baseline values. Time 0, time at which ozone would have been introduced into the chamber in ozone-exposed animals, but in these animals ozone was never introduced, and animals breathed only filtered air in the chamber. Baseline values are mean values in 20-min period before time 0.

Figure 2 shows the effect of a 90-min O3 exposure on ventilatory parameters in O3-exposed rats. O3 caused a decrease in VE in both Veh- and Cap-treated rats, but there was a significant effect of Cap treatment on this response (P < 0.018). In Cap-treated rats, a significant decrease in VE occurred within 10 min of O3 exposure, whereas a significant decrease in VE was not observed in the Veh-treated animals until after 70 min of O3 exposure.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2.   Effects of ozone (2 parts/million) on VE, breathing frequency (f), tidal volume (VT), inspiratory time (TI), and expiratory time (TE). Results are expressed as %changes from baseline values (mean values in 20-min period before ozone exposure) and are means ± SE of data from 8 Veh-treated (open circle ) and 8 Cap-treated () rats. Compared with baseline: * P < 0.05; ** P < 0.01.

In both groups, the change in VE was primarily the result of a decrease in VT (Fig. 2), and, again, there was a significant effect of Cap treatment on O3-induced changes in VT. (P < 0.012). VT decreased earlier in the Cap-treated rats (30 min) than in the Veh-treated controls (70 min). There was substantial variability in the f response to O3 exposure among rats. Some rats showed an increase in f, whereas others showed a decrease. For Veh-treated rats, the mean effect was no net change over time, whereas for the Cap-treated rats, there was a statistically significant decrease in f 10 min after O3 exposure but not at any other time point (Fig. 2). Overall, there was no significant effect of Cap treatment on f. Even though f was not altered by O3 exposure, the timing of the breath was altered. In particular, TI increased in the first 10 min of O3 exposure and then decreased to values well below baseline over the next 60 min (Fig. 2). In contrast, TE increased over the course of O3 exposure (Fig. 2), although the changes were much more variable among animals. There were no statistically significant differences between Cap- and Veh-treated animals in the changes in TI and TE induced by O3 exposure.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The results of the present study demonstrate that combined inhibition of NK1 and NK2 tachykinin receptors during exposure to O3 augments neutrophil influx into the lungs and airways (Table 1). In contrast, combined inhibition of NK1 and NK2 receptors does not result in the development of airway hyperresponsiveness after O3 exposure (Table 2). Neonatal Cap pretreatment also resulted in greater O3-induced neutrophil influx and protein extravasation (Table 4). In addition, rats treated neonatally with Cap had more pronounced decreases in VE during O3 exposure than did control rats (Fig. 2).

Sterner-Kock et al. (37) have previously reported that, in Wistar rats treated neonatally with Cap, 1 ppm O3 exposure for 8 h caused more severe interstitial inflammation and epithelial necrosis, more protein in BAL fluid, and increased neutrophils in BAL fluid compared with Veh-treated rats. We also observed more neutrophils and more protein in BAL fluid in Cap-treated Sprague-Dawley rats (Table 4) exposed to 1 ppm O3 for only 3 h. These observations with Cap-treated animals suggest that, in two different strains of rat, C fibers protect the lungs against O3-induced injury and consequent inflammation. The observation that, after O3 exposure, treatment with TKRAs also increases the recovery of neutrophils in BAL fluid (Table 1) suggests that tachykinins mediate at least part of this protective effect provided by C fibers.

We had hypothesized that one way in which C fibers might be mediating these protective effects was through changes in VE. In particular, we reasoned that stimulation of C fibers by O3 might lead to a decrease in VE in normal rats, which would not be present in the Cap-treated rats. The net effect would be that the Cap-treated rats received a greater dose of O3, resulting in greater inflammatory responses. Our results do not support this hypothesis. After a delay of ~70 min, VE did decrease to ~70% of values measured in the 20-min period immediately before O3 exposure in Veh-treated rats (Fig. 2), consistent with other reports (1, 25). However, VE decreased earlier and to a greater extent in the Cap-treated rats (Fig. 2). Tepper et al. (38) reported similar results in Cap-treated guinea pigs exposed to O3. Thus the increased inflammatory response to O3 in Cap-treated rats (Table 4) is not the result of a greater inhaled dose of O3. In contrast, increased inflammation develops despite a lower inhaled dose.

The observations that VE and VT decreased to a greater extent in Cap- than in Veh-treated rats and that changes in the timing of the breath were not altered by Cap treatment indicate that C fibers are not the sensory arm of the changes in VE or ventilatory timing evoked by O3 in rats. What is responsible for these changes? Other receptors, for example, rapidly adapting pulmonary stretch receptors and upper airway receptors, are also stimulated during O3 exposure (1, 4). In dogs, data obtained during CO2 rebreathing after O3 exposure suggest that slowly adapting pulmonary stretch receptors are sensitized by O3 (31), although direct recordings from slowly adapting pulmonary stretch receptors do not indicate any consistent activation of these fibers by O3 (4). The effect of vagotomy or vagal cooling on ventilatory responses to O3 in rats has not been evaluated, but in awake dogs O3 exposure still results in decreases in VT and TI, even in dogs with both cervical vagi cooled to 0°C (31), suggesting that nonvagal mechanisms may be important. For example, it is possible that decreases in VE evoked by O3 are the result of changes in metabolic rate. Both our laboratory and others have reported a decrease in O2 consumption, core body temperature, and heart rate during O3 exposure in rats (15, 25, 32). Furthermore, the decrease in VE induced by O3 occurs with only a very small change in arterial partial pressure of CO2 (40). VE declined fairly slowly after initiation of O3 exposure (Fig. 2). In addition, when the O3 flow into the chamber was turned off, VE remained low for approximately another 25 min before gradually returning to preexposure levels (data not shown). This time course suggests that formation and release of certain chemical mediators may be necessary to evoke changes in VE. For example, O3 exposure causes increased tumor necrosis factor-alpha (TNF-alpha ) expression in alveolar macrophages (28). TNF-alpha is a cryogenic factor (21) and could contribute to the decreases in core body temperature and O2 consumption observed.

It is not apparent why VE decreases more in the Cap-treated rats, but one possibility is that increased inflammation and injury, caused by loss of protective effects of tachykinins released from the peripheral ends of the neurons, result in increased generation of mediators such as TNF-alpha . Our laboratory (15) has previously reported that the decreased core body temperature and heart rate that occur in response to O3 are not different in Cap- and Veh-treated rats, suggesting that metabolic rate decreases to the same extent in both groups. However, De Sanctis et al. (5) have reported a decreased ventilatory response to hypoxic gas mixtures in Cap-treated rats, probably resulting from loss of tachykinins in the neurons subtending the carotid bodies. It is possible that loss of responsiveness to hypoxia allows the Cap-treated animals to decrease VE to a greater extent than O2 consumption.

O3 causes damage to the airway epithelium including vacuolation, sloughing, and necrosis (37), and there is reason to believe that tachykinin effects on the airway epithelium could mediate the protective role of these peptides during O3 exposure. Baluk et al. (2) reported that 85% of SP-immunoreactive sensory axons in the rat respiratory tract are located in close proximity to the epithelium, and Sertl et al. (34) showed that SP receptors were concentrated in the epithelium of the central airways in the rat lung. Tachykinins stimulate epithelial cell migration and proliferation (17). Finally, in cultured bronchial epithelial cells, pretreatment with SP decreases permeability to mannitol, a marker of tight junction permeability. Furthermore, O3-induced increases in epithelial permeability were attenuated in cells treated with SP (48). These data suggest that SP maintains epithelial cell integrity otherwise damaged by O3. Weakened epithelial integrity caused by O3 in the absence of tachykinins could reflect enhanced O3-induced injury and result in the generation of increased neutrophil chemotactic factors.

Tachykinins also have effects on airway blood flow and mucous secretion that may protect the airways from O3-induced damage. In rat airways, injection of SP increases microvascular blood flow through NK1 receptors (29), and mucosal application of SP stimulates mucous secretion in an isolated trachea preparation (46). Increased blood flow to airway mucosa could facilitate removal of O3 from the airway surface, and increased mucous secretion could contribute to the protection by covering the epithelium and reducing the concentration of O3 that actually reaches the lung surface.

Exposure to O3 (1 ppm for 3 h) did not cause any change in airway responsiveness to inhaled aerosolized methacholine in Veh (control)-treated rats (Table 2). These results are similar to reports by our laboratory and others of rats of the same strain exposed to the same concentrations of O3 (15, 39) and measured at the same point in time after O3 exposure, although airway hyperresponsiveness can be induced in rats with higher concentrations of O3 or in other strains (9, 42). However, our laboratory (15) has previously reported that, even though O3 exposure did not induce an increase in airway responsiveness in Veh-treated rats, the same concentration and time of O3 exposure did increase responsiveness in Cap-treated rats. In contrast, combined inhibition of NK1 and NK2 receptors throughout O3 exposure and methacholine challenge did not result in airway hyperresponsiveness (Table 2). These results suggest that tachykinins per se are not involved in the development of O3-induced hyperresponsiveness in rats, even though C fibers are. It is unlikely that the lack of effect of the TKRAs on airway hyperresponsiveness reflects inadequate receptor antagonism. Treatment with CP-99994 and SR-48968 caused a log shift in the dose of SP required to elicit a change in blood pressure (Table 3). The observation that TKRA treatment also altered the BAL cell profile (Table 1) further supports the efficacy of the receptor antagonists. One possible explanation for the augmentation of airway responsiveness by Cap pretreatment but not by tachykinin-receptor inhibition is that substances other than tachykinins present in C fibers are important. Possible candidates are calcitonin gene-related peptide and nitric oxide (NO). Both are colocalized with tachykinins in C fibers and act as bronchodilators (3, 7, 11). Absence of these potent bronchodilators coreleased from C-fiber endings on O3 stimulation might be responsible for augmented airway responsiveness in Cap-pretreated rats.

There were also differences between TKRA treatment and neonatal Cap treatment in the effects on O3-induced BAL protein changes. BAL protein concentration was significantly increased in O3-exposed, Cap-treated rats compared with Veh-treated rats, but TKRAs did not alter the effects of O3 on BAL protein, despite significant increases in BAL neutrophils (Table 1). These results suggest that substances other than tachykinins present in C fibers may act to counteract the O3-induced changes in vascular permeability that lead to changes in protein. NO has been reported to maintain pulmonary vascular endothelial integrity (11, 20), and, in guinea pig airways, inhibition of endogenous NO resulted in enhancement of vagally induced plasma albumin leakage (20). Lack of the endogenous NO present in C-fiber endings may thus contribute to the increased plasma protein leakage induced by O3 exposure in Cap-treated animals.

There is evidence of species differences in the effect of C fibers on O3-induced airway responsiveness. In guinea pigs, depletion of neuropeptides by Cap treatment attenuates O3-induced airway hyperresponsiveness (38). In rats, Cap pretreatment promotes airway hyperresponsiveness after O3 exposure (15). One potential explanation for this species difference is the differing bronchoconstrictor potency of tachykinins. In guinea pigs, SP and NKA are both potent bronchoconstrictors (19), whereas, in rats, depending on the strain, the tachykinins either have a weak constrictor potential (16) or actually cause dilation (6). In contrast, effects of Cap pretreatment on lung injury induced by O3 appear to be similar in the two species. Both in this study in rats (Table 4) and in the study of Tepper et al. (38) in guinea pigs, increased protein in BAL fluid was observed in Cap-treated animals compared with Veh-treated controls. Similar results were obtained by Sterner-Kock et al. (37). The accumulation of protein in BAL has been shown to be a sensitive indicator of O3-induced pulmonary damage (43). Hence, our data and those of others support the hypothesis that C fibers act to attenuate O3-induced pulmonary damage, at least in these two species. Similarly, evidence for a role for C fibers in inhibiting the development of airway injury and/or inflammation has also been obtained with other inhaled irritants. More severe lung injury and/or inflammation in the airways has been observed in C-fiber-ablated rats after chronic sulfur dioxide (22, 23) or hydrogen sulfide (30) exposure, and in Cap-treated guinea pigs after acute acrolein inhalation (41).

In summary, our results show that, in rats, more neutrophils are recovered by BAL when tachykinin-receptor antagonists are administered throughout O3 exposure. These results are consistent with observations reported herein and with other reports of greater inflammation after O3 exposure in Cap-treated rats lacking C fibers, compared with Veh-treated controls (37). The greater inflammatory response does not appear to be the result of a greater inhaled dose of O3, because VE was decreased in Cap-treated rats relative to Veh-treated controls during O3 exposure. Together, the results support the hypothesis that tachykinins released from peripheral ending of C fibers mediate at least part of the protective effects of these afferents against O3-induced pulmonary inflammation.

    ACKNOWLEDGEMENTS

The authors thank Roslyn Hennessey for excellent technical assistance, and express their appreciation of the late William Skornik for assistance with the O3 exposures.

    FOOTNOTES

This study was supported by National Institutes of Health Grants HL-19170 and ES-00002.

Address for reprint requests: S. A. Shore, Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115 (E-mail: sshore{at}hsph.harvard.edu).

Received 27 October 1997; accepted in final form 27 March 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Arito, H., M. Takahashi, T. Isawaki, and I. Uchiyama. Age-related changes in ventilatory and heart rate responses to acute O3 exposure in the conscious rat. Ind. Health 35: 78-86, 1997[Medline].

2.   Baluk, P., J. A. Nadel, and D. M. McDonald. Substance P-immunoreactive sensory axons in the rat respiratory tract: a quantitative study of their distribution and role in neurogenic inflammation. J. Comp. Neurol. 319: 586-598, 1992[Medline].

3.   Cadieux, A., C. Lanoue, P. Sirois, and J. Barabe. Carbamylcholine- and 5-hydroxytryptamine-induced contraction in rat isolated airways: inhibition by calcitonin gene-related peptide. Br. J. Pharmacol. 101: 193-199, 1990[Medline].

4.   Coleridge, J. C., H. M. Coleridge, E. S. Schelegle, and J. F. Green. Acute inhalation of ozone stimulates bronchial C fibers and rapidly adapting receptors in dogs. J. Appl. Physiol. 74: 2345-2352, 1993[Abstract/Free Full Text].

5.   De Sanctis, G. T., F. H. Y. Green, and J. E. Remmers. Ventilatory responses to hypoxia and hypercapnia in awake rats pretreated with capsaicin. J. Appl. Physiol. 70: 1168-1174, 1991[Abstract/Free Full Text].

6.   Devillier, P., G. M. Acke, C. Advenier, J. Marsac, D. Regoli, and N. Frossard. Activation of an epithelial neurokinin NK-1 receptor induces relaxation of rat trachea through release of prostaglandin E2. J. Pharmacol. Exp. Ther. 263: 767-772, 1992[Abstract/Free Full Text].

7.   Dupuy, P. M., S. A. Shore, J. M. Drazen, C. Frostell, W. A. Hill, and W. M. Zapol. Bronchodilator action of inhaled nitric oxide in guinea pigs. J. Clin. Invest. 90: 421-428, 1992.

8.   Emonds-Alt, X., C. Advenier, T. Croci, L. Manara, G. Neliat, M. Poncelet, V. Proietto, V. Santucci, P. Soubrie, D. V. Broeck, P. Vilain, G. L. Fur, and J.-C. Breliere. SR 48986, a neurokinin (NK2) receptor antagonist. Regul. Pept. 46: 31-36, 1993[Medline].

9.   Evans, T. W., J. J. Brokaw, K. F. Chung, J. A. Nadel, and D. M. McDonald. O3-induced bronchial hyperresponsiveness in the rat is not accompanied by neutrophil influx or increased vascular permeability. Am. Rev. Respir. Dis. 138: 140-144, 1988[Medline].

10.   Folkesson, R., A. Neil, and L. Terenius. Enzyme-linked immunosorbent assay of substance P and its metabolite SP1-7. A comparison with RIA. J. Neurosci. Methods 14: 169-176, 1985[Medline].

11.   Gaston, B., J. M. Drazen, J. Loscalzo, and J. S. Stamler. The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care Med. 149: 538-551, 1994[Abstract].

12.   Hazbun, M. E., R. Hamilton, A. Holian, and W. L. Eschenbacher. Ozone-induced increases in substance P and 8-epi-prostaglandin F2a in the airways of human subjects. Am. J. Respir. Cell Mol. Biol. 9: 568-572, 1993.

13.   Hong, J.-L., I. W. Rodger, and L.-Y. Lee. Cigarette smoke-induced bronchoconstriction: cholinergic mechanisms, tachykinins, and cyclooxygenase products. J. Appl. Physiol. 78: 2260-2266, 1995[Abstract/Free Full Text].

14.   Jancso, G., E. Kiraly, and A. Jancso-Gaabor. Pharmacologically induced selective degeneration of chemosensitive primary sensory neurons. Nature 270: 741-743, 1977[Medline].

15.   Jimba, M., W. A. Skornik, C. R. Killingsworth, N. C. Long, J. D. Brain, and S. A. Shore. Role of C fibers in physiological responses to ozone in rats. J. Appl. Physiol. 78: 1757-1763, 1995[Abstract/Free Full Text].

16.  Joos, G., J. Kips, R. Pauwels, and V. D. Straeten. The effect of tachykinins on the conducting airways of the rat. Arch. Int. Pharmacodyn. 280, Suppl: 176-190, 1986.

17.   Kim, J. S., K. F. Rabe, H. Magnussen, J. Green, and S. R. White. Migration and proliferation of guinea pig and human airway epithelial cells in response to tachykinins. Am. J. Physiol. 269 (Lung Cell Mol. Physiol. 13): L119-L126, 1995[Abstract/Free Full Text].

18.   Lilly, C. M., T. R. Bai, S. A. Shore, A. E. Hall, and J. M. Drazen. Neuropeptide content of lungs from asthmatic and nonasthmatic patients. Am. J. Respir. Crit. Care Med. 151: 548-553, 1995[Abstract].

19.   Lilly, C. M., J. M. Drazen, and S. A. Shore. Peptidase modulation of airway effects of neuropeptides. Proc. Soc. Exp. Biol. Med. 203: 388-404, 1993[Abstract].

20.   Liu, S., H.-P. Kuo, M. N. Sheppard, P. J. Barnes, and T. W. Evans. Vagal stimulation induces increased pulmonary vascular permeability in guinea pig. Am. J. Respir. Crit. Care Med. 149: 744-750, 1994[Abstract].

21.   Long, N. C., S. L. Kunkel, A. J. Vander, and M. J. Kluger. Antiserum against tumor necrosis factor enhances lipopolysaccharide fever in rats. Am. J. Physiol. 258 (Regulatory Integrative Comp. Physiol. 27): R332-R337, 1990[Abstract/Free Full Text].

22.   Long, N. C., J. G. Martin, R. Pantano, and S. A. Shore. Airway hyperresponsiveness in a rat model of chronic bronchitis: role of C-fibers. Am. J. Respir. Crit. Care Med. 155: 1222-1229, 1997[Abstract].

23.   Long, N. C., and S. A. Shore. Effects of capsaicin pretreatment on bronchoalveolar cells recovered from rats during SO2 induction of chronic bronchitis (Abstract). Am. J. Respir. Crit. Care Med. 147: A72, 1993.

24.   Lundberg, J. M., T. Hokfelt, C. R. Martling, A. Saria, and C. Cuello. Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man. Cell Tissue Res. 235: 251-261, 1984[Medline].

25.   Mautz, W. J., and C. Bufalino. Breathing pattern and metabolic rate responses of rats exposed to ozone. Respir. Physiol. 76: 69-78, 1989[Medline].

26.   McLean, S., A. Ganong, P. A. Seymour, R. M. Snider, M. C. Desai, T. Rosen, D. K. Bryce, K. P. Longo, L. S. Reynolds, G. Robinson, A. W. Schmidt, C. Siok, and J. Heym. Pharmacology of CP-99,994; a nonpeptide antagonist of the tachykinin neurokinin-1 receptor. J. Pharmacol. Exp. Ther. 267: 472-479, 1993[Abstract/Free Full Text].

27.   Nagy, J. I., S. P. Hunt, L. L. Iversen, and P. C. Emson. Biochemical and anatomical observations on the degeneration of peptide-containing primary afferent neurons after neonatal capsaicin. Neuroscience 6: 1923-1934, 1981[Medline].

28.   Pendino, K. J., R. L. Shuler, J. D. Laskin, and D. L. Laskin. Enhanced production of interleukin-1, tumor necrosis factor-alpha , and fibronectin by rat lung phagocytes following inhalation of a pulmonary irritant. Am. J. Respir. Cell Mol. Biol. 11: 279-286, 1994[Abstract].

29.   Piedimonte, G., J. I. Hoffman, W. K. Husseini, W. L. Hiser, and J. A. Nadel. Effect of neuropeptides released from sensory nerves on blood flow in the rat airway microcirculation. J. Appl. Physiol. 72: 1563-1570, 1992[Abstract/Free Full Text].

30.   Prior, M., F. Green, A. Lopez, A. Balu, G. T. De Sanctis, and G. Fick. Capsaicin pretreatment modifies hydrogen sulphide-induced pulmonary injury in rats. Toxicol. Pathol. 18: 279-288, 1990[Medline].

31.   Sasaki, K., J. A. Nadel, and H. L. Hahn. Effect of ozone on breathing in dogs: vagal and nonvagal mechanisms. J. Appl. Physiol. 62: 15-26, 1987[Abstract/Free Full Text].

32.   Scheel, L. D., O. J. Dobrogorski, J. T. Mountain, J. L. Svirbely, and H. E. Stokinger. Physiologic, biochemical, immunologic and pathologic changes following ozone exposure. J. Appl. Physiol. 14: 67-80, 1959[Abstract/Free Full Text].

33.   Schelegle, E. S., M. L. Carl, H. M. Coleridge, J. C. Coleridge, and J. F. Green. Contribution of vagal afferents to respiratory reflexes evoked by acute inhalation of ozone in dogs. J. Appl. Physiol. 74: 2338-2344, 1993[Abstract/Free Full Text].

34.   Sertl, K., C. J. Wiedermann, M. L. Kowalski, S. Hurtado, J. Plutchok, I. Linnoila, C. B. Pert, and M. A. Kaliner. Substance P: the relationship between receptor distribution in rat lung and the capacity of substance P to stimulate vascular permeability. Am. Rev. Respir. Dis. 138: 151-159, 1988[Medline].

35.   Skrabanek, P., and D. Powell. Blood pressure regulation and other smooth muscle effects. In: Substance P. Montreal, Canada: Eden, 1983, vol. 3, p. 46-52.

36.   Solway, J., B. M. Kao, J. E. Jordan, B. Gitter, I. W. Roger, J. J. Howbert, L. E. Alger, J. Necheles, A. R. Leff, and A. Garland. Tachykinin receptor antagonists inhibit hyperpnea-induced bronchoconstriction in guinea pigs. J. Clin. Invest. 92: 315-323, 1993.

37.   Sterner-Kock, A., K. R. Vesely, M. Y. Stovall, E. S. Schelegle, J. F. Green, and D. M. Hyde. Neonatal capsaicin treatment increases the severity of ozone-induced lung injury. Am. J. Respir. Crit. Care Med. 153: 436-443, 1996[Abstract].

38.   Tepper, J. S., D. L. Costa, S. Fitzgerald, D. L. Doerfler, and P. A. Bromberg. Role of tachykinins in ozone-induced acute lung injury in guinea pigs. J. Appl. Physiol. 75: 1404-1411, 1993[Abstract/Free Full Text].

39.   Tepper, J. S., D. L. Costa, and J. R. Lehmann. Extrapolation of animal data to humans: homology of pulmonary physiological responses with O3 exposure. In: Toxicology of the Lung (2nd ed.), edited by D. E. Gardner, J. D. Crapo, and R. O. McClellan. New York: Raven, 1993, p. 217-251.

40.   Tepper, J. S., M. J. Weister, M. F. Weber, and M. G. Menache. Measurements of cardiopulmonary response in awake rats during acute exposure to near-ambient concentrations of ozone. J. Appl. Toxicol. 10: 7-15, 1990[Medline].

41.   Turner, C. R., R. B. Stow, S. D. Talerico, E. P. Christian, and J. C. Williams. Protective role for neuropeptides in acute pulmonary response to acrolein in guinea pigs. J. Appl. Physiol. 75: 2456-2465, 1993[Abstract/Free Full Text].

42.   Uchida, D. A., C. A. Ballowe, C. G. Irvin, and G. L. Larsen. Assessment of airway responsiveness to inhaled methacholine and effects of short term O3 exposure in aged Fischer-344 rats. HEI Communication 1: 20-23, 1992.

43.   US Environmental Protection Agency. Air Quality Criteria for Ozone and Other Photochemical Oxidants. Research Triangle Park, NC: US Environmental Protection Agency, 1986, vol. IV (EPA-600/8-84-020dF)

44.   Von Neergard, K., and K. Wirz. Uber eine methode zur messung der lungenelastizitat am lebenden menshen, insbesondere beim emphysem. Z. Klin. Med. 105: 35-50, 1927.

45.   Von Neergard, K., and K. Wirz. Die messung der stroemungs wiederstaende in den atemwegen des menschen, insbesondere bei asthma und emphysem. Z. Klin. Med. 105: 52-82, 1927.

46.   Wagner, U., H. C. Fehmann, D. Bredenbroker, F. Yu, P. J. Barth, and P. Vonwichert. Galanin and somatostatin inhibition of substance P-induced airway mucous secretion in rat. Neuropeptides 28: 59-64, 1995[Medline].

47.   Weister, M. J., T. B. Williams, E. King, M. G. Menache, and F. J. Muller. Ozone uptake in awake Sprague-Dawley rats. Toxicol. Appl. Pharmacol. 89: 429-437, 1987[Medline].

48.   Yu, X. Y., B. J. Undem, and E. W. Spannhake. Protective effects of substance P on ozone-induced bronchial epithelial permeability (Abstract). Am. J. Respir. Crit. Care Med. 151: A497, 1995.

J APPL PHYSIOL 85(2):442-450
8570-7587/98 $5.00 Copyright © 1998 the American Physiological Society


This article has been cited by other articles:


Home page
 Eur Respir JHome page
K.G. Tournoy, K.O. De Swert, P.G. Leclere, R.A. Lefebvre, R.A. Pauwels, and G.F. Joos
Modulatory role of tachykinin NK1 receptor in cholinergic contraction of mouse trachea
Eur. Respir. J., January 1, 2003; 21(1): 3 - 10.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. A. SHORE, I. N. SCHWARTZMAN, B. LE BLANC, G. G. KRISHNA MURTHY, and C. M. DOERSCHUK
Tumor Necrosis Factor Receptor 2 Contributes to Ozone-induced Airway Hyperresponsiveness in Mice
Am. J. Respir. Crit. Care Med., August 15, 2001; 164(4): 602 - 607.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
R. M. GRAHAM, M. FRIEDMAN, and G. W. HOYLE
Sensory Nerves Promote Ozone-induced Lung Inflammation in Mice
Am. J. Respir. Crit. Care Med., July 15, 2001; 164(2): 307 - 313.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
S. A. Shore, J. H. Abraham, I. N. Schwartzman, G. G. K. Murthy, and J. D. Laporte
Ventilatory responses to ozone are reduced in immature rats
J Appl Physiol, June 1, 2000; 88(6): 2023 - 2030.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takebayashi, T.
Right arrow Articles by Shore, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takebayashi, T.
Right arrow Articles by Shore, S. A.

HOME HELP