Journal of Applied Physiology Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 91: 371-378, 2001;
8750-7587/01 $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 ISI Web of Science
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 ISI Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wu, Z.-X.
Right arrow Articles by Dey, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, Z.-X.
Right arrow Articles by Dey, R. D.
Vol. 91, Issue 1, 371-378, July 2001

Role of intrinsic airway neurons in ozone-induced airway hyperresponsiveness in ferret trachea

Zhong-Xin Wu1, David F. Maize Jr.1,3, Brian E. Satterfield1, David G. Frazer2, Jeff S. Fedan2, and Richard D. Dey1

1 Department of Anatomy, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown 26506; 2 Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505; and 3 Department of Pharmaceutical Sciences, Nesbitt School of Pharmacy at Wilkes University, Wilkes-Barre, Pennsylvania 18766


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure to ozone (O3) enhances airway responsiveness, which is mediated partly by the release of substance P (SP) from airway neurons. In this study, the role of intrinsic airway neurons in O3-induced airway responses was examined. Ferrets were exposed to 2 ppm O3 or air for 1 h. Reactivity of isolated tracheal smooth muscle to cholinergic agonists was significantly increased after O3 exposure, as were contractions to electrical field stimulation at 10 Hz. Pretreatment with CP-99994, a neurokinin type 1 receptor antagonist, partially abolished the O3-induced reactivity to cholinergic agonists and electrical field stimulation. The O3-enhanced airway responses were present in tracheal segments cultured for 24 h, a procedure shown to deplete sensory nerves while maintaining viability of intrinsic airway neurons, and all the enhanced smooth muscle responses were also diminished by CP-99994. Immunocytochemistry showed that the percentage of SP-containing neurons in longitudinal trunk and the percentage of neurons innervated by SP-positive nerve fibers in superficial muscular plexus were significantly increased at 1 h after exposure to O3. These results suggest that enhanced SP levels in airway ganglia contribute to O3-induced airway hyperresponsiveness.

airway smooth muscle; sensory nerves; neurokinin receptors; tachykinins; substance P


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE EXPOSURE TO OZONE (O3), one of the major air pollutants in urban areas, is frequently associated with airway hyperresponsiveness (AHR) (18-20, 34). Although airway smooth muscle contraction is mediated by acetylcholine (ACh) binding to atropine-sensitive M3-muscarinic receptors (27), separate mechanisms have been proposed as mediating the induction of AHR, including altered M2 function (39), release of arachidonic acid-derived mediators (4), inhibition of neutral endopeptidase (30), release of tachykinins from sensory nerves (15), and inhibition of release of epithelium-derived relaxing factor (11). Several studies have shown that increased airway smooth muscle responsiveness after O3 exposure is mediated by the release of endogenous tachykinins from capsaicin-sensitive nerve fibers in airway wall (18, 19, 34).

Neuropeptides, such as substance P (SP), neurokinin A (NKA), and calcitonin gene-related peptide (CGRP), are localized in the peripheral endings of the nonmyelinated C-fibers innervating the airways and are synthesized in sensory nerve cell bodies located in the jugular and nodose ganglia (17, 21). Other neuropeptides, such as vasoactive intestinal peptide (VIP) and nitric oxide (NO), are associated with neurons of intrinsic airway ganglia located in the adventitia of airway walls (9, 12). However, recent studies have shown that SP may also be present in airway ganglia (6, 8, 14). A substantial portion of the innervation of airway smooth muscle, glands, and blood vessels in the airways originates from neurons with cell bodies in intrinsic airway ganglia (8, 10). Although most innervation of airway epithelium originates from neurons with cell bodies located in nodose or jugular ganglia (7, 17), nerve fibers from cell bodies in intrinsic airway ganglia may also project to the airway epithelium (10), providing a potential neural pathway for local modulation of airway responses.

The role of SP released from neurons in airway ganglia is unknown. Inhalation of chemical irritants triggers the local release of neuropeptides from sensory nerves (22, 37). Neuropeptides, especially SP in the airway, are known to produce potent effects on airway smooth muscle tone (33) and alter neurotransmission through airway ganglia (26, 36). Inhalation of irritants such as antigens (13) and toluene diisocyanate (16) increases SP levels in sensory neurons projecting to the airways. However, whether SP released from intrinsic airway neurons contributes to AHR after inhaled irritants has not been determined. Therefore, the purpose of this study was to investigate the possible involvement of intrinsic airway neurons in the development of AHR induced by O3 exposure.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Female nonalbino ferrets (Marshall Farms, North Rose, NY; 250-500 g body wt) were housed two to four per cage with access to food and water ad libitum in an American Association for Accreditation of Laboratory Animal Care-accredited facility. Female ferrets were used, because they adapt better than males to group housing. The ferrets were euthanized by inhalation of 100% CO2 for 3 min in a sealed exposure chamber. All procedures were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and were also approved by the West Virginia University Animal Care and Use Committee.

O3 exposure. All O3 exposures were done at 2 ppm in a 12 × 12-in. stainless steel-and-glass chamber for 1 h. O3 was produced by passing hospital-grade air through a drying and high-efficiency particle filter and then through an ultraviolet light source. The O3 concentration in the chamber was measured by chemiluminescence with a calibrated O3 analyzer (model OA 350-2R, Forney, Carrollton, TX) and adjusted every 10 s by an on-line computer. A separate group of animals was subjected to air exposure, in which procedures identical to those described above were followed, except O3 was not delivered to the mixing chamber. To determine the possible involvement of endogenously released SP, some ferrets were given one injection of CP-99994 (1.0 mg/kg ip), a neurokinin (NK) type 1 (NK1)-receptor antagonist, 30 min before O3 or air exposure. The dose of this antagonist was determined on the basis of previous findings (35, 37, 38, 40).

Cultured tracheal segments. To examine intrinsic innervation in tracheas depleted of sensory fibers, organotypic cultures of tracheas were used following a modification of our previously described technique (10). Ferrets were exposed to 2 ppm O3 or air for 1 h as described above. Immediately after exposure, tracheas were removed and washed with cold culture medium (described below), placed in a petri dish with culture medium, and cut into 10-mm-long segments beginning with the caudal end. After a second wash, the segments were placed directly on the bottom of petri dishes containing fresh culture medium. In some of the cultures, CP-99994 (final concentration 10 mM) was added to the culture media and maintained throughout the experiment to determine the role of SP in intrinsic airway neurons. The antagonist concentration was based on previous studies (1). The petri dishes were then placed in a controlled-atmosphere culture chamber and gassed with 45% O2-5% CO2-50% N2. The chamber was placed on a rocker and incubated at 37°C for 24 h. After culture, smooth muscle responses were measured in the segments. The culture media was CMRL 1066 containing 0.1 µg/ml hydrocortisone hemisuccinate, 1 µg/ml recrystalized bovine insulin, 60 µg/ml penicillin G (100 U/ml), 10 µg/ml amphotericin B, 100 µg/ml streptomycin, and 5% inactivated fetal calf serum.

Measurement of tissue contraction in vitro. Fresh tracheal segments from ferrets 1 h after air or O3 exposure and from the 24-h cultures were cut into 3-mm-wide strips, mounted in holders, and maintained in gassed (95% O2-5% CO2) modified Krebs-Henseleit solution at 37°C with a composition (in mM) as follows: 113 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgSO4, 24 NaHCO3, 1.2 KH2PO4, and 5.7 glucose, pH 7.4. The strips were tied at each end with 4-0 silk and positioned between the rings of platinum electrodes in tissue holders. Each holder was anchored in a 10-ml water-jacketed organ bath, and the top string was attached to a force-displacement transducer connected to a recorder (Gould Instruments, Valley View, OH). Strips were equilibrated for 60 min at a resting tension of 1.0 g, determined in preliminary studies to be optimal for contraction, during which time the modified Krebs-Henseleit solution in the baths was changed every 15 min. After equilibration, cumulative concentration-response curves for ACh and methacholine (MCh) were constructed for separate strips by adding a series of concentrations of ACh or MCh to the bath in half-log-increment concentrations ranging from 10-9 to 10-3 M. The next concentration was not added until the previous response reached a plateau. After concentration-response curves were completed, electrical field stimulation (EFS)-induced responses were obtained with a stimulator (model S48, Grass Instruments, W. Warwick, Richmond, VA). Frequency-response curves were constructed by increasing the frequency from 0.3 to 30 Hz using a submaximum voltage of 120 V, 0.2-ms pulse duration, and 10-s train duration. Between each stimulation period, 10 min were allowed for the previous response to return to baseline. EFS-induced contractions were normalized by the percent response of each tissue to 10-3 M ACh (%ACh response). In some experiments, atropine (10-6 M) was added to Krebs solution to verify that the responses elicited by EFS were mediated by the release of ACh from cholinergic neurons. In initial experiments, contractions to EFS in noncultured and cultured tracheal segments were totally abolished after treatment with 10-6 M atropine (data not shown).

Immunocytochemistry. In a separate group of ferrets exposed using the same O3 exposure protocol, tracheas were removed 1 h after O3 or air exposure, fixed in picric acid-formaldehyde fixative for 3 h (32), and rinsed three times with a 0.1 M phosphate-buffered saline containing 0.15% Triton X-100. The tracheas were dissected and frozen in isopentane, cooled with liquid nitrogen, and stored in airtight bags at -80°C. The tracheas were oriented with the dorsal surface uppermost so the tracheal muscle would be sectioned in a coronal plane.

Cryostat sections (12 µm thick) were collected on gelatin-coated coverslips and dried briefly at room temperature. Immunocytochemical procedures for localizing neuropeptides in neurons and nerve fibers are identical to those described by us previously (6, 7). Briefly, cryostat sections on coated coverslips were covered with SP antibody diluted 1:100, incubated in a humid chamber at 37°C for 30 min, and rinsed with a 1% bovine serum albumin + phosphate-buffered saline containing 0.15% Triton X-100 three times, with 5 min allowed for each rinse. The sections were then covered with fluorescein isothiocyanate-labeled goat anti-rabbit antibody diluted 1:100, incubated at 37°C for 30 min, and rinsed. Then the same procedures were used to label VIP using mouse-anti-VIP (1:100) and goat anti-mouse labeled with rhodamine (1:100). VIP labeling was done to allow efficient identification of superficial muscular plexus (SMP) neurons, which are typically difficult to locate but can be easily visualized immunocytochemically, because 90% are VIP immunoreactive. After all immunocytochemical procedures were conducted, the coverslips were mounted with Fluoromount and observed with a fluorescence microscope equipped with fluorescein (excitation wavelengths 455-500 nm, emission wavelengths >510 nm) and rhodamine (excitation 540-504 nm, emission >580 nm). Controls consisted of testing the specificity of primary antiserum by absorption with 1 µg/ml of the specific antigen. Nonspecific background labeling was determined by omission of primary antiserum.

To measure fluorescence intensity in longitudinal trunk (LT) neurons, images were digitally recorded using an AX 70 microscope (Olympus America, Melville, NY) with the SPOT 2 (Diagnostics Instruments, Sterling Heights, MI). Fluorescence intensity of SP was measured using commercial image processing software (Optimas 6.5, Media Cybernetics, Silver Spring, MD). The intensity recordings were calibrated using the InSpeck Green (505/515) Microscope Image Intensity Calibration Kit (Molecular Probes, Eugene, OR). The LT neurons were identified by drawing the perimeter of the cell, and the fluorescence intensity was reported as gray level for each neuron. Neurons with a gray level <50 were considered negative, because they were at or below the general background. Fluorescence intensities of >= 50 were counted as labeled neurons. To measure SP innervation of SMP neurons (identified by VIP colocalization), all identifiable SMP neurons were scored as either innervated or not innervated. All LT and SMP neurons were evaluated in every fifth section collected from serial sections, usually amounting to a total of 10-15 sections analyzed.

Data analysis. Unless otherwise stated, values are means ± SE. Contractions elicited by EFS are expressed as a percentage of the maximal contraction elicited by ACh. Contractions to ACh and MCh were normalized as a percentage of the respective maximal responses for each agonist. EC50 values for ACh and MCh were calculated using a four-parameter logistic curve fit (Sigmoidal, SigmaPlot 2000) and are presented with 95% confidence intervals. Force development was expressed by normalizing force (g) divided by the wet weight of the tissue. LT neurons are expressed as percent SP-positive cell bodies, and SMP neurons are expressed as percent SP-innervated cell bodies. Statistical analyses of immunocytochemisty and EC50 were performed using Student's t-test. Statistical analysis of EFS was performed using two-way repeated-measures analysis of variance. One factor between the groups was O3 exposure; the other factor within the group was EFS effect. When the main effect was considered significant at P < 0.05, pairwise comparisons were made with a post hoc analysis (Fisher's least significant difference). P < 0.05 was considered significant, and n represented the number of animals studied.

Materials. ACh chloride, MCh chloride, atropine sulfate, hydrocortisone hemisuccinate, amphotericin B, and recrystalized bovine insulin were obtained from Sigma Chemical (St. Louis, MO). Penicillin G, streptomycin, fetal calf serum, and CMRL 1066 were obtained from GIBCO (Grand Island, NY). CP-99994 was obtained from Pfizer (Groton, CT). SP antibody was obtained from Peninsula (Belmont, CA). Mouse-anti-VIP monoclonal antibody was a gift from Dr. John Porter (University of Texas Southwestern Medical School, Dallas, TX). Fluorescein isothiocyanate-labeled goat anti-rabbit antibody was obtained from ICN Immunobiologicals (Costa Mesa, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of O3 on airway responsiveness in noncultured trachea. The initial experiments were intended to demonstrate that O3 exposure increases airway smooth muscle sensitivity to ACh and MCh. The cumulative dose-response curves for ACh and MCh were markedly shifted to the left after exposure to O3 (Fig. 1, A and B), and the EC50 values for ACh and MCh (Table 1) were decreased by 69% and 61% in O3-exposed animals. Exposure to O3 also increased airway response for EFS. A leftward shift in the frequency-response curve to EFS was observed after O3 exposure (Fig. 1C), and contraction produced by EFS at 10 Hz was significantly increased by 28% after O3 exposure.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1.   Cumulative dose-response curve for acetylcholine (ACh, A) and methacholine (MCh, B) and frequency-response curve for electrical field stimulation (EFS, C) in tracheal smooth muscle after exposure to air (open circle ) or 2.0 ppm O3 () for 1 h. Contractions are plotted as percentages of the maximum response. Responses for EFS are plotted as percentages of maximum response to ACh. Values are means ± SE; n = 6. *Significant difference between control and O3-exposed animals, P < 0.05.


                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Effect of O3 on cumulative dose-response curves for ACh and MCh in trachea smooth muscle

Effects of NK1 antagonist in tracheal strips. In separate experiments, NK1 receptors were blocked with CP-99994 to test the involvement of SP in O3-induced AHR. In control ferrets pretreated with saline, the dose-response curves and EC50 values for ACh and MCh (Fig. 2, A and C, Table 2) demonstrated the expected changes (Fig. 1) after O3 exposure: a shift in the cumulative dose-response curves for ACh and MCh to the left of control and a decrease in EC50. A significant increase in EFS-stimulated contraction at 10 Hz was also observed (Fig. 2E). There were no effects of CP-99994 on ACh or MCh dose-response curves obtained from strips in air-exposed animals (Table 2), nor was EFS-induced contraction at 10 Hz affected (Fig. 2F). However, the increase in reactivity to ACh and MCh (Fig. 2, B and D, Table 2) and the potentiated response to 10-Hz EFS stimulation (Fig. 2F) were partly abolished by CP-99994 treatment. EC50 values for ACh and MCh decreased by only 43% and 40% after O3 exposure and pretreatment with CP-99994 compared with 72% and 66% after pretreatment with saline. In O3-exposed ferrets, EFS-stimulated airway contraction at 10 Hz increased by 12% after pretreatment with CP-99994 compared with 31% after pretreatment with saline. The results indicated that pretreatment with CP-99994 attenuates O3-induced responses to ACh, MCh, and EFS.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2.   Effects of saline (A, C, and E) and CP-99994 (B, D, and F) on cumulative dose-response curve for ACh (A and B) and MCh (C and D) and frequency-response curve for EFS (E and F) in tracheal strips from ferrets after exposure to air (open circle ) or 2.0 ppm O3 () for 1 h. Values are means ± SE; n = 5. NS, not significant. *Significant difference between control and O3-exposed animals, P < 0.05.


                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Effect of CP-99994 treatment on cumulative dose-response curves for ACh and MCh

Effects of NK1 antagonist in cultured tracheal strips. The next series of studies was done to examine the contribution of SP from intrinsic airway neurons to O3-induced AHR. Previous studies have shown that innervation of smooth muscle by intrinsic airway neurons remains intact during short-term culture but that SP-containing sensory neurons mostly degenerate (10). Therefore, tracheal segments from ferrets exposed to air or O3 were maintained in organotypic culture with saline or CP-99994 for 24 h. After culture, dose-response curves for ACh and MCh (Fig. 3, A and C) were shifted to the left, and EC50 values (Table 3) were significantly less in tracheal segments cultured with saline from O3-exposed ferrets. O3 exposure also increased EFS-stimulated contractions at 10 and 30 Hz (Fig. 3E) in tracheal segments cultured with saline. However, the increase in reactivity to ACh and MCh (Fig. 3, B and D) and the potentiated responses to 10- and 30-Hz EFS stimulation (Fig. 3F) were partly abolished by CP-99994 treatment. EC50 values for ACh and MCh decreased by only 48% and 47% after O3 exposure in culture in the CP-99994 group compared with 74% and 77% in culture in the saline group (Table 3). EFS-stimulated airway contractions at 10 and 30 Hz increased only by 11% and 8%, respectively, after O3 in tracheas cultured with CP-99994 compared with 33% and 34% in tracheas cultured with saline. There were no significant effects of CP-99994 on ACh or MCh dose-response curves or EFS-induced contraction (Fig. 3F) in strips from air-exposed animals (Table 3).


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3.   Effects of saline (A, C, and E) and CP-99994 (B, D, and F) on cumulative dose-response curve for ACh (A and B) and MCh (C and D) and frequency-response curve for EFS (E and F) in cultured tracheal strips from ferrets after exposure to air (open circle ) or 2.0 ppm O3 () for 1 h. Values are means ± SE; n = 6. *Significant difference between control and O3-exposed animals, P < 0.05.


                              
View this table:
[in this window]
[in a new window]
  		Table 3.   Effect of CP-99994 treatment on cumulative dose-response curves for ACh and MCh in cultured tracheal strips

Changes in immunoreactive SP-containing intrinsic airway neurons. Because the previous experiments suggested that O3 exposure might alter SP expression of intrinsic airway neurons, studies were done to examine SP levels in the nerve cell bodies of the ganglia of the LT and the extent of SP innervation in cell bodies of the SMP. About 20% of the LT cell bodies labeled for SP in air-exposed ferrets (Figs. 4A and 5A) and ~50% of the SMP neurons were innervated by SP-containing nerve fibers (Figs. 4C and 5B). After exposure to O3, >60% of the cell bodies in the LT contained SP (Fig. 4B) and ~90% of the cell bodies in the SMP were innervated by SP-containing nerve fibers (Figs. 4D and 5B).


View larger version (75K):
[in this window]
[in a new window]
  		Fig. 4.   Fluorescence photomicrographs of substance P (SP)-immunoreactive nerve cell bodies and fibers in longitudinal trunk (LT, A and B) and superficial muscular plexus (SMP, C and D) 1 h after O3 or air exposure. A: a few SP-immunoreactive LT neurons are seen in the neurons of the control ganglia. Several cell bodies are negative for SP. B: 1 h after O3 exposure, most of the LT nerve cell bodies contain SP immunoreactivity. C: unlabeled cell bodies in the SMP of control ferret. One cell body is surrounded by a few SP-immunoreactive fibers that form putative synaptic boutons on the cell membrane. D: 1 h after O3 exposure, several SP-immunoreactive nerve fibers are in close association with the unlabeled SMP nerve cell bodies. Magnification: ×285 (A and B) and ×588 (C and D).



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of exposure to air (open bars) and 2.0 ppm O3 (solid bars) for 1 h on SP-containing nerve cell bodies in LT (A) and SP innervation of airway neurons in SMP (B). Values are means ± SE; n = 6. *Significant difference between control and O3-exposed animals, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results obtained from this study show that acute exposure of young ferrets to O3 produces increased airway responsiveness to cholinergic agonists and enhances smooth muscle contraction during EFS. The elevated airway smooth muscle responses were attenuated by treatment with an NK1-receptor antagonist before O3 exposure, indicating that endogenously released SP was involved. A role for tachykinins in causing elevated smooth muscle responses in the airways has been reported, but most previous studies indicate that the source of the tachykinins is sensory neurons innervating the airways (18, 22). Our results are unique, because they suggest that SP levels in intrinsic airway neurons of the LT and innervation density of SMP neurons are increased. Although the involvement of SP released from sensory nerves was not entirely excluded in this study, the finding that an NK1-dependent AHR is maintained after 24 h in culture, a procedure shown to cause the loss of SP from airway projections of sensory nerves, suggests that sensory nerves are not the only source for SP in these airways. Our observation that O3-enhanced airway responses were diminished in tracheal strips cultured with CP-99994 for 24 h suggests that elevated airway responses to O3 exposure are mediated partly through release of SP from LT neurons and the resulting action of SP either directly on airway smooth muscle or indirectly through modification of SMP neurons.

It should be pointed out that the SP-containing nerve fiber around SMP neurons identified in the present study could have originated from sensory neurons or from LT neurons. Although our studies evaluated airway responsiveness after 24 h in culture, at a time when sensory nerves were probably not present, exposure to O3 occurred in vivo when sensory nerves were still intact. This experimental design does not entirely eliminate the possibility that O3-induced cholinergic hyperresponsiveness may have resulted from a prolonged effect of SP released by sensory nerve fibers that were viable during the early hours of the culture period. However, SP normally undergoes rapid degradation through the action of neutral endopeptidase in the airways. Therefore, it is expected that the only SP effect after 24 h in culture would result from active release by viable neurons. Because sensory fibers should be substantially degraded by 24 h in culture, the only viable neurons would be neurons in airway ganglia. However, additional studies are needed to determine whether O3-induced SP released by sensory nerve terminals early in the culture period may produce long-lasting effects, resulting in cholinergic hyperresponsiveness.

The precise neural pathways mediating the enhanced cholinergic responses after O3 exposure are not clear from the present study. Possible explanations require an evaluation of the intrinsic neural pathways in the ferret airways. Previous anatomic and neurophysiological studies in the airways have identified neural circuits capable of mediating the cholinergic responses after O3-enhanced SP production in LT neurons. Mitchell and Coburn (25) showed that LT neurons activate individual smooth muscle cells in ferret trachea through cholinergic mechanisms. Our laboratory has demonstrated that LT neurons are predominantly cholinergic (5) and that LT neurons project directly to airway smooth muscle (41). These studies suggest that cholinergic pathways from LT neurons to smooth muscle could affect airway smooth muscle directly. One possible way enhanced SP production in LT ganglia could be involved in O3-induced cholinergic hyperresponsiveness is by modulated cholinergic sensitivity in airway smooth muscle. We hypothesize that O3 exposure causes enhanced or de novo SP synthesis in and release from LT neurons. When LT neurons are activated during or subsequent to O3 exposure, ACh and SP are coreleased, with SP producing enhanced smooth muscle sensitivity to ACh. Known modulatory effects of SP include the enhancement of cholinergic sensitivity in airway smooth muscle (2, 19, 33, 34, 36).

A second neural pathway potentially involved in O3-enhanced cholinergic airway smooth muscle responsiveness is the projection from neurons of LT ganglia to neurons of SMP ganglia. Our laboratory showed recently that LT neurons project to and form close, possibly synaptic, connections with VIP- and NO synthase (NOS)-containing cell bodies in SMP ganglia (41). The findings in the present study indicate that O3 exposure increases SP innervation of these putative VIP/NOS neurons in the SMP, suggesting that SP may modulate the production or release of VIP or NO in SMP neurons. Our laboratory previously showed that >90% of the neurons in SMP ganglia contain VIP and/or NOS. The effect of SP on airway neurons has not been investigated in ferrets. In guinea pigs, however, activation of NK1 receptors produces enhanced smooth muscle responsiveness mediated through airway ganglia (36). In another study, capsaicin-induced membrane depolarization of guinea pig airway neurons was inhibited by NK3-receptor antagonists but unaffected by NK1-receptor antagonists (26). Taken together, these studies suggest that SP release may affect actions of VIP or NO, neurotransmitters associated with the inhibitory nonadrenergic noncholinergic (iNANC) innervation of airway smooth muscle. The exact mechanism remains unknown, since activation of iNANC nerves would seemingly reduce airway responsiveness, and further studies are needed to determine the effects of SP on iNANC neurons. A recent study showed that viral exposure causes a reduction of iNANC innervation with associated enhancement of airway smooth muscle responsiveness (3), but a possible role of SP in mediating iNANC responses was not investigated.

The potential role of tachykinins such as SP in mediating inflammation, vascular permeability, and smooth muscle contraction in the airway is well known (23). Although SP is generally considered a sensory neuropeptide in the airway and has been associated with neurogenic inflammation mediated through sensory pathways, our laboratory has demonstrated previously that SP is also synthesized in the airway neurons (6, 8). Stimulation of sensory nerve afferents by inhalation of irritants is known to trigger the release of neuropeptides from these afferent endings (22, 24, 37). Pretreatment of animals with a high doses of capsaicin, which is known to cause degeneration of C fibers and deplete SP and NKA in the lung, reduces the magnitude of O3-induced AHR (18, 19), clearly implicating the involvement of SP in sensory neurons as a mediator of O3-induced AHR. However, much of the evidence implicating SP as a mediator of altered airway responsiveness does not differentiate between sensory and intrinsic airway neurons as the source of SP. Our finding that pretreatment with the NK1-receptor antagonist CP-99994 in vivo partly abolished O3-enhanced cholinergic and EFS-mediated contractility emphasizes an important role for SP in O3-induced AHR.

Ollerenshaw and co-workers (29) demonstrated that SP nerve fiber density was increased in airway smooth muscle of patients with severe asthma, and SP and CGRP nerve fiber density was increased in human airway epithelium from subjects with persistent nonproductive cough (28). We know of no direct evidence that SP is present in intrinsic airway neurons of humans, although the persistence of SP fibers in lungs from transplant recipients suggests an intrinsic origin for some SP-positive nerves (31). Some studies show that neuronal levels of SP and preprotachykinin mRNA are increased in airway sensory neurons after exposure to toluene diisocyanate (16) or antigen (13). Thus it is clear that airway neurons are able to adapt to a variety of conditions associated with enhanced airway responsiveness by increasing SP production and elevating the levels of SP available for release in the airway wall. The present study clearly demonstrates that O3 inhalation alters SP levels and innervation of intrinsic airway neurons as well.

In conclusion, the results obtained from this study show that exposure to O3 induces elevated SP levels in and around airway neurons at the same time that SP-dependent airway smooth muscle responses are increased. Furthermore, pretreatment with the NK1-receptor antagonist CP-99994 in vivo attenuates O3-enhanced airway responsiveness to cholinergic stimulation and EFS, suggesting that SP plays an important role in O3-induced AHR. O3-enhanced airway responses were also diminished in tracheal strips cultured with CP-99994 for 24 h, indicating that SP may be released from intrinsic airway neurons, although additional studies are needed. Thus these findings show that O3 exposure enhances SP levels in intrinsic airway neurons and suggest that neurons in airway ganglia may contribute to O3-induced AHR.


    ACKNOWLEDGEMENTS

The authors are grateful to Dr. G. Hobbs (Dept. of Statistics, West Virginia University) for statistical analysis. The authors also thank Pfizer for the supply of CP-99994.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant RO1 HL-35812.

Address for reprint requests and other correspondence: R. D. Dey, Dept. of Anatomy, PO Box 9128, West Virginia University, Morgantown, WV 26506 (E-mail: rdey{at}hsc.wvu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 November 2000; accepted in final form 21 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Agani, FH, Kuo NT, Chang CH, Dreshaj IA, Farver CF, Krause JE, Ernsberger P, Haxhiu MA, and Martin RJ. Effect of hyperoxia on substance P expression and airway reactivity in the developing lung. Am J Physiol Lung Cell Mol Physiol 273: L40-L45, 1997[Abstract/Free Full Text].

2.   Campos, MG, Segura P, Vargos MH, Vanda B, Ponce-Monter H, Selman M, and Montano LM. O3-induced airway hyperresponsiveness to noncholinergic system and other stimuli. J Appl Physiol 73: 354-361, 1992[Abstract/Free Full Text].

3.   Colasurdo, GN, Hemming VG, Prince GA, Gelfand AS, Loader JE, and Larsen GL. Human respiratory syncytial virus produces prolonged alterations of neural control in airways of developing ferrets. Am J Respir Crit Care Med 157: 1506-1511, 1998[Abstract/Free Full Text].

4.   Daniel, EE, and O'Byrne P. Effect of inflammatory mediators on airway nerves and muscle. Am Rev Respir Dis 143, Suppl: S3-S5, 1991[ISI][Medline].

5.   Dey, RD, and Altemus JB. Distribution of cholinergic and VIP-containing neurons in ferret tracheal plexus (Abstract). Am Rev Respir Dis 143: A362, 1991.

6.   Dey, RD, Altemus JB, Rodd AB, Mayer B, Said SI, and Coburn RF. Neurochemical characterization of intrinsic neurons in ferret tracheal plexus. Am J Respir Cell Mol Biol 14: 207-216, 1996[Abstract].

7.   Dey, RD, Altemus JB, Zervos I, and Hoffpauir J. Origin and colocalization of CGRP- and SP-reactive nerves in cat airway epithelium. J Appl Physiol 68: 770-778, 1990[Abstract/Free Full Text].

8.   Dey, RD, Hoffpauir J, and Said SI. Co-localization of vasoactive intestinal peptide- and substance P-containing nerves in cat bronchi. Neuroscience 24: 275-281, 1988[ISI][Medline].

9.   Dey, RD, Mayer B, and Said SI. Colocalization of vasoactive intestinal peptide and nitric oxide synthase in neurons of the ferret trachea. Neuroscience 54: 839-843, 1993[ISI][Medline].

10.   Dey, RD, Satterfield B, and Altemus JB. Innervation of tracheal epithelium and smooth muscle by neurons in airway ganglia. Anat Rec 254: 166-172, 1999[Medline].

11.   Fedan, JS, Millecchia LL, Johnston RA, Rengasamy A, Hubbs A, Dey RD, Yuan L-X, Watson D, Goldsmith WT, Reynolds JS, Orisini L, Dortch-Carnes J, Cutler D, and Frazer DG. Effect of ozone treatment on airway reactivity and epithelium-derived relaxing factor in guinea pigs. J Pharmacol Exp Ther 293: 724-734, 2000[Abstract/Free Full Text].

12.   Fischer, A, and Hoffmann B. Nitric oxide synthase in neurons and nerve fibers of lower airways and in vagal sensory ganglia of man: correlation with neuropeptides. Am J Respir Crit Care Med 154: 209-216, 1996[Abstract].

13.   Fischer, A, McGregor GP, Saria A, Philippin B, and Kummer W. Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic airway inflammation. J Clin Invest 98: 2284-2291, 1996[ISI][Medline].

14.   Fontan, JJ, Cortright DN, Krause JE, Velloff CR, Karpitskyi VV, Carver TW, Shapiro SD, and Mora BN. Substance P and neurokinin-1 receptor expression by intrinsic airway neurons in the rat. Am J Physiol Lung Cell Mol Physiol 278: L344-L355, 2000[Abstract/Free Full Text].

15.   Hsiue, T-R, Garland A, Ray DW, Hershenson MB, Leff AR, and Solway J. Endogenous sensory neuropeptide release enhances nonspecific airway responsiveness in guinea pigs. Am Rev Respir Dis 146: 148-153, 1992[ISI][Medline].

16.   Hunter, DD, Satterfield BE, Huang J, Fedan JS, and Dey RD. Toluene diisocyanate enhances substance P in sensory neurons innervating the nasal mucosa. Am J Respir Crit Care Med 161: 543-549, 2000[Abstract/Free Full Text].

17.   Hunter, DD, and Undem BJ. Identification and substance P content of vagal afferent neurons innervating the epithelium of the guinea pig trachea. Am J Respir Crit Care Med 159: 1943-1948, 1999[Abstract/Free Full Text].

18.   Joad, JP, Kott KS, and Bric JM. The local C-fiber contribution to ozone-induced effects on the isolated guinea pig lung. Toxicol Appl Pharmacol 141: 561-567, 1996[ISI][Medline].

19.   Koto, H, Aizawa H, Takata S, Inoue H, and Hara N. An important role of tachykinins in ozone-induced airway hyperresponsiveness. Am J Respir Crit Care Med 151: 1763-1769, 1995[Abstract].

20.   Lee, LY, Bleecker ER, and Nadel JA. Effects of ozone on the bronchomotor response to inhaled histamine aerosol in dogs. J Appl Physiol 43: 626-631, 1977[Abstract/Free Full Text].

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

22.   Lundberg, JM, and Saria A. Capsaicin-induced desensitization of airway mucosa to cigarette smoke, mechanical and chemical irritants. Nature 302: 251-253, 1983[Medline].

23.   Maggi, CA, Giachetti A, Dey RD, and Said SI. Neuropeptides as regulators of airway function: vasoactive intestinal peptide and the tachykinins. Physiol Rev 75: 277-322, 1995[Free Full Text].

24.   Martins, MA, Shore SA, and Drazen JM. Release of tachykinins by histamine, methacholine, PAF, LTD4, and substance P from guinea pig lungs. Am J Physiol Lung Cell Mol Physiol 261: L449-L455, 1991[Abstract/Free Full Text].

25.   Mitchell, HW, and Coburn RF. Multiple motor pathways to single smooth muscle cells in the ferret trachea. J Physiol (Lond) 456: 557-574, 1992[Abstract/Free Full Text].

26.   Myers, AC, and Undem BJ. Electrophysiological effects of tachykinins and capsaicin on guinea-pig bronchial parasympathetic ganglion neurones. J Physiol (Lond) 470: 665-679, 1993[Abstract/Free Full Text].

27.   Nadel, JA. Autonomic regulation of airway smooth muscle. In: Physiology and Pharmacology of the Airways, , edited by Nadel JA.. New York: Dekker, 1980, p. 217-257.

28.   O'Connell, F, Springall DR, Moradoghli-Haftvani A, Krausz T, Price D, Fuller RW, Polak JM, and Pride NB. Abnormal intraepithelial airway nerves in persistent unexplained cough? Am J Respir Crit Care Med 152: 2068-2075, 1996[Abstract].

29.   Ollerenshaw, SL, Jarvis D, Sullivan CE, and Woolcock AJ. SP immunoreactive nerves in airways from asthmatics and nonasthmatics. Eur Respir J 4: 673-682, 1991[Abstract].

30.   Sekizawa, K, Tamaoki J, Nadel JA, and Borson DB. Enkephalinase inhibitor potentiates substance P- and electrically induced contraction in ferret trachea. J Appl Physiol 63: 1401-1405, 1987[Abstract/Free Full Text].

31.   Springall, DR, Polak JM, Howard L, Power RF, Krausz T, Manickam S, Banner NR, Khagani A, Rose M, and Yacoub MH. Persistence of intrinsic neurones and possible phenotypic changes after extrinsic denervation of human respiratory tract by heart-lung transplantation. Am Rev Respir Dis 141: 1538-1546, 1990[ISI][Medline].

32.   Stefanini, M, de Martino C, and Zamboni L. Fixation of ejaculated spermatozoa for electron microscopy. Nature 216: 173-174, 1967[Medline].

33.   Tanaka, DT, and Grunstein MM. Maturation of neuromodulatory effect of substance P in rabbit airways. J Clin Invest 85: 345-350, 1990.

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

35.   Watson, JW, Gonsalves SF, Fossa AA, McLean S, Seeger T, Obach S, and Andrews PLR The anti-emetic effects of CP-99,994 in the ferret and the dog: role of the NK1 receptor. Br J Pharmacol 115: 84-94, 1995[ISI][Medline].

36.   Watson, N, Maclagan J, and Barnes PJ. Endogenous tachykinins facilitate transmission through parasympathetic ganglia in guinea-pig trachea. Br J Pharmacol 109: 751-759, 1993[ISI][Medline].

37.   Wu, Z-X, and Lee L-Y. Airway hyperresponsiveness induced by chronic exposure to cigarette smoke in guinea pigs: role of tachykinins. J Appl Physiol 87: 1621-1628, 1999[Abstract/Free Full Text].

38.   Wu, Z-X, Morton RF, and Lee LY. Role of tachykinins in ozone-induced airway hyperresponsiveness to cigarette smoke in guinea pigs. J Appl Physiol 83: 958-965, 1997[Abstract/Free Full Text].

39.   Yost, BL, Gleich GJ, and Fryer AD. Ozone-induced hyperresponsiveness and blockade of M2-muscarinic receptors by eosinophil major basic protein. J Appl Physiol 87: 1272-1278, 1999[Abstract/Free Full Text].

40.   Zaman, S, Woods AJ, Watson JW, Reynolds DJM, and Andrews PLR The effect of the NK1 receptor antagonist CP-99,994 on emesis and c-Fos protein induction by loperamide in the ferret. Neuropharmacology 39: 316-323, 2000[ISI][Medline].

41.   Zhu, W, and Dey RD. Projections and pathways of VIP- and NOS-containing airway neurons in ferret trachea. Am J Respir Cell Mol Biol. 24: 38-43, 2001[Abstract/Free Full Text].

J APPL PHYSIOL 91(1):371-378
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
Z.-X. Wu and R. D. Dey
Nerve growth factor-enhanced airway responsiveness involves substance P in ferret intrinsic airway neurons
Am J Physiol Lung Cell Mol Physiol, July 1, 2006; 291(1): L111 - L118.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
H.-Y. Huang and Y.-L. Lai
Lipopolysaccharide Induces Preprotachykinin Gene Expression
Am. J. Respir. Cell Mol. Biol., November 1, 2003; 29(5): 606 - 612.
[Abstract] [Full Text]

Home page
 J. Appl. Physiol.Home page
Z.-X. Wu, B. E. Satterfield, and R. D. Dey
Substance P released from intrinsic airway neurons contributes to ozone-enhanced airway hyperresponsiveness in ferret trachea
J Appl Physiol, August 1, 2003; 95(2): 742 - 750.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
Z.-X. Wu, B. E. Satterfield, J. S. Fedan, and R. D. Dey
Interleukin-1beta -induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway
Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L909 - L917.
[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 ISI Web of Science
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 ISI Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wu, Z.-X.
Right arrow Articles by Dey, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, Z.-X.
Right arrow Articles by Dey, R. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online