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Department of Neurobiology and Anatomy, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia
Submitted 29 August 2005 ; accepted in final form 30 January 2006
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ABSTRACT |
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airway smooth muscle contraction; muscarinic agonists; neurokinin receptor; airway innervation
SP acts as a neuromodulator increasing the cholinergic sensitivity of airway smooth muscle (8) and increasing the excitability of airway neurons (32), suggesting that SP release may mediate development of AHR. SP also has a direct effector action on smooth muscle contractility and vascular permeability in some species (3, 28). SP is localized in the peripheral endings of nerves innervating the lung and airways and originates in nerve cell bodies located in sensory ganglia (13, 26). Recent studies found that SP-containing nerve fibers innervating the lung and airways also originate in nerve cell bodies located in intrinsic airway ganglia (12, 15). The intrinsic airway ganglia are organized to form two layers in some species: the longitudinal trunk (LT) ganglia located in a pair of nerve trunks that extend nearly the entire length of the trachea and a second layer of ganglia closely associated with the tracheal muscle and arranged as a diffuse network named the superficial muscular plexus (SMP) (1, 19). Our previous studies (30, 46, 48, 49) showed that the nerve plexus on the dorsal surface of the ferret trachea is an ideal model system in which to study intrinsic airway neurons. In ferrets, most nerve cell bodies in the LT ganglia are cholinergic and do not normally contain many neurons expressing nitric oxide synthase, SP, or vasoactive intestinal peptide (VIP) (12, 14, 15). Cell bodies in the superficial muscular plexus contain predominantly VIP and nitric oxide (NO), with a small population containing SP (12, 15).
Our recent studies (45, 48) reported that exposure to ozone increases production and release of SP from intrinsic neurons of ferret airway. Because exposure to irritant or allergen challenge elevates NGF level in the lavage fluid (40, 43) and NGF enhances production of SP in sensory ganglia (16, 25), we hypothesize that SP production in airway may be regulated by NGF. Therefore, the purpose of this study was to evaluate the effect of NGF on AHR and determine whether these effects are mediated through enhanced synthesis and release of SP from the intrinsic airway ganglia.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In Vivo NGF Treatment
Ferrets were anesthetized with ketamine (25 mg/kg) and xylazine (2 mg/kg) in a single intraperitoneal injection. An 18-gauge steel tube 15 cm in length was marked to indicate when the tip reached the carina, connected to a 1-ml tuberculin syringe filled with NGF (200 µg/ml, Santa Cruz Biotechnology) or saline, and inserted through the oral cavity and pharynx into the trachea. NGF or saline (0.4 ml) was instilled into the trachea and deposited at four equal intervals along the trachea from immediately superior to the carina to immediately inferior to the larynx. Twenty-four hours after NGF or saline treatment, ferrets were killed and tracheas were removed and cut into several segments to measure smooth muscle contractions and for immunocytochemistry. The 24 -h time point was selected based on our time course of toluene diisocyanate (TDI)-induced NGF release in rat nasal mucosa (43, 44) and the study by Hunter et al. (25) showing that TDI induced increased SP synthesis in guinea pig airways neurons at 24 h.
In Vitro Tracheal Segments Cultured 24 h
Organotypic cultures of tracheas from normal ferrets were used, following a modification of our previously described technique (15, 45, 46). Under sterile conditions, tracheas were removed and washed with cold culture medium. The tissue was then placed in a petri dish with culture medium and cut into 10-mm-long segments beginning at the carina. After a second wash, the segments were placed directly on the bottom of petri dishes containing fresh culture medium containing NGF (final concentration 1 µg/ml) or saline. In some experiments, CP-99994 (3 x 10–6 M) was added to the culture medium 30 min before addition of NGF or saline and maintained throughout the experiment to determine the role of SP in intrinsic airway neurons. This approach was done to avoid possible long-term neuromodulatory effects of enhanced SP synthesis and release that might occur during the 24-h NGF treatment period. The prolonged treatment also facilitates penetration of the antagonist to nerve terminals that might be deeper in the smooth muscle layer. According to manufacturer's specifications, the liquid neurokinin antagonist is stable for up to 30 days in 37°C. The antagonist concentration was based on our previous studies (46). The petri dishes were then placed in a controlled-atmosphere culture chamber and gassed with a mixture of 95% O2 and 5% CO2. 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 medium consisted of 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% heat-inactivated fetal calf serum.
Measurement of Tracheal Smooth Muscle Contraction
Tracheal smooth muscle reactivity was evaluated by measuring contractile responses to methacholine (MCh) or electrical field stimulation (EFS). MCh responses measure smooth muscle responses to the applied agonist, whereas EFS evaluates cholinergic responses resulting from the release of ACh from airway nerves. Tracheal segments from in vivo NGF or saline treatment and from segments cultured for 24 h with NGF or saline 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 of (mM) 113 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgSO4, 24 NaHCO3, 1.2 KH2PO4, and 5.7 glucose, pH 7.4. The detailed procedures were described previously (46, 48). Briefly, the strips were tied at each end with 4-0 silk, positioned between the rings of platinum electrodes attached to tissue holders, and equilibrated for 60 min at a resting tension of 1.0 g. After equilibration, cumulative concentration-response curves for MCh were constructed by adding a series of concentrations of 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 the MCh solution was completely washed out and smooth muscle tension returned to baseline, EFS experiments were carried out. Frequency-response curves were constructed by increasing the frequency from 1 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 was allowed for the previous response to return to baseline. EFS-induced contractions were normalized as a percentage of the response to 10–3 M MCh (% MCh response).
Morphometric Analysis
The goal of the morphometric analysis was to evaluate SP levels in nerve cell bodies of neurons in airway ganglia and to measure changes in nerve fiber density (NFD) in tracheal smooth muscle. This approach involves 1) immunocytochemical processing of tracheal tissues, followed by separate morphometric approaches to measure 2) SP immunofluorescence intensity of nerve cell bodies in airway ganglia and 3) NFD in tracheal smooth muscle.
Immunocytochemistry. The procedures for immunocytochemical demonstration of SP-like immunoreactivity were described previously (15, 46, 48). Briefly, tracheal segments from in vivo NGF or saline treatment and segments from culture for 24 h with NGF or saline were fixed in picric acid-formaldehyde fixative for 3 h and rinsed three times with a 0.1 M phosphate-buffered saline solution containing 0.3% Triton X-100 (PBS-TX), frozen in isopentane, cooled with liquid nitrogen, and stored at –80°C. Cryostat sections (12-µm thickness) were collected on gelatin-coated coverslips and dried briefly at room temperature. Cryostat sections were then covered with SP antibody diluted 1:200, incubated in a humid chamber at 37°C for 30 min, and rinsed with a 1% bovine serum albumin-PBS-TX solution three times. 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. After all immunocytochemical procedures were conducted, the coverslips were mounted with fluoromount and observed with a fluorescence microscope.
Fluorescence intensity of SP-containing nerve cell bodies.
Fluorescence intensity of SP-like immunofluorescence was measured in LT and SMP ganglion cell bodies. Images were digitally recorded with an AX 70 microscope (Olympus America, Melville, NY) with SPOT 2 (Diagnostics Instruments, Sterling Heights, MI), and fluorescence intensity was measured with commercial image processing software (Optimas 6.5, Media Cybernetics, Silver Spring, MD). The intensity recordings were calibrated with the InSpeck Green (505/515) microscope image intensity calibration kit (Molecular Probes, Eugene, OR). The cell bodies were identified by drawing the perimeter of the cell, and the fluorescence intensity was reported as gray level on a scale of 256 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. All identifiable neurons in LT and SMP ganglia were evaluated in every fifth section collected from serial sections, usually amounting to a total of 10–15 sections analyzed per ferret. The data are expressed separately for the LT and SMP ganglia as the percentage of SP-positive neurons compared with total number of neurons observed.
NFD in tracheal smooth muscle. For measuring NFD in tracheal smooth muscle, images of SP-containing nerve fibers were collected in series with the Zeiss LSM 510 confocal microscope. A series of images representing all of the tracheal smooth muscle in a section were collected in digital files, saved to an internal database, and measured with Optimas software. The smooth muscle regions were outlined to measure total cross-sectional area of smooth muscle. SP-positive nerve fibers were identified by segmentation using threshold gray levels with the Optimas software. NFD was then calculated as percentage of SP-immunoreactive nerve fiber area based on the total cross-sectional area of smooth muscle. At least 10 measurements were made for each section, and 15 sections were measured in each animal.
Data Analysis
Unless otherwise stated, results are expressed as means ± SE. Contractions elicited by EFS were expressed as a percentage of the maximal contraction elicited by MCh. Contractions to MCh were normalized as a percentage of the respective maximal responses. The half-maximum concentration (EC50) values for MCh were calculated with a four-parameter logistic curve fit (Sigmoidal, SigmaPlot 2000) and are presented with the 95% confidence interval in parentheses. Force development was expressed by normalizing force (g) divided by the wet weight of the tissue. LT and SMP neurons were expressed as % SP-positive cell bodies. Nerve fiber density was expressed as % area of SP-immunoreactive nerve fibers in the total area of the smooth muscle. Statistical analyses of immunocytochemistry and EC50 were performed with Student's t-test. Statistical analysis of EFS was performed with two-way repeated-measures ANOVA. One factor between the groups was NGF treatment, and another 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). A P value <0.05 was considered significant, and n represents the number of animals studied.
Materials
MCh chloride, atropine sulfate, hydrocortisone hemisuccinate, amphotericin B, and recrystalized bovine insulin were obtained from Sigma (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). Fluorescein isothiocyanate-labeled goat anti-rabbit antibody was obtained from ICN Immunobiologicals (Costa Mesa, CA).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The initial experiments examined the effect of in vivo NGF treatment on tracheal smooth muscle contractility. Cumulative concentration-response curves for MCh were not significantly shifted (Fig. 1A), and the EC50 value for the control group was not significantly different from that for the NGF treatment group (Table 1). However, NGF increased contractile responses to EFS. A leftward shift in the frequency-response curve was observed in NGF-treated animals (Fig. 1B), and contractions produced by EFS at 10 and 30 Hz were significantly increased in the NGF treatment group (10 Hz: 33.57 ± 2.44%; 30 Hz: 40.12 ± 2.78%) compared with the control group (10 Hz: 27.24 ± 2.14%; 30 Hz: 33.33 ± 2.31%). The contractions to MCh and EFS were totally abolished after treatment with 10–6 M atropine in both control and NGF treatment groups (Fig. 1).
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To examine the contribution of intrinsic airway neurons on NGF-enhanced airway responsiveness, tracheal segments were maintained in organotypic culture and treated with NGF or saline for 24 h. Our previous studies (46) showed that culture for 24 h causes a significant anatomic and functional loss of SP-containing sensory fibers while maintaining viability of intrinsic airway neurons. After culture, tracheal smooth muscle contractility to MCh and EFS was assessed. Cumulative concentration-response curves for MCh were not significantly shifted in segments cultured with NGF compared with saline controls (Fig. 2A, Table 2). However, contractions produced by EFS at 10 and 30 Hz were increased significantly in tracheal segments cultured with NGF (10 Hz: 32.54 ± 1.98%; 30 Hz: 39.66 ± 2.52%) compared with the control group (10 Hz: 25.32 ± 1.78%; 30 Hz: 31.25 ± 2.16%) (Fig. 2B). The contractions to MCh and EFS in cultured tracheal segments were also totally abolished by treatment with 10–6 M atropine in both control and NGF treatment groups (Fig. 2).
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First, the effects of in vitro NGF on immunoreactive SP of intrinsic airway were examined. SP nerve fibers and SP-containing cell bodies were present within the neural plexuses of trachea in tracheal segments cultured with saline (control) and NGF (Fig. 3). About 32% of the LT cell bodies (Fig. 3A and Fig. 4A) and about 38% of the SMP neurons (Fig. 3C and Fig. 4B) labeled SP positive in control segments. In tracheal segments cultured with NGF, about 58% of the cell bodies in the LT (Fig. 3B and Fig. 4A) and nearly 69% of the cell bodies in the SMP (Fig. 3D and Fig. 4B) neurons contained SP. Also, SP NFD was significantly increased from 0.23% in control tracheal segments (Fig. 3E and Fig. 4C) to 0.43% in segments cultured with NGF (Fig. 3F and Fig. 4C).
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A separate set of experiments examined the involvement of SP in NGF-enhanced airway responsiveness by blocking the neurokinin 1 (NK1) receptor. The EFS-stimulated contractions at 10 and 30 Hz (Fig. 6A) demonstrated expected changes in cultured tracheal segments cultured 24 h with NGF. The contractions produced by EFS at 10 and 30 Hz were significantly increased in tracheal segments cultured with NGF after treatment with saline (Fig. 6A). However, pretreatment with the NK1 antagonist CP-99994 attenuated the NGF-enhanced contractile responses to EFS (Fig. 6B). There was no significant difference in responses to the same frequency of EFS between segments cultured with saline and NGF after treatment with CP-99994.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Previous studies have found that circulating NGF levels are elevated in asthmatic patients (4, 33), suggesting a possible link between NGF expression and asthma. NGF has also been implicated in the development of AHR in animal models (2, 21). SP is generally considered a sensory neuropeptide in the airways and has been associated with AHR. We demonstrated previously (12, 24) that SP is synthesized in sensory neurons and intrinsic airway neurons. Irritant exposure increases SP levels in nerve cell bodies and fibers of intrinsic airway nerves innervating tracheal smooth muscle (45, 48), but the exact mechanism of irritant-enhanced SP expression in neurons of intrinsic airway ganglia is not clear. Inhalation of irritants also enhances levels of NGF in the airway (44). Recent investigations in our laboratory (43) showed that treating the nasal cavity with K252a, a nonspecific tyrosine kinase inhibitor previously shown to inhibit or interfere with NGF activity, reduced the irritant-induced increases in SP-positive cell bodies in the sensory neurons, indicating that NGF mediates regulation of SP synthesis in airway sensory neurons during irritant exposure. NGF produces upregulation of SP expression in airway A
sensory neurons (25). The effect of NGF on intrinsic airway ganglia has not been studied as extensively as that on sensory neurons, although our recent studies (48) showed that ozone inhalation enhances SP content in ferret airway neurons that normally do not express this neuropeptide, confirming that airway neurons are capable of exhibiting plasticity to irritants. Although endogenous NGF may contribute to the maintenance of basal levels of SP production in sensory neurons, endogenous NGF may not be required to support SP production in intrinsic airway neurons. In preliminary data (47), we showed that antibody inhibition of NGF did not alter smooth muscle EFS responses in control, cultured airways, although responses after ozone exposure were attenuated. These findings suggest that endogenous NGF is not needed to support SP production of intrinsic airway neuron but ozone-induced NGF release mediates the increase in SP production in intrinsic airway neurons. Thus we hypothesized that enhanced NGF in the airways may upregulate SP expression in intrinsic airway neurons. The immunocytochemical data in the present study demonstrate that SP expression in LT and SMP neurons was increased in tracheas cultured with NGF, directly indicating that NGF enhances SP expression in intrinsic airway ganglia.
The present study also showed that NGF enhances tracheal airway smooth muscle responsiveness to EFS. The contractile responses to EFS at 10 and 30 Hz were 25% and 31%, respectively, in control animals; these values represent the percentage of contraction compared with maximal contraction from pharmacological doses of MCh. After NGF treatment, contractions produced by EFS increased to 32% and 40% (Fig. 2B), respectively. Thus the changes of contractions from 25% to 32% and from 31% to 40% were 28% and 29% increases, respectively, after NGF treatment. These changes are almost identical to the changes observed with ozone exposure in our previous studies (45, 48). Although it is difficult to translate EFS contraction in a smooth muscle strip to a physiological parameter such as airway resistance, it might be argued that a 28% enhancement in airway contraction occurring after an irritant exposure or NGF release could result in a 28% reduction in airway caliber and substantial increase in airway resistance.
One possible mechanism that may explain our finding that NGF enhances tracheal smooth muscle responsiveness to EFS is that NGF increases SP levels in airway neurons. Our data showing that the SP receptor antagonist CP-99994 significantly attenuated the effect of NGF on EFS responses suggest the involvement of SP release as the mediator of NGF action on airway contractile responses. However, although SP is a known bronchoconstrictor in some species (3, 29), direct action of SP on smooth muscle does not appear to be an important effect in this study because our findings that administration of atropine completely blocks airway smooth muscle responsiveness to EFS even after NGF treatment demonstrate that smooth muscle contraction in ferret trachea is entirely atropine sensitive. Thus the logical explanation of the NGF effect is that SP alters cholinergic responsiveness. Previous studies have shown that SP enhances cholinergic responsiveness either through a direct effect on sensitivity of airway smooth muscle (39) or by enhancing ACh release from parasympathetic nerve terminals (32, 37, 42). In ferrets, EFS is known to produce airway smooth muscle contraction only through ACh release from parasympathetic nerve terminals (46, 48). Our present findings show that NGF enhances only tracheal smooth muscle responsiveness to EFS and does not increase airway contractions to cholinergic agonists, suggesting that NGF-enhanced SP alters ACh release from parasympathetic nerve terminals. Although the exact mechanism of enhanced ACh release from parasympathetic terminals by NGF was not determined in the present study, the finding that the SP receptor antagonist CP-99994 significantly attenuated the effect of NGF on EFS responses in cultured trachea supports the idea that NGF induces SP upregulation that then modulates ACh release through NK1-dependent binding. There are no reports showing that neurokinin A enhances EFS responsiveness by increasing ACh release from parasympathetic nerve terminals. Our preliminary data also showed that the NK2 receptor antagonist SR-48968 did not affect the enhanced contraction to EFS in ferrets after irritant exposure. Thus we did not continue to investigate what appeared to be negative findings of NK2 receptor involvement in the present study.
The increased SP in intrinsic airway neurons is certainly not the only possible mechanism responsible for NGF-induced AHR. Recent studies have demonstrated that NGF upregulates NK1 receptor expression in the lungs and anti-NGF antibody inhibits NK1 receptor upregulation in respiratory syncytial virus-infected lungs (23), indicating that enhanced NK1 receptor expression in the airway smooth muscle may cause AHR. There is also another possibility that NGF directly affects airway smooth muscle. A recent study by Freund-Michel et al. (20) reported that NGF directly induces proliferation of human airway smooth muscle cells through activation of Trk A receptor. The hypertrophy of smooth muscle cell may contribute to AHR.
Another possible mechanism of NGF-enhanced tracheal airway smooth muscle responsiveness to EFS may involve complex communications of the intrinsic airway neurons. The neurons of airway ganglia not only represent the cholinergic system, they also represent the inhibitory nonadrenergic, noncholinergic (iNANC) system. Both NO and VIP, potent relaxant mediators, are putative transmitters of the iNANC system (17, 31). In control ferrets, LT neurons contain predominantly ACh and cell bodies in the SMP contain predominantly VIP and NO, with a small population containing SP (13, 49). LT neurons project to and appear to synapse with SMP neurons (49). In normal conditions, most of the LT neurons produce ACh, and it would be assumed that the projections to the SMP ganglia release ACh, potentially activating the iNANC neurons to release NO and VIP. This arrangement may serve to balance the constrictor actions of ACh at airway ganglia (36, 41). A previous study found that sensory neuropeptide depletion augments iNANC VIP responses in the airway (38). After NGF treatment, levels of SP production in LT and SMP neurons are increased. Thus it is possible that the increased SP production downregulates the iNANC responses, attenuating the VIP- and NO-mediated cholinergic responses. Such an effect would lead to an increase in tracheal smooth muscle contraction to EFS and result in AHR. A few studies have associated the attenuation of VIP with asthma or increased smooth muscle responsiveness (10, 18). In humans, the levels of VIP are reduced and the levels of SP are increased in individuals with severe asthma (7, 35).
In conclusion, our results show that NGF treatment increases SP levels in and around airway neurons and tracheal smooth muscle. At the same time, airway smooth muscle responses to EFS are increased. This effect is maintained in tracheal segments cultured for 24 h. Administration of CP-99994, an antagonist of the NK1 receptor, attenuates the NGF-induced airway responses in cultured tracheal segments. The findings indicate that NGF induces AHR by enhancing SP expression in airway neurons.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-induced hyperresponsiveness to [Sar9,Met(O2)11]substance P in isolated human bronchi. Eur J Pharmacol 379: 87–95, 1999.[CrossRef][Web of Science][Medline] mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle. J Clin Invest 104: 657–667, 1999.[Web of Science][Medline]-induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway. Am J Physiol Lung Cell Mol Physiol 283: L909–L917, 2002.This article has been cited by other articles:
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  M. S. Hazari, J. H. Pan, and A. C. Myers Nerve growth factor acutely potentiates synaptic transmission in vitro and induces dendritic growth in vivo on adult neurons in airway parasympathetic ganglia Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L992 - L1001. [Abstract] [Full Text] [PDF]
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) or nerve growth factor (NGF, 
) treatment. Responses to MCh are plotted as % of the maximum response. Responses to EFS are plotted as % of the maximum response to MCh. 
, Airway responses to same challenge after administration of 10–6 M atropine (data of control and NGF treatment are totally overlapped). Values are means ± SE; n = 6. *Significant difference between saline and NGF treatment, P 
0.05.
