Influence of maturation on constrictive response to stimulation of C-fiber afferents in isolated guinea pig airways

Z.-X. Wu,* Q. H. Yang,* T. Ruan, and L.-Y. Lee

Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky 40536

Submitted 19 May 2003 ; accepted in final form 10 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated whether the airway constrictive response to stimulation of bronchopulmonary C-fiber afferents is altered during the maturation process. Isometric tension was measured in airway rings isolated from three tracheobronchial locations (intrathoracic trachea and main and hilar bronchi) and compared in mature [M, 407 ± 10 (SE) g body wt, n = 36] and immature (IM, 161 ± 5 g body wt, n = 35) guinea pigs. Our results showed no difference in the ACh (10–5 M)- or KCl (40 mM)-induced contraction between M and IM groups, regardless of the airway location. In sharp contrast, the concentration-response curves of 10–8–10–6 M capsaicin were distinctly lower in IM hilar bronchi; for example, response to the same concentration of capsaicin (10–6 M) was 89.2 ± 15.3% of the response to 10–5 M ACh in IM and 284.7 ± 43.2% in M animals. Similar, but smaller, differences in the bronchoconstrictive response to capsaicin between IM and M groups were also observed in the trachea and main bronchus. Electrical field stimulation induced airway constriction in all three locations in M and IM groups. However, after administration of 10–6 M atropine and 10–6 M propranolol, electrical field stimulation-induced contraction was significantly smaller in the hilar bronchus of IM than M animals, and this difference was not prevented by pretreatment with 5 x 10–5 M indomethacin. Although radioimmunoassay showed no difference in the tissue content of substance P between M and IM airways, the constrictive responses to exogenous substance P and neurokinin A were markedly greater in M airways at all three locations. In conclusion, the constriction of isolated airways evoked by C-fiber stimulation was significantly weaker in the IM guinea pigs, probably because of a less potent effect of tachykinins on the airway smooth muscle.

airway smooth muscle; bronchoconstriction; capsaicin; tachykinins; airway reflex


THE IMPORTANT ROLE OF BRONCHOPULMONARY unmyelinated (C-fiber) afferents in regulation of airway functions has been extensively documented in various species, including humans (8, 24). Activation of these sensory nerve endings by inhaled irritants or endogenous chemical mediators can elicit reflex bronchoconstriction via a centrally mediated cholinergic mechanism (2, 8, 24). In addition, stimulation of C-fiber afferents by chemical stimulants, such as capsaicin, can also trigger the local release of tachykinins from the sensory terminals. The potent biological effects of tachykinins on a number of effector cells in the airways have been extensively documented (3, 28, 38). Among these effects, an excitatory effect of tachykinins on the airway smooth muscle is well recognized and seems to be particularly potent in rodents, such as the guinea pig (7, 24).

It has been reported that the pulmonary chemoreflexes elicited by stimulation of C-fiber afferents in the lungs are not fully developed in the early postnatal stage of life in cats and rabbits (20, 42). These observations led to speculation that maturation of the C-fiber sensory endings occurs later in postnatal development (42). In contrast, pulmonary chemoreflexes were clearly demonstrated shortly after birth in pigs and dogs (2). Although reflex bronchoconstriction elicited by stimulation of bronchopulmonary C-fibers has been reported in newborn animals (2, 33), the constrictive responses of isolated airways to C-fiber stimulation have not been quantitatively compared between mature (M) and immature (IM) animals. Furthermore, the relative contributions of cholinergic and tachykininergic mechanisms to the neural regulation of bronchomotor tone vary substantially in the longitudinal direction within the tracheobronchial tree; the cholinergic mechanism dominates in the trachea, whereas tachykinin-mediated bronchoconstriction is more pronounced in the more distal airways, such as hilar bronchi (13, 26). Whether this longitudinal variation in the neurotransmitters and regulatory mechanisms of airway smooth muscle tone holds true in IM animals is not known.

In light of the existing knowledge and unanswered questions described above, this study was carried out to determine whether there is a difference in the airway constrictive responses to stimulation of C-fiber afferents between M and IM animals and, if there is a difference, to determine the mechanism(s) that probably contributes to this difference. Furthermore, we investigated whether this difference in the bronchomotor response to C-fiber stimulation between M and IM animals varies at different locations along the tracheobronchial tree. To answer these questions, we used an isolated airway preparation from guinea pigs.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The procedures were performed in accordance with the recommendations for the care and use of laboratory animals of the National Institutes of Health and were approved by the University of Kentucky Institutional Animal Care and Use Committee.

This study was carried out in IM [161 ± 5 (SE) g body wt, 8–13 days old, n = 35] and M (407 ± 10 g body wt, >8 wk old, n = 36) male Hartley guinea pigs. We chose these ages for the two groups because the average weaning age of guinea pigs is 3 wk and the sexual maturity age is 4–6 wk (37).

Isolated airway preparations. Guinea pigs were killed by a blow to the head. The lungs, along with the trachea, were rapidly removed after a thoracotomy and immersed into cold oxygenated Krebs solution (in mM: 118 NaCl, 2.5 CaCl2, 1.2 MgSO4, 24.9 NaHCO3, 1.2 KH2PO4, 4.7 KCl, 5.6 glucose, and 12.6 HEPES) (27). Airway rings (3 mm long) were dissected from the following locations of the tracheobronchial tree: intrathoracic trachea, left main bronchus, and hilar bronchi of left and right lungs (Fig. 1); data obtained from the two hilar bronchi were averaged for each animal. Each airway ring was mounted on a Plexiglas holder, which was then immersed in a 5-ml glass tissue bath (model 158306, Radonti) filled with Krebs solution that was maintained at 37°C and aerated continuously with 95% O2-5% CO2. We measured the isometric contractile force generated by the airway ring by a force-displacement transducer (model FT03C, Grass) and recorded the response continuously on a polygraph (model 79D, Grass). An initial resting tension of 0.5 g was applied and maintained by continuous readjustment during an equilibration period (90 min) before any measurement and also after pharmacological treatments (e.g., after indomethacin) throughout the experiment. This resting tension was determined in our preliminary studies; it produced the maximal and most reproducible airway constrictive response to C-fiber stimulation by capsaicin.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1. A: force generated by isometric contraction of isolated airway rings in response to 10–5 M ACh in mature (M; solid bars, n = 36) and immature (IM; open bars, n = 35) guinea pigs. B: responses to 40 mM KCl in M (n = 7) and IM (n = 7) guinea pigs. Values are means ± SE. {dagger}Significantly different from corresponding data for trachea. Inset: schematic drawing of tracheobronchial tree of the guinea pig. Shaded areas show locations where airway rings were dissected: trachea (a), main bronchus (b), and hilar bronchus (c).

 
The constrictive response of the airway ring to 10–5 M acetylcholine (ACh) was determined first in each preparation to establish an internal standard contraction; data obtained in preliminary experiments showed that this concentration of ACh produced a reproducible and near-maximal response to ACh in airways of all three locations. To determine whether the smooth muscle content was different between M and IM airways, the constrictive response of the airway ring to 40 mM KCl was also determined.

Cumulative concentration-response curves to capsaicin and tachykinins. Cumulative concentration-response curves of the airway rings to 10–8–10–6 M capsaicin were obtained by successive increases () of the concentration of capsaicin solution every 2–10 min or until after the response approached a plateau. Capsaicin-induced contractions were then expressed as a percentage of the contraction induced by 10–5 M ACh. To determine whether the constrictive responses to capsaicin were generated by the endogenously released tachykinins, the highest concentration of capsaicin (10–6 M) was applied 60 min after the isolated airways were pretreated with a combination of 3 x 10–7 M CP-99994, a selective antagonist of the neurokinin (NK) type 1 (NK1) receptor (31), and 10–7 M SR-48968, a selective antagonist of the NK2 receptor (11), in M (n = 3) and IM guinea pigs (n = 4).

In separate groups of M and IM animals, cumulative concentration-response curves to 10–9–10–6 M substance P (SP) and 10–10–10–7 M NKA were obtained in a manner similar to that described above and also expressed as a percentage of the contraction induced by 10–5 M ACh.

Electrical field stimulation. Electrical field stimulation (EFS) was applied to determine the contractile responses of the airway rings to endogenous release of neurotransmitters from airway nerves. Each airway ring was placed between a pair of 20-mm-long platinum electrodes that were built in the tissue holder, positioned in parallel, and separated by a distance of 6 mm. EFS was produced by a stimulator (model SD4, Grass) that delivered 1-ms 40-V square-wave pulses for 10 s. The responses to different intensities of EFS were determined by successive increases in the stimulation frequency (5, 10, 15, and 20 Hz); >=20 min were allowed between two consecutive stimulations. The nonadrenergic noncholinergic (NANC) component of the EFS-induced contraction was then determined by repeating the same protocols after pretreatments with 10–6 M atropine and 10–6 M propranolol. To determine whether endogenous cyclooxygenase metabolites are involved in the NANC responses, the same protocols were repeated after pretreatments with 10–6 M atropine, 10–6 M propranolol, and 5 x 10–5 M indomethacin in the same airway rings.

Radioimmunoassay of SP-like immunoreactivity in tracheobronchial tissues. Procedures for radioimmunoassay (RIA) of SP-like immunoreactivity (SP-LI) in airway tissue have been previously described in detail (23). Briefly, the intrathoracic trachea and bronchi were rapidly removed from each animal as described above. The specimen was weighed and boiled (95°C) for 10 min in distilled water, and the homogenate was transferred to polypropylene tubes and centrifuged at 40,000 g at 4°C for 20 min. Supernatant fractions were removed and partially purified with C18 Sep-Pak columns. After the columns were washed four times with 5 ml of 0.1% trifluoroacetic acid, SP was eluted from columns with 60% acetonitrile in 0.1% trifluoroacetic acid. The samples were lyophilized and reconstituted in RIA buffer containing 0.1 M phosphate buffer (pH 7.4), 0.1% NaCl, and 0.1% Triton X-100. For the measurement of SP, 200 µl of sample were incubated at 4°C for 24 h with 100 µl of SP antibody (Peninsula Laboratories). Standard curves were established with synthetic SP. 125I-SP (100 µl; Amersham) was added to each tube for incubation for 24 h at 4°C; then 100 µl of goat anti-rabbit IgG (Peninsula Laboratories) and 100 µl of normal rabbit serum were added for 2 h at room temperature. Finally, 0.5 ml of RIA buffer was added, the sample was centrifuged at 1,700 g at 4°C for 20 min, and the gamma radioactivity in the remaining pellet was counted.

Experimental protocols. Four series of experiments were performed to answer the following questions: 1) Is there a difference in the constrictive responses of the airway rings to capsaicin between M and IM guinea pigs? If so, is the difference in the response to capsaicin between M and IM animals dependent on the location in the tracheobronchial tree? Furthermore, what is the role of the endogenously released tachykinins in this difference in the constrictive responses? 2) Is there a difference in the EFS-induced NANC airway responses between M and IM guinea pigs at different airway locations? 3) Are the constrictive responses of airway rings to exogenously applied tachykinins (SP and NKA) in IM guinea pigs different from those in M animals? 4) Is there a difference in the tissue content of SP in the airways between M and IM animals?

Statistical analysis. Concentration responses to capsaicin, SP, and NKA were analyzed by a three-way analysis of variance (ANOVA; age x airway location x dose). A two-way ANOVA was applied to analyze the data obtained in the studies of responses to ACh and KCl (age x airway location) and to EFS (age x indomethacin treatment). When the ANOVA showed a significant interaction, pairwise comparisons were made with a post hoc analysis (Fisher's least significant difference). RIA data were analyzed by a one-way ANOVA. Values are means ± SE. P < 0.05 was considered significant.

Materials. Capsaicin (10–3 M; Sigma) was prepared in a vehicle of 10% Tween 80, 10% ethanol, and 80% isotonic saline. SP (10–3 M; Peninsula Laboratories) and NKA (10–3 M; Peninsula Laboratories) were prepared in ethanol. CP-99994 (10–3 M; kindly provided by Pfizer, Groton, CT) and SR-48968 (10–3 M; kindly provided by Sanofi Recherche, Montpellier Cedex, France) were dissolved in polyethylene glycol (average mol wt 200; Sigma). Indomethacin (5 x 10–3 M; Sigma) was prepared in 4% sodium bicarbonate solution. All these concentrated solutions were kept in small (50- to 200-µl) aliquots at –20°C and then diluted in Krebs solution to the desired concentrations just before each experiment.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Concentration-response curves of isolated airways to capsaicin. ACh (10–5 M) induced a stronger contraction in tracheae and main bronchi than in hilar bronchi of M (n = 36) and IM (n = 35) guinea pigs (Fig. 1). However, there was no difference in the ACh-induced contraction between M and IM guinea pigs, regardless of the airway location (Fig. 1A). Similarly, 40 mM KCl also induced an intense, but reversible, contraction in the airway rings, and there was no difference in the responses between M (n = 7) and IM (n = 7) guinea pigs at any of the three airway locations (Fig. 1B).

Cumulative concentration-response curves of isolated airways to 10–8–10–6 M capsaicin are shown in Fig. 2; the capsaicin-induced airway contraction was concentration dependent in all three tracheobronchial locations in M (n = 15, P < 0.05) and IM (n = 14, P < 0.05) guinea pigs. Clearly, the airway constriction induced by a given concentration of capsaicin was markedly stronger in the M group. This difference is particularly pronounced in hilar bronchi; e.g., the response to 10–6 M capsaicin was 78.0 ± 13.4% of the response to 10–5 M ACh in IM animals and 231.6 ± 29.9% (P < 0.001) in M animals. Similar, but smaller, differences between IM and M animals were also observed in main bronchi (59.3 ± 9.8% and 112.2 ± 14.7% in IM and M, respectively, P < 0.01) and tracheae (33.0 ± 5.8% and 96.8 ± 10.4% in IM and M, respectively, P < 0.01). The responses to >10–6 M capsaicin were not determined, because a poor reversibility was found in some of the airway specimens at higher concentrations in our preliminary trials.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Concentration-response curves to 10–8–10–6 M capsaicin (Cap) in trachea, main bronchus, and hilar bronchus of M ({bullet}, n = 15) and IM ({circ}, n = 14) guinea pigs. Capsaicin-induced contractions are expressed as percentages of contraction induced by 10–5 M ACh. Values are means ± SE. *Significant difference between M and IM. {dagger}Significantly different from corresponding data for trachea. Data from M ({blacktriangledown}, n = 3) and IM ({triangledown}, n = 4) guinea pigs after pretreatment with 3 x 10–7 M CP-99994 and 10–7 M SR-48968 for 60 min are shown.

 
In M animals, there was a distinctly greater constrictive response to capsaicin in the hilar bronchi than in the tracheae or main bronchi (Fig. 2), whereas this difference is considerably less in IM animals.

In M (n = 3) and IM (n = 4) guinea pigs, pretreatment of the airway rings with a combination of 3 x 10–7 M CP-99994 and 10–7 M SR-48968 for 60 min completely eliminated the contraction evoked by the highest concentration (10–6 M) of capsaicin (Fig. 2).

EFS-evoked NANC contraction of isolated airways. EFS evoked a biphasic response in isolated airways in M (n = 7) and IM (n = 7) guinea pigs. The response was reproducible if >20 min elapsed between two consecutive stimulations (Fig. 3).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Representative traces of responses of isolated airways to electrical field stimulation (EFS) in M (A) and IM (B) guinea pigs. Two consecutive control responses separated by 30 min (a and b) and after pretreatment with 10–6 M atropine and 10–6 M propranolol (c) are shown. {bullet}, EFS application.

 
In main and hilar bronchi, EFS evoked an initial vigorous contraction that rapidly declined, followed by a lower-magnitude, but long-lasting, contraction. The initial constrictive responses to EFS were not significantly different between M and IM animals in main (P > 0.1) or hilar bronchi (P > 0.05; Fig. 3). After pretreatments with 10–6 M atropine and 10–6 M propranolol to block the cholinergic and adrenergic pathways, respectively, the initial response was completely eliminated, but the delayed contraction persisted (Fig. 3). The delayed NANC contraction was significantly greater in hilar bronchi of M than of IM animals: at 10-Hz stimulation frequency, 58.9 ± 8.3% and 24.7 ± 3.9% of control contraction in M and IM, respectively (P < 0.01; Figs. 3 and 4). Furthermore, this difference between M and IM hilar bronchi was not abolished by pretreatment with indomethacin: at the same stimulation frequency after indomethacin, 45.3 ± 7.0% and 13.6 ± 2.8% of control contraction in M and IM, respectively (P < 0.01; Fig. 4). Overall, the intensity of the NANC contraction in the hilar bronchus was significantly greater than that in the main bronchus of M guinea pigs (P < 0.01, 1-way ANOVA), but such a difference was not found in IM guinea pigs (Fig. 4).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4. Nonadrenergic noncholinergic (NANC) responses of isolated airway rings to EFS (40 V, 1-ms duration for 10 s) at different stimulation frequencies in M ({bullet}, n = 7) and IM ({circ}, n = 7) guinea pigs. A: before indomethacin (5 x 10–5 M). B: after indomethacin. NANC response was measured as the area enclosed by the curve (see Fig. 3) over a 5-min period starting at onset of EFS and expressed as percentage of control response (before atropine and propranolol) to EFS; control response in trachea was measured as combined areas of contraction and relaxation. Inhibitory NANC responses in trachea are expressed by negative values. Values are means ± SE. *Significant difference between M and IM. {dagger}Significant difference between corresponding data before and after indomethacin in the same airway specimen.

 
In M tracheae, EFS produced an initial abrupt contraction followed by a more sustained relaxation (Fig. 3A). Similar initial contraction was also observed in IM tracheae, but the delayed relaxation was absent or substantially smaller (Fig. 3B). After pretreatments with 10–6 M atropine and 10–6 M propranolol, the initial contraction was completely eliminated in M and IM tracheae, and the delayed relaxation was not significantly affected in the former (Fig. 3). Pretreatment with indomethacin consistently reduced the resting tension in M and IM tracheae. When the baseline tension was readjusted to 0.5 g after indomethacin, the EFS-induced NANC inhibitory effect was significantly attenuated in M tracheae; the difference in the inhibitory NANC (iNANC) responses between M and IM tracheae was substantially reduced after the indomethacin pretreatment (Fig. 4).

Concentration-response curves of isolated airways to tachykinins. Our data (Fig. 2) indicated that endogenously released tachykinins were responsible for the capsaicin-induced constriction of isolated airways in M and IM animals. To determine whether the greater response to capsaicin in M airways was due to a higher sensitivity to tachykinins, we compared the effect of exogenously applied tachykinins on isolated airways in M (n = 9) and IM (n = 9) animals. Cumulative concentration-response curves to 10–9–10–6 M SP were markedly higher in the M than in the IM airways, regardless of the tracheobronchial locations (Fig. 5). Surprisingly, there was no detectable difference in the airway responses to SP among the three locations in M or IM animals (Fig. 5). A clear difference between M and IM animals was also found in the concentration-response curves to 10–10–10–7 M NKA, which is similar to the response to SP, except NKA showed a 10-fold higher potency in all three airway locations (Fig. 6).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. Concentration-response curves to 10–9–10–6 M substance P (SP) in trachea, main bronchus, and hilar bronchus of M ({bullet}, n = 9) and IM ({circ}, n = 9) guinea pigs. SP-induced contractions are expressed as percentages of contraction induced by 10–5 M ACh. Values are means ± SE. *Significant difference between M and IM.

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6. Concentration-response curves to 10–10–10–7 M neurokinin A (NKA) in trachea, main bronchus, and hilar bronchus of M ({bullet}, n = 9) and IM ({circ}, n = 9) guinea pigs. NKA-induced contractions are expressed as percentages of contraction induced by 10–5 M ACh. Values are means ± SE. *Significant difference between M and IM.

 
RIA of SP-LI in tracheobronchial tissues. Tissues of trachea and bronchi were pooled for the RIA. The results showed no significant difference in the SP-LI of the tracheobronchial tissues between M and IM guinea pigs: 8.2 ± 1.2 and 12.3 ± 1.7 pg/g tissue in M and IM, respectively (P > 0.05, n = 6). Because of limited sensitivity of the RIA, a minimal tissue mass was required for the analysis. Thus we were unable to determine the tissue contents of SP separately at the three different airway locations.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Results of this study clearly show that the constrictive responses of isolated airways to capsaicin were significantly weaker in all three locations in the tracheobronchial tree of IM guinea pigs than in M animals. Similarly, the EFS-induced NANC contraction in hilar bronchi was significantly smaller in IM than in M animals. The difference between M and IM animals in their responses to capsaicin was completely eliminated by pretreatment with selective antagonists of the NK1 and NK2 receptors, indicating a critical role for the endogenously released tachykinins in the response mechanism(s). Furthermore, the fact that the constrictive responses to exogenous SP and NKA were significantly weaker in the isolated airway rings of IM animals lends additional support to this conclusion.

Vagal bronchopulmonary C-fibers represent ~75% of the vagal afferents innervating the respiratory tract (1) and play an important role in the regulation of airway functions in physiological and pathophysiological conditions (3, 8, 24). Stimulation of these C-fiber afferents is known to induce bronchoconstriction via two different mechanisms in various species: 1) reflex bronchoconstriction mediated through the cholinergic pathways in intact animals (2, 8, 17) and 2) a slower-onset, but more sustained, bronchoconstriction via the release of tachykinins (17, 27, 28); the latter seems to be a more dominant factor in guinea pig airways (7, 24, 28). SP and NKA are two major types of tachykinins identified in the mammalian respiratory tract (21, 28, 34, 38). These neuropeptides are primarily localized in the C-fiber afferent nerves and are synthesized in the cell bodies of these neurons in the nodose and intracranial jugular ganglia (23, 35). The majority of them are then transported along the axon toward the sensory terminals, where they are stored in large-granular vesicles and released on a surge of calcium influx triggered by membrane depolarization (28, 34). SP and NKA are frequently colocalized and coreleased in the same neurons, probably because they are derived from the same precursor gene, the preprotachykinin A (22).

Three distinct types of G protein-coupled receptors mediate the action of tachykinins and are located in the plasma membrane of effector cells: NK1, NK2, and NK3 receptors. The NK1 and NK2 receptors are more abundantly found in the respiratory tract and have distinct preferential affinities for SP and NKA, respectively (21, 34). In particular, the NK2 receptor is expressed primarily on the airway smooth muscle, and its activation induces bronchoconstriction in most species, including humans (3, 21, 28, 34, 38). The NK2 receptor is also found on postganglionic prejunctional cholinergic nerve terminals, and its activation can facilitate ACh release and smooth muscle contraction (15). The NK1 receptor is present on a number of cells in the airways, including the smooth muscle, but the bronchoconstrictive effect of NK1 receptor activation is weak compared with that of the NK2 receptor in guinea pigs (14, 21). Recent studies have further indicated the involvement of the NK3 receptor in facilitating the synaptic transmission at the airway parasympathetic ganglion (5, 9). In this study, the concentration-response curves of SP and NKA are distinctly lower in the IM airways at all three airway locations (Figs. 5 and 6), clearly indicating a lower potency of the excitatory effect of these tachykinins on the airway smooth muscles. These results do not agree with the previous observation by Tanaka and Grunstein (40), who found no significant difference in the direct constrictive effect of SP on the tracheal smooth muscle between M and IM (2-wk-old) rabbits. However, our data are in general agreement with data reported previously from an in vivo study by Tokuyama et al. (41). These investigators demonstrated that the increases in total lung resistance evoked by the same intravenous doses of SP and NKA were significantly lower in IM guinea pigs at the same age as those used in this study than in M animals. The responsiveness of tracheal smooth muscles to tachykinins also increases progressively with age in piglets over the first 10 wk of life (16), but the more potent bronchoconstrictive effect of SP than of NKA in piglets is different from that in guinea pig airways (10, 16). The possible mechanism(s) contributing to such a difference between M and IM airways is not known. One possibility is a lower density and/or expression of the NK receptors on the airway smooth muscle of the IM animals. Alternatively, a reduced potency may be related to a difference in the properties of NK receptors (e.g., affinity) (44) or a change at certain site(s) in the signal transduction pathways (32). A decrease in response could also be related to the smaller mass of smooth muscles (30) or a difference in the contractile protein composition in the IM airways (39). However, this possibility seems unlikely, because the intensity of airway constriction generated by the same concentration of KCl was not significantly different between M and IM animals at any of the three airway locations (Fig. 1).

Capsaicin, a potent and selective stimulant of C-fiber afferents in the airways (8, 24), can constrict the isolated airway via the local release of tachykinins. A significantly weaker contractile response to capsaicin of the isolated airway rings in the IM animals (Fig. 2) can be explained, at least in part, by the lower potency of tachykinins on the smooth muscles (Figs. 5 and 6). A weaker constrictive response could also be due to a smaller quantity of tachykinins being synthesized and stored in the C-fiber terminals in the IM airways. However, our data obtained from the RIA experiments, indicating no difference in the SP-LI in the airway tissue between M and IM animals, fail to support such a possibility. The difference in the response to capsaicin may also be related to a lower sensitivity of C-fiber endings in IM animals. Thus a given concentration of capsaicin would evoke a lower intensity of stimulation of the C-fibers and less release of tachykinins. Indeed, it has been shown that the pulmonary chemoreflex responses elicited by stimulation of pulmonary C-fibers are poorly developed in newborns: a sixfold increase in the dose of phenyldiguanide, a chemical stimulant of C-fiber afferents, was required in 1-wk-old kittens to elicit the same reflex responses (i.e., apnea, rapid shallow breathing, and bradycardia) seen in adult cats (20). Similarly, sensitivity of pulmonary chemoreflex elicited by lactic acid is considerably lower during the 1st wk of life in rabbits than in adulthood (42). Although reflex bronchoconstriction elicited by capsaicin was clearly demonstrated in newborn dogs and piglets, the threshold doses were higher than the dose normally applied in adult animals (2). On the other hand, a lack of difference in the reflex response to phenyldiguanide between newborn piglets and adult pigs has also been described (36). Presumably, the difference in the excitability of the reflex response in the early postnatal stage of life is related, at least in part, to the fact that the degree of maturity of newborns differs among species (4, 43). A quantitative comparison is further hindered by the differences in the maturation rate and the equivalent age between species (4, 43, 44). Furthermore, whether the difference occurs at the level of sensory receptors or at other components of the reflex arc remains to be determined.

In isolated main and hilar bronchi, EFS evoked a biphasic constrictive response: an initial rapid and intense contraction followed by a lower-magnitude, but more sustained, contraction (Fig. 3). After pretreatment with atropine and propranolol, the initial contraction was eliminated, but the long-lasting constriction remained. This atropine-resistant constrictive response to EFS is known as the excitatory NANC (eNANC) response (19), and tachykinins are believed to be the neurotransmitters (28, 38). In M animals, the EFS-evoked eNANC response was much more pronounced in the hilar bronchus than the main bronchus or trachea (Figs. 3 and 4). Similarly, the same dose of capsaicin (10–7–10–6 M) also generated a much greater contraction of the hilar bronchus in M animals (Fig. 2). These results are in agreement with previous reports of a potent NANC bronchoconstrictive effect on the hilar bronchus, but not on the trachea, of the guinea pig (13). Interestingly, there was no detectable difference in the constrictive responses to exogenous tachykinins (SP and NKA) among the different locations of the tracheobronchial tree in the M animals (Figs. 5 and 6). Together, these results seem to suggest that a larger quantity of tachykinins is released from the C-fiber terminals innervating the hilar bronchi than from the C-fiber terminals innervating the trachea. Unfortunately, our RIA data do not provide the critical information required for the comparison of the tissue contents of these neuropeptides at different airway locations of the M animals. Alternatively, the difference in the constrictive response could be influenced by a difference in the rate of degradation of these neuropeptides after release from the nerve endings among different locations (29, 34, 38). Whether all or any of these factors contribute to the observed difference in the M guinea pigs could not be determined in this study. Such a longitudinal difference in the bronchoconstriction evoked by C-fiber stimulation was not as evident in the IM guinea pigs (Figs. 24). Furthermore, the EFS-induced NANC contraction of hilar bronchi was significantly weaker in IM than in M animals (Figs. 3 and 4), presumably because of the weaker responses of the airway smooth muscle to tachykinins (Figs. 5 and 6). Indomethacin pretreatment reduced, but did not abolish, the NANC contractile response to EFS in the hilar bronchi of M animals (Fig. 4), suggesting an involvement of the cyclooxygenase metabolites of arachidonic acid in the eNANC responses (18, 19).

In sharp contrast, the contractile responses of the isolated airways to the same concentration of ACh (10–5 M) were almost identical between M and IM animals in all three airway locations (Fig. 1). In addition, there was no significant difference between M and IM animals, regardless of the airway location, in the initial bronchoconstrictive response to EFS, which was totally abolished by pretreatment with atropine (Fig. 3). These observations seem to suggest that the cholinergic innervation of the airway smooth muscle is more fully developed than the C-fiber afferents in the IM animals. This is in general agreement with the finding of early maturation of the airway cholinergic responses reported by others in larger mammals (12).

In isolated tracheal rings of the M guinea pigs, EFS evoked an initial contraction followed immediately by a more sustained relaxation (Fig. 3). The contraction was completely eliminated by pretreatment with atropine, whereas the smooth muscle relaxation was not significantly altered by propranolol. This adrenergic antagonist-resistant bronchodilation induced by EFS has been termed the iNANC response (19, 25) and is believed to be mediated primarily through the action of nitric oxide and/or vasoactive intestinal peptide (19, 25). Our results show that the iNANC response of the trachea to EFS is clearly more pronounced in M animals (Figs. 3 and 4). These results are in general agreement with a previous report by Chitano et al. (6) that the EFS-induced NANC relaxation of tracheal strips was significantly weaker in guinea pigs between 1 and 3 wk of age than in their adult counterparts. These investigators further concluded that the smaller iNANC response in the IM animals was due to the inhibition of smooth muscle relaxation by cyclooxygenase metabolites (6). Indeed, our results showed that the difference in iNANC responses between M and IM tracheae was markedly attenuated after pretreatment with indomethacin (Fig. 3). However, the EFS-induced relaxation was not increased by indomethacin in IM tracheae as demonstrated in their study (6). This discrepancy may be related to readjustment of the resting tension of the airway ring to the control level after indomethacin treatment in our experiment. The precise mechanisms underlying the action of cyclooxygenase metabolites that contributes to the NANC inhibitory effect in M tracheae (6, 19, 26) cannot be determined in this study.

In conclusion, the constriction of isolated airways evoked by C-fiber stimulation is significantly weaker in the IM guinea pigs. The difference in the constrictive responses to capsaicin was completely eliminated by pretreatment with selective antagonists of the NK receptors. The NANC contraction in hilar bronchi induced by EFS was also distinctly stronger in M animals. In addition, the responses to exogenous SP and NKA were markedly weaker in the IM airways. Together, these results suggest that a less potent effect of tachykinins on the airway smooth muscles is responsible for the weaker bronchoconstrictive response to C-fiber afferent stimulation in IM animals.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-58686 and HL-63797.


    ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Qihai Gu, Robert Morton, and Wen-Bin Yang for technical assistance and to Dr. Mary K. Rayens for statistical analysis.

Present addresses: Z. X. Wu, Dept. of Neurobiology and Anatomy, West Virginia University, Morgantown, WV 26506; Q. H. Yang, Dept. of Pharmacology, Zhejiang University, Hangzhou, Zhejiang 310006, PRC.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L.-Y. Lee, Dept. of Physiology, Univ. of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536-0298 (E-mail: lylee{at}uky.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.

* Z.-X. Wu and Q. H. Yang contributed equally to the study. Back


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Agostoni E, Chinnock JE, Daly MB, and Murray JG. Functional and histological studies of the vagus nerve and its branches to the heart, lungs and abdominal viscera in the cat. J Physiol 135: 182–205, 1957.[ISI]
  2. Anderson JW and Fisher JT. Capsaicin-induced reflex bronchoconstriction in the newborn. Respir Physiol 93: 13–27, 1993.[CrossRef][ISI][Medline]
  3. Barnes PJ. Neuropeptides in human airways: function and clinical implications. Am Rev Respir Dis 136: S77–S83, 1987.[ISI][Medline]
  4. Burri PH. Fetal and postnatal development of the lung. Annu Rev Physiol 46: 617–628, 1984.[CrossRef][ISI][Medline]
  5. Canning BJ, Reynolds SM, Anukwu LU, Kajekar R, and Myers AC. Endogenous neurokinins facilitate synaptic transmission in guinea pig airway parasympathetic ganglia. Am J Physiol Regul Integr Comp Physiol 283: R320–R330, 2002.[Abstract/Free Full Text]
  6. Chitano P, Cox CM, and Murphy TM. Relaxation of guinea pig trachealis during electrical field stimulation increases with age. J Appl Physiol 92: 1835–1842, 2002.[Abstract/Free Full Text]
  7. Coleridge HM and Coleridge JCG. Airway axon reflexes—where now? Int Union Physiol Sci 10: 91–96, 1995.
  8. Coleridge JC and Coleridge HM. Afferent vagal C fibre innervation of the lungs and airways and its functional significance. Rev Physiol Biochem Pharmacol 99: 1–110, 1984.[ISI][Medline]
  9. Daoui S, Cognon C, Naline E, Emonds-Alt X, and Advenier C. Involvement of tachykinin NK3 receptors in citric acid-induced cough and bronchial responses in guinea pigs. Am J Respir Crit Care Med 158: 42–48, 1998.[ISI][Medline]
  10. Dreshaj IA, Martin RJ, Miller MJ, and Haxhiu MA. Responses of lung parenchyma and airways to tachykinin peptides in piglets. J Appl Physiol 77: 147–151, 1994.[Abstract/Free Full Text]
  11. Emonds-Alt X, Vilain P, Goulaouic P, Proietto V, Van Broeck D, Advenier C, Naline E, Neliat G, Le Fur G, and Breliere JC. A potent and selective non-peptide antagonist of the neurokinin A (NK2) receptor. Life Sci 50: L101–L106, 1992.
  12. Fisher JT, Brundage KL, Waldron MA, and Connelly BJ. Vagal cholinergic innervation of the airways in newborn cat and dog. J Appl Physiol 69: 1525–1531, 1990.[Abstract/Free Full Text]
  13. Grundstrom N, Andersson RG, and Wikberg JE. Pharmacological characterization of the autonomous innervation of the guinea pig tracheobronchial smooth muscle. Acta Pharmacol Toxicol 49: 150–157, 1981.[ISI][Medline]
  14. Hajj AM, Burki NK, and Lee LY. Role of tachykinins in sulfur dioxide-induced bronchoconstriction in anesthetized guinea pigs. J Appl Physiol 80: 2044–2050, 1996.[Abstract/Free Full Text]
  15. Hall AK, Barnes PJ, Meldrum LA, and Maclagan J. Facilitation by tachykinins of neurotransmission in guinea-pig pulmonary parasympathetic nerves. Br J Pharmacol 97: 274–280, 1989.[Abstract]
  16. Haxhiu-Poskurica B, Haxhiu MA, Kumar GK, Miller MJ, and Martin RT. Tracheal smooth muscle responses to substance P and neurokinin A in the piglet. J Appl Physiol 72: 1090–1095, 1992.[Abstract/Free Full Text]
  17. Hong JL, Rodger IW, and Lee LY. Cigarette smoke-induced bronchoconstriction: cholinergic mechanisms, tachykinins, and cyclooxygenase products. J Appl Physiol 78: 2260–2266, 1995.[Abstract/Free Full Text]
  18. Ito Y. Prejunctional control of excitatory neuroeffector transmission by prostaglandins in the airway smooth muscle tissue. Am Rev Respir Dis 143: S6–S10, 1991.[ISI][Medline]
  19. Johansson-Rydberg IG, Andersson RG, and Grundstrom N. The modulatory effects of prostaglandins on both excitatory and inhibitory non-adrenergic non-cholinergic neurotransmission in guinea-pig airways. Acta Physiol Scand 144: 439–449, 1992.[ISI][Medline]
  20. Kalia M. Visceral and somatic reflexes produced by J pulmonary receptors in newborn kittens. J Appl Physiol 41: 1–6, 1976.[Abstract/Free Full Text]
  21. Khawaja AM and Rogers DF. Tachykinins: receptor to effector. Int J Biochem Cell Biol 28: 721–738, 1996.[CrossRef][ISI][Medline]
  22. Krause JE, Chirgwin JM, Carter MS, Xu ZS, and Hershey AD. Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A. Proc Natl Acad Sci USA 84: 881–885, 1987.[Abstract]
  23. Kwong K, Wu ZX, Kashon ML, Krajnak KM, Wise PM, and Lee LY. Chronic smoking enhances tachykinin synthesis and airway responsiveness in guinea pigs. Am J Respir Cell Mol Biol 25: 299–305, 2001.[Abstract/Free Full Text]
  24. Lee LY and Pisarri TE. Afferent properties and reflex functions of bronchopulmonary C-fibers. Respir Physiol 125: 47–65, 2001.[CrossRef][ISI][Medline]
  25. Li CG and Rand MJ. Evidence that part of the NANC relaxant response of guinea-pig trachea to electrical field stimulation is mediated by nitric oxide. Br J Pharmacol 102: 91–94, 1991.[Abstract]
  26. Linden A, Ullman A, Skoogh BE, and Lofdahl CG. Non-adrenergic, non-cholinergic regulation of guinea-pig airway smooth muscle—indomethacin-induced changes and segmental differences. Pulm Pharmacol 4: 170–176, 1991.[CrossRef][ISI][Medline]
  27. Lou YP, Lee LY, Satoh H, and Lundberg JM. Postjunctional inhibitory effect of the NK2 receptor antagonist, SR 48968, on sensory NANC bronchoconstriction in the guinea-pig. Br J Pharmacol 109: 765–773, 1993.[Abstract]
  28. Lundberg JM and Saria A. Polypeptide-containing neurons in airway smooth muscle. Annu Rev Physiol 49: 557–572, 1987.[CrossRef][ISI][Medline]
  29. Martins MA, Shore SA, and Drazen JM. Capsaicin-induced release of tachykinins: effect of enzyme inhibitors. J Appl Physiol 70: 1950–1956, 1991.[Abstract/Free Full Text]
  30. Matsuba K and Thurlbeck WM. A morphometric study of bronchial and bronchiolar walls in children. Am Rev Respir Dis 105: 908–913, 1972.[ISI][Medline]
  31. McLean S, Ganong A, Seymour PA, Snider RM, Desai MC, Rosen T, Bryce DK, Longo KP, Reynolds LS, Robinson G, Schmidt AW, Siok C, and Heym J. Pharmacology of CP-99994; a nonpeptide antagonist of the tachykinin neurokinin-1 receptor. J Pharmacol Exp Ther 267: 472–479, 1993.[Abstract]
  32. Nakanishi S. Mammalian tachykinin receptors. Annu Rev Neurosci 14: 123–136, 1991.[CrossRef][ISI][Medline]
  33. Nault MA, Vincent SG, and Fisher JT. Mechanisms of capsaicin- and lactic acid-induced bronchoconstriction in the newborn dog. J Physiol 515: 567–578, 1999.[Abstract/Free Full Text]
  34. Piedimonte G. Tachykinin peptides, receptors, and peptidases in airway disease. Exp Lung Res 21: 809–834, 1995.[ISI][Medline]
  35. Riccio MM, Kummer W, Biglari B, Myers AC, and Undem BJ. Interganglionic segregation of distinct vagal afferent fibre phenotypes in guinea-pig airways. J Physiol 496: 521–530, 1996.[Abstract]
  36. Schleman M, Gootman N, and Gootman PM. Cardiovascular and respiratory responses to right atrial injections of phenyl diguanide in pentobarbital-anesthetized newborn piglets. Pediatr Res 13: 1271–1274, 1979.[Abstract]
  37. Sisk DB. Physiology. In: The Biology of the Guinea Pig, edited by Wagner JE and Manning PJ. New York: Academic, 1976, p. 63–98.
  38. Solway J and Leff AR. Sensory neuropeptides and airway function. J Appl Physiol 71: 2077–2087, 1991.[Abstract/Free Full Text]
  39. Sparrow MP and Mitchell HW. Contraction of smooth muscle of pig airway tissues from before birth to maturity. J Appl Physiol 68: 468–477, 1990.[Abstract/Free Full Text]
  40. Tanaka DT and Grunstein MM. Maturation of neuromodulatory effect of substance P in rabbit airways. J Clin Invest 85: 345–350, 1990.[ISI][Medline]
  41. Tokuyama K, Yokoyama T, Morikawa A, Mochizuki H, Kuroume T, and Barnes PJ. Attenuation of tachykinin-induced airflow obstruction and microvascular leakage in immature airways. Br J Pharmacol 108: 23–29, 1993.[Abstract]
  42. Trippenbach T. Pulmonary reflexes and control of breathing during development. Biol Neonate 65: 205–210, 1994.[CrossRef][ISI][Medline]
  43. Waldron MA and Fisher JT. Neural control of airway smooth muscle in the newborn. In: Developmental Neurobiology of Breathing, edited by Haddad GG and Farber JP. New York: Dekker, 1991, p. 483–518.
  44. Wills-Karp M. Effects of ageing upon airways smooth muscle contractility. In: Airways Smooth Muscle: Development and Regulation of Contractility, edited by Raeburn D and Giembycz MA. Basel: Birkhauser Verlag, 1994, chapt. 7, p. 185–218.




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
287/1/L168    most recent
00156.2003v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Google Scholar
Articles by Wu, Z.-X.
Articles by Lee, L.-Y.
Articles citing this Article
PubMed
PubMed Citation
Articles by Wu, Z.-X.
Articles by Lee, L.-Y.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.