Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky 40536
Submitted 19 May 2003 ; accepted in final form 10 February 2004
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ABSTRACT |
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airway smooth muscle; bronchoconstriction; capsaicin; tachykinins; airway reflex
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.
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METHODS |
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This study was carried out in IM [161 ± 5 (SE) g body wt, 813 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 46 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.
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Cumulative concentration-response curves to capsaicin and tachykinins.
Cumulative concentration-response curves of the airway rings to 108106 M capsaicin were obtained by successive increases () of the concentration of capsaicin solution every 210 min or until after the response approached a plateau. Capsaicin-induced contractions were then expressed as a percentage of the contraction induced by 105 M ACh. To determine whether the constrictive responses to capsaicin were generated by the endogenously released tachykinins, the highest concentration of capsaicin (106 M) was applied 60 min after the isolated airways were pretreated with a combination of 3 x 107 M CP-99994, a selective antagonist of the neurokinin (NK) type 1 (NK1) receptor (31), and 107 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 109106 M substance P (SP) and 1010107 M NKA were obtained in a manner similar to that described above and also expressed as a percentage of the contraction induced by 105 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 106 M atropine and 106 M propranolol. To determine whether endogenous cyclooxygenase metabolites are involved in the NANC responses, the same protocols were repeated after pretreatments with 106 M atropine, 106 M propranolol, and 5 x 105 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 (103 M; Sigma) was prepared in a vehicle of 10% Tween 80, 10% ethanol, and 80% isotonic saline. SP (103 M; Peninsula Laboratories) and NKA (103 M; Peninsula Laboratories) were prepared in ethanol. CP-99994 (103 M; kindly provided by Pfizer, Groton, CT) and SR-48968 (103 M; kindly provided by Sanofi Recherche, Montpellier Cedex, France) were dissolved in polyethylene glycol (average mol wt 200; Sigma). Indomethacin (5 x 103 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.
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RESULTS |
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Cumulative concentration-response curves of isolated airways to 108106 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 106 M capsaicin was 78.0 ± 13.4% of the response to 105 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 >106 M capsaicin were not determined, because a poor reversibility was found in some of the airway specimens at higher concentrations in our preliminary trials.
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In M (n = 3) and IM (n = 4) guinea pigs, pretreatment of the airway rings with a combination of 3 x 107 M CP-99994 and 107 M SR-48968 for 60 min completely eliminated the contraction evoked by the highest concentration (106 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).
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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 109106 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 1010107 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).
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DISCUSSION |
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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 (107106 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 (105 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.
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GRANTS |
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ACKNOWLEDGMENTS |
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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.
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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.
* Z.-X. Wu and Q. H. Yang contributed equally to the study.
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REFERENCES |
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