Main

SP is an 11-amino acid NK which has been identified in the vagus nerve(1) and in nerve fibers supplying the airways in both adult and immature animals(1, 2). Although its precise physiologic role in the airways is unknown, there now exists substantial evidence that SP, in addition to its direct contractile effect on ASM, may also modulate the presynaptic release of ACh from airway nerve fibers(3–6).

In considering the mechanism for SP's contractile effects, it is often assumed that the peptide binds to a single population of NK receptors located on either ASM or neural elements. However, the preponderance of evidence suggests that the NK receptor in nonpulmonary tissues is heterogeneous(7) and is composed of at least three different subtype receptors identified as NK-1, NK-2, and NK-3(8–10). In this connection, the most convincing evidence that the NK receptor is heterogeneous has come from recent successes in identifying the molecular weights of the NK-1 and NK-2 receptors(9, 11) as well as actual cDNA cloning of the NK-2 receptor(12).

Relatively little is known about the localization and function of these NK subtype receptors in immature airways. We previously reported that SP contracted immature ASM both in vivo and in vitro(6, 13). As in the adult rabbit(4, 5), we found evidence that the contractile action to SP in immature rabbit airways was in part cholinergically mediated. In these earlier studies, it was assumed that SP acted on a homogeneous population of NK receptors whose primary difference lay in their location on either the ASM or neural elements. However, in light of recent reports demonstrating the heterogeneity of the SP receptor in adult animal airways(14, 15), this latter assumption requires closer scrutiny.

Accordingly, our present study was designed to meet the following objectives in isolated TSM and BSM segments obtained from 2-wk-old rabbits:1) evaluate the airway contractile action of SP in the absence and presence of effective inhibition of NEP with PHOS, 2) identify regional airway differences in SP sensitivity or efficacy in the presence of effective NEP inhibition, 3) determine whether SP's cholinergic component accounts for these regional differences, and 4) investigate the NK subtype receptors responsible for SP-induced contraction. Our findings indicate that the noncholinergic contractions produced by SP in immature rabbit airways are potentiated by PHOS and are likely mediated by NK-1 and NK-2 subtype receptors.

METHODS

TSM and BSM segments of approximately 1 cm in length were isolated from 2-wk-old New Zealand White rabbits. The animals were anesthetized using ketamine (35 mg/kg of body weight) and xylazine (5 mg/kg of body weight) and killed by the injection of an air embolus into a systemic vein. The euthanasia methods were approved by the Duke University Institutional Animal Care and Use Committee. ASM segments with intact epithelia were obtained after careful dissection of lung parenchyma. TSM segments were obtained at the level of the carina and BSM segments from the first and second order bronchi of both lungs. The contractile responses of these BSM segments did not vary significantly to either cholinergic or peptidergic agonists, and their data were pooled for the purposes of statistical comparison. Each ASM segment was cleaned of loose connective tissue and then placed in siliconized Harvard 15-mL organ baths and supported longitudinally by firm stainless steel wire triangular supports. The lower support was attached to a glass hook at the base of the organ bath, and the upper support was attached via a gold chain to a Grass FT.03C isometric force transducer. The latter was mounted on a rack and pinion clamp so that the individual resting lengths of the TSM and BSM segments could be adjusted. The tissues were bathed in a modified Krebs-Ringer solution containing (in mM): 125 NaCl; 14 NaHCO3; 2.25 CaCl2: 2 H2O; 1.46 MgSO4: 7 H2O; 4 KCl; 1.2 NaH2PO4:H2O; and 4 g/L dextrose. The baths were aerated with a 95% O2-5% CO2 gas mixture, and a pH of 7.38 ± 0.05 was established for the duration of each experiment. The temperature of the baths was maintained at 37°C.

The ASM segments were equilibrated in the baths for 1 h, during which time they were passively stretched on several occasions to a tension of 3 g, and the baths rinsed with fresh buffer solution. The electrical field stimulation was delivered by a Grass S488D stimulator, connected to a stimulus isolation unit (Grass SIU5), and applied transmurally across the tissues by means of parallel platinum-plate electrodes. The optimal resting lengths(L0) for TSM and BSM were established by assessing their maximal contractile response to the following standard electrical field stimulus: 8 V/cm, 2-ms pulse duration, 30 Hz stimulus frequency, at a current density of 82 mA/cm2. In preliminary experiments, we found that electrical current densities in excess of 100 mA/cm2 resulted in tissue damage. Typically L0 was achieved at a resting tension between 0.5 and 1.0 g. The tissues were subsequently placed under theirL0 conditions and dose-response curves to the agonists were obtained using final bath concentrations ranging from 10-9 to 3 × 10-5 M. Cumulative dose-response curves were generated by administering increasing doses of SP after the previous dose produced a plateau response. After a stable response was obtained to the highest dose of agonist, the organ baths were thoroughly rinsed with fresh buffer solution. The tissues were allowed to recover for 20 min between each dose-response curve.

To evaluate the influence of progressive NEP inhibition on SP-induced contraction in upper and lower airway regions, we examined in TSM and BSM segments the effect of increasing concentrations of PHOS (10-8 to 10-5 M) on the contractile responses induced by 3 × 10-7 M SP. In a series of preliminary experiments, we found that the dose-response relationships to carbachol, a cholinergic agonist, were unaffected by 1× 10-5 M PHOS. These latter findings are in agreement with those previously reported in adult guinea pigs(16).

To assess possible regional differences in NK reactivity due to ACh metabolism, we examined the effect of 10-5 M NEO, an AChase inhibitor, on the ASM contractile response to SP in the presence of 3 × 10-6 M PHOS. To examine SP-induced contractions in the absence of its cholinergic component(6), SP dose-response curves were obtained in the presence of 3 × 10-6 M PHOS, 10-5 M NEO, and 10-5 M ATR, a cholinergic receptor antagonist. At this concentration, ATR completely inhibited the contractile response induced by 10-4 M carbachol.

Noncumulative dose-response curves to Septide (Peninsula Labs., Belmont, CA) (10-8 to 3 × 10-6 M), a highly selective NK-1 agonist(17), were separately determined in ASM segments after treatment with 3 × 10-6 M PHOS, 10-5 M NEO, and 10-5 M ATR. To assess the possible contribution of NK-3 receptors in these airways, we examined the contractile actions of Senktide (Peninsula Labs.) (10-8 to 3 × 10-6 M), a selective NK-3 receptor agonist(18).

To determine the contribution of NK-1 receptors in the airway contractile response to SP, we first examined the effect of competitive inhibition of NK-1 function using a competitive NK-1 antagonist, GR 82334 (10-6 M). We confirmed the findings of NK-1 receptor contribution by selectively inactivating the NK-1 airway receptor with NK-1 specific agonists(15, 18). In the present study, NK-1 receptor function was inactivated by pretreating the ASM segments with either Septide(10-6 M) or with GR 82334 (10-6 M).

Drugs used in this study included NKA, SP, Septide, GR 82334, and Senktide from Peninsula Labs. and ATR, PHOS, NEO, and carbachol from Sigma Chemical Co., St. Louis.

At the end of each experiment, the airway contractile responses were normalized to the active tensions produced by 10-4 M carbachol(i.e.,% carbachol) which elicited a near-maximal contractile response in each tissue. Where possible, the dose-response curves were further characterized by determining agonist efficacy (i.e., the maximal tension (Tmax) produced by the agonist) and sensitivity as established by a logarithmic transformation of the agonist concentration which produced 50% of the Tmax (i.e. log ED50).

Statistical analyses of these data were performed using either 2-tailed paired or unpaired t tests. In those instances where a clearTmax response was not attained, the linear regions of the dose-response curves were compared using an analysis of covariance(19). In this latter analysis, simple linear regressions were fitted to the data sets by the least squares method. Statistical significance was then determined between the relative positions and slopes of these simple linear regressions. Significance in the F statistic,Felev, was taken as evidence of a significant shift in the dose-response relationship relative to the initial (i.e. control) curve. To determine whether a shift in a dose-response relationship was parallel, the slopes of these regression lines were compared using theF statistic, Fslope. Significance of theF statistics was ascertained from standard statistical tables(19). A p value of 0.05 or less was considered significant.

RESULTS

The contractile actions of SP in TSM and BSM segments are depicted inFig. 1. Here, SP-induced contractions are normalized to the contractile responses to 10-4 M carbachol and plotted as a function of increasing peptide concentration. At the highest administered concentration of the peptide (i.e. 3 × 10-5 M), SP was more reactive in BSM (80.3 ± 3.8% carbachol) than in TSM (42.8 ± 4.6% carbachol, p < 0.001). An accurate determination of the ED50 was not possible because a clear Tmax response to SP was not consistently attained in the BSM segments. Accordingly, the linear sections of the dose-response relationships (i.e. between 10-8 M and 10-5 M) were compared by using analysis of covariance. Over this latter range of concentrations, SP was more reactive in BSM than TSM segments as indicated by the Fslope = 7.11;df = 1, 84: p < 0.01; and Felev = 8.50; df = 1, 85; p < 0.01.

Figure 1
figure 1

Comparison of SP contractile responses in BSM and TSM in the absence of NEP inhibition. Isometric tensions are expressed as a percentage (±SE) of respective near-maximal contractile response to carbachol (10-4 M). Final bath concentrations of SP are expressed in log [M]; n = 10.

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As illustrated in Fig. 2, progressive inhibition of NEP with increasing concentrations of PHOS (10-8 M to 10-5 M) enhanced the contractile responses to 10-7 M SP. Here, the airway contractile response to 10-7 M SP is expressed as a percentage of the respective initial control response (i.e. before PHOS administration) and the data plotted against the bath concentration of cumulatively administered PHOS. The maximal effect of the peptidase inhibitor is attained in both tissues at a bath concentration of about 3 × 10-6 M. In this connection, the addition of PHOS produced a parallel shift in the SP-induced contractile responses in BSM relative to TSM segments(Fslope = 2.50; df = 1, 32; p = NS:Felev = 12.58; df = 1, 33; p < 0.01).

Figure 2
figure 2

Enhancement of the contractile responses to 10-7 M SP in BSM and TSM by PHOS (10-8 to 3 × 10-5 M). Isometric tensions are expressed as a percentage (±SE) of respective near-maximal contractile response to carbachol (10-4 M). Final bath concentrations of PHOS are expressed in log [M]; n = 6.

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The effect of 3 × 10-6 M PHOS on SP-induced contractions is depicted in Fig. 3. The mean ± SE contractile responses to 3 × 10-5 M SP increased significantly in the presence of PHOS in both TSM segments (81.4 ± 1.8% carbacholversus 42.8 ± 4.6% carbachol, p < 0.001) and BSM responses (99.3 ± 2.2% carbachol versus 80.3 ± 3.8% carbachol, p < 0.01). In this connection, theTmax response to SP was greater (p < 0.01) in BSM segments (99.3 ± 2.2% carbachol) than in TSM segments (81.4± 1.8% carbachol). After NEP inhibition, SP was also more potent in BSM segments (n = 5) than in TSM segments as indicated by respective mean ± SE log ED50 values of -8.04 ± 0.19 Mversus -6.79 ± 0.39 M (p < 0.05).

Figure 3
figure 3

SP contractile responses in BSM and TSM segments obtained in the presence of 3 × 10-6 M PHOS. Isometric tensions are expressed as a percentage (±SE) of respective near-maximal contractile response to carbachol (10-4 M). Final bath concentrations of SP are expressed in log [M]; n = 5.

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The cholinergic contribution to SP-induced contractions is depicted inFig. 4. As can be seen, SP is more potent in BSM segments than in TSM segments after treatment with both 3 × 10-6 M PHOS and 10-5 M NEO; as evidenced by their respective mean ± SE log ED50 values of -8.20 ± 0.03 M versus -6.94 ± 0.17 M (p < 0.001). The significant difference (p < 0.01) between the Tmax response in BSM segments (96.9± 1.1% carbachol) versus TSM segments (91.5 ± 1.5% carbachol) persisted after AChase inhibition. In the presence of 10-5 M ATR, the mean ± SE Tmax responses to SP in TSM segments before (91.2 ± 1.9% carbachol) and after ATR treatment (95.0± 1.6% carbachol) were similar (p = 0.17). In contrast, theTmax response to SP in BSM segments decreased significantly(p < 0.05) in the presence of ATR from 96.9 ± 1.1% carbachol to 91.5 ± 3.3% carbachol. As a result, the small regional differences in the Tmax responses to SP in these airways noted previously were eliminated (p = 0.32) after ATR treatment. On the other hand, SP remained significantly more potent (p < 0.001) in BSM segments versus TSM segments after the treatment with 10-5 M ATR (log ED50: -8.02 ± 0.11 M versus-6.43 ± 0.08 M, respectively).

Figure 4
figure 4

Effects of 10-5 M ATR on the contractile responses to SP obtained in BSM segments and TSM segments in the presence of 3× 10-6 M PHOS and 10-5 M NEO. Isometric tensions are expressed as a percentage (±SE) of respective near-maximal contractile response to carbachol (10-4 M). Final bath concentrations of SP are expressed in log [M]; n = 10.

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To evaluate the possible contribution of the NK-3 receptor on SP-mediated contractions in these airways, we examined the dose-response relationships to Senktide, a highly selective NK-3 agonist(18). As depicted in Fig. 5, Senktide behaved as a very weak agonist in both TSM and BSM segments after pretreatment of the tissues with 3× 10-6 M PHOS and 10-5 M NEO.

Figure 5
figure 5

Comparison of Senktide contractile responses in BSM and TSM segments obtained in the presence of 3 × 10-6 M PHOS and 10-5 M NEO. Isometric tensions are expressed as a percentage(±SE) of respective near-maximal contractile response to carbachol(10-4 M). Final bath concentrations of Senktide are expressed in log[M]; n = 6.

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To assess a potential NK-2 receptor contribution, we examined the ASM contractile responses to NKA, a potent, but nonselective NK-2 agonist(20, 21). As seen in Fig. 6, NKA is a potent agonist when administered in the presence of PHOS, NEO, and ATR. However, in contrast to SP, there were no significant regional differences to NKA in either the Tmax responses (TSM: 87.9± 2.4% carbachol versus BSM: 93.6 ± 2.6% carbachol,p = 0.11) or log ED50 values (TSM: -8.18 ± 0.07 Mversus BSM: -7.94 ± 0.11 M, p = 0.08).

Figure 6
figure 6

NKA contractile responses in BSM and TSM segments obtained in the presence of 3 × 10-6 M PHOS, 10-5 M NEO, and 10-5 M ATR. Isometric tensions are expressed as a percentage(±SE) of respective near-maximal contractile response to carbachol(10-4 M). Final bath concentrations of NKA are expressed in log [M];n = 10.

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To assess potential regional differences in the distribution of NK-1 receptors, we examined ASM contractile responses to Septide, a highly selective NK-1 agonist(17, 18). As illustrated in Fig. 7, Septide in the presence of 3 × 10-6 M PHOS, 10-5 M NEO, and 10-5 M ATR progressively increased the active tension in BSM segments; whereas in TSM segments, Septide behaved only as a weak agonist. The Tmax response (mean± SE) to Septide was significantly greater (p < 0.001) in BSM segments (78.5 ± 6.0% carbachol) than in the TSM segments (9.1± 3.6% carbachol). Septide was also more potent in BSM segments than in TSM segments after treatment with PHOS, NEO, and ATR as evidenced by their respective mean ± SE log ED50 values of -7.27 ± 0.15 Mversus -6.75 ± 0.20 M (p < 0.05).

Figure 7
figure 7

Comparison of Septide contractile responses in BSM and TSM segments obtained in the presence of 3 × 10-6 M PHOS, 10-5 M NEO, and 10-5 M ATR. Isometric tensions are expressed as a percentage (±SE) of respective near-maximal contractile response to carbachol (10-4 M). Final bath concentrations of agonists are expressed in log [M]; n = 8.

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In view of these regional differences in NK-1 activity, we considered the possibility that NK-1 receptor function might be responsible for the regional differences in SP reactivity. To explore this latter possibility, we first examined the effect of GR 82334, a selective NK-1 antagonist, on the ASM contractile responses to SP obtained in the presence of PHOS, NEO, and ATR. As seen in Fig. 8, the presence of GR 82334 significantly affected the potency, but not the Tmax responses to SP. However, despite the large shift in BSM potency, significant regional differences in SP responsiveness persisted.

Figure 8
figure 8

Comparison of SP contractile responses in BSM and TSM segments after inactivation of NK-1 receptors with GR 82334 (10-6 M). Experiments were conducted in the presence of 3 × 10-6 M PHOS, 10-5 M NEO, and 10-5 M ATR. Isometric tensions are expressed as a percentage (±SE) of respective near-maximal contractile response to carbachol (10-4 M). Final bath concentrations of SP are expressed in log [M]; n = 8.

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In a previous study conducted in adult rabbit airways, we showed that selective inactivation of NK-1 receptors occurred in the continuing presence of Septide, a potent NK-1 agonist(15). In the present study, we found that the NK-1 contractile response could be completely eliminated after pretreating the tissues for prolonged exposure (>15 min) to Septide (data not shown). To further assess the contribution of NK-1 receptors in the airway contractile responses to SP, the SP-induced contractions were subsequently obtained in ASM segments which were pretreated with 10-6 M Septide. The results of these studies are depicted inFig. 9. Here, the ASM contractile responses to SP are normalized to their respective responses to their Tmax responses and plotted as a function of increasing SP bath concentration. We found that the SP dose-response relationship in TSM segments was virtually unaffected by the presence of 10-6 M Septide. In contrast, after inactivation of NK-1 function with Septide, the SP dose-response relationship in BSM segments diminished as evidenced by a significant decrease (p< 0.002) in the mean ± SE Tmax response to SP (69.5± 7.1% carbachol) compared with the corresponding contractile response in TSM segments (91.3 ± 3.4% carbachol). Moreover, SP sensitivity in these BSM segments also decreased significantly (p < 0.01) in the presence of Septide as evidenced by an increase in the log ED50 concentration to -6.56 ± 0.32 M. This observation, together with the finding that SP sensitivity in TSM segments was similar (p = 0.11) in the absence (-6.43 ± 0.08 M) or presence (-6.39 ± 0.09 M) of Septide, indicate that inhibition of NK-1 airway receptors with Septide effectively eliminated the regional differences in SP sensitivity (mean± SE log ED50) noted previously (i.e. -6.56 ± 0.32 M, BSM versus -6.39 ± 0.09 M, TSM; p = 0.60).

Figure 9
figure 9

Comparison of SP contractile responses in BSM and TSM segments after inactivation of NK-1 receptors with 10-6 M Septide. Experiments were conducted in the presence of 3 × 10-6 M PHOS, 10-5 M NEO and 10-5 M ATR. Isometric tensions are expressed as a percentage (±SE) of respective near-maximal contractile response to carbachol (10-4 M). Final bath concentrations of SP are expressed in log [M]; n = 8.

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DISCUSSION

The present study was designed to elucidate the mechanisms underlying SP-induced contraction in immature rabbit airways by quantitatively assessing its bronchoconstrictor actions on BSM and TSM segments. Before inactivation of endogenous NEP and Achase, we found that: 1) SP produced dose-dependent increases in isometric tension in both TSM and BSM segments and2) clear Tmax responses were not attained in BSM segments even at relatively high peptide concentration (3 × 10-5 M). One possible explanation for this latter finding is that local degradation of the peptide resulted in a nonequilibrium state existing between the biophase and the bathing medium(19). With respect to this latter possibility, several investigators have shown that the airway contractile response to SP is augmented in the presence of effective NEP inhibition(16, 22, 23). After inactivation of NEP with PHOS, SP became more sensitive in BSM than in TSM segments and was nearly equipotent with the contractile responses elicted by 10-4 M carbachol. In this connection, it should be noted that the effects of increasing PHOS concentration on SP-mediated contractile responses in 2-wk-old airways are very similar to those we have reported in adult rabbit airways(15). These observations suggest that NEP likely plays an important role in the regulation of SP reactivity in immature airways and that its sensitivity to PHOS inhibition is relatively well conserved with age.

In considering several mechanisms which might account for the apparent regional difference in SP reactivity observed in the presence of PHOS, we first examined the possibility that regional differences in SP-induced constriction might involve a cholinergic mechanism(6). In this connection, it has been shown that AChase 1) can degrade SP(24) and 2) is relatively diminished in immature airways(25). Thus, the regional differences in SP reactivity seen in airway tissues pretreated with PHOS might represent local changes in AChase activity. To examine this latter possibility, we first inhibited endogenous AChase activity with 10-5 M NEO but found that SP remained significantly more potent in BSM than in TSM (Fig. 3). To assess the possibility that differences in postsynaptic cholinergic reactivity might have accounted for the persistence of the regional differences with SP contractility, we conducted additional studies in the presence of PHOS, NEO, and 10-5 M ATR. The persistence of regional airway differences in SP reactivity after effective inhibition of airway cholinergic receptors with ATR supports the notion that the regional differences in SP-mediated contractions cannot be attributed to the peptide's cholinergic component alone. In this respect, there appears to be substantial agreement in the airway responses to SP in these immature airways with those we previously reported in adult airways(15).

Accordingly, we considered the likelihood that differences in SP-mediated contractility could be explained on the basis of regional differences in NK receptor populations. To assess the potential contribution of the NK-3 subtype receptor(18), we examined the airway contractile responses to Senktide, a very selective NK-3 receptor agonist(18). Although we conducted the study only in the presence of PHOS and NEO without ATR, we found that Senktide had very little activity in these 2-wk-old airways. The observation that Senktide has little effect on 2-wk-old rabbit airways suggests that the NK-3 receptor does not play a major role in the regulation of ASM contraction. This finding is in agreement with an earlier report conducted in human bronchi(26). Moreover, when the dose response to Senktide was repeated in the presence of 10-5 M ATR, the weak contractile responses to the agonist was virtually eliminated in both TSM and BSM segments (data not shown). Thus, it appears very unlikely that this particular NK subtype receptor contributes significantly to the observed regional airway differences in SP reactivity.

In contrast to the weak contractile responses to Senktide, NKA was a very potent agonist in both TSM and BSM. Our data support the notion that functional NK-2 receptors exist in both TSM and BSM in 2-wk-old rabbits. On the other hand, in the presence of PHOS, NEO, and ATR, there were no significant regional differences in either the NKA Tmax responses or log ED50 values. Thus, although the NK-2 receptor may contribute to the airway contractile responses to SP, it seems unlikely that activation of the NK-2 receptor can account for regional differences in SP-induced contraction.

These observations, together with the previously noted similarities between immature and adult airway contractile responses to SP, suggested that possibilities that regional differences in NK-1 reactivity might, as we previously found in the adult rabbit model(15), explain the regional differences in SP-mediated contractility in these immature rabbit airways. As a first approximation of this latter hypothesis, we examined the effects of 10-6 M GR 82334, a selective, competitive NK-1 antagonist(27) on the contractile responses to SP obtained in the presence of PHOS, NEO, and ATR. We found that GR 82334 had a profound inhibitory effect on BSM contractility to SP, but had only a modest inhibitory action on the contractile responses to SP in TSM segments. We considered that higher concentrations of GR 82334 might have eliminated the regional differences to SP altogether, but the peptide's inhibitory action on NK-2 function at higher concentrations (data not shown), together with the prohibitive cost of these higher peptide concentrations, caused us to consider an alternative method to assess NK-1 contribution in these tissues.

In the presence of PHOS, NEO, and ATR, we found that Septide, a highly selective NK-1 agonist was a potent agonist in BSM, but not in TSM segments. This observation confirms our earlier report of regional NK-1 differences in adult rabbit airways(15), Using an approach developed in adult rabbit airways, we found that these regional differences in SP sensitivity could also be eliminated after inactivation of the NK-1 receptor with Septide (data not shown)(15).

After inactivation of the NK-1 receptor using Septide, we found that the TSM contractile responses to SP did not change significantly. In contrast, de-activation of NK-1 function in BSM segments with 3 × 10-6 M Septide produced a significant “rightward” shift in the SP dose-response relationship. As a result, the regional differences in SP responsiveness were eliminated after inactivation of the NK-1 receptor.

In conclusion, the present study was concerned with assessing the contractile action of SP in airways isolated from 2-wk-old rabbits and with elucidating the mechanisms underlying its bronchoactive role. Our results show that 1) inhibition of endogenous NEP significantly increases SP contractility in both TSM and BSM, and after inhibition of NEP, SP reactivity is greater in BSM than in TSM; 2) regional differences in SP sensitivity are not due to cholinergic influences; 3) neither the weak NK-3 activity in these airways nor the equipotent contractile responses to NKA can account for the regional contractile differences to SP;4) in contrast, NK-1 activity was significantly greater in BSM than TSM segments; and 5) inactivation of NK-1 receptors with either GR 82334, a competitive NK-1 antagonist or NK-1 receptor inactivation with Septide, significantly reduced or eliminated the regional airway differences in SP sensitivity. These findings, together with previous observations in adult airways(14, 15, 22), strongly support the notion that the regional differences in SP reactivity in 2-wk-old rabbit airways are the result of regional differences in NK-1 receptor function. This study, when considered in the light of different mechanisms which may affect airway reactivity, further suggests that both peptide metabolism and NK receptor heterogeneity may play important roles in modulating SP-induced contractions in immature airways.