Am J Physiol Lung Cell Mol Physiol 301(1): L91-L98

PMC: PMC3129899

PMID: 21515660

Neurokinin-neurotrophin interactions in airway smooth muscle

MATERIALS AND METHODS

Rat ASM Cells

The techniques for isolating ASM cells have been described previously (41, 50). All procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee. Briefly, following an overdose of adult Sprague Dawley rats (200–250 g body wt) with 120 mg/kg pentobarbital sodium, the trachea and lungs were rapidly excised and placed in oxygenated Krebs physiological buffer. The first- and second-generation bronchi were then excised, cleaned of excess debris, and cut longitudinally to expose the airway lumen. The epithelial layer was then removed by blunt dissection, after which the ASM layer was excised. The ASM samples were finely minced in ice-cold Ca^{-free Hanks' balanced salt solution (“0” Ca}^{HBSS), and cells were isolated using collagenase (}41, 50). Following ovomucoid/albumin separation, cell pellets were resuspended in DMEM-F-12 medium with 10% FBS (DMEM complete), centrifuged, resuspended, and seeded overnight in culture flasks or eight-well cover glass-bottomed LabTek dishes in DMEM complete maintained at 37°C (5% CO₂-95% air). Cells were serum deprived for at least 24 h, and experiments were performed within 48 h thereafter. For Western analysis, cells were grown in 100-mm plastic petri dishes. To verify that the cell isolation and maintenance procedure did not result in dedifferentiation, periodic quality control studies were performed to verify sustained expression of smooth muscle markers such as myosin heavy chain and smooth muscle actin, and lack of other markers such as fibroblast-specific protein and caldesmon.

Western Analysis

Rat ASM cells from petri dish cultures were used for measurement of BDNF across different experimental protocols. Cells were rinsed two times with HBSS, harvested, and subjected to sonication for 5 s in cell lysis buffer (Cell Signaling Technologies, Beverly, MA) containing 1 μM phenylmethylsulfonyl fluoride, and the resultant supernatant was assayed for protein using the DC protein assay (Bio-Rad, Hercules, CA). Samples were then subjected to SDS-PAGE using the Criterion Gel System (Bio-Rad) and a 4–15% gradient gel. The gels were run at a current of 200 V for 60 min followed by transfer to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked for 1 h in 5% nonfat dry milk in TBS containing 0.1% Tween 20 followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h. Blots were then visualized by exposure to BioMax XAR film (Eastman Kodak, Rochester, NY) using Supersignal West Dura substrate (Pierce Biotechnology, Rockford, IL).

Oxygen Exposures

Rat ASM cells were divided into two groups based on oxygen levels during the cell culture/maintenance process: normoxia (21% O₂-5% CO₂, as in Rat ASM Cells) and hyperoxia (50% O₂-5% CO₂). Although in previous studies we have used a very high concentration of oxygen (95%), we selected a lower concentration to more realistically represent current clinical standards for the use of high inspired oxygen in the neonatal intensive care unit (16).

For the hyperoxia group, oxygen concentrations were continuously monitored while a mixture of room air and 100% O₂ was introduced in the cell culture incubator to maintain an average concentration of 50% O₂. To ensure that normobaric conditions were maintained with continued flow of gases in the chamber, a commercial barometer was used to verify maintenance of pressure levels (97.8 ± 2 kPa locally). Cells were maintained at normoxia or hyperoxia for 24 h (see specific protocols).

[Ca^]_i Imaging

The techniques for [Ca^]_i imaging of ASM cells have been described previously (40). Rat ASM cells were incubated in 5 μM fura 2-AM (Invitrogen) and visualized using a real-time fluorescence imaging system (MetaFluor; Universal Imaging, Downingtown, PA) on a Nikon Diaphot inverted microscope equipped with a Micromax 12-bit camera system (Princeton Instruments) and Lambda 10–3 shutter system. Fura 2 was alternately excited at 340 and 380 nm approximately every 0.75 s (1.33 Hz), and emissions at 510 nm were used to calculate [Ca^]_i as previously described (3). Cells were perfused with HBSS, and the baseline [Ca^]_i level was established in ∼25 cells/protocol using software-defined regions of interest.

Statistical Analysis

Pair-wise comparisons were performed across groups using two-way ANOVA with repeated comparisons and a Bonferroni correction as appropriate. For statistical analysis, “n” values represented numbers of animals. Unless stated otherwise, cells were derived from 5 animals, with at least 20 cells/animal being tested within each protocol. Separate sets of cells were used for each protocol. Statistical significance was tested at the P < 0.05 level. Values are reported as means ± SE.

Rat ASM Cells

Western Analysis

Oxygen Exposures

[Ca^]_i Imaging

Statistical Analysis

RESULTS

NK Receptors, BDNF, and TrkB in Rat ASM Cells

Western analyses of rat ASM cells (n = 5) demonstrated expression of both NK₁ and NK₂ receptors (sc-15323, sc-28951; 1 μg/ml; Santa Cruz). Furthermore, full-length TrkB receptors (AB51190, 1 μg/ml; Abcam) were detected in rat ASM cells (Fig. 1A). Western analysis demonstrated baseline presence of BDNF (AB3655, 1 μg/ml; Abcam). Overnight (24 h) exposure to 10 nM SP significantly increased the level of BDNF (P < 0.05; Fig. 1A) as well as that of full-length TrkB receptors (P < 0.05; Fig. 1A). Blockade of NK₂ receptors using MEN-10376 (1 μM) prevented the SP-induced increase in BDNF expression by rat ASM cells, whereas NK₁ receptor antagonism (RP-67580; 5 nM) had no effect (P < 0.05 for NK₂ receptor antagonist; Fig. 1A).

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Fig. 1.

Neurokinins (NKs) and neurotrophins in rat airway smooth muscle (ASM) cells. A: rat ASM cells normally express the neurotrophin brain-derived neurotrophic factor (BDNF) and its high-affinity full-length (FL) receptor tropomyosin-related kinase (TrkB). Exposure (24 h) to 10 nM substance P (SP) significantly increased BDNF and TrkB expression in rat ASM cells, an effect prevented by blockade, especially of NK₂ receptors (NK2R) (MEN-10376; 1 μM), compared with NK₁ receptor (NK1R) blockade (RP-67580; 5 nM). B: isolated rat ASM cells maintained under conditions of normoxia (21% O₂-5% CO₂) expressed both NK₁ and NK₂ receptors as well as TrkB FL. Exposure to 50% hyperoxia (5% CO₂, isobaric conditions; 24 h) did not substantially alter NK₁ or NK₂ receptor expression, whereas BDNF and TrkB FL expression was increased significantly. Western blots are representative of data from 4–5 animals. Values are means ± SE. P < 0.05, significant SP or hyperoxia effect (*) and significant inhibitor effect (#). White spaces between blots represent noncontiguous gel lanes.

Overnight exposure of rat ASM cells to 50% hyperoxia did not substantially alter the expression of NK₁ or NK₂ receptors (Fig. 1B). In contrast, expression of BDNF as well as TrkB was significantly increased by hyperoxia compared with normoxic controls (P < 0.05; Fig. 1B).

[Ca^]_i Responses to Agonists

In fura 2-loaded ASM cells not exposed to SP (24 h) or BDNF (30 min) (i.e., controls), exposure to 1 μM ACh resulted in a typical [Ca^]_i response with a higher peak Ca^{followed by decline to a lower plateau level (}Fig. 2, A and B).

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Fig. 2.

SP effects on intracellular Ca^{concentration ([Ca}^]_i) regulation in rat ASM cells. A: in ASM cells loaded with the fluorescent, ratiometric Ca^{indicator fura 2, exposure to 1 μM ACh resulted in a typical [Ca}^]_i response with a higher peak Ca^{followed by decline to a lower plateau level above baseline. Exposure (24 h) to 10 nM SP significantly increased peak and plateau [Ca}^]_i responses. Blockade of NK₂ receptors (MEN-10376, 1 μM) prevented SP-induced enhancement of [Ca^]_i peak and plateau responses (B) while application of NK₁ receptor antagonist (RP-67580, 5 nM) had no significant effect. Tracings in A are representative of data from 5 animals. Values are means ± SE. P < 0.05, significant SP effect (*) and significant inhibitor effect (#).

Effect of SP.

Overnight (24 h) exposure to 10 nM SP significantly increased the amplitude (peak Ca^{relative to baseline) and plateau (relative to baseline) [Ca}^]_i responses to 1 μM ACh (n = 5 animals; Fig. 2). Blockade of NK₁ receptors using RP-67580 (5 nM) had minimal effect on enhancement of the [Ca^]_i response to 1 μM ACh following prior SP exposure, whereas MEN-10376 (NK₂ receptor antagonist, 1 μM) prevented this increase (P < 0.05; Fig. 2B). In cells not exposed to SP, neither NK receptor antagonist had any significant effect on [Ca^]_i responses to ACh (Fig. 2B).

Mechanisms of SP action.

Previous studies have already established that [Ca^]_i regulation in ASM involves both Ca^{influx and intracellular Ca}^{release (}12, 25, 27, 36). Accordingly, in separate experiments (n = 5), the relative contribution of influx vs. release in enhancement of [Ca^]_i following 24 h of SP exposure was examined. Following 24 h exposure to 10 nM SP or vehicle only, ASM cells were loaded with fura 2, and baseline [Ca^]_i levels were recorded. Extracellular Ca^{was then removed by exposure to zero Ca}^{HBSS for 5 min and then 1 μM ACh was added in the continued absence of extracellular Ca}^{. In ASM cells not exposed to SP (vehicle controls), removal of extracellular Ca}^{substantially blunted the [Ca}^]_i response to ACh (P < 0.05; Fig. 3, A and B), indicating a large influx component. Prior 24 h exposure to SP only slightly enhanced the response to ACh in the absence of extracellular Ca⁽Fig. 3B).

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Fig. 3.

Role of Ca^{influx in SP enhancement of [Ca}^]_i responses to ACh. A: in ASM cells, prior removal of extracellular Ca^{substantially blunted the subsequent [Ca}^]_i response to ACh, reflected by a decrease in the peak amplitude (B). This effect was greater in cells exposed to SP, indicating SP effects being largely mediated by enhanced influx, with partial effects on sarcoplasmic reticulum (SR) Ca^{release [reflected by a maintained significant difference (albeit smaller) in peak [Ca}^]_i responses even in 0 extracellular Ca⁽B)]. In a separate set of experiments, removal of extracellular Ca^{following the peak [Ca}^]_i response to ACh was used to determine the contribution of influx (C). Compared with cells not exposed to SP, removal of extracellular Ca^{resulted in a greater reduction of the plateau Ca}^{levels (reflecting influx) in cells exposed to SP (}D). Values are means ± SE. P < 0.05, significant effect of SP (*) and significant effect of 0 Ca^{Hanks' balanced salt solution (HBSS; †).}

To further verify that enhanced [Ca^]_i following prior SP exposure involves influx [rather than sarcoplasmic reticulum (SR) Ca^{release], two sets of additional experiments were performed (}n = 5). In the first set of studies in cells exposed overnight (24 h) to vehicle or SP, [Ca^]_i responses to ACh in the presence of 2 mM extracellular Ca^{were initiated. When the [Ca}^]_i levels had already peaked, extracellular Ca^{was removed rapidly, and the extent of reduction in Ca}^{levels to a plateau level was determined (as an indicator of the influx component). We found that removal of extracellular Ca}^{produced a greater reduction in [Ca}^]_i levels during the plateau phase in cells previously exposed to SP compared with vehicle controls (P < 0.05; Fig. 3, C and D).

In the second set of experiments in cells exposed overnight (24 h) to vehicle or SP, the SR was functionally isolated using 1 mM LaCl₃ for 5 min (to nonspecifically inhibit plasma membrane Ca^{fluxes), and [Ca}^]_i responses to ACh were evaluated. As expected in both groups of cells, the lack of influx resulted in overall smaller [Ca^]_i responses to ACh (Fig. 4A). However, the effect of SP on peak or plateau [Ca^]_i levels was now absent (Fig. 4A), suggesting that most of the effect of enhanced [Ca^]_i responses following prior exposure to SP involves Ca^influx.

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Fig. 4.

Mechanisms of enhanced influx by SP. In ASM cells, nonspecific blockade of plasma membrane Ca^{fluxes with 1 mM lanthanum chloride significantly reduced both peak and plateau responses to ACh. The effect of La}^{was greater in cells exposed to SP (}A), suggesting that SP effects were mediated via influx. Separately, the extent of store-operated Ca^{entry (SOCE) was evaluated using previously described techniques (}3) and showed enhanced SOCE in SP-exposed cells (verified by a substantial decrease in influx with 1 μM La^{) (}B). SP effect on SOCE involves increased expression of STIM1 and TRPC3 (but not Orai1) (C). Values are means ± SE. P < 0.05, significant effect of SP (*) and significant effect of La^{(#). White spaces between blots represent noncontiguous gel lanes.}

To determine the contribution of store-operated Ca^{entry (SOCE) to SP effects, we used previously published protocols (}3, 40) to deplete SR Ca^{stores using cyclopiazonic acid, and induce SOCE. The extent of SOCE was increased significantly in ASM cells exposed previously to SP for 24 h (}P < 0.05; Fig. 4B). Corroborating Western analysis of rat ASM cell lysates demonstrated increased expression of STIM1 (Novus NB110–60547) and TRPC3 (Alomone ACC016), proteins involved in SOCE (37, 57) (P < 0.05; Fig. 4C), but not Orai1 (Alomone ACC060). To rule out any confounding effects of SP on muscarinic receptor expression, we verified that the M₃ subtype was unaltered by overnight exposure to SP (data not shown).

Role of BDNF.

A premise of our study was that ASM cells produce and secrete BDNF that then has autocrine effects (mediated via TrkB) in terms of [Ca^]_i regulation. To verify this, ASM cells were exposed overnight (24 h) to the tyrosine kinase inhibitor K252a (100 nM; to prevent downstream TrkB signaling) or to the BDNF chelating chimeric compound TrkB-F_c (1 μg/ml; to prevent autocrine effect of secreted BDNF). Under these conditions, [Ca^]_i responses to 1 μM ACh were slightly smaller than control ASM cells, suggesting a baseline BDNF release (however, statistical significance was not achieved; Fig. 5A). However, in ASM cells exposed overnight (24 h) to 10 nM SP, where K252a or TrkB-F_c was additionally present (introduced 30 min before SP), the enhancing effect of SP on [Ca^]_i responses to ACh was blunted significantly by either compound (P < 0.05; n = 5; Fig. 5A), suggesting SP-induced release of BDNF.

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Fig. 5.

Role of BDNF in SP effects on [Ca^]_i responses. A: in ASM cells, inhibition of tyrosine kinase activity using K252a, or chelation of extracellular BDNF (presumably released from ASM cells) using TrkB-F_c, resulted in small reductions of [Ca^]_i responses to ACh. These data suggest that baseline BDNF release and activity are small. However, in cells exposed to SP (where [Ca^]_i responses were enhanced), both K252a and TrkB-F_c substantially blunted the effect of SP, suggesting enhanced BDNF release following SP. B: based on the finding that SP increases TrkB expression (Fig. 1), the effect of exogenous BDNF (30 min acute exposure) was determined to be enhanced in SP-exposed cells. Values are means ± SE. P < 0.05, significant effect of SP (*), significant BDNF effect (‡), and significant antagonist effect (#).

Based on our finding of increased TrkB expression following SP exposure (Fig. 1), we determined the effect of exogenous BDNF on [Ca^]_i responses. Even in cells not exposed to SP, 30 min exposure to 10 nM BDNF increased baseline [Ca^]_i and subsequent [Ca^]_i responses to ACh compared with cells not exposed to BDNF (P < 0.05; n = 5; Fig. 5B). The enhancing effect of BDNF (30 min) on the [Ca^]_i response was even greater in cells exposed overnight (24 h) to 10 nM SP (P < 0.05; Fig. 5B). As expected, K252a significantly blunted BDNF enhancement of [Ca^]_i (P < 0.05; Fig. 5B).

Effect of Hyperoxia on [Ca^]_i Responses

Exposure of rat ASM cells for 24 h to 50% oxygen increased subsequent [Ca^]_i responses to 1 μM ACh (P < 0.05 compared with 21% normoxic controls; n = 5 all groups; Fig. 6). Separately, hyperoxia also potentiated the effects of 10 nM SP (24 h, introduced just before hyperoxia) on [Ca^]_i responses to ACh (P < 0.05; Fig. 6). Furthermore, in hyperoxia-exposed cells, the acute (30 min) effects of BDNF on [Ca^]_i responses to ACh were enhanced (P < 0.05; Fig. 7). Finally, the effects of combining SP (24 h) and BDNF (30 min) were also increased when hyperoxia was introduced (P < 0.05). The presence of K252a or TrkB-F_c during hyperoxia exposure significantly blunted the effects of hyperoxia on [Ca^]_i responses to 1 μM ACh (P < 0.05; Fig. 7). Both inhibitors had substantially smaller (and largely insignificant) effects on [Ca^]_i responses in the absence of hyperoxia or SP.

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Fig. 6.

Effect of hyperoxia on [Ca^]_i regulation. Exposure of rat ASM cells for 24 h to 50% oxygen increased subsequent [Ca^]_i responses to 1 μM ACh compared with 21% normoxic controls. Hyperoxia also potentiated the effects of 10 nM BDNF (30 min) or 10 nM SP (24 h, introduced just before hyperoxia) on [Ca^]_i responses to ACh. Hyperoxia also enhanced the effects of combining SP and BDNF on [Ca^]_i responses. Vertical axis indicates average peak [Ca^]_i response to 1 μM ACh. P < 0.05, significant hyperoxia effect (*) and significant drug effect vs. control (normoxia or hyperoxia) (†). #Significant agonist or inhibitor effect of the two agents used to inhibit BNDF signaling (K252A and TrkB-F_c); the presence of these substances significantly inhibited the BNDF-induced increase on Ca^{response to acetylcholine (over and above the substance P effect).}

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Fig. 7.

Schematic of SP-BDNF interactions in ASM cells. In ASM cells, SP acts especially via NK₂ receptors to enhance [Ca^]_i responses to bronchoconstrictor agonist by enhancing Ca^{influx (e.g., SOCE). Separately, the growth factor BDNF acutely enhances [Ca}^]_i responses to agonist, acting via its high-affinity receptor TrkB. SP-induced enhancement of [Ca^]_i involves increased BDNF production/secretion by ASM cells and enhanced TrkB expression. In the presence of SP, increased BDNF further enhances [Ca^]_i, thus potentially contributing to increased bronchoconstriction. Hyperoxia potentiates such interactions by enhancing the BDNF/TrkB system.

NK Receptors, BDNF, and TrkB in Rat ASM Cells

Fig. 1.

[Ca^]_i Responses to Agonists

Fig. 2.

Effect of SP.

Mechanisms of SP action.

Fig. 3.

Fig. 4.

Role of BDNF.

Fig. 5.

Effect of SP.

DISCUSSION

The present study demonstrates that a positive feedback relationship exists between signaling by the tachykinin SP and the NT BDNF in contributing to enhanced ASM [Ca^]_i. In rat ASM cells, SP acts especially via NK₂ receptors to increase BDNF release and TrkB expression, resulting in enhanced [Ca^]_i responses to agonist via increased Ca^{influx. ASM-derived BDNF has an autocrine effect in further enhancing [Ca}^]_i, thus contributing to increased ASM contractility. Moderate hyperoxia potentiates the effects of SP on BDNF and its further effects on ASM [Ca^]_i regulation. A schematic of our findings is shown in Fig. 7.

Neural regulation of airway tone is an important aspect of airway reactivity throughout life (7, 9, 10). In this regard, the bronchoconstrictive arm of NANC regulation involves tachykinins [neurokinin A and B and SP (19)]. SP can act via a direct contractile effect on ASM NK receptors or potentiate presynaptic ACh release at cholinergic terminals (13, 19). The results of the present study focus on the former mechanism and suggest a role for BDNF in mediating the direct effects of SP on ASM.

BDNF, well-recognized in neuronal growth and development, acts via high-affinity TrkB receptor tyrosine kinase (5, 8, 23, 28) with activation of downstream signaling enzymes and adaptor proteins (8, 28). In addition to such classical genomic effects, BDNF can act rapidly within minutes to regulate events such as synaptic transmission (8, 30, 33, 39), involving changes in [Ca^]_i (29–31). Both BDNF and TrkB have now been identified in a range of lung cell types, including nerves and ASM (recently reviewed in Refs. 43 and 48). For example, BDNF is expressed in airway preganglionic neurons in the brain stem (60), as well as in airway nerves of different species (45, 52). NTs may serve as a link between airway inflammation and airway hyperresponsiveness in diseases such as asthma (17, 34, 35, 42, 49, 53, 54). The mechanisms by which such a link is mediated are still under investigation, but two aspects are relevant here: a potential role for SP and the importance of ASM itself.

The present study demonstrates two effects of SP that serve to increase [Ca^]_i (and thus contractility) of ASM. First, even under conditions of normoxia, with 24 h of exposure, SP acts largely via NK₂ receptors to enhance [Ca^]_i responses to agonist, predominantly via enhanced Ca^{influx. Interestingly, this effect appears to involve altered protein expression (evidenced by changes in TRPC3 and STIM1). Regulation of [Ca}^]_i in ASM involves both SR Ca^{release and Ca}^{influx (occurring via several mechanisms) (}3, 12, 25, 27, 36, 40), with the typical biphasic response to agonists such as ACh represented by an initial peak (SR Ca^{release) and a lower plateau (SR release and Ca}^{influx). SP could therefore influence several mechanisms. In pilot studies, we found that SP does not influence muscarinic receptor expression, suggesting a downstream target for enhancing [Ca}^]_i. Here, SP appears to have a substantial effect on SOCE. This is evidenced by enhanced Ca^{influx in response to store depletion in ASM cells exposed to SP, an effect prevented by a low concentration of La}^{. SP further seems to enhance expression of proteins that are involved in SOCE per se, especially STIM1 (}37, 57). SP may also enhance influx via other mechanisms, since 1 mM La^{(which nonspecifically inhibits Ca}^{influx) produced greater reduction in the plateau phase of [Ca}^]_i responses to ACh following SP exposure. Here, our finding of enhanced TRPC3 expression is significant, since this protein may be involved in SOCE (57) as well as non-SOCE-mediated Ca^{entry (}56). Whether SP affects expression of other TRPC isoforms [e.g., TRPC6, which can interact with TRPC3 (15)] in modulating Ca^{influx remains to be established, with the caveat that species- and cell-specific differences in their expression or contribution to influx need to be considered.}

The second effect of SP is that on BDNF expression and signaling. The link between SP and NTs has been examined only to a limited extent. Furthermore, the focus to date has been on the effects of NGF, which increases SP content in mouse subepithelial sensory nerves (17, 26) and in vagal afferents (11). There is now evidence (including our own) that, in addition to nerves, ASM also expresses NTs as well as Trk and p75NTR receptors. Furthermore, NTs such as BDNF can increase agonist-induced [Ca^]_i and force responses in ASM (40, 42) and enhance the effects of inflammatory cytokines (42). Accordingly, an interplay between SP and NTs can serve to modulate ASM contractility. Here, the effect of SP on ASM expression of BDNF and TrkB is relevant and novel. Considering previous data on BDNF enhancement of ASM [Ca^]_i (40, 42), such increase in BDNF/TrkB expression alone should contribute to the SP-induced increase in [Ca^]_i (regardless of SP effects on other mechanisms). This is evidenced by the blunting of SP effects on [Ca^]_i when BDNF is chelated using TrkB-F_c or BDNF action is suppressed via K252a. These data suggest that SP induces BDNF secretion, which, in turn, has autocrine effects on ASM to enhance [Ca^]_i. Interestingly, in pilot studies, we found that BDNF does not alter SP signaling (e.g., NK receptor expression is unchanged). Thus BDNF appears to be downstream of SP in regulating [Ca^]_i in ASM. The relevance of our findings lies in the enhancement of this relationship with hyperoxia, contributing to the observed increase in [Ca^]_i.

Previous studies have evaluated the effects of hyperoxia on lung expression of NTs and their receptors (58, 59). For example, in 5-day-old rat pups, 1 wk of 95% O₂ exposure increases ASM expression of BDNF and TrkB (but not TrkA; highlighting a role for BDNF, rather than NGF where much previous work has focused) (59). These findings, along with other data showing BDNF expression in airway preganglionic neurons and hyperoxia-induced increase in whole lung ACh content (an effect inhibited by the tyrosine kinase inhibitor K252a), would suggest that the BDNF/TrkB pathway is also involved in the hyperoxia-induced increase in cholinergic outflow to the airways, as demonstrated by Sopi et al. (51) in vitro. Furthermore, in neonatal rats, hyperoxia increases expression of the preprotachykinin gene, which encodes SP, as well as SP itself (2). In turn, SP enhances electrical field stimulation-induced airway contractility. Accordingly, enhanced tachykinin signaling may be important in increased airway reactivity following hyperoxia. While supportive of this conclusion, the results of the present study address the importance and underlying mechanisms of a direct effect of SP on ASM (rather than an indirect effect via enhanced cholinergic outflow). Such effect is mediated via increased Ca^{influx, as well as enhanced production of the NT BDNF. Again, under conditions of hyperoxia, BDNF appears to be downstream of SP such that chelating BDNF or preventing its action substantially blunts the effect of hyperoxia in terms of [Ca}^]_i.

The relevance of our findings lies in the detrimental effects of hyperoxia on the lung, especially early in development. Airway patency is critical for adequate gas exchange in the lung, yet positive airway pressure or hyperoxic conditions, as commonly seen in ventilated patients, can be detrimental to both adults and neonates. Premature birth and neonatal lung injury, especially that induced by exposure to high oxygen concentrations, are associated with chronic neonatal lung injury (or BPD) and higher incidence of childhood asthma. Accordingly, the mechanisms by which hyperoxia influences airway reactivity are a topic of research investigation. Considering the importance of NK signaling in control of airway tone (10, 14, 20, 38), and hyperoxia effects on NK signaling, our novel data showing that both SP and hyperoxia effects involve BDNF point to a potentially new target for interference with mechanisms that enhance airway contractility.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute (NHLBI) R01 Grants HL-56470 (R. J. Martin, Y. S. Prakash, S. I. A. Zaidi) and HL-088029 (Y. S. Prakash) and by a Clinical Innovator Award from the Flight Attendants Medical Research Institute (Y. S. Prakash). A. Stewart was supported via NHLBI Grant R25 HL-092621. L. Meuchel is supported by the Mayo Graduate School, Rochester, MN.

DISCLOSURES

No conflicts of interest are declared by the authors.

Departments of ^{Physiology & Biomedical Engineering and}

^{Anesthesiology, Mayo Clinic College of Medicine, Rochester, Minnesota; and}

^{Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, Ohio}

^{Corresponding author.}

Address for reprint requests and other correspondence: Y. S. Prakash, 4-184 W Jos SMH, Mayo Clinic College of Medicine, Rochester, MN 55905 (e-mail: ude.oyam@sy.hsakarp).

Received 2010 Sep 9; Accepted 2011 Apr 19.

Abstract

Neurally derived tachykinins such as substance P (SP) play a key role in modulating airway contractility (especially with inflammation). Separately, the neurotrophin brain-derived neurotrophic factor (BDNF; potentially derived from nerves as well as airway smooth muscle; ASM) and its tropomyosin-related kinase receptor, TrkB, are involved in enhanced airway contractility. In this study, we hypothesized that neurokinins and neurotrophins are linked in enhancing intracellular Ca^{concentration ([Ca}^]_i) regulation in ASM. In rat ASM cells, 24 h exposure to 10 nM SP significantly increased BDNF and TrkB expression (P < 0.05). Furthermore, [Ca^]_i responses to 1 μM ACh as well as BDNF (30 min) effects on [Ca^]_i regulation were enhanced by prior SP exposure, largely via increased Ca^{influx (}P < 0.05). The enhancing effect of SP on BDNF signaling was blunted by the neurokinin-2 receptor antagonist MEN-10376 (1 μM, P < 0.05) to a greater extent than the neurokinin-1 receptor antagonist RP-67580 (5 nM). Chelation of extracellular BDNF (chimeric TrkB-F_c; 1 μg/ml), as well as tyrosine kinase inhibition (100 nM K252a), substantially blunted SP effects (P < 0.05). Overnight (24 h) exposure of ASM cells to 50% oxygen increased BDNF and TrkB expression and potentiated both SP- and BDNF-induced enhancement of [Ca^]_i (P < 0.05). These results suggest a novel interaction between SP and BDNF in regulating agonist-induced [Ca^]_i regulation in ASM. The autocrine mechanism we present here represents a new area in the development of bronchoconstrictive reflex response and airway hyperreactive disorders.

Keywords: bronchial smooth muscle, oxygen, hyperoxia, brain-derived neurotrophic factor, tropomyosin related kinase, calcium, substance P

Abstract

preterm birth and neonatal lung injury are associated with bronchopulmonary dysplasia (BPD) and a high incidence of childhood asthma (1, 21). While BPD is likely multifactorial in origin, a well-characterized model for BPD is neonatal hyperoxic exposure in rat pups, which is associated with increased cholinergically mediated airway contractile responses in vitro and in vivo (6, 24, 59). In this regard, neurally mediated bronchoconstriction involves cholinergic mechanisms as well as nonadrenergic/noncholinergic (NANC) pathways (9, 44, 55) mediated via subepithelial C-fiber sensory afferents that release tachykinins [neurokinin A and B and substance P (SP) (19)]. SP can elicit a direct contractile effect on airway smooth muscle (ASM) via neurokinin (NK) receptors or potentiate presynaptic ACh release at cholinergic terminals. Thus SP can substantially enhance airway reactivity and contribute to diseases such as asthma or BPD.

Neurotrophins (NTs), polypeptides long known to support neuronal growth and development, are increasingly being identified in nonneuronal tissues, including the lung (4, 26, 47). NTs [such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and their receptors (the high-affinity Trk and the low-affinity p75NTR)] have been identified in a range of cell types within the lung, including immune cells, epithelium, ASM, and nerves (22, 34, 43, 45). There is increasing evidence that NTs may serve as a link between airway inflammation and airway hyperresponsiveness in diseases such as asthma (34). In this regard, by virtue of being growth factors for neurons, NTs could potentially affect neural control of airway tone. NTs and their receptors have been localized to airway innervation (reviewed in Refs. 17 and 18), with BDNF expressed in brain stem airway preganglionic neurons (60). NTs can increase the SP content of sensory nerves (17) and of vagal afferents (32, 46). There is now evidence that, in addition to nerves, ASM also expresses and responds to NTs (43). For example, BDNF increases agonist-induced [Ca^]_i and force responses in ASM (40). Thus ASM appears to be a key target of NTs in their contribution to airway hyperreactivity. Accordingly, an interplay between SP and NTs can serve to modulate not only neural activity but also ASM.

With this background, we previously found that, in 5-day-old rat pups, 1 wk of continuous hyperoxia (95% O₂) increases BDNF and BDNF tropomyosin-related kinase receptor (TrkB) levels, especially in peribronchial smooth muscle, raising the possibility that upregulation of NTs may contribute to airway reactivity in pathophysiological states associated with neonatal lung injury (58, 59). In a physiological correlate, hyperoxia increased electrical field-stimulated contraction (and impaired relaxation) of lung strips in vitro, effects blunted by tyrosine kinase inhibition but enhanced by exogenous BDNF (51).

In the present study, we tested the hypothesis that a positive feedback relationship exists between NKs and NTs in contributing to enhancement of ASM contractility, which underlies the effect of hyperoxia on contractility. Using rat ASM cells and real-time fluorescence intracellular Ca^{concentration ([Ca}^]_i) imaging, we examined the mechanisms by which SP modulates BDNF expression and signaling and, conversely, the effect of BDNF on NK signaling, with the overall effect of such interactions on [Ca^]_i response to bronchoconstrictor agonist in the settings of normoxia vs. hyperoxia.

ACKNOWLEDGMENTS

Some results in this manuscript have been presented in abstract form at the 2010 American Thoracic Society International Meeting in New Orleans, LA.

ACKNOWLEDGMENTS

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Neurokinin-neurotrophin interactions in airway smooth muscle

MATERIALS AND METHODS

Rat ASM Cells

Western Analysis

Oxygen Exposures

[Ca]i Imaging

Statistical Analysis

Rat ASM Cells

Western Analysis

Oxygen Exposures

[Ca]i Imaging

Statistical Analysis

RESULTS

NK Receptors, BDNF, and TrkB in Rat ASM Cells

[Ca]i Responses to Agonists

Effect of SP.

Mechanisms of SP action.

Role of BDNF.

Effect of Hyperoxia on [Ca]i Responses

NK Receptors, BDNF, and TrkB in Rat ASM Cells

[Ca]i Responses to Agonists

Effect of SP.

Mechanisms of SP action.

Role of BDNF.

Effect of SP.

Mechanisms of SP action.

Role of BDNF.

Effect of Hyperoxia on [Ca]i Responses

DISCUSSION

GRANTS

DISCLOSURES

Abstract

ACKNOWLEDGMENTS

REFERENCES

References

[Ca^]_i Imaging

[Ca^]_i Imaging

[Ca^]_i Responses to Agonists

Effect of Hyperoxia on [Ca^]_i Responses

[Ca^]_i Responses to Agonists

Effect of Hyperoxia on [Ca^]_i Responses