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Am J Physiol Lung Cell Mol Physiol 291: L447-L456, 2006. First published April 28, 2006; doi:10.1152/ajplung.00501.2005
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Neurotrophin effects on intracellular Ca2+ and force in airway smooth muscle

Y. S. Prakash,1,2 Adeyemi Iyanoye,2 Binnaz Ay,1 Carlos B. Mantilla,1,2 and Christina M. Pabelick1

1Department of Anesthesiology and 2Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 26 November 2005 ; accepted in final form 21 March 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neurotrophins [e.g., brain-derived neurotrophic factor (BDNF), neurotrophin 4 (NT4)], known to affect neuronal structure and function, are expressed in nonneuronal tissues including the airway. However, their function is unclear. We examined the effect of acute vs. prolonged neurotrophin exposure on regulation of airway smooth muscle (ASM) intracellular Ca2+ concentration ([Ca2+]i): sarcoplasmic reticulum (SR) Ca2+ release and Ca2+ influx (specifically store-operated Ca2+ entry, SOCE). Human ASM cells were incubated for 30 min in medium (control) or 1 or 10 nM BDNF, NT3, or NT4 (acute exposure) or overnight in 1 nM BDNF, NT3, or NT4 (prolonged exposure) and imaged after loading with the Ca2+ indicator fura-2 AM. [Ca2+]i responses to ACh, histamine, bradykinin, and caffeine and SOCE following SR Ca2+ depletion were compared across cell groups. Force measurements were performed in human bronchial strips exposed to neurotrophins. Basal [Ca2+]i, peak responses to all agonists, SOCE, and force responses to ACh and histamine were all significantly enhanced by both acute and prolonged BDNF exposure (smaller effect of NT4) but decreased by NT3. Inhibition of the BDNF/NT4 receptor trkB by K252a prevented enhancement of [Ca2+]i responses. ASM cells showed positive immunostaining for BDNF, NT3, NT4, trkB, and trkC (NT3 receptor). These novel data demonstrate that neurotrophins influence ASM [Ca2+]i and force regulation and suggest a potential role for neurotrophins in airway diseases.

brain-derived neurotrophic factor; neurotrophin 4; neurotrophin 3; sarcoplasmic reticulum; capacitative calcium entry

NEUROTROPHINS ARE MOLECULES originally described on the basis of their effects on neuronal cells (4, 15, 17, 18, 24, 34, 56, 61). In the nervous system, neurotrophins support the development and survival of neurons, controlling differentiation, morphology, function, and gene expression. Neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4) exert their effects by binding to receptors (e.g., p75NTR, trkA, trkB, trkC) (8, 22, 56). The trk proteins are high-affinity tyrosine kinase receptors that autophosphorylate and activate several signaling cascades, whereas the p75NTR is a low-affinity receptor.

There is growing evidence that nonneuronal cells also respond to neurotrophins (32, 33, 48, 49, 53, 55, 60). Recent studies (33, 48, 49, 60) provide the first direct evidence of neurotrophin and neurotrophin receptor expression in different lung components. In this regard, inflammatory cells express neurotrophin receptors (32, 50), raising the possibility that inflammation (e.g., relevant to the airway) is modulated by neurotrophins. Indeed, altered neurotrophin and receptor expression has been observed in pathological lung states such as asthma, allergy, and lung cancer (16, 33, 38, 39, 47, 51, 57) as well as in neonatal bronchial smooth muscle exposed to hyperoxia (60). A recent study found that inflammatory cytokines alter the expression of neurotrophins and their receptors in airway smooth muscle (ASM) (25). However, the mechanisms by which neurotrophins may affect the airway or other components of the respiratory system are still under investigation.

Given that changes in bronchial and bronchiolar smooth muscle are a major component of airway hyperreactivity in diseases such as asthma, the recent work on neurotrophins in the lung raises the exciting possibility that neurotrophins are potential mediators of airway inflammation during asthma and lung injury. ASM tone represents a balance between bronchoconstriction and bronchodilation. In this regard, cytosolic (intracellular) Ca2+ concentration ([Ca2+]i) is a key determinant of ASM tone (21, 41, 52). In the present study, we hypothesized that neurotrophins influence [Ca2+]i regulation in the airway, thus targeting a potential mechanism by which neurotrophins may modulate lung tissue function.

Elevation of [Ca2+]i by bronchoconstrictors such as acetylcholine (ACh) and histamine involves both Ca2+ release from sarcoplasmic reticulum (SR) stores and plasma membrane Ca2+ influx. In ASM, Ca2+ influx occurs through both voltage-gated (59) and receptor-gated (37) channels. In addition, we recently demonstrated (1, 40) the existence of controlled Ca2+ influx in response to SR Ca2+ depletion, thus allowing for replenishment of intracellular Ca2+ stores [store-operated Ca2+ entry (SOCE); also termed capacitative Ca2+ entry; Refs. 42, 45, 46]. In the present study, we examined the presence of neurotrophins and their receptors in human ASM cells and investigated the role of neurotrophins in modulation of two specific mechanisms that contribute to [Ca2+]i responses in ASM: SR Ca2+ release and Ca2+ influx, specifically SOCE. In separate experiments, we examined the effect of neurotrophins on contractile responses of human bronchial smooth muscle to agonists. We selected three neurotrophins (BDNF, NT3, and NT4) that are known to work via different receptors: trkB (BDNF, NT4) and trkC (NT3).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of human ASM cells. Human ASM cells were obtained from Cambrex BioScience (Walkersville, MD) and cultured in a humidified atmosphere of 5% CO2-95% O2 at 37°C with smooth muscle growth medium (SmGM-2; Cambrex) with 5% fetal bovine serum, penicillin, and streptomycin. On reaching 70–90% confluence, cells were passaged, plated for use in experiments, and/or saved in cryopreservation medium for future use. All Ca2+ imaging and gel electrophoresis experiments were performed on cells from less than five passages. For Ca2+ imaging, cells were plated onto borosilicate coverglass-bottomed culture chambers. The presence of smooth muscle cells was confirmed by immunofluorescence for smooth muscle {alpha}-actin. Samples that did not produce [Ca2+]i responses to ACh were excluded from analysis.

Neurotrophin expression in ASM. ASM cells were fixed in 2% formaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 15 min. Cells were then briefly permeabilized with 0.3% Triton-X in 0.1 M PB for 10 min, washed in PB, and incubated for 30 min in 4% normal donkey serum. Cells were then incubated overnight at 4°C in PB only (unstained control), 1 µg/ml rabbit IgG (staining control), or anti-BDNF, anti-NT3, anti-NT4, anti-TrkB, or anti-TrkC (all rabbit host antibodies; Santa Cruz Biotechnology) in PB. Cells were then washed three times in PB and incubated for 2 h in Cy3-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch). Labeled cells were visualized with an Olympus FluoView laser scanning confocal microscope equipped with a Kr laser (568-nm line, emission 590 nm) and a x40 oil immersion objective lens.

Quantitative comparison of fluorescent stains is difficult given confounding factors such as differences in antibody concentration and affinities, nonspecific binding, and tissue autofluorescence. To allow for at least qualitative comparisons across different stains, a fixed laser intensity was selected and photomultiplier gain and background for the confocal imaging system were adjusted until images of the staining control cells had pixel intensities of >10% of maximum. From that point on, laser intensity, gain, and background were fixed, and all other (positively stained) cells were imaged with these parameters. Positive staining was assumed when pixel intensities were >25% of maximum. Adjustment of parameters was performed only when the above staining protocols were repeated on a different day with a separate batch of cells.

Neurotrophin exposure. ASM cells were incubated overnight (prolonged exposure) at 37°C in SmGM-2 growth medium (control), or 1 nM human recombinant BDNF, NT3, or NT4 (R&D Systems) reconstituted in growth medium. The neurotrophin concentration was selected based on pilot experiments in which we found that 100 pM BDNF or NT4 had no significant effect on [Ca2+]i regulation, whereas 10 nM or higher neurotrophin (especially BDNF) resulted in significant cell death with prolonged exposure. For acute exposure, ASM cells already loaded with a Ca2+ indicator (see below) were exposed to 10 nM BDNF, NT3, or NT4 for 30 min at 37°C. In pilot studies, we found that 1 nM BDNF had minimal effects on [Ca2+]i responses to different agonists. Accordingly, only 10 nM neurotrophins were used for acute exposure. Cells were immediately used for [Ca2+]i imaging experiments after acute neurotrophin exposure.

[Ca2+]i imaging. ASM cells were incubated in 5 µM fura-2 AM (Molecular Probes, Eugene OR) and visualized with a real-time fluorescence imaging system (MetaFluor; Universal Imaging, Downingtown, PA) on a Nikon Diaphot inverted microscope (Fryer Instruments, Edina, MN). Cells were initially perfused with HBSS [2.5 mM Ca2+, room temperature (25°C)], and a baseline [Ca2+]i level was established. A custom-built fluid level controller allowed cell perfusion with rapid exchange of perfusate (<300 ms). [Ca2+]i responses of 25 cells per chamber were obtained for individual, software-defined regions of interest. Fura-2-loaded cells were alternately excited at 340 and 380 nm with a Lambda 10-2 filter changer (Sutter Instrument). Fluorescence emissions were collected separately for each wavelength with a 510-nm barrier filter. Images were acquired with a Micromax 12-bit camera system (Princeton Instruments). The ratio of fura-2 emissions when excited at 340 and 380 nm was calculated approximately every 0.75 s (image acquisition rate of 1.33 Hz) and used to calculate [Ca2+]i. [Ca2+]i was calculated from the ratio of intensities at 340 nm and 380 nm by using a calibration curve as previously described (1, 40).

[Ca2+]i response to agonist. ASM cells exposed to medium only (control), BDNF, NT3, or NT4 (acute or prolonged exposure) were loaded with fura-2 AM (as above). Cells were washed in HBSS for 2–3 min to verify cell stability, and extracellular Ca2+ was subsequently removed by exposure to zero-Ca2+ HBSS (5 mM EGTA). The cells were then exposed to 10 nM, 100 nM, or 1 µM ACh in zero-Ca2+ HBSS to evaluate the effect of neurotrophins on SR Ca2+ release. For acute exposure, ACh responses were first evaluated in cells that were then washed and exposed to neurotrophin for 30 min, followed by a repeat ACh exposure (same concentration). Separate sets of cells were used for each neurotrophin and ACh concentration.

In a second set of experiments on ASM cells exposed to medium or neurotrophin (10 nM acute or 1 nM prolonged exposure only), 100 nM, 1 µM, or 10 µM histamine was used instead of ACh. In a third set of experiments, 1 nM, 10 nM, or 100 nM bradykinin was used.

[Ca2+]i response to caffeine. Control and BDNF-, NT3-, or NT4-exposed cells loaded with fura-2 were washed in HBSS and exposed to zero-Ca2+ HBSS for at least 5 min. Cells were then exposed to 5 mM caffeine to induce SR Ca2+ release via ryanodine receptor (RyR) channels.

Store-operated Ca2+ entry. The protocol for activation of SOCE in ASM was published recently by our group (1, 40). As in previous studies, ASM cells (control, BDNF, NT3, or NT4) were perfused with HBSS, followed by removal of extracellular Ca2+ by exposure to zero-Ca2+ HBSS for 5 min. There is certainly evidence for non-SOCE Ca2+ influx in ASM occurring via L-type Ca2+ channels (59). Therefore, to ensure that L-type Ca2+ channels were not activated during the SOCE protocol, cells were also exposed to 1 µM nifedipine and 10 mM KCl (clamping membrane potential) in zero-Ca2+ HBSS. Cells were then rapidly exposed to 1 µM cyclopiazonic acid [CPA; inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)] in zero-Ca2+ HBSS (and nifedipine and KCl), resulting in passive SR depletion with continued SR Ca2+ leak [likely from both inositol 1,4,5-trisphosphate (IP3)- and ryanodine-sensitive SR stores]. As in previous studies (1, 40), a gradual elevation of [Ca2+]i was typically noted that eventually reached a plateau or started trending downward (because plasma membrane Ca2+ efflux was not inhibited). At this point, HBSS (with 2.5 mM Ca2+) was rapidly reintroduced (in the continued presence of CPA, nifedipine, and KCl) and the observed [Ca2+]i response was measured. For acute neurotrophin exposure, a control SOCE response was first performed, CPA, nifedipine, and KCl were then washed off with HBSS (15 min), and cells were exposed to neurotrophin for 30 min. The SOCE protocol was then repeated.

Effect of neurotrophin receptor blockade. To determine whether the effects of neurotrophins on [Ca2+]i were mediated via their receptors, ASM cells were first exposed to 100 nM K252a for 30 min, followed by acute exposure to medium or neurotrophin (BDNF, NT3, or NT4). Previous studies found K252a to specifically inhibit tyrosine-specific protein kinases (26). We recently (35) used this substance to demonstrate neurotrophin effects via trk receptors in diaphragm muscle. The protocols for ACh (1 µM only), caffeine, and SOCE were then performed as described above. In pilot studies, we found that overnight exposure to K252a resulted in significant cell loss (due to unclear reasons), as well as a significantly elevated [Ca2+]i level in many cells. Therefore, we did not examine whether K252a exposure reversed alterations in [Ca2+]i regulation following overnight neurotrophin exposure.

Effect of neurotrophins on force response. Human bronchi were obtained from specimens incidental to patient surgery and discarded by surgical pathology (approved by the Mayo Foundation Institutional Review Board). Airway tissues were immersed in ice-cold HBSS, and smooth muscle was freed from adherent tissue with a dissecting microscope. Samples for force measurements were excised, cleared of adventitia, suspended in force transducer-based tissue organ baths between platinum plate electrodes, and constantly perfused with PSS at 37°C. Optimal length for contraction was determined by repeated exposure to 1 µM ACh with intervening washes. A final 1 µM ACh contraction was performed for comparison to subsequent contractions. Strips were then exposed to 1 µM ACh or to 100 µM histamine. For "acute" exposure, ACh or histamine contractions were verified in strips at optimal length, and the strips were then washed with PSS and exposed to 10 nM BDNF, NT3, or NT4 for 1 h, followed by a second contraction to ACh or histamine. Force responses before and after neurotrophin exposure were compared.

Statistical analysis. Comparisons were performed across groups with independent Student's t-test or two-way ANOVA as appropriate. Repeated comparisons were tested with a Bonferroni correction. At least 10 cells per group or 3 ASM strip samples were used in each comparison. Statistical significance was tested at the P < 0.05 level. Values are reported as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Neurotrophin expression in ASM. Paraformaldehyde-fixed and permeabilized human ASM cells displayed significant immunostaining for BDNF as detected by Cy3 fluorescence imaging (Fig. 1). There was considerable membrane staining for BDNF, with additional patchy intracellular and significant nuclear areas of bright fluorescence. Compared with the intense staining for BDNF, ASM cells showed mostly nuclear immunostaining for NT4, with considerably weaker membrane staining, but considerably above background (Fig. 1). Although NT3 was also localized to nucleus, there was much stronger membrane staining compared with NT4. The common, high-affinity receptor for BDNF and NT4, trkB, was detected at both plasma membrane and nucleus. In some cells, patchy cytosolic staining (above background) was also detected (Fig. 1). trkC was mostly localized to membrane and cytosol.


Figure 1
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  		Fig. 1. Expression of neurotrophins in human airway smooth muscle (ASM) cells. Fixed and permeabilized ASM cells were immunostained for brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), as well as the receptor-associated tyrosine kinases TrkB and TrkC, and visualized with a Cy3-conjugated secondary antibody and epifluorescence microscopy. Cells exposed to isotype immunoglobulin instead of primary antibody are shown as staining control. Background level was set with unstained cells. Bar, 50 µm.

 
Neurotrophin effects on [Ca2+]i response to ACh. In control ASM cells, exposure to ACh typically resulted in a characteristic "biphasic" [Ca2+]i response, with an initial, significantly higher peak followed by a sustained elevation significantly above baseline (representative tracings in Fig. 2). ACh at 100 nM resulted in increased peak and plateau [Ca2+]i compared with 10 nM (P < 0.05); however, only peak [Ca2+]i level increased with 1 µM ACh compared with 100 nM ACh (P < 0.05; data not shown).


Figure 2
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  		Fig. 2. Representative tracings of intracellular Ca2+ concentration ([Ca2+]i) responses of ASM cells to acetylcholine (ACh, A), histamine (B), and bradykinin (C) after acute exposure to neurotrophins. [Ca2+]i responses were first evaluated in separate groups of ASM cells by exposure to 1 µM ACh, 10 µM histamine, or 1 nM bradykinin. After a wash in HBSS, cells were exposed to 10 nM BDNF, NT3, or NT4 in HBSS for 30 min and [Ca2+]i responses to the same agonist were reevaluated.

 
Baseline [Ca2+]i levels in ASM cells ranged between 80 and 110 nM (84 ± 9 nM). Acute exposure (30 min) to 10 nM BDNF resulted in a slow and significant increase in baseline [Ca2+]i levels (95–166 nM, 129 ± 19 nM for 10 nM; P < 0.05). Subsequent exposure to 100 nM or 1 µM ACh in zero extracellular Ca2+ resulted in significantly higher peak and plateau [Ca2+]i responses compared with control cells exposed to medium only (Fig. 2; summary data of peak responses in Fig. 3). Compared with these effects of BDNF, acute exposure to 10 nM NT4 had a small but significant enhancing effect on the [Ca2+]i response to 1 µM ACh (Figs. 2 and 3). In contrast to BDNF and NT4, acute exposure to 10 nM NT3 resulted in a decrease in the [Ca2+]i response to ACh (Figs. 2 and 3).


Figure 3
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  		Fig. 3. Effect of acute neurotrophin exposure on [Ca2+]i response to agonist. Exposure to 10 nM BDNF, and to a lesser extent NT4, resulted in enhanced [Ca2+]i responses to ACh (A), histamine (B), and bradykinin (C). In contrast, NT3 blunted [Ca2+]i responses. There was no clear agonist concentration-dependent trend in neurotrophin effects. *Significant difference from control; #significant difference from BDNF. Values are means ± SE.

 
Overnight exposure to 1 nM BDNF resulted in a significantly elevated baseline [Ca2+]i level (88–174 nM, 139 ± 17 nM) compared with control cells exposed overnight to medium only (94–122 nM, 107 ± 9 nM; P < 0.05). Subsequent exposure to 10 nM, 100 nM, or 1 µM ACh in zero extracellular Ca2+ resulted in significantly greater peak as well as plateau [Ca2+]i responses (summary of peak responses shown in Fig. 4). Compared with acute BDNF exposure, these effects of prolonged exposure were greater at lower ACh concentrations (P < 0.05). Compared with BDNF, overnight exposure to NT4 increased peak and plateau [Ca2+]i responses to 100 nM and 1 µM ACh to a lesser extent; however, these effects were significantly more pronounced than acute NT4 exposure at the same ACh concentration (P < 0.05). In contrast to these effects of BDNF and NT4, overnight exposure to 1 nM NT3 significantly decreased the [Ca2+]i response to ACh at all concentrations (P < 0.05; Fig. 4).


Figure 4
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  		Fig. 4. Effect of prolonged neurotrophin exposure on [Ca2+]i response to agonist. Exposure to 1 nM BDNF or NT4 resulted in significant enhancement of [Ca2+]i responses to ACh (A), histamine (B), and bradykinin (C). As with acute exposure, NT3 blunted [Ca2+]i responses. Overall prolonged neurotrophin effects even at the lower concentration of 1 nM were greater than those with 10 nM acute exposure (significances not shown). *Significant difference from control; #significant difference from BDNF. Values are means ± SE.

 
Neurotrophin effects on [Ca2+]i response to histamine. In control ASM cells, histamine produced a transient [Ca2+]i elevation. The peak of the [Ca2+]i response increased with increasing histamine concentration (data not shown). Compared with control cells, acute exposure (30 min) to 10 nM BDNF and NT4 both resulted in significantly increased peak [Ca2+]i response to all concentrations of histamine (Fig. 3). As with ACh, in contrast to BDNF and NT4, acute exposure to 10 nM NT3 resulted in a decrease in the [Ca2+]i response to histamine at 1 and 10 µM (Fig. 3). With overnight exposure, 1 nM BDNF and NT4 both increased [Ca2+]i responses to histamine at all concentrations, whereas NT3 decreased the responses at all histamine concentrations (P < 0.05; Fig. 4).

Neurotrophin effects on [Ca2+]i response to bradykinin. Bradykinin, like histamine, produced transient [Ca2+]i responses that increased with agonist concentration. Compared with control, acute BDNF or NT4 exposure (30 min) significantly increased peak [Ca2+]i response to all concentrations of bradykinin (Fig. 3), whereas acute exposure to NT3 significantly decreased the [Ca2+]i response to 1 and 10 nM bradykinin (Fig. 3). With overnight exposure, BDNF and NT4 increased [Ca2+]i responses to bradykinin at all concentrations, whereas NT3 decreased the responses at all agonist concentrations (P < 0.05; Fig. 4).

Neurotrophin effects on [Ca2+]i response to caffeine. The typical transient [Ca2+]i response to 5 mM caffeine was noted in ASM cells from all groups. Acute exposure to 10 nM BDNF (but not 1 nM) followed by 5 mM caffeine resulted in significantly higher peak [Ca2+]i response compared with control (Fig. 5). Compared with BDNF, acute exposure to 10 nM NT4 had no effect on the [Ca2+]i response to caffeine. In contrast, 10 nM NT3 significantly decreased the [Ca2+]i response to caffeine. Prolonged (overnight) exposure to 1 nM BDNF resulted in a significantly greater [Ca2+]i response to caffeine compared with both control and acute BDNF exposure (P < 0.05; Fig. 5). Compared with BDNF, prolonged NT4 exposure did not significantly increase the peak of the [Ca2+]i response to caffeine. Indeed, in some cells NT4 exposure resulted in a smaller caffeine response. In contrast to BDNF, overnight exposure to NT3 decreased the [Ca2+]i response to caffeine (P < 0.05, Fig. 5).


Figure 5
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  		Fig. 5. Effects of acute and prolonged exposure to neurotrophins on [Ca2+]i response to caffeine. ASM cells were exposed for 30 min to 1 or 10 nM BDNF, NT4, or NT3 (acute exposure, A) or overnight to 1 nM neurotrophin (B). Peak [Ca2+]i responses to 5 mM caffeine were then evaluated. Although both acute and prolonged BDNF exposure enhanced caffeine responses, NT3 blunted the [Ca2+]i transients. *Significant difference from neurotrophin exposure control; #significant difference from BDNF (P < 0.05).

 
Neurotrophin effects on SOCE. Reintroduction of extracellular Ca2+ after SR Ca2+ depletion by CPA in zero Ca2+ resulted in SOCE in all cell groups. In control cells, the amplitude of SOCE was >50% of the CPA response, considerably higher than that reported in our earlier studies in porcine ASM (1). Acute exposure to 1 and 10 nM BDNF both resulted in significant increases in the extent of SOCE compared with control (Fig. 6). In contrast, acute exposure to NT4 had no effect, whereas both 1 and 10 nM NT3 inhibited SOCE. Prolonged exposure to 1 nM BDNF or NT4 resulted in a significantly greater SOCE compared with both control and acute BDNF exposure (P < 0.05; Fig. 6). Compared with BDNF, the effect of NT4 was smaller. In contrast, NT3 inhibited SOCE (P < 0.05, Fig. 6).


Figure 6
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  		Fig. 6. Effect of acute and prolonged exposure to neurotrophins on store-operated Ca2+ entry (SOCE). A: extracellular Ca2+ was removed by exposure to zero-Ca2+ HBSS (representative tracing shown). Potential Ca2+ influx via voltage-gated Ca2+ channels was minimized by exposure to nifedipine and KCl (to clamp membrane potential). Sarcoplasmic reticulum (SR) Ca2+ was then depleted by exposure to cyclopiazonic acid (CPA). In the continued presence of CPA, extracellular Ca2+ was reintroduced to trigger SOCE. NT, neurotrophin. B: to examine acute effects of neurotrophin, 1 or 10 nM BDNF, NT4, or NT3 was introduced for 30 min after SR Ca2+ was depleted with CPA. BDNF, but not NT4, increased SOCE. In contrast, NT3 inhibited SOCE. CTL, control. C: in ASM cells exposed overnight to neurotrophins, SOCE (triggered as in control cells) was significantly increased by both BDNF and NT4 but inhibited by NT3. *Significant difference from neurotrophin exposure control; #significant difference from BDNF (P < 0.05).

 
Effect of neurotrophin receptor blockade. Preexposure to 100 nM K252a for 30 min followed by acute exposure to 10 nM BDNF substantially decreased BDNF-induced enhancement of [Ca2+]i responses to 1 µM ACh and caffeine and that of SOCE after CPA (Fig. 7). The acute effects of NT4 on responses to ACh, caffeine, or SOCE following SR Ca2+ depletion were not significantly different from control in the presence of K252a. In contrast, the effects of NT3 on ACh and caffeine were inhibited by preexposure to K252a.


Figure 7
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  		Fig. 7. Effect of K252a on neurotrophin enhancement of [Ca2+]i responses. ASM cells were preexposed to the Trk inhibitor K252a at 100 nM for 30 min, followed by acute exposure to 10 nM BDNF or NT4. K252a substantially reversed neurotrophin-induced enhancement (BDNF, NT4) as well as inhibition (NT3) of [Ca2+]i responses (if present) to 1 µM ACh and caffeine and that of SOCE following CPA. *Significant effect of K252a (P < 0.05).

 
Effect of neurotrophins on force response. Human ASM strips stretched to optimal length produced sustained force responses to 1 µM ACh. Within a strip, compared with the control response to ACh or histamine, 1-h exposure to 10 nM BDNF significantly enhanced the force response to subsequent exposure to ACh or histamine. However, NT4 effects were inconsistent, with increased force responses to histamine in two strips but to ACh only in one strip. In contrast, NT3 consistently decreased the force response to ACh and histamine (P < 0.05; Fig. 8).


Figure 8
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  		Fig. 8. Effect of neurotrophins on force responses of human ASM strips to ACh and histamine. Strips were stretched to optimal length with repeated ACh contractions, and force responses to 1 µM ACh and 10 µM histamine were evaluated. Samples were then exposed for 1 h to 10 nM BDNF, NT4, or NT3, and agonist responses were reevaluated. Representative tracings for 1 µM ACh are shown in A. B: BDNF enhanced force responses, whereas NT3 diminished the responses to both ACh and histamine. *Significant difference from control (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we report novel data on the effects of acute and prolonged neurotrophin exposure on [Ca2+]i regulation and force responses in human ASM. Our results indicate that even brief exposure to some neurotrophins (BDNF, NT4) results in enhancement of SR Ca2+ release, likely via both IP3 and RyR channels, and increased SOCE triggered by SR Ca2+ depletion. In contrast, NT3 generally diminishes agonist-induced [Ca2+]i and force responses. Such effects appear to be mediated via the receptors trkB and trkC. Overall, these novel data lay the foundation for examination of the role of neurotrophins in the airway under both normal and pathological conditions.

Neurotrophins in the lung. Neurotrophins, a family of polypeptide growth factors that share common structural features, are known to have profound effects on development, differentiation, maintenance, and repair of the nervous system in vertebrates (4, 15, 17, 18, 24, 34, 56, 61). Neurotrophins are known to act via their corresponding high-affinity tyrosine kinase (Trk) receptor subtypes as well as a common low-affinity receptor p75NTR (8, 22, 56). Recent data demonstrate that neurotrophins and Trk receptors are expressed in nonneural tissues, including the lung (32, 33, 48, 49, 53, 55, 60). Indeed, there is significant interest in the role of neurotrophins in gastrointestinal disorders (36, 54) and coronary artery disease (13). However, less is known about neurotrophins in the lung. Ricci et al. (48) provided initial, direct evidence of neurotrophin and receptor expression in different lung components in humans, including lung innervation and ASM itself. In this regard, our immunocytochemical evidence is completely consistent. Furthermore, we now provide evidence that both neurotrophins (i.e., BDNF, NT3, and NT4) and their receptors (i.e., trkB, trkC) are indeed expressed in ASM cells.

With accumulating evidence for expression of neurotrophins in the lung, their physiological role is actively being investigated. Neurotrophins may be derived from several sources, including immune cells (5, 32, 50), suggesting a role in airway inflammation. In an ovalbumin-sensitized mouse model of asthma, BDNF mRNA expression is increased in airway infiltrates and lavages (6). In humans, sputum or bronchoalveolar lavage samples from patients with allergic and reactive airway diseases show high levels of neurotrophins (16, 33, 38, 39, 47, 51, 57). These data suggest that neurotrophins may contribute to immunologic aspects of airway diseases and thus link airway inflammation and airway hyperreactivity.

The mechanisms by which neurotrophins participate in the pathophysiology of airway hyperreactivity are still being investigated. Recent studies suggest that neurotrophins may modulate neural influences on the airway, increasing contractility. For example, inhibition of neurokinin-1 receptors blocks NGF-induced airway hyperreactivity (12). In rat pups subjected to hypoxic stress (a model for bronchopulmonary dysplasia), increased BDNF mRNA and protein expression was noted in peribronchial smooth muscle (60). In the ovalbumin mouse model inhibition of BDNF appears to blunt airway hyperreactivity, whereas in vitro contraction of bronchial rings by electrical field stimulation is inhibited (6). However, in these previous studies, direct effects of neurotrophins on ASM were not examined. Braun et al. (6) found that airway responses to methacholine challenge were not significantly affected by BDNF inhibition; however, actual neurotrophin levels at the level of ASM are difficult to determine. In contrast to BDNF enhancement of airway contractility, one study found that prolonged NT3 exposure of mouse trachea diminished the responsiveness to electric field stimulation in vitro (2). The findings in our study of BDNF, NT3, and NT4 expression suggest that, even under normal conditions, ASM-derived neurotrophins may have an autocrine or paracrine effect; however, trkB and trkC expression may also allow ASM to react to neurotrophins derived from other sources such as neurons or immune cells. Furthermore, the differential effects of BDNF vs. NT3 suggest that, regardless of their source, neurotrophins may modulate both airway contraction and relaxation, even under normal circumstances. In this regard, the opposing effects of BDNF vs. NT3 are consistent with previous studies.

We found differences in the expression and distribution of different neurotrophins and their receptors. Such differences may partly underlie differences in effects of different neurotrophins on Ca2+ and force. For example, ASM cells showed mostly nuclear staining for NT4, whereas trkB was detected at both plasma membrane and nucleus. BDNF and NT4 differed in the extent of their effects on [Ca2+]i regulation. It is possible that BDNF expression and release by an ASM cell are involved in [Ca2+]i regulation of the same (autocrine) or adjacent (paracrine) cells, whereas NT4 mediates nuclear effects not related to Ca2+, such as regulation of gene and protein expression. The mixed distribution of NT3 and trkC may then suggest both plasma membrane and nuclear effects of this neurotrophin. These issues remain to be clarified in future studies.

[Ca2+]i regulation in ASM. In ASM, SR Ca2+ release occurs via both IP3 receptor channels (10) and RyR channels (23). Although the initial [Ca2+]i response to agonists such as ACh and even histamine largely involves SR Ca2+ release, maintenance of [Ca2+]i (i.e., a plateau phase above baseline) involves both sustained Ca2+ influx and continued SR Ca2+ release (23, 44). Ca2+ influx, which may serve to maintain SR Ca2+ stores, is known to occur in ASM via voltage-gated (59) and receptor-gated (20, 37) channels, as well as in response to SR Ca2+ depletion (i.e., SOCE) (1, 19, 40).

Effect of neurotrophins on [Ca2+]i regulation. Given the relatively recent identification of neurotrophins in the airway, there are no previous data on neurotrophin effects on [Ca2+]i regulation in smooth muscle. Nonetheless, evidence from other cell types (especially neurons) show both "slow" effects following prolonged exposure and "rapid" effects, although the rapidity of such effects may depend on the actual cell type (see Ref. 82 for review). Exogenously applied neurotrophin (as in this study) rapidly increases [Ca2+]i in hippocampal neurons, predominantly via Ca2+ release from intracellular stores (3, 7, 58). Furthermore, BDNF-induced increase in [Ca2+]i in neurons is mediated via trkB, phospholipase C activation, IP3 production, and Ca2+ release via IP3-sensitive stores (31). The results of the present study demonstrating that the peak [Ca2+]i response to ACh, histamine, and bradykinin are all enhanced by 30-min exposure, especially to BDNF and to a lesser extent by NT4, are certainly indicative of neurotrophin enhancement of SR Ca2+ release in ASM. Because ACh- and histamine-induced SR Ca2+ release involves IP3 (10). it is possible that the observed enhancement is mediated, at least in part, via IP3 receptor channels. Furthermore, enhancement of the Ca2+ response to caffeine (an RyR channel agonist) by acute exposure to BDNF demonstrates that neurotrophins also modulate Ca2+ release via RyR channels, a previously unreported result.

Neurotrophin effects on [Ca2+]i may also involve Ca2+ influx. In pontine neurons of the newborn rat, BDNF increases influx via canonical transient receptor potential (TRPC)3 channels (30). Furthermore, BDNF-induced Ca2+ transients in hippocampal slices are inhibited by blockers of voltage-gated Ca2+ channels (27). Accordingly, enhancement of ACh-induced sustained [Ca2+]i levels by neurotrophins in the current study is consistent, given previous reports of both voltage-gated and receptor-operated Ca2+ channels being present in ASM (37, 59). Previous studies showed that SOCE is likely mediated via TRPC channels. We previously reported (1) that ASM express multiple TRPC isoforms, including TRPC3, which is expressed to the greatest extent. In this study, we found that both BDNF and NT4 enhance CPA-induced SOCE in ASM. Therefore, our novel data suggest another potential mechanism by which neurotrophins influence [Ca2+]i.

Data from other cell types demonstrate that cellular effects of neurotrophins are mediated via the high-affinity Trk receptors or the pan-neurotrophin receptor p75 (8, 22, 56). Downstream effects of BDNF, NT3, and NT4 are mediated via Trk dimerization and autophosphorylation resulting in activation of a number of intracellular signaling pathways that may be cell- and receptor subtype specific. In the present study, we did not specifically examine these downstream pathways. Nevertheless, our data demonstrating that the effects of all three neurotrophins are significantly blunted by K252a indicate that, even in ASM, neurotrophin effects on [Ca2+]i are likely mediated via Trk receptors.

As mentioned above, there is emerging evidence for rapid effects of neurotrophin on cell signaling. Given only limited data on neurotrophins and smooth muscle, it is obviously difficult to define what constitutes acute vs. prolonged effects of neurotrophin on ASM on a temporal scale. Nonetheless, in the present study, we found that relatively short exposure (30 min) has significantly less effect than overnight exposure to the same neurotrophin concentration on several aspects of [Ca2+]i regulation. The mechanisms underlying such differences remain to be investigated. Possibilities include 1) enhanced expression of Ca2+ regulatory proteins with prolonged neurotrophin expression and thus increased Ca2+ fluxes following agonist stimulation and 2) modification of Ca2+ regulatory protein function (e.g., TRPC, SR Ca2+ release channels, SERCA) with prolonged Trk-mediated phosphorylation.

Although BDNF and NT4 activate the same receptor (trkB), we found BDNF effects to be greater than those with NT4. Although surprising, previous studies have found these two neurotrophins not to be interchangeable in terms of their effects. For example, replacing the BDNF coding sequence with NT4 in mice is lethal, whereas NT4 replacement of BDNF is not (14). Furthermore, BDNF and NT4 differ in their support of both sensory and motor neurons (29). Furthermore, BDNF, but not NT4, induces the formation of the early gene c-fos protein (29). These studies suggest that although BDNF and NT4 act through a common receptor, trkB, they differ either in the extent of trkB activation or in subsequent, downstream effects. Such differences may explain our results with BDNF vs. NT4 in ASM, and they remain to be investigated further. In contrast to the enhancing effects of BDNF and NT4 on Ca2+ regulatory mechanisms, the inhibitory effect of NT3, another novel finding, is also of interest, suggesting that differential neurotrophin effects on Ca2+ regulation may exist under normal circumstances. Indeed, BDNF and NT3 differ in their effects on neuronal ion channel expression and function (43). Furthermore, BDNF-mediated potentiation of neurotransmitter release required Ca2+ influx, whereas NT3-mediated potentiation requires release of intracellular Ca2+ stores (43). Overall, these data emphasize the importance of examining the roles of different neurotrophins in cellular function.

Effect of neurotrophins on force regulation. Our novel data on BDNF and NT4 enhancement (but NT3 diminution) of agonist-induced force production in human ASM underline a physiological effect of neurotrophins. A previous study found that methacholine challenge of ovalbumin-sensitized mice is not altered by BDNF inhibition (6); however, neurotrophin concentrations at the level of ASM were indeterminate. Furthermore, mice and human airways may differ in neurotrophin effects. On the other hand, our data with NT3 in human ASM are consistent with those of Bachar et al. (2) on NT3 decrement of force in mouse trachea.

The mechanisms by which neurotrophins affect force regulation in ASM are not known. In addition to just increasing [Ca2+]i, neurotrophins may affect Ca2+ sensitivity for force generation. Indeed, in other cell systems, neurotrophins influence rho-kinase (11), which, in turn, influences myosin light chain phosphatase (9). However, this effect appears to involve the low-affinity p75NTR receptor. Although examination of these pathways was beyond the scope of the present study, our results nonetheless suggest that neurotrophins may activate diverse pathways in ASM, resulting in [Ca2+]i and force regulation.

In summary, the present study provides novel data demonstrating that neurotrophins can either enhance or inhibit [Ca2+]i and force in ASM. Although all of the mechanisms affected by neurotrophins remain to be determined, we propose that neurotrophins such as BDNF and NT4, acting via their receptor (trkB), enhance both SR Ca2+ release and Ca2+ influx, thus accentuating the [Ca2+]i response to agonist in ASM. In contrast, NT3, acting via trkC, may inhibit [Ca2+]i regulatory mechanisms. Force regulation by neurotrophins may involve other pathways that remain to be explored. Given the emerging evidence of altered neurotrophin expression in the diseased lung, these data provide a foundation for examining the physiological role of neurotrophins in the airway.


   GRANTS
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 ABSTRACT
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 DISCUSSION
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This work was supported by the Foundation for Anesthesia Education and Research and National Institute of General Medical Sciences Minority Supplemental Grant GM-56686 (A. Iyanoye).


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the support and advice of Drs. Gary C. Sieck, Physiology and Biomedical Engineering, Mark E. Wylam, Pulmonary and Critical Care Medicine, and Keith A. Jones, Mayo Clinic. The technical assistance of Kathleen Street and Ailing Xu is gratefully acknowledged.


   FOOTNOTES
 

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: prakash.ys{at}mayo.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.


   REFERENCES
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