European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Pulmonary, gastrointestinal and urogenital pharmacology
Stimulation of cannabinoid CB1 receptors prevents nerve-mediated airway hyperreactivity in NGF-induced inflammation in mouse airways Turgut Emrah Bozkurt a,b,n, Olivia Larsson c, Mikael Adner a a
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Hacettepe University, Faculty of Pharmacy, Department of Pharmacology, 06100 Sihhiye, Ankara, Turkey c Division of ENT Disease, CLINTEC, Karolinska Institutet, Stockholm, Sweden b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 October 2015 Received in revised form 11 February 2016 Accepted 15 February 2016
Cannabinoids are known to inhibit neuronal activity and have significant immunomodulatory effects which suggest a role in inflammatory airway diseases. In the present study, we tested the hypothesis that cannabinoids have both acute and chronic modulatory effects on nerve-mediated contractions in NGFinduced airway inflammation. Contractions induced by electrical field stimulation (EFS) were examined in tracheal segments isolated from male BALB/c mice. Tissues were both used fresh or after four days of culture with NGF to induce airway inflammation, and further exposed to cannabinoid receptor agonists. In order to evaluate nerve density, tracheal segments were also examined by immunohistochemistry after in vitro treatments. The CB1 receptor agonists ACEA and ACPA inhibited the constant train EFSinduced contractions in both fresh and NGF-exposed tracheas, an effect that could be blocked by the CB1 receptor antagonist AM251. Culturing the tissues with NGF up-regulated the frequency-dependent EFScontractions in isolated tracheas. This up-regulation could be inhibited by concomitant treatment with ACEA or ACPA. The treatment with NGF and/or ACEA did not affect the potency or the maximum response to carbachol. In histological sections, it was recognized that the enhanced effect induced by NGF was associated with an increase in nerve density, which, similarly, could be prevented by ACEA treatment. This study shows that stimulation of cannabinoid CB1 receptors modifies the increase of neuronal activity and density in NGF-induced airway inflammation and directly inhibits cholinergic contractions in the airways by a presynaptic mechanism. These findings indicate a protective role of CB1 receptors in airway inflammation. & 2016 Elsevier B.V. All rights reserved.
Keywords: Cannabinoid Airway Airway inflammation Airway hyperreactivity Nerve growth factor
1. Introduction Asthma is a chronic inflammatory disease of the airways, associated with respiratory symptoms such as wheezing, cough, chest tightness and shortness of breath. Airway hyperresponsiveness is a characteristic feature of asthma which occurs through nonspecific irritants or pharmacological agonists (Bousquet et al., 2000; Wardlaw et al., 2002). It has been suggested that impairment of autonomic nerve function, as well as neurogenic inflammation contributes to the pathogenesis of airway hyperresponsiveness (Barnes, 2001; McGovern and Mazzone, 2014). Cannabinoids are biologically active compounds produced in the plant Cannabis sativa and endogenously synthesized as a regulatory system affecting mainly neuronal activity in mammals (Dewey, 1986; Stefano et al., 1996). In earlier studies, it has been n Corresponding author at: Hacettepe University, Faculty of Pharmacy, Department of Pharmacology, 06100 Sihhiye, Ankara, Turkey. E-mail address: [email protected] (T.E. Bozkurt).
shown that smoking or ingestion of cannabinoids produces bronchodilation in the airways of both healthy volunteers and asthmatic patients (Tashkin et al., 1973, 1974; Vachon et al., 1973), suggesting a therapeutic role for these compounds in airway diseases. Cannabinoids mainly exert their effects through two seven transmembrane, G-protein coupled receptors, namely CB1 and CB2 (Devane et al., 1988; Howlett, 2002; Pertwee et al., 2010). CB1 receptors are mainly distributed in the central and peripheral nervous system and nerve terminals, whereas CB2 receptors are located in peripheral tissues, primarily in the immune cells and immune-related organs. The previously reported inhibitory properties on neuronal cholinergic contractions in isolated smooth muscle preparations, such as mouse stomach (Mule et al., 2007) and rat trachea (Yousif and Oriowo, 1999), have recently also been observed in human airways (Grassin-Delyle et al., 2014). These findings indicate the involvement of cannabinoids in modulating the function of cholinergic nerves and hence airway smooth muscle tone during inflammatory conditions. In the present study, we tested the hypothesis that
http://dx.doi.org/10.1016/j.ejphar.2016.02.045 0014-2999/& 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Bozkurt, T.E., et al., Stimulation of cannabinoid CB1 receptors prevents nerve-mediated airway hyperreactivity in NGF-induced inflammation in mouse airways. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.045i
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cannabinoids, in addition to the acute effects on neurogenic contractions of the airways, also affected neural pathways in an experimental model of airway inflammation. Nerve growth factor (NGF), a prototypical neurotrophin, which is increased in blood and airways of allergic and asthmatic patients (Bonini et al., 1996; Undem et al., 1999), was used as an inflammatory stimulus in our study. Therefore, we characterized the action of cannabinoids in a model where NGF was used to induce chronic inflammation. For the first time the effect of the cannabinoid receptors using the CB1 receptor agonists ACEA and ACPA, the CB2 receptor agonist JWH133 and the CB1 antagonist AM251 are reported for cholinergic nerve-mediated contractions on inflammation-induced airway hyperreactivity in isolated mouse trachea.
2. Materials and methods 2.1. Tissue preparations The study was approved by the Swedish Animal Experimentation Ethical Review Board. 10–12 weeks-old male BALB/c mice were killed by cervical dislocation. Whole tracheae were rapidly removed and placed into either Krebs-Henseleit solution for experiments on fresh segments or into Dulbecco's Modified Eagle's Medium (DMEM; 4500 mg/l, D-glucose, 110 mg/l, sodium pyruvate, 584 mg/l, L-glutamine), supplemented with 100 U ml/1 penicillin and 100 mg/ml streptomycin for cultured tissues. Each trachea was thereafter dissected free from connective tissue under sterile conditions and the proximal part was used since it has been shown to be sensitive to NGF (Bachar et al., 2004). The tracheal segments were used directly after dissection (fresh) or placed individually in 300 ml DMEM-containing 96-well plates (Ultra-low attachment; Sigma, St. Louis, MO, U.S.A.) for organ culture under culture conditions at 37 °C in humidified 5% CO2 in air for four days (Adner et al., 2002). During the culture period the segments were exposed to NGF (100 ng/ml; (Bachar et al., 2004) and the cannabinoid receptor agonists, and were transferred into new wells containing fresh media and drugs every day. 2.2. Organ bath experiments Tracheal smooth muscle reactivity was analyzed in temperature-controlled (37 °C) myographs (Organ Bath Model 700MO, J.P. Trading, Aarhus, Denmark) containing Krebs-Henseleit buffer solution composed of (mM) Na þ 143, K þ 5.9, Ca2 þ 1.5, Mg2 þ 2.5, Cl 128, H2 PO−4 1.2, SO−4 2 1.2, HCO−3 25 and D-glucose 10. The solution was continuously equilibrated with 5% CO2 and 95% O2 resulting in a pH of 7.4. The tracheal segments were mounted on two L-shaped metal prongs. One prong was connected to a force–displacement transducer for continuous recording of isometric tension by the Chart Software (AD Instruments Ltd, Hastings, U.K.). The other prong was connected to a displacement device, allowing adjustment of the distance between the two parallel prongs.
P. Trading, Aarhus, Denmark). Initially, the segments were given five consecutive training impulses of 4 Hz, 55 mA, 0.8 ms pulse duration, which also were used for the constant train of EFS. Later, segments were given either a constant train of EFS with 4 Hz or a 2 log EFS series of 0.2–25.6 Hz. For the latter, each impulse lasted 1 min, and was followed by 1.5 min recovery period. All parameters were optimized to produce stable and reproducible contractions. These parameters induced cholinergic contraction responses of tracheas, as they were completely blocked by atropine (100 nM). The effect of cannabinoid CB1 and CB2 receptor agonists were tested on constant EFS-induced contraction responses. ACEA (Arachidonyl-2′-chloroethylamide) and ACPA (N-(Cyclopropyl)5Z,8Z,11Z,14Z-eicosatetraenamide) were used as selective CB1 agonists and JWH133 ((6aR,10aR)-3-(1,1-Dimethylbutyl)6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) was used as a selective CB2 agonist. AM251 (N-(Piperidin-1-yl)-5-(4iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) was used as a CB1 receptor antagonist. CB1 and CB2 agonists were added to the organ bath after the acquisition of stable cholinergic contractions (corresponding to four to six contractions). All antagonists were added 30 min before the EFS response was started. Segments were incubated for 30 min with 3 mM of the cyclooxygenase (COX) inhibitor indomethacin before carbachol and EFS responses. 2.4. Immunohistochemistry Neuronal density in tracheal segments was determined by immunohistochemical detection of neurofilament. Segments were post-fixed in 4% phosphate buffered paraformaldehyde, embedded in paraffin and subsequently cut into 5 mm sections on a microtome. Following heat-mediated antigen retrieval in citrate buffer, sections were permeabilized with 1% Triton-X100 and blocked for non-specific binding with 10% normal donkey serum and 1% bovine serum albumin (BSA). Neurofilament was detected using a chicken anti-neurofilament primary antibody (1:1500) and an AlexaFluors555-conjugated goat anti-IgY secondary antibody (1:200). Images of stained tracheal segments were captured on a Nikon TE-2000U microscope, connected to a Nikon Digital Sight DS-U1 camera. Fluorescence intensity was determined using ImageJ (Image Processing and Analysis in Java) software. 2.5. Analysis All data is expressed as mean values 7S.E.M. Contractile responses to carbachol are expressed in mN, and since this agonist induces contraction close to the maximal capacity of the tissue, EFS responses were expressed as a percentage of the maximal contraction to carbachol. Agonist concentration-effect curve data from individual tissues were fitted to the Hill equation (GraphPad Prism, San Diego, U.S.A). Statistical analysis was performed using one-way analysis of variance (ANOVA), and if the null hypothesis was rejected (P o0.05), Dunnett's post-test was performed. 2.6. Materials
2.3. Experimental protocol Tissues were equilibrated for one h under 0.8 mN resting tension by slowly stretching. The contractile capacity of each tissue segment was tested by two exposures to 60 mM KCl. Following a 30 min resting period, carbachol (1 nM to 100 mM) concentrationeffect curves were obtained on each segment by cumulative adding to the organ bath. After a washout period of one h, electrical field stimulation (EFS) responses were elicited in the myograph, by placing electrodes delivering electrical stimulation on opposite sides of the tracheal segments (Current Stimulator Model CS200, J.
Dulbecco's Modified Eagle's Medium, carbachol and atropine were obtained from Sigma (St. Louis, MO, U.S.A.); penicillin and streptomycin were obtained from Life Technologies (Gathisburg, MD, U.S.A.); recombinant mouse β-NGF was obtained from R&D Systems (Abingdon, U.K.); ACEA (Arachidonyl2′-chloroethylamide), ACPA (N-(Cyclopropyl)-5Z,8Z,11Z,14Z-eicosatetraenamide), JWH133 ((6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran), AM251 (N-(Piperidin-1-yl)-5(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3carboxamide) were obtained from Tocris. The primary
Please cite this article as: Bozkurt, T.E., et al., Stimulation of cannabinoid CB1 receptors prevents nerve-mediated airway hyperreactivity in NGF-induced inflammation in mouse airways. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.045i
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anti-neurofilament antibody (AB5539) was obtained from Merck Millipore (Darmstadt, Germany). The secondary goat anti-IgY antibody (ab150174) was obtained from Abcam (Cambridge, UK).
3. Results 3.1. Acute effects of cannabinoid receptor agonists on nerve-mediated contractions in isolated mouse trachea The direct effect of cannabinoids on EFS responses was first investigated by administration of the CB1 receptor agonists ACEA and ACPA or the CB2 receptor agonist JWH133 during a stable response of constant train stimulation in fresh mouse tracheal segments. The CB1 receptor agonists (ACEA, ACPA) significantly inhibited the constant train EFS contractions in fresh tracheal segments whereas the CB2 receptor agonist (JWH133) had no effect (Fig. 1). In contrast, ACEA, ACPA and JWH133 had no direct effect on the carbachol-induced contractions (pD2 values are 7.19 70.21, 7.11 70.07, 7.247 0.13 and 7.09 70.16 for control, ACEA, ACPA and JWH133 respectively), supporting a prejunctional mode of action. 3.2. The influence of NGF-treatment on the direct effect of cannabinoid receptor activation on EFS stimulation and on the muscarinic receptor-induced smooth muscle responses Next we examined if the direct effect of cannabinoids on fresh trachea were affected after culture for four days with and without NGF treatment. In these experiments, similar inhibition of EFS with constant train stimulation by ACEA was observed in the segments cultured with NGF as in fresh segments and also in the segments treated with NGFþ ACEA and ACEA alone (Fig. 2A). When further characterizing the receptors involved in this effect after culture, both ACEA and ACPA caused a similar inhibition of constant train EFS contractions as in fresh segments, and this effect was blocked by the CB1 receptor antagonist AM251 (Fig. 2B and C). To evaluate the influence of NGF-treatment on the muscarinic smooth muscle responses, concentration-response curves for carbachol were performed in tracheal segments that had been cultured for four days. None of the treatments, i.e. NGF, NGFþ ACEA or ACEA, caused alterations of neither maximum amplitude nor potency for carbachol (Fig. 2D).
Fig. 1. ACEA, ACPA and JWH133-induced inhibition of the constant train (4 Hz) EFS-induced contractions in fresh tracheal segments. Data are presented as Mean 7 S.E.M. (n¼ 6).
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3.3. The effect of cannabinoid receptor agonist treatment on nervemediated contractions of isolated mouse trachea after NGF-induced in vitro airway inflammation To study the response of cannabinoids on NGF-induced inflammation, the tracheal segments were cultured for 4 days and thereafter exposed to step-wise increments of frequencies of EFS, which increases the potential to detect the effects of the treatments. During this period, the segments were exposed to NGF, with parallel controls without NGF, in the absence or presence of CB1 receptor agonist ACEA, ACPA or the CB2 receptor agonist JWH133, or the agonists alone. In the tracheal segments, EFS induced a frequency-dependent contraction after four days of culture (Fig. 3A–C). Exposure to NGF during culture caused a reproducible increase in the frequency-dependent contractions induced by EFS that were significant for the intermediate frequencies (0.8–12.8 Hz). The response induced by NGF was inhibited when segments were co-cultured with either ACEA (Fig. 3A) or ACPA (Fig. 3B). The NGF-induced increase was unaffected when the tissues were co-cultured with JWH133 (Fig. 3C). 3.4. The effect of cannabinoid receptor CB1 agonist treatment on nerve density in isolated mouse trachea after NGF-induced in vitro airway inflammation We hypothesized that the inhibitory effect of CB1 receptor agonist on the NGF-induced increase in EFS involved inhibition of NGF-induced neuronal growth. The nerve density in cultured tracheal segments was therefore examined by quantitative immunohistochemistry (Fig. 4A and B). Four days of culture with NGF significantly increased the density of nerves in the tracheal segments when compared with control treated segments (Fig. 4A and B). Concomitant treatment with ACEA completely prevented the NGF-induced increase of nerve density, whereas ACEA alone had no effect (Fig. 4A and B).
4. Discussion In the present study, the effect of cannabinoid receptor stimulation on airway hyperreactivity was investigated in an experimental model where inflammation was induced by exposure of mouse trachea to NGF for four days. Using an in vitro organ culture technique, we show that the NGF treatment up-regulates electrical field stimulation (EFS)-induced cholinergic contractions in the proximal part of the mouse trachea, an effect that could be prevented by simultaneous culture with selective cannabinoid CB1 receptor agonists. Furthermore, direct stimulation through CB1 receptors inhibited the EFS-induced cholinergic contractions in a similar manner both in fresh trachea and after culture, independently of NGF treatment or CB1 receptor activation during the culture period. To verify that cannabinoids can influence EFS in mouse airways, the effect was first investigated on fresh tracheal segments from mice exposed to EFS with a constant train of 4 Hz. The contraction for the constant train was stable for more than an h and therefore suitable for studying direct effects of different treatments. Moreover, this effect could, as previously described, be blocked by atropine, implicating that it was mediated through activation of cholinergic nerves (Bachar et al., 2004). Administration of the CB1 receptor agonists ACEA or ACPA during the constant train period caused a marked inhibition of the contractile responses, suggesting a presynaptic modulatory role of these receptors. This effect is likely to occur through the inhibition of acetylcholine release as described in guinea pig trachea and human bronchus (Grassin-Delyle et al., 2014; Spicuzza et al.,
Please cite this article as: Bozkurt, T.E., et al., Stimulation of cannabinoid CB1 receptors prevents nerve-mediated airway hyperreactivity in NGF-induced inflammation in mouse airways. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.045i
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Fig. 2. Acute effect of ACEA on constant train (4 Hz) stimulation responses in tracheal segments cultured for 4 days with NGF, NGFþ ACEA, ACEA (A). The effect of CB1 receptor antagonist AM251 on the acute inhibitory effect of ACEA (B) and ACPA (C) on constant train (4 Hz) stimulation responses. Carbachol-induced concentrationdependent contraction responses in tracheal segments cultured for 4 days with NGF, NGFþACEA, ACEA (D). Data are presented as Mean 7 S.E.M. (n¼ 6).
2000). That the effect is mediated by CB1 receptor is indicated both because ACEA and ACPA are highly selective CB1 receptor agonists possessing nearly 2000 times greater affinity to CB1 receptors over CB2 (Hillard et al., 1999), and also because the acute inhibitory effect of these agonists on EFS-contractions was blocked by the CB1 antagonist AM251. This contrast to the regulatory role of cannabinoids on neural control in guinea pig airways which has been shown to be mediated by TRPV1 rather than cannabinoid receptors (Nieri et al., 2003a, 2003b). On the other hand, it has been shown that CB2 receptor activation can reduce cholinergic neurotransmission in the mouse stomach by playing a prejunctional modulatory role on the cholinergic excitatory transmission (Mule et al., 2007). However CB2 receptor selective agonist JWH133 failed to inhibit the EFS-induced cholinergic cannabinoids further supporting that the inhibitory response of CB1 agonists in our experiments is mediated through the cannabinoid CB1 receptors. Recently, the CB1 receptor-mediated inhibitory effect of EFS-induced contractions has also been shown in human bronchus (Grassin-Delyle et al., 2014). Thus, our findings in mouse airways indicate that stimulation of presynaptic CB1 receptors can be an important mechanism to directly modify neurogenic contractility of the airways by acting on cholinergic nerves. After four days treatment the inhibitory effects of ACEA and ACPA on EFS-induced contractions were similar to those seen in fresh segments. As the effect of both agonists was inhibited by the CB1 receptor antagonist AM251, the role of CB1 receptor in inhibition of constant train EFS was further confirmed. Moreover, the effect by ACEA was neither influenced by the treatment of NGF nor by exposure of segments to ACEA during the four days culture. Thus, the direct effect of ACEA or ACPA in tracheal segments which is present also in segments cultured with CB1 receptor agonists, indicates that this acute direct effect is reversible and is not affected by tachyphylaxis. In the present study, treatment of the tracheas with NGF for four days caused an up-regulation of the intermediate frequencies
of the EFS-induced contractions in the mouse trachea. The culture period of four days has earlier shown to maintain both contractile and relaxant properties of the smooth muscle (Adner et al., 2002). In this study, treatment during this time period with either NGF, ACEA or both together did not alter the smooth muscle activation of muscarinic receptors, indicating a presynaptic effect for NGF during the culture procedure. This phenomenon was earlier described by Bachar and colleagues and was explained by an increase of neuronal fibres in the trachea (Bachar et al., 2004). It is possible that this peripheral effect of NGF can be the cause of the NGFenhanced nerve-mediated airway hyperresponsiveness observed in mice and guinea-pigs (Braun et al., 1998, 2001; de Vries et al., 1999). The NGF-induced up-regulation of the contractions was completely abolished when the tracheas were co-cultured with either of the CB1 receptor agonists ACEA or ACPA. As the effect was not prevented by CB2 agonist JWH133 when co-cultured with NGF, the compiled data support that this effect was mediated through the CB1 receptor. This contrasts with the earlier findings in guinea pig where CB2 receptor stimulation has shown to inhibit neurogenic airway inflammation (Yoshihara et al., 2005). In a recent study, using guinea pig tracheal segments, it was shown that four days culture with TNFα caused an increased contraction to EFS stimulation and that this effect was decreased by both CB1 and CB2 receptor activation (Makwana et al., 2015). However, this effect was demonstrated when the cannabinoids where given 30 min before EFS stimulation and caused a decrease below the effect for the control segments cultured with saline. Thus, this is the first study showing that long-term stimulation with CB1 receptor activation modulates specifically the inflammation-induced increase of neural-induced contraction. It has previously been shown that NGF causes an increase in nerve density in mouse tracheal segments (Bachar et al., 2004). Thus, one mechanism behind the specific reduction for CB1 receptor agonists on the NGF-induced increase could be an
Please cite this article as: Bozkurt, T.E., et al., Stimulation of cannabinoid CB1 receptors prevents nerve-mediated airway hyperreactivity in NGF-induced inflammation in mouse airways. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.045i
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Fig. 3. EFS-induced frequency-dependent contraction responses in the tracheal segments cultured for 4 days with NGF, NGFþ ACEA, ACEA (A), NGF, NGFþ ACPA, ACPA (B) or NGF, NGFþJWH133, JWH133 (C). Data are presented as Mean 7 S.E.M. (n ¼6–9).
inhibition of nerve density. This study confirmed and extended the documentation by demonstrating that NGF caused a remarkable increase in nerve density, supporting an important effect for NGFinduced inflammation. Moreover, treatment with NGF together with ACEA restored the nerve density to control levels, whereas treatment with ACEA alone had no effect. Thus, these data clearly support the specific effect of CB1 receptor stimulation on NGFinduced inflammation and most likely explain the altered responses for the functional data. Our data show that the CB1 receptor has at least two separate effects, one for acute inhibition of cholinergic contractions and another for prevention of NGF-mediated hyperreactivity in mouse trachea. That the direct effect was not affected by the long-term
stimulation during culture indicates different signaling pathways participating for the two events (Fig. 5). As with many G-protein coupled receptors the intracellular activation of the CB1 receptor has been described to be complex (Turu and Hunyady, 2010). Although the main activity is considered to be generated through Gi/o proteins, diverse actions such as inhibition of cAMP, modulation of ion channels and activation of mitogen-activated proteins (MAPKs) been shown. Speculating, it is possible that the direct inhibitory effect on the EFS-stimulation acts through inhibition of cAMP or ion channel modulation, as these pathways are rapid in action. On the other hand, MAPK activation with subsequent transcriptional regulation takes longer and may be the mechanism by which CB1 agonists inhibit NGF-induced nerve growth. One
Please cite this article as: Bozkurt, T.E., et al., Stimulation of cannabinoid CB1 receptors prevents nerve-mediated airway hyperreactivity in NGF-induced inflammation in mouse airways. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.045i
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Fig. 4. Fluorescence intensity measured from immunohistochemical preparations of fresh tracheal segments or tracheal segments cultured for 4 days with NGF, NGFþ ACEA or ACEA (A). Immunofluorescence images taken from proximal part of fresh tracheal segments, or segments cultured for 4 days with ACEA, NGF or NGFþACEA (B). Pictures were taken at 20 magnification. The scale bar represents 100 mm. Data are presented as Mean 7 S.E.M. (n¼ 6).
treatment of asthma and other inflammatory airway diseases by reducing airway hyperreactivity.
Conflict of interest The authors declare no conflict of interests.
Acknowledgements This study was supported by The Swedish Heart and Lung Foundation (Grant numbers 20130532, 20130636), Konsul Th C Berghs Research Foundation, the ChAMP project, COST BM1201, the Centre for Allergy Research at Karolinska Institutet, Karolinska Institutet, Hacettepe University Scientific Research Projects Coordination Unit (Project ID:1649) and The Scientific and Technological Research Council of Turkey (TUBITAK) (2219 Program).
References
Fig. 5. The two distinct effects of CB1 receptors on airway cholinergic nerves. Stimulation of CB1 receptors can acutely inhibit nerve-mediated airway contractions and can attenuate neural growth induced by NGF and thus prevent airway hyperreactivity (CB1: Cannabinoid CB1 receptor; Ach: Acetylcholine; EFS: Electrical field stimulation; NGF: Nerve growth factor; MR: Muscarinic receptor; TrkA: Tyrosine kinase A receptor; p75: p75 neurotrophin receptor).
protein downstream of MAPK is AKT, a protein that has been shown to be involved in the inhibitory effect of ACEA in NGF-induced bladder hyperreactivity in rats (Wang et al., 2015), may be of importance for the neural growth. Taken together, the present study in mouse trachea indicate that CB1 receptor activation can directly inhibit cholinergic contractile responses by a presynaptic mechanism and, additionally, can persistently modify neuronal airway hyperreactivity by an action involving inhibition of neuronal growth induced by NGF. In view of previous observations in human airways, it can be concluded that CB1 receptors qualify as preventive targets for
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Stimulation of cannabinoid CB1 receptors prevents nerve-mediated airway hyperreactivity in NGF-induced inflammation in mouse airways Turgut Emrah Bozkurt a,b,n, Olivia Larsson c, Mikael Adner a a
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Hacettepe University, Faculty of Pharmacy, Department of Pharmacology, 06100 Sihhiye, Ankara, Turkey c Division of ENT Disease, CLINTEC, Karolinska Institutet, Stockholm, Sweden b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 October 2015 Received in revised form 11 February 2016 Accepted 15 February 2016
Cannabinoids are known to inhibit neuronal activity and have significant immunomodulatory effects which suggest a role in inflammatory airway diseases. In the present study, we tested the hypothesis that cannabinoids have both acute and chronic modulatory effects on nerve-mediated contractions in NGFinduced airway inflammation. Contractions induced by electrical field stimulation (EFS) were examined in tracheal segments isolated from male BALB/c mice. Tissues were both used fresh or after four days of culture with NGF to induce airway inflammation, and further exposed to cannabinoid receptor agonists. In order to evaluate nerve density, tracheal segments were also examined by immunohistochemistry after in vitro treatments. The CB1 receptor agonists ACEA and ACPA inhibited the constant train EFSinduced contractions in both fresh and NGF-exposed tracheas, an effect that could be blocked by the CB1 receptor antagonist AM251. Culturing the tissues with NGF up-regulated the frequency-dependent EFScontractions in isolated tracheas. This up-regulation could be inhibited by concomitant treatment with ACEA or ACPA. The treatment with NGF and/or ACEA did not affect the potency or the maximum response to carbachol. In histological sections, it was recognized that the enhanced effect induced by NGF was associated with an increase in nerve density, which, similarly, could be prevented by ACEA treatment. This study shows that stimulation of cannabinoid CB1 receptors modifies the increase of neuronal activity and density in NGF-induced airway inflammation and directly inhibits cholinergic contractions in the airways by a presynaptic mechanism. These findings indicate a protective role of CB1 receptors in airway inflammation. & 2016 Elsevier B.V. All rights reserved.
Keywords: Cannabinoid Airway Airway inflammation Airway hyperreactivity Nerve growth factor
1. Introduction Asthma is a chronic inflammatory disease of the airways, associated with respiratory symptoms such as wheezing, cough, chest tightness and shortness of breath. Airway hyperresponsiveness is a characteristic feature of asthma which occurs through nonspecific irritants or pharmacological agonists (Bousquet et al., 2000; Wardlaw et al., 2002). It has been suggested that impairment of autonomic nerve function, as well as neurogenic inflammation contributes to the pathogenesis of airway hyperresponsiveness (Barnes, 2001; McGovern and Mazzone, 2014). Cannabinoids are biologically active compounds produced in the plant Cannabis sativa and endogenously synthesized as a regulatory system affecting mainly neuronal activity in mammals (Dewey, 1986; Stefano et al., 1996). In earlier studies, it has been n Corresponding author at: Hacettepe University, Faculty of Pharmacy, Department of Pharmacology, 06100 Sihhiye, Ankara, Turkey. E-mail address: [email protected] (T.E. Bozkurt).
shown that smoking or ingestion of cannabinoids produces bronchodilation in the airways of both healthy volunteers and asthmatic patients (Tashkin et al., 1973, 1974; Vachon et al., 1973), suggesting a therapeutic role for these compounds in airway diseases. Cannabinoids mainly exert their effects through two seven transmembrane, G-protein coupled receptors, namely CB1 and CB2 (Devane et al., 1988; Howlett, 2002; Pertwee et al., 2010). CB1 receptors are mainly distributed in the central and peripheral nervous system and nerve terminals, whereas CB2 receptors are located in peripheral tissues, primarily in the immune cells and immune-related organs. The previously reported inhibitory properties on neuronal cholinergic contractions in isolated smooth muscle preparations, such as mouse stomach (Mule et al., 2007) and rat trachea (Yousif and Oriowo, 1999), have recently also been observed in human airways (Grassin-Delyle et al., 2014). These findings indicate the involvement of cannabinoids in modulating the function of cholinergic nerves and hence airway smooth muscle tone during inflammatory conditions. In the present study, we tested the hypothesis that
http://dx.doi.org/10.1016/j.ejphar.2016.02.045 0014-2999/& 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Bozkurt, T.E., et al., Stimulation of cannabinoid CB1 receptors prevents nerve-mediated airway hyperreactivity in NGF-induced inflammation in mouse airways. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.045i
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cannabinoids, in addition to the acute effects on neurogenic contractions of the airways, also affected neural pathways in an experimental model of airway inflammation. Nerve growth factor (NGF), a prototypical neurotrophin, which is increased in blood and airways of allergic and asthmatic patients (Bonini et al., 1996; Undem et al., 1999), was used as an inflammatory stimulus in our study. Therefore, we characterized the action of cannabinoids in a model where NGF was used to induce chronic inflammation. For the first time the effect of the cannabinoid receptors using the CB1 receptor agonists ACEA and ACPA, the CB2 receptor agonist JWH133 and the CB1 antagonist AM251 are reported for cholinergic nerve-mediated contractions on inflammation-induced airway hyperreactivity in isolated mouse trachea.
2. Materials and methods 2.1. Tissue preparations The study was approved by the Swedish Animal Experimentation Ethical Review Board. 10–12 weeks-old male BALB/c mice were killed by cervical dislocation. Whole tracheae were rapidly removed and placed into either Krebs-Henseleit solution for experiments on fresh segments or into Dulbecco's Modified Eagle's Medium (DMEM; 4500 mg/l, D-glucose, 110 mg/l, sodium pyruvate, 584 mg/l, L-glutamine), supplemented with 100 U ml/1 penicillin and 100 mg/ml streptomycin for cultured tissues. Each trachea was thereafter dissected free from connective tissue under sterile conditions and the proximal part was used since it has been shown to be sensitive to NGF (Bachar et al., 2004). The tracheal segments were used directly after dissection (fresh) or placed individually in 300 ml DMEM-containing 96-well plates (Ultra-low attachment; Sigma, St. Louis, MO, U.S.A.) for organ culture under culture conditions at 37 °C in humidified 5% CO2 in air for four days (Adner et al., 2002). During the culture period the segments were exposed to NGF (100 ng/ml; (Bachar et al., 2004) and the cannabinoid receptor agonists, and were transferred into new wells containing fresh media and drugs every day. 2.2. Organ bath experiments Tracheal smooth muscle reactivity was analyzed in temperature-controlled (37 °C) myographs (Organ Bath Model 700MO, J.P. Trading, Aarhus, Denmark) containing Krebs-Henseleit buffer solution composed of (mM) Na þ 143, K þ 5.9, Ca2 þ 1.5, Mg2 þ 2.5, Cl 128, H2 PO−4 1.2, SO−4 2 1.2, HCO−3 25 and D-glucose 10. The solution was continuously equilibrated with 5% CO2 and 95% O2 resulting in a pH of 7.4. The tracheal segments were mounted on two L-shaped metal prongs. One prong was connected to a force–displacement transducer for continuous recording of isometric tension by the Chart Software (AD Instruments Ltd, Hastings, U.K.). The other prong was connected to a displacement device, allowing adjustment of the distance between the two parallel prongs.
P. Trading, Aarhus, Denmark). Initially, the segments were given five consecutive training impulses of 4 Hz, 55 mA, 0.8 ms pulse duration, which also were used for the constant train of EFS. Later, segments were given either a constant train of EFS with 4 Hz or a 2 log EFS series of 0.2–25.6 Hz. For the latter, each impulse lasted 1 min, and was followed by 1.5 min recovery period. All parameters were optimized to produce stable and reproducible contractions. These parameters induced cholinergic contraction responses of tracheas, as they were completely blocked by atropine (100 nM). The effect of cannabinoid CB1 and CB2 receptor agonists were tested on constant EFS-induced contraction responses. ACEA (Arachidonyl-2′-chloroethylamide) and ACPA (N-(Cyclopropyl)5Z,8Z,11Z,14Z-eicosatetraenamide) were used as selective CB1 agonists and JWH133 ((6aR,10aR)-3-(1,1-Dimethylbutyl)6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) was used as a selective CB2 agonist. AM251 (N-(Piperidin-1-yl)-5-(4iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) was used as a CB1 receptor antagonist. CB1 and CB2 agonists were added to the organ bath after the acquisition of stable cholinergic contractions (corresponding to four to six contractions). All antagonists were added 30 min before the EFS response was started. Segments were incubated for 30 min with 3 mM of the cyclooxygenase (COX) inhibitor indomethacin before carbachol and EFS responses. 2.4. Immunohistochemistry Neuronal density in tracheal segments was determined by immunohistochemical detection of neurofilament. Segments were post-fixed in 4% phosphate buffered paraformaldehyde, embedded in paraffin and subsequently cut into 5 mm sections on a microtome. Following heat-mediated antigen retrieval in citrate buffer, sections were permeabilized with 1% Triton-X100 and blocked for non-specific binding with 10% normal donkey serum and 1% bovine serum albumin (BSA). Neurofilament was detected using a chicken anti-neurofilament primary antibody (1:1500) and an AlexaFluors555-conjugated goat anti-IgY secondary antibody (1:200). Images of stained tracheal segments were captured on a Nikon TE-2000U microscope, connected to a Nikon Digital Sight DS-U1 camera. Fluorescence intensity was determined using ImageJ (Image Processing and Analysis in Java) software. 2.5. Analysis All data is expressed as mean values 7S.E.M. Contractile responses to carbachol are expressed in mN, and since this agonist induces contraction close to the maximal capacity of the tissue, EFS responses were expressed as a percentage of the maximal contraction to carbachol. Agonist concentration-effect curve data from individual tissues were fitted to the Hill equation (GraphPad Prism, San Diego, U.S.A). Statistical analysis was performed using one-way analysis of variance (ANOVA), and if the null hypothesis was rejected (P o0.05), Dunnett's post-test was performed. 2.6. Materials
2.3. Experimental protocol Tissues were equilibrated for one h under 0.8 mN resting tension by slowly stretching. The contractile capacity of each tissue segment was tested by two exposures to 60 mM KCl. Following a 30 min resting period, carbachol (1 nM to 100 mM) concentrationeffect curves were obtained on each segment by cumulative adding to the organ bath. After a washout period of one h, electrical field stimulation (EFS) responses were elicited in the myograph, by placing electrodes delivering electrical stimulation on opposite sides of the tracheal segments (Current Stimulator Model CS200, J.
Dulbecco's Modified Eagle's Medium, carbachol and atropine were obtained from Sigma (St. Louis, MO, U.S.A.); penicillin and streptomycin were obtained from Life Technologies (Gathisburg, MD, U.S.A.); recombinant mouse β-NGF was obtained from R&D Systems (Abingdon, U.K.); ACEA (Arachidonyl2′-chloroethylamide), ACPA (N-(Cyclopropyl)-5Z,8Z,11Z,14Z-eicosatetraenamide), JWH133 ((6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran), AM251 (N-(Piperidin-1-yl)-5(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3carboxamide) were obtained from Tocris. The primary
Please cite this article as: Bozkurt, T.E., et al., Stimulation of cannabinoid CB1 receptors prevents nerve-mediated airway hyperreactivity in NGF-induced inflammation in mouse airways. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.045i
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anti-neurofilament antibody (AB5539) was obtained from Merck Millipore (Darmstadt, Germany). The secondary goat anti-IgY antibody (ab150174) was obtained from Abcam (Cambridge, UK).
3. Results 3.1. Acute effects of cannabinoid receptor agonists on nerve-mediated contractions in isolated mouse trachea The direct effect of cannabinoids on EFS responses was first investigated by administration of the CB1 receptor agonists ACEA and ACPA or the CB2 receptor agonist JWH133 during a stable response of constant train stimulation in fresh mouse tracheal segments. The CB1 receptor agonists (ACEA, ACPA) significantly inhibited the constant train EFS contractions in fresh tracheal segments whereas the CB2 receptor agonist (JWH133) had no effect (Fig. 1). In contrast, ACEA, ACPA and JWH133 had no direct effect on the carbachol-induced contractions (pD2 values are 7.19 70.21, 7.11 70.07, 7.247 0.13 and 7.09 70.16 for control, ACEA, ACPA and JWH133 respectively), supporting a prejunctional mode of action. 3.2. The influence of NGF-treatment on the direct effect of cannabinoid receptor activation on EFS stimulation and on the muscarinic receptor-induced smooth muscle responses Next we examined if the direct effect of cannabinoids on fresh trachea were affected after culture for four days with and without NGF treatment. In these experiments, similar inhibition of EFS with constant train stimulation by ACEA was observed in the segments cultured with NGF as in fresh segments and also in the segments treated with NGFþ ACEA and ACEA alone (Fig. 2A). When further characterizing the receptors involved in this effect after culture, both ACEA and ACPA caused a similar inhibition of constant train EFS contractions as in fresh segments, and this effect was blocked by the CB1 receptor antagonist AM251 (Fig. 2B and C). To evaluate the influence of NGF-treatment on the muscarinic smooth muscle responses, concentration-response curves for carbachol were performed in tracheal segments that had been cultured for four days. None of the treatments, i.e. NGF, NGFþ ACEA or ACEA, caused alterations of neither maximum amplitude nor potency for carbachol (Fig. 2D).
Fig. 1. ACEA, ACPA and JWH133-induced inhibition of the constant train (4 Hz) EFS-induced contractions in fresh tracheal segments. Data are presented as Mean 7 S.E.M. (n¼ 6).
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3.3. The effect of cannabinoid receptor agonist treatment on nervemediated contractions of isolated mouse trachea after NGF-induced in vitro airway inflammation To study the response of cannabinoids on NGF-induced inflammation, the tracheal segments were cultured for 4 days and thereafter exposed to step-wise increments of frequencies of EFS, which increases the potential to detect the effects of the treatments. During this period, the segments were exposed to NGF, with parallel controls without NGF, in the absence or presence of CB1 receptor agonist ACEA, ACPA or the CB2 receptor agonist JWH133, or the agonists alone. In the tracheal segments, EFS induced a frequency-dependent contraction after four days of culture (Fig. 3A–C). Exposure to NGF during culture caused a reproducible increase in the frequency-dependent contractions induced by EFS that were significant for the intermediate frequencies (0.8–12.8 Hz). The response induced by NGF was inhibited when segments were co-cultured with either ACEA (Fig. 3A) or ACPA (Fig. 3B). The NGF-induced increase was unaffected when the tissues were co-cultured with JWH133 (Fig. 3C). 3.4. The effect of cannabinoid receptor CB1 agonist treatment on nerve density in isolated mouse trachea after NGF-induced in vitro airway inflammation We hypothesized that the inhibitory effect of CB1 receptor agonist on the NGF-induced increase in EFS involved inhibition of NGF-induced neuronal growth. The nerve density in cultured tracheal segments was therefore examined by quantitative immunohistochemistry (Fig. 4A and B). Four days of culture with NGF significantly increased the density of nerves in the tracheal segments when compared with control treated segments (Fig. 4A and B). Concomitant treatment with ACEA completely prevented the NGF-induced increase of nerve density, whereas ACEA alone had no effect (Fig. 4A and B).
4. Discussion In the present study, the effect of cannabinoid receptor stimulation on airway hyperreactivity was investigated in an experimental model where inflammation was induced by exposure of mouse trachea to NGF for four days. Using an in vitro organ culture technique, we show that the NGF treatment up-regulates electrical field stimulation (EFS)-induced cholinergic contractions in the proximal part of the mouse trachea, an effect that could be prevented by simultaneous culture with selective cannabinoid CB1 receptor agonists. Furthermore, direct stimulation through CB1 receptors inhibited the EFS-induced cholinergic contractions in a similar manner both in fresh trachea and after culture, independently of NGF treatment or CB1 receptor activation during the culture period. To verify that cannabinoids can influence EFS in mouse airways, the effect was first investigated on fresh tracheal segments from mice exposed to EFS with a constant train of 4 Hz. The contraction for the constant train was stable for more than an h and therefore suitable for studying direct effects of different treatments. Moreover, this effect could, as previously described, be blocked by atropine, implicating that it was mediated through activation of cholinergic nerves (Bachar et al., 2004). Administration of the CB1 receptor agonists ACEA or ACPA during the constant train period caused a marked inhibition of the contractile responses, suggesting a presynaptic modulatory role of these receptors. This effect is likely to occur through the inhibition of acetylcholine release as described in guinea pig trachea and human bronchus (Grassin-Delyle et al., 2014; Spicuzza et al.,
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Fig. 2. Acute effect of ACEA on constant train (4 Hz) stimulation responses in tracheal segments cultured for 4 days with NGF, NGFþ ACEA, ACEA (A). The effect of CB1 receptor antagonist AM251 on the acute inhibitory effect of ACEA (B) and ACPA (C) on constant train (4 Hz) stimulation responses. Carbachol-induced concentrationdependent contraction responses in tracheal segments cultured for 4 days with NGF, NGFþACEA, ACEA (D). Data are presented as Mean 7 S.E.M. (n¼ 6).
2000). That the effect is mediated by CB1 receptor is indicated both because ACEA and ACPA are highly selective CB1 receptor agonists possessing nearly 2000 times greater affinity to CB1 receptors over CB2 (Hillard et al., 1999), and also because the acute inhibitory effect of these agonists on EFS-contractions was blocked by the CB1 antagonist AM251. This contrast to the regulatory role of cannabinoids on neural control in guinea pig airways which has been shown to be mediated by TRPV1 rather than cannabinoid receptors (Nieri et al., 2003a, 2003b). On the other hand, it has been shown that CB2 receptor activation can reduce cholinergic neurotransmission in the mouse stomach by playing a prejunctional modulatory role on the cholinergic excitatory transmission (Mule et al., 2007). However CB2 receptor selective agonist JWH133 failed to inhibit the EFS-induced cholinergic cannabinoids further supporting that the inhibitory response of CB1 agonists in our experiments is mediated through the cannabinoid CB1 receptors. Recently, the CB1 receptor-mediated inhibitory effect of EFS-induced contractions has also been shown in human bronchus (Grassin-Delyle et al., 2014). Thus, our findings in mouse airways indicate that stimulation of presynaptic CB1 receptors can be an important mechanism to directly modify neurogenic contractility of the airways by acting on cholinergic nerves. After four days treatment the inhibitory effects of ACEA and ACPA on EFS-induced contractions were similar to those seen in fresh segments. As the effect of both agonists was inhibited by the CB1 receptor antagonist AM251, the role of CB1 receptor in inhibition of constant train EFS was further confirmed. Moreover, the effect by ACEA was neither influenced by the treatment of NGF nor by exposure of segments to ACEA during the four days culture. Thus, the direct effect of ACEA or ACPA in tracheal segments which is present also in segments cultured with CB1 receptor agonists, indicates that this acute direct effect is reversible and is not affected by tachyphylaxis. In the present study, treatment of the tracheas with NGF for four days caused an up-regulation of the intermediate frequencies
of the EFS-induced contractions in the mouse trachea. The culture period of four days has earlier shown to maintain both contractile and relaxant properties of the smooth muscle (Adner et al., 2002). In this study, treatment during this time period with either NGF, ACEA or both together did not alter the smooth muscle activation of muscarinic receptors, indicating a presynaptic effect for NGF during the culture procedure. This phenomenon was earlier described by Bachar and colleagues and was explained by an increase of neuronal fibres in the trachea (Bachar et al., 2004). It is possible that this peripheral effect of NGF can be the cause of the NGFenhanced nerve-mediated airway hyperresponsiveness observed in mice and guinea-pigs (Braun et al., 1998, 2001; de Vries et al., 1999). The NGF-induced up-regulation of the contractions was completely abolished when the tracheas were co-cultured with either of the CB1 receptor agonists ACEA or ACPA. As the effect was not prevented by CB2 agonist JWH133 when co-cultured with NGF, the compiled data support that this effect was mediated through the CB1 receptor. This contrasts with the earlier findings in guinea pig where CB2 receptor stimulation has shown to inhibit neurogenic airway inflammation (Yoshihara et al., 2005). In a recent study, using guinea pig tracheal segments, it was shown that four days culture with TNFα caused an increased contraction to EFS stimulation and that this effect was decreased by both CB1 and CB2 receptor activation (Makwana et al., 2015). However, this effect was demonstrated when the cannabinoids where given 30 min before EFS stimulation and caused a decrease below the effect for the control segments cultured with saline. Thus, this is the first study showing that long-term stimulation with CB1 receptor activation modulates specifically the inflammation-induced increase of neural-induced contraction. It has previously been shown that NGF causes an increase in nerve density in mouse tracheal segments (Bachar et al., 2004). Thus, one mechanism behind the specific reduction for CB1 receptor agonists on the NGF-induced increase could be an
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Fig. 3. EFS-induced frequency-dependent contraction responses in the tracheal segments cultured for 4 days with NGF, NGFþ ACEA, ACEA (A), NGF, NGFþ ACPA, ACPA (B) or NGF, NGFþJWH133, JWH133 (C). Data are presented as Mean 7 S.E.M. (n ¼6–9).
inhibition of nerve density. This study confirmed and extended the documentation by demonstrating that NGF caused a remarkable increase in nerve density, supporting an important effect for NGFinduced inflammation. Moreover, treatment with NGF together with ACEA restored the nerve density to control levels, whereas treatment with ACEA alone had no effect. Thus, these data clearly support the specific effect of CB1 receptor stimulation on NGFinduced inflammation and most likely explain the altered responses for the functional data. Our data show that the CB1 receptor has at least two separate effects, one for acute inhibition of cholinergic contractions and another for prevention of NGF-mediated hyperreactivity in mouse trachea. That the direct effect was not affected by the long-term
stimulation during culture indicates different signaling pathways participating for the two events (Fig. 5). As with many G-protein coupled receptors the intracellular activation of the CB1 receptor has been described to be complex (Turu and Hunyady, 2010). Although the main activity is considered to be generated through Gi/o proteins, diverse actions such as inhibition of cAMP, modulation of ion channels and activation of mitogen-activated proteins (MAPKs) been shown. Speculating, it is possible that the direct inhibitory effect on the EFS-stimulation acts through inhibition of cAMP or ion channel modulation, as these pathways are rapid in action. On the other hand, MAPK activation with subsequent transcriptional regulation takes longer and may be the mechanism by which CB1 agonists inhibit NGF-induced nerve growth. One
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Fig. 4. Fluorescence intensity measured from immunohistochemical preparations of fresh tracheal segments or tracheal segments cultured for 4 days with NGF, NGFþ ACEA or ACEA (A). Immunofluorescence images taken from proximal part of fresh tracheal segments, or segments cultured for 4 days with ACEA, NGF or NGFþACEA (B). Pictures were taken at 20 magnification. The scale bar represents 100 mm. Data are presented as Mean 7 S.E.M. (n¼ 6).
treatment of asthma and other inflammatory airway diseases by reducing airway hyperreactivity.
Conflict of interest The authors declare no conflict of interests.
Acknowledgements This study was supported by The Swedish Heart and Lung Foundation (Grant numbers 20130532, 20130636), Konsul Th C Berghs Research Foundation, the ChAMP project, COST BM1201, the Centre for Allergy Research at Karolinska Institutet, Karolinska Institutet, Hacettepe University Scientific Research Projects Coordination Unit (Project ID:1649) and The Scientific and Technological Research Council of Turkey (TUBITAK) (2219 Program).
References
Fig. 5. The two distinct effects of CB1 receptors on airway cholinergic nerves. Stimulation of CB1 receptors can acutely inhibit nerve-mediated airway contractions and can attenuate neural growth induced by NGF and thus prevent airway hyperreactivity (CB1: Cannabinoid CB1 receptor; Ach: Acetylcholine; EFS: Electrical field stimulation; NGF: Nerve growth factor; MR: Muscarinic receptor; TrkA: Tyrosine kinase A receptor; p75: p75 neurotrophin receptor).
protein downstream of MAPK is AKT, a protein that has been shown to be involved in the inhibitory effect of ACEA in NGF-induced bladder hyperreactivity in rats (Wang et al., 2015), may be of importance for the neural growth. Taken together, the present study in mouse trachea indicate that CB1 receptor activation can directly inhibit cholinergic contractile responses by a presynaptic mechanism and, additionally, can persistently modify neuronal airway hyperreactivity by an action involving inhibition of neuronal growth induced by NGF. In view of previous observations in human airways, it can be concluded that CB1 receptors qualify as preventive targets for
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