Airway Cholinergic Nerves Research Articles

Differentiation and on axon-guidance chip culture of human pluripotent stem cell-derived peripheral cholinergic neurons for airway neurobiology studies.

Airway cholinergic nerves play a key role in airway physiology and disease. In asthma and other diseases of the respiratory tract, airway cholinergic neurons undergo plasticity and contribute to airway hyperresponsiveness and mucus secretion. We currently lack human in vitro models for airway cholinergic neurons. Here, we aimed to develop a human in vitro model for peripheral cholinergic neurons using human pluripotent stem cell (hPSC) technology. hPSCs were differentiated towards vagal neural crest precursors and subsequently directed towards functional airway cholinergic neurons using the neurotrophin brain-derived neurotrophic factor (BDNF). Cholinergic neurons were characterized by ChAT and VAChT expression, and responded to chemical stimulation with changes in Ca2+ mobilization. To culture these cells, allowing axonal separation from the neuronal cell bodies, a two-compartment PDMS microfluidic chip was subsequently fabricated. The two compartments were connected via microchannels to enable axonal outgrowth. On-chip cell culture did not compromise phenotypical characteristics of the cells compared to standard culture plates. When the hPSC-derived peripheral cholinergic neurons were cultured in the chip, axonal outgrowth was visible, while the somal bodies of the neurons were confined to their compartment. Neurons formed contacts with airway smooth muscle cells cultured in the axonal compartment. The microfluidic chip developed in this study represents a human in vitro platform to model neuro-effector interactions in the airways that may be used for mechanistic studies into neuroplasticity in asthma and other lung diseases.

A Rho-kinase inhibitor, Y-27632, reduces cholinergic contraction but not neurotransmitter release

This study examined the effects of the selective Rho-kinase inhibitor Y-27632 [(+)-( R)- trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride]) on cholinergic nerve-mediated contraction and neurotransmitter release in murine and guinea-pig isolated tracheal preparations. In tracheal preparations obtained from both species, Y-27632 shifted carbachol concentration–effect curves to the right and reduced the maximal contractile response. Repeated electrical field stimulation (EFS) evoked transient, consistent and reproducible contractions in murine and guinea-pig tracheal preparations. Y-27632 inhibited these cholinergic nerve-mediated contractions in a concentration-dependent manner. EFS (0.1–30 Hz) elicited frequency-dependent cholinergic nerve-mediated contractile responses. In murine tracheal preparations, Y-27632 (3 μM and 10 μM) shifted frequency–response curves to EFS to the right by 5.5 and 13.0 fold respectively and markedly reduced the maximal contractile response. In murine and guinea-pig tracheal preparations loaded with [ 3H]-choline, Y-27632 (10 μM) significantly increased the EFS-induced outflow of radioactivity from airway cholinergic nerves by 27% and 54% respectively. Thus, Y-27632 inhibited both carbachol-induced and cholinergic nerve-mediated contractile responses. Conversely, Y-27632 increased neurotransmitter release from airway cholinergic nerves. However, since antagonism of acetylcholine-induced contraction by Y-27632 overwhelmed the increased neurotransmitter release, the overall effect of this Rho-kinase inhibitor was to inhibit cholinergic nerve-mediated contraction.

Adhesion-dependent interactions between eosinophils and cholinergic nerves.

Eosinophils adhere to airway cholinergic nerves and influence nerve cell function by releasing granule proteins onto inhibitory neuronal M(2) muscarinic receptors. This study investigated the mechanism of eosinophil degranulation by cholinergic nerves. Eosinophils were cocultured with IMR32 cholinergic nerve cells, and eosinophil peroxidase (EPO) or leukotriene C(4) (LTC(4)) release was measured. Coculture of eosinophils with nerves significantly increased EPO and LTC(4) release compared with eosinophils alone. IMR32 cells, like parasympathetic nerves, express the adhesion molecules vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 (ICAM-1). Inhibition of these adhesion molecules alone or in combination significantly inhibited eosinophil degranulation. IMR32 cells also significantly augmented the eosinophil degranulation produced by formyl-Met-Leu-Phe. Eosinophil adhesion to IMR32 cells resulted in an ICAM-1-mediated production of reactive oxygen species via a neuronal NADPH oxidase, inhibition of which significantly inhibited eosinophil degranulation. Additionally, eosinophil adhesion increased the release of ACh from IMR32 cells. These neuroinflammatory cell interactions may be relevant in a variety of inflammatory and neurological conditions.

Eosinophil adhesion to cholinergic nerves via ICAM-1 and VCAM-1 and associated eosinophil degranulation.

In vivo, eosinophils localize to airway cholinergic nerves in antigen-challenged animals, and inhibition of this localization prevents antigen-induced hyperreactivity. In this study, the mechanism of eosinophil localization to nerves was investigated by examining adhesion molecule expression by cholinergic nerves. Immunohistochemical and functional studies demonstrated that primary cultures of parasympathetic nerves express vascular cell adhesion molecule-1 (VCAM-1) and after cytokine pretreatment with tumor necrosis factor-alpha and interferon-gamma intercellular adhesion molecule-1 (ICAM-1). Eosinophils adhere to these parasympathetic neurones after cytokine pretreatment via a CD11/18-dependent pathway. Immunohistochemistry and Western blotting showed that a human cholinergic nerve cell line (IMR-32) expressed VCAM-1 and ICAM-1. Inhibitory experiments using monoclonal blocking antibodies to ICAM-1, VCAM-1, or CD11/18 and with the very late antigen-4 peptide inhibitor ZD-7349 showed that eosinophils adhered to IMR-32 cells via these adhesion molecules. The protein kinase C signaling pathway is involved in this process as a specific inhibitor-attenuated adhesion. Eosinophil adhesion to IMR-32 cells was associated with the release of eosinophil peroxidase and leukotriene C(4). Thus eosinophils adhere to cholinergic nerves via specific adhesion molecules, and this leads to eosinophil activation and degranulation; this may be part of the mechanism of eosinophil-induced vagal hyperreactivity.

Modulation of ACh release from airway cholinergic nerves in horses with recurrent airway obstruction.

To evaluate the functional status of neuronal alpha2-adrenoceptors (ARs) and beta2-ARs on ACh release in horses with recurrent airway obstruction (RAO), we examined the effects of the physiological agonists epinephrine (Epi) and norepinephrine (NE) and the beta2-agonists RR- and RR/SS-formoterol on ACh release from airway cholinergic nerves of horses with RAO. Because SS-formoterol, a distomer of the beta2-agonist, increases ACh release from airways of control horses only after the autoinhibitory muscarinic receptors are blocked by atropine, we also tested the hypothesis that if there is an M2-receptor dysfunction in equine RAO, SS-formoterol should increase ACh release even in the absence of atropine. ACh release was evoked by electrical field stimulation and measured by HPLC. Epi and NE caused less inhibition of ACh release in horses with RAO than in control horses. At the catecholamine concentration achieved during exercise (10(-7) M), the inhibition induced by Epi and NE was 10.8 +/- 13.2 and 3.4 +/- 6.8%, respectively, in equine RAO versus 41.0 +/- 6.4 and 27.1 +/- 5.6%, respectively, in control horses. RR- and RR/SS-formoterol (10(-8) to 10(-5) M) increased ACh release to a similar magnitude as that in control horses. These results indicate that neuronal beta2-ARs are functioning; however, the alpha2-ARs are dysfunctional in the airways of horses with RAO in response to circulating catecholamines. SS-formoterol (10(-8) to 10(-5) M) facilitated ACh release in horses with RAO even in the absence of atropine. Addition of atropine did not cause significantly more augmentation of ACh release over the effect of SS-formoterol alone. The magnitude of augmentation in horses with RAO in the absence of atropine was similar to that in control horses in the presence of atropine. The latter observations could be explained by neuronal muscarinic-autoreceptor dysfunction in equine RAO.

Regulation of the expression and function of the M2 muscarinic receptor.

Since the cloning and expression of many of the G protein-coupled receptors during the 1980s, there has been a massive increase in our understanding of many aspects of their function. The use of molecular biology to engineer and express mutant receptors has made it possible to determine key amino acids involved in receptor function. Although advances in molecular biology have contributed greatly to our understanding of the pharmacology and structure of the five subtypes of muscarinic receptor, much remains to be learned about the factors that regulate their expression and function. This review by El-Bdaoui Haddad and Jonathan Rousell describes the current state of awareness and highlights recent advances made in the elucidation of the mechanisms involved in muscarinic receptor regulation. Because most is known about the regulation of expression of the M2 receptor subtype, particular attention will be paid to it. Furthermore, this receptor subtype plays an important role in regulating acetylcholine output from airway cholinergic nerves, and there is substantial evidence from studies both in vivo and in vitro in human and animal models that these receptors are dysfunctional in asthma.

Immunocytochemical detection of endothelin receptors in rat cultured airway nerves.

Endothelin-1 (ET-1) has been shown to potentiate cholinergic neurotransmission in human bronchus as well as in airways from a variety of animal species, suggesting that ET receptors exist prejunctionally on airway cholinergic nerves. We have successfully isolated and maintained rat tracheal para-sympathetic neurons in culture. Most cultured cells were associated with specific fluorescence for the nerve cell marker protein gene product 9.5 (PGP 9.5). These cultures contained a high proportion of parasympathetic neurons. Importantly, specific immunofluorescent antibodies for ETB receptors were colocalized with those for PGP 9.5. Therefore, for the first time, ETB receptors have been shown to exist on airway parasympathetic neurons in culture.

Origin and modulation of ACh release from rat airway cholinergic nerves

The release of acetylcholine (ACh) from airway parasympathetic nerves was studied in rat trachea. We established stimulus parameters, examined the role of extracellular Ca2+, and investigated the origin of the released ACh by use of vesamicol, an inhibitor of ACh uptake in synaptic vesicles. The role of muscarinic autoreceptors and prostanoids on ACh release was also studied. Tracheal rings were incubated in Krebs-Henseleit solution containing neostigmine and guanethidine with or without atropine. ACh release was measured by high-performance liquid chromatography with electrochemical detection. ACh release was dependent on frequency (0.5-16 Hz), voltage (10-25 V), and pulse duration (0.5-4 ms). At 4 Hz, one-fifth of electrical field stimulation-induced ACh release was extracellular Ca2+ independent and vesamicol resistant, indicating its nonvesicular origin. Three-fifths were Ca2+ dependent and vesamicol sensitive, indicating that it was newly synthesized, and one-fifth was Ca2+ dependent but vesamicol resistant, indicating its origin from prestored vesicles. At 16 Hz, two-fifths were nonvesicular and three-fifths were newly synthesized. Blockade of the muscarinic autoreceptor by atropine potentiated the release of ACh four- to fivefold. Neither of the cyclooxygenase inhibitors indomethacin or meclofenamate nor exogenous prostaglandin E2 affected ACh release, indicating that inhibitory prostanoids do not modulate ACh release.

Excitatory prejunctional β 2-adrenoceptor distribution within equine airway cholinergic nerves

We examined the effect of activation of β 2-adrenoceptor (AR) by isoproterenol (ISO) on acetylcholine (ACh) release evoked by electrical field stimulation (EFS: 20 V, 0.5 Hz, 0.5 msec) from cholinergic nerves in five regions of equine airways. We also tested if the effect of ISO was dependent on epithelium or prostanoids by examining the effect of ISO on ACh release in the presence and absence of epithelium or cyclooxygenase inhibition. Trachealis strips or bronchial rings were preincubated for 60 min with 10 −7 M atropine, 10 −6 M neostigmine, and 10 −5 M guanethidine. The ACh amount was measured by high-performance liquid chromatography with electrochemical detection. Isoproterenol (10 −8−10 −6 M) augmented ACh release throughout the whole airway in a concentration-dependent manner. Rubbing off the epithelium potentiated EFS-induced ACh release but neither epithelium removal nor cyclooxygenase inhibition affected the magnitude of ISO-induced augmentation of ACh release. These results indicate that in equine airway parasympathetic nerves: (1) excitatory prejunctional β 2-adrenoceptors are distributed throughout the tracheobronchial tree; (2) the function of this excitatory β 2-adrenoceptor is independent of endogenous prostanoids and epithelium; and (3) there is an epithelium-derived factor that inhibits ACh release.

Prejunctional muscarinic autoreceptors on horse airway cholinergic nerves

Muscarinic autoreceptors on horse airway cholinergic nerves were studied by examining the effects of muscarinic receptor antagonists on electrical field stimulation (EFS)-induced acetylcholine (ACh) release in trachealis preparations. All the antagonists including atropine (non-selective), pirenzepine (M 1-selective), AF-DX 116 (M 2-selective), and hexahydrosiladifenidol (M 3-selective) augmented ACh release concentration-dependently. The augmentation was not due to displacement of ACh molecules from tissue receptors into the bath liquid because incubation with atropine after EFS had no influence on the measured amount of ACh. Hexahydrosiladifenidol was more potent in inhibiting ACh-induced muscle contraction, which is known to be mediated by M 3 receptors, than in augmenting ACh release. The maximal ACh release rate in response to the selective antagonists was much less than that following atropine. Furthermore, the concentrations of the selective antagonists required to augment ACh release far exceeded their K ds for m 1, M 2, or M 3 receptors. These observations suggest that the muscarinic autoreceptors on horse airway cholinergic nerves may belong to a novel subtype.

Acetylcholine release from airway cholinergic nerves in horses with heaves, an airway obstructive disease.

The present study was conducted to determine if acetylcholine (ACh) release from airway cholinergic nerves is increased and if modulation of ACh release by prejunctional receptors is altered in horses with heaves, an obstructive airway disease characterized by airway inflammation and bronchospasm. Trachealis strips and bronchial segments of normal horses and horses affected with heaves were suspended in 2-ml tissue baths. ACh release was induced by electrical field stimulation and the bath ACh content was measured by high performance liquid chromatography (HPLC) with electrochemical detection. In response to electrical field stimulation, the rate of ACh release from cholinergic nerves innervating the trachea was not significantly different between the two groups of horses. The nonselective muscarinic receptor antagonist atropine augmented ACh release to the same extent in both groups, indicating that there is no dysfunction of muscarinic autoreceptors on cholinergic nerves of large airways from horses with heaves. Compared with the data from normal horses, the inhibitory effect of the alpha 2-adrenoceptor agonist clonidine on ACh release was lacking in the bronchi and less potent in the trachea, suggesting that the prejunctional alpha 2-adrenoceptors are dysfunctional in horses with heaves. Neither exogenous prostaglandin E2 (PGE2) nor the cyclooxygenase inhibitor indomethacin influenced ACh release from the bronchi, suggesting that a decrease in airway mucosal PGE2 production reported previously in horses with heaves does not alter ACh release.

Acetylcholine release from airway cholinergic nerves in horses with heaves, an airway obstructive disease.

The present study was conducted to determine if acetylcholine (ACh) release from airway cholinergic nerves is increased and if modulation of ACh release by prejunctional receptors is altered in horses with heaves, an obstructive airway disease characterized by airway inflammation and bronchospasm. Trachealis strips and bronchial segments of normal horses and horses affected with heaves were suspended in 2-ml tissue baths. ACh release was induced by electrical field stimulation and the bath ACh content was measured by high performance liquid chromatography (HPLC) with electrochemical detection. In response to electrical field stimulation, the rate of ACh release from cholinergic nerves innervating the trachea was not significantly different between the two groups of horses. The nonselective muscarinic receptor antagonist atropine augmented ACh release to the same extent in both groups, indicating that there is no dysfunction of muscarinic autoreceptors on cholinergic nerves of large airways from horses with heaves. Compared with the data from normal horses, the inhibitory effect of the alpha 2-adrenoceptor agonist clonidine on ACh release was lacking in the bronchi and less potent in the trachea, suggesting that the prejunctional alpha 2-adrenoceptors are dysfunctional in horses with heaves. Neither exogenous prostaglandin E2 (PGE2) nor the cyclooxygenase inhibitor indomethacin influenced ACh release from the bronchi, suggesting that a decrease in airway mucosal PGE2 production reported previously in horses with heaves does not alter ACh release.

Catecholamine affects acetylcholine release in trachea: alpha 2-mediated inhibition and beta 2-mediated augmentation

We investigated the effects of catecholamines on acetylcholine (ACh) release from equine airway parasympathetic nerves. Trachealis strips were suspended in 2-ml tissue baths with Krebs-Henseleit solution containing atropine (10(-7) M), neostigmine (10(-6) M), and guanethidine (10(-5) M). Electrical field stimulation (20 V, 0.5 ms, 0.5 Hz, for 15 min) was applied, and ACh was measured by high-performance liquid chromatography with electrochemical detection. Epinephrine (Epi) and norepinephrine (NE) inhibited ACh release in a concentration-dependent manner. Inhibition was attenuated by the alpha 2-adrenoceptor antagonist idazoxan (10(-6) M) but not by the alpha 1-antagonist prazosin (10(-6) M). After alpha 2-blockade with idazoxan (10(-5) to 10(-4) M), Epi but not NE augmented ACh release. Isoproterenol (10(-7) to 10(-5) M) increased ACh release, an effect that was reversed by the beta 2-adrenoceptor antagonist ICI-118,551 (10(-5) M) but not by the beta 1-adrenoceptor antagonist atenolol (10(-5) M). Our results indicate that horse airway cholinergic nerves are modulated by both alpha 2-inhibitory and beta 2-excitatory adrenoceptors, with the former being predominant.

ACh release from horse airway cholinergic nerves: effects of stimulation intensity and muscle preload.

This study was conducted to determine the effects of stimulation parameters and muscle preload on acetylcholine (ACh) release induced by electrical field stimulation (EFS) of horse airway cholinergic nerves. Trachealis strip bundles were prepared and suspended in 2-ml tissue baths. The tissues were stimulated three to five times for 30 min each. Increasing frequency (0.5-16 Hz) and voltage (5-20 V) increased ACh release; increasing pulse duration (0.5-3 ms) had only a minor effect. Alterations in muscle preload (2-20 g) had no effect on ACh release. ACh release was fairly constant for up to five repeated stimulation periods with the same EFS parameters. Stimulation of the tissues for 15 min released the same amount of ACh as 30 min if the amount was expressed as picomoles per gram per minute, suggesting that ACh release rate was constant during the 30-min period of stimulation. Atropine (10(-6) M) potentiated the release of ACh four- to fivefold, presumably by removing the autoinhibitory effect of ACh on the cholinergic nerves. Tetrodotoxin (10(-6) M) abolished the EFS-induced ACh release.

Effect of bradykinin on airway neural responses in vitro.

We investigated the effects of bradykinin (BK) on airway excitatory nonadrenergic noncholinergic (e-NANC) and cholinergic nerves in vitro. Neural responses were elicited by electrical field stimulation in guinea pig airways in vitro before and after the addition of BK (10(-10)-10(-7) M). Captopril (10(-5) M) and phosphoramidon (10(-6) M) were added to prevent degradation of BK, and all neural responses were measured in the presence of indomethacin (10(-5) M) and propranolol (10(-6) M). BK potentiated e-NANC responses in bronchi in a concentration-dependent manner (10(-10)-10(-7) M) without changing concentration-response curves to exogenously applied substance P (10(-10)-10(-5) M). BK significantly potentiated e-NANC neural constrictor responses by 22 +/- 7% at 10(-8) M (mean +/- SE, n = 5, P < 0.05) and 32 +/- 7% at 10(-7) M (n = 8, P < 0.01), compared with changes in time-matched control tissues (7 +/- 2%, n = 8). The potentiation of e-NANC responses by BK was abolished by pretreatment with a specific B2-receptor antagonist, HOE 140 (10(-7) M). Cholinergic constrictor responses elicited to electrical field stimulation were not affected by the addition of BK (up to 10(-7) M). These results suggest that BK potentiates e-NANC bronchoconstrictor responses prejunctionally via a B2-receptor.

The effect of methoctramine on airway cholinergic nerves in the guinea-pig

The effect of methoctramine on airway cholinergic nerves in the guinea-pig

Inhibitory Muscarinic Receptors on Airway Cholinergic Nerves in Man: Evidence for a Defect in Asthma

Inhibitory Muscarinic Receptors on Airway Cholinergic Nerves in Man: Evidence for a Defect in Asthma

Selective autonomic stimulation of canine trachealis with dimethylphenylpiperazinium.

The response of canine tracheal muscle to autonomic stimulation with 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) was studied isometrically in 39 dogs in vivo. Intra-arterial (ia) DMPP (2.5 X 10(-4) to 2.5 X 10(-2) mg/kg) caused selective dose related contraction [maximum 30.1 +/- 6.5 gram-force (gf)/cm] due to regional stimulation of parasympathetic ganglia. This contraction was blocked by regional administration of atropine 10(-3) mg/kg ia and hexamethonium 5 X 10(-2) mg/kg ia. Nonselective intravenous (iv) administration of DMPP 2.5 X 10(-2) mg/kg caused parasympathetic tracheal contraction [+13.4 +/- 1.64 gf/cm] followed by later sympathetic relaxation [-11.8 +/- 2.3 gf/cm]; 0.5 mg/kg iv atropine abolished contraction but did not affect relaxation. The role of the adrenal gland vs. direct sympathetic innervation in producing tracheal relaxation after sympathetic stimulation was also studied. Tracheal relaxation to 2.5 X 10(-2) mg/kg iv DMPP was -18.2 +/- 4.0 gf/cm before adrenalectomy (ADX) and -4.3 +/- 0.9 gf/cm afterward (P less than 0.001). In contrast, tracheal contraction resulting from alpha-adrenergic stimulation after 2.5 X 10(-2) mg/kg iv DMPP in beta-blocked (BB) dogs was not significantly altered by ADX. At 2.5 X 10(-1) mg/kg iv DMPP, the alpha-adrenergic contractile response was still 70% of the response prior to ADX. We conclude that sympathetic tracheal relaxation in dogs is predominantly mediated by circulating catecholamine from the adrenal gland, but that alpha-adrenergic contraction after BB results predominantly from direct sympathetic innervation and is not greatly augmented by adrenal secretion. We also report a new method for selective stimulation of airway cholinergic nerves in vivo without systemic effects.