Published ahead of print on October 11, 2004, doi:10.1164/rccm.200405-600OC
© 2005 American Thoracic Society doi: 10.1164/rccm.200405-600OC
A Novel Role for Tachykinin Neurokinin-3 Receptors in Regulation of Human Bronchial Ganglia NeuronsDepartment of Medicine, The Johns Hopkins University, Baltimore, Maryland; Department of Pharmacology, The University of Western Australia, Perth, Western Australia, Australia; and Respiratory and Inflammation Center of Excellence for Drug Discovery, GlaxoSmithKline, King of Prussia, Pennsylvania Correspondence and requests for reprints should be addressed to Allen C. Myers, Ph.D., The Johns Hopkins Asthma and Allergy Center, 5501 Bayview Boulevard, 1A.62, Baltimore, MD 21224. E-mail: amyers{at}jhmi.edu
The neuropeptide tachykinins and their receptors have been implicated in the pathogenesis of lung disease, although the role of the tachykinin neurokinin-3 receptor has not been elucidated. Using confocal microscopy, we identified tachykinin neurokinin-3 receptors on human bronchial parasympathetic ganglion neurons. Electrophysiologic recordings demonstrated that activation of sensory nerve fibers, either by antidromic stimulation or capsaicin, depolarized these neurons. This response was mimicked by exogenously applied tachykinin neurokinin-3 receptor–selective agonist, senktide analogue, but not significantly by tachykinin neurokinin-1 or neurokinin-2 receptor–selective agonists. Responses to endogenous tachykinins or exogenous selective tachykinin neurokinin-3 receptor activation with senktide analogue were inhibited by the selective tachykinin neurokinin-3 receptor antagonists, SB 223412 or SB 235375. We provide the first evidence that tachykinin neurokinin-3 receptors regulate human bronchial parasympathetic ganglion neurotransmission by activation of a peripheral reflex. This pathway may play a significant role in controlling bronchomotor tone and air flow to the lung.
Key Words: airways • asthma • bronchoconstriction • chronic obstructive pulmonary disease • neurokinins Investigations of the lower airways of mammals demonstrate rich networks of afferent and efferent nerves that have a broad range of neurotransmitters and functions (1). The coordinated signaling between these networks triggers normal homeostatic responses including control of bronchomotor tone, cough, mucous secretion, and increases in bronchial microvascular permeability. In pulmonary diseases, such as asthma and chronic obstructive pulmonary disease (COPD), these responses are exaggerated to the point where they contribute significantly to airway pathologies including mucus hypersecretion, bronchial edema, enhanced bronchomotor tone, and airway hyperreactivity. For example, inhaled anticholinergics are used for symptomatic relief for COPD (2), highlighting the key role of parasympathetic nerves. Communication between these nerve networks, both at the level of the central nervous system and within the trachea and bronchi, is not clearly defined, however, especially in human airways. The tachykinins are a family of sensory neuropeptides, whose main members include substance P, neurokinin-A, and neurokinin-B, which produce their diverse effects by three distinct receptors, designated tachykinin neurokinin-1 receptors (NK-1R), NK-2R, and NK-3R. The tachykinins are found in sensory nerves in the lung and contract airway smooth muscle, mainly by interaction with NK-2Rs (3), whereas the vascular and proinflammatory effects are mediated predominantly by the NK-1R (4). Based on the elevated levels of the substance P and neurokinin-A in patients with asthma and COPD, and the myriad of effects of the tachykinins on pulmonary cells, it has been proposed that they may play a role in the pathophysiology of these diseases, by an interaction with NK-1Rs and NK-2Rs (4, 5). The presence and role of NK-3Rs in the human lung remains to be determined. Strong evidence for an important role for tachykinins in the regulation of parasympathetic ganglion neurotransmission has been revealed recently in guinea pig airways (6). Importantly, these data show NK-3R–mediated tachykinin-induced modulation of lower airway ganglion neurotransmission (6), associated with cholinergic airway smooth muscle contraction (7), and direct activation of parasympathetic neurons associated with airway smooth muscle relaxation (8). No role for NK-3R in such responses has been identified in the human lung, however, despite the fact that there is a relatively dense neurokinin-containing innervation in the intrinsic parasympathetic ganglia in human airways (9, 10). This led us to determine whether similar receptors exist in human bronchial ganglia neurons and whether they had a potential role to play in the pathophysiology of lung disease. The present study reports for the first time the immunohistochemical detection of human airway ganglion cell NK-3Rs and their role in the modulation of peripheral reflex parasympathetic neurotransmission. The NK-3R may represent a novel therapeutic approach for the treatment of lung diseases, such as COPD, which are characterized by aberrant parasympathetic drive. Some results in this study have been previously reported in the form of an abstract (11).
Tissue Preparation Methods for electrophysiologic recording and visualization of bronchial ganglia neurons have been described recently (12, 13). Bronchi dissected from the lungs of 28 human organ donors were transferred to warmed (37°C) oxygenated Krebs bicarbonate buffer, equilibrated with 95% O2–5% CO2 (pH 7.4); the composition of Krebs buffer was (in millimolar): NaCl, 136; KCl, 5.6; MgCl2, 1.2; CaCl2, 2.2; NaH2PO2, 1.2; NaHCO3, 14.3, and dextrose, 11. The average age of the organ donors was 41 ± 7 years (range of 20–62 years; 13 women, 15 men). The lungs were obtained by The National Disease Research Institute (Philadelphia, PA), and shipped to our laboratory within 24 hours of removal. This tissue is from deceased, deidentified organ donors and has been approved for research by The Johns Hopkins University Human Subjects Research Committee. Using intracellular recording techniques (12, 13), synaptic input was verified by the presence of peribronchial nerve-stimulated fast excitatory postsynaptic potentials (12), which were subsequently blocked with hexamethonium (100 µM); atropine (0.1 µM) and CP 99994 (1 µM) were also added at this time to block muscarinic and NK-1 receptors, respectively. The responses to nerve stimulation, bath-applied (1- to 2-minute exposure) tachykinin receptor agonists, or capsaicin were recorded before and after treatment (  30 minutes) with the NK-3R–selective antagonists SB 235375 (1 µM) or SB 223412 (1 µM). In separate experiments, the tissue was incubated for longer periods of time ( 1 hour) with either a lower concentration of NK-3R antagonist (SB223412, 0.1 µM) or with the NK-2R antagonist SR 48,968 (0.1 µM) before nerve stimulation, capsaicin, or agonist application.
Confocal Microscopy Studies Ganglia were then washed repeatedly at 4°C over a 24-hour period in wash buffer (0.1% Triton X-100; Tris-buffered saline: 50 mM Tris-HCL, 100 mM NaCl, pH 7.4). Incubation with secondary antibodies (goat antimouse Alexa Fluor 488 and goat antirabbit Alexa Fluor 546; 1:250; Molecular Probes, Eugene, OR) was conducted overnight at 4°C in the dark. Preparations were again washed over a 24-hour period in wash buffer and then examined with a BioRad MRC1024 laser scanning confocal microscope (Hemmel Hempstead, UK) using sequential imaging for dual emission protocols (14).
Data Presentation and Statistical Analysis
Drugs and Reagents
Confocal Microscopy Immunofluorescent staining of NK-3R in whole mount preparations of human isolated bronchial parasympathetic ganglia (n = 4) was examined by confocal microscopy. An antibody to PGP 9.5 was used as a marker of neurons including ganglion cell bodies. As expected, cell bodies and their processes were positive for PGP 9.5 staining (red) (Figure 1A). NK-3R staining (green) was also seen in these ganglion structures (Figure 1B). Instead of staining the entire structure as observed with PGP 9.5, however, NK-3R appeared as localized, punctate staining, associated with some, but not all, of the neuronal cell bodies. When the images for PGP 9.5 and NK-3R staining were computer-superimposed, areas of colocalized PGP 9.5 and NK-3R appear in yellow (Figure 1C). When the NK-3R antibody was incubated with its blocking peptide, there was no staining even though the PGP 9.5 staining was still visible in red (data not shown).
Electrophysiology Electrophysiologic current clamp recordings were made from the somas of intrinsic ganglia neurons as previously described (12); the active and passive membrane properties (e.g., resting membrane potentials and membrane resistance) were similar to those reported in that study, with ranges of –31 to –68 mV, and 10 to 70 M  , respectively.
Effects of nerve stimulation.
The NK-3R–selective antagonists SB 235375 (6, 15) or SB 223412 (6, 16) essentially abolished nerve-stimulated membrane depolarization. In paired preparations, in the presence of SB 235375 (1 µM, n = 4) or SB 223412 (1 µM, n = 3), antidromic stimulation of sensory nerves evoked a 0.5 ± 1 mV depolarization (Figure 2B) of the resting potential (p < 0.05) when compared with predrug control responses (4.4 ± 0.9 mV for SB 235375 and 3.8 ± 1.1 mV for SB 2223412; Figure 2C) with no change in the membrane resistance. In separate, unpaired experiments, ganglia were perfused with 0.1 µM SB 223412 for greater than or equal to 1 hour and nerve stimulation had little effect on four neurons; depolarization in the presence of SB 223412 was 0.5 ± 0.5 mV (n = 4; not shown). Similar observations were made with 0.1 µM SB 235375 (n = 2; not shown).
In three preparations examined, continuous superfusion (
Effects of capsaicin.
Effects of tachykinin receptor agonists. The responses to nerve stimulation and capsaicin application were mimicked by the NK-3R–selective agonist, senktide analogue. Senktide analogue (0.1 µM, 8–10 ml, 1 minute) caused a 4.3 ± 2 mV (n = 3, range of 3–6 mV) depolarization of human bronchial ganglion neurons (Figure 4A); a second application of senktide analogue (0.1 µM, 8–10 ml), 30 minutes after the first application, elicited a 3 ± 1 mV depolarization (p > 0.05 when compared with the first application).
In a separate series of experiments, a single application of senktide analogue (0.1 µM, 8–10 ml, 1 minute) caused a 6.4 ± 2 mV membrane depolarization (range of 5–9 mV) in six neurons; three other neurons did not depolarize in response to initial application of senktide analogue and were not further studied (not shown). SB 235375 (1 µM; 30-minute perfusion pretreatment), essentially abolished the response to senktide analogue (0.1 µM, 8–20 ml, 1–2 minutes) in four of the responding neurons; depolarization in the presence of SB 235375 was 1 ± 1 mV (p < 0.05 compared with first application; n = 4). SB 223412 (1 µM) had a similar effect on senktide analogue (0.1 µM, 8–20 ml, 1–2 minutes) depolarizations in two neurons (not shown). In separate, unpaired experiments, ganglia were perfused with 0.1 µM SB 223412 for greater than or equal to 60 minutes and senktide analogue (0.1 µM, 8–20 ml, 1–2 minutes) had little or no effect on four neurons; depolarization in the presence of SB 223412 was 0.7 ± 0.5 mV (n = 4; not shown). Similar observations were made with 0.1 µM SB 235375 (n = 2; not shown). The NK-1R–selective agonist ASM-substance P (0.01 ± 1 µM, 8–10 ml, 1 minute) did not depolarize the resting potential, but at concentrations of 0.1 and 1 µM increased the membrane resistance 23 ± 11% (range of 12–44%) in three of six neurons (Figure 4C); this effect was blocked with the NK-1 receptor antagonist CP 99994 (1 µM; not shown). Neurokinin-A (0.1 µM, 8–10 ml, 1 minute; n = 4) or the NK-2R–selective agonist ßAla-8-neurokinin-A–4–10 (1 µM, 8–10 ml; n = 2) had no effect on the resting membrane potential or input resistance (not shown).
Although the effects and potential pathophysiologic roles of tachykinins and the NK-1Rs and NK-2Rs in the mammalian lung have been extensively studied (4, 5, 17), there is limited information on NK-3Rs, in particular, in human lung. Indeed, only very low levels of NK-3R have ever been reported in human lung (18). The present study is the first to identify the location and define the function of NK-3Rs in a subpopulation of human bronchial parasympathetic ganglion neurons. These data provide evidence for a unique neuromodulatory pathway with the potential to significantly influence airway caliber in disease, and may represent a novel therapeutic approach (i.e., NK-3R antagonism) for lung disease characterized by dysfunction in neuronal inputs (e.g., COPD) (2). The size and location of these NK-3R–containing cells are very similar to principal neurons in human bronchial parasympathetic ganglia (12). Intracellular electrophysiologic recordings provided convincing evidence for peripheral reflex regulation of parasympathetic nerve activity in human bronchus. Given that the parasympathetic autonomic nervous system provides the dominant neural efferent drive to critical submucosal tissues, including airway smooth muscle, the negative impact of up-regulated NK-3R–mediated enhancement of a reflex is likely to be significant, particularly in airway diseases involving airflow limitation, such as asthma and COPD. The predominant role of the parasympathetic system in controlling lung function is highlighted by the use of inhaled anticholinergics, such as ipratropium or tiotropium, for symptomatic relief in COPD (2). Direct recording of cellular electrical activity revealed that activation of the capsaicin-sensitive nerves innervating the bronchus evoked a marked membrane depolarization of airway ganglion neurons. The cells that responded to nerve stimulation were most likely principal neurons and not interneurons, because fast excitatory postsynaptic potentials were recorded in these cells (12). That the observed response is caused by antidromic stimulation of sensory nerves came from evidence that (1) the response is associated with tachykinins; (2) the response is not inhibited by a cholinergic receptor antagonist (e.g., hexamethonium or atropine); and (3) we can mimic the response with capsaicin, a compound known to cause the release of the transmitter from sensory nerves. Evidence that these responses were caused by NK-3R activation, and were not secondary to NK-1, NK-2, nicotinic, or muscarinic receptor activation (19), was clearly demonstrated by the lack of effect of NK-1R or NK-2R antagonism, hexamethonium, or atropine, respectively, on nerve-stimulated depolarizations. In addition, this observation is supported by the lack of significant effect of selective NK-1R or NK-2R agonists. Furthermore, antidromic sensory nerve stimulation and capsaicin-induced membrane depolarization were mimicked by the NK-3R–selective agonist senktide analogue. Importantly, all of these responses were attenuated by the NK-3R antagonist SB 223412 (16) and abolished by the closely related analogue SB 235375 (15). The time course and changes in membrane resistance associated with the depolarizations of human bronchial ganglia neurons by senktide analogue were similar to those reported for guinea pig bronchial neurons (20). Unlike guinea pig bronchial neurons, however, activation of NK-3R in human ganglia depolarized the neurons to action potential threshold (Figure 3). In the human airway ganglia, only a subpopulation of neurons responded to nerve stimulation, capsaicin, or senktide analogue, correlating with the anatomic observation that only a subpopulation of neurons was immunoreactive for the NK-3R. Such results suggest either that the sensory nerve fibers have degraded or, possibly, heterogeneity within the population of neurons within a ganglion. Previous studies have demonstrated that activation of tachykinin receptors alters cholinergic contractions of the lower airways of rabbits (21) and guinea pigs (7, 22), with such effects usually associated with postganglionic mechanisms. The postganglionic effects of neurokinins on both the cholinergic nerves and on the airway smooth muscle have been shown to be caused exclusively by activation of NK-1R and NK-2R (21, 22). A unique role for NK-3R activation has been recently reported for guinea pig lower airway parasympathetic ganglia where NK-3R activation does not elicit action potentials in ganglia neurons but does potentiate synaptic transmission and cholinergic contractions (6). The results in the present study are entirely distinct from these reported effects of tachykinins on guinea pig airway nerves and describe an additional, novel pathway for peripheral reflex regulation of smooth muscle tone in the lower airway. Conceptually, the peripheral reflex may be a mechanism for local regulation of airflow to the lung, such that when a stimulus is localized to one bronchial segment, parasympathetic tone is altered only in that segment. The present study provides morphologic and electrophysiologic evidence for NK-3R on principal neurons in human bronchial parasympathetic ganglia and also provides the first reported evidence of a tachykinin-mediated peripheral reflex in any human autonomic ganglion. Based on the present studies, tachykinins released from sensory nerves in airway ganglia may activate NK-3R and have an important role in localized neural regulation of airflow to the lungs. That NK-3R are restricted to parasympathetic nerves indicates their potential to mediate tachykinin-induced transmission within the ganglion (23), without affecting normal function of sensory nerves or neurotransmitters released from parasympathetic ganglion nerve terminals. These findings demonstrate an unrealized role for NK-3Rs in the modulation of aberrant parasympathetic drive in the lower airways.
The authors acknowledge the important contributions to this work from Dr. Paul Rigby and Ms. Lisa Spalding from the Biomedical Confocal Microscopy Research Centre, Pharmacology Unit, School of Medicine and Pharmacology, The University of Western Australia, and from Ms. Holly K Rohde, The Johns Hopkins University. The authors also thank Dr. James Krause (Neurogen) for providing the NK-3R antibody.
Supported in part by a grant from the National Heart, Lung and Blood Institute of the National Institutes of Health, Bethesda, Maryland (A.C.M.), and by GlaxoSmithKline, King of Prussia, Pennsylvania. Conflict of Interest Statement: A.C.M. received materials, peptides and receptor antagonists from SmithKlineBeecham (now GlaxoSmithKline), and the Department in which these studies were performed receives a nonbinding contract from GlaxoSmithKline, and GlaxoSmithKline has had a nonbinding, not for services, contract ranging from $50,000 to $300,000 per year with the Division of Allergy and Clinical Immunology in the Department of Medicine at The Johns Hopkins University since 1990 for access to intellectual input and testing of compounds. This agreement does not deny investigators the right to examine data independently nor does it prevent submission of manuscripts for publication without first obtaining the consent of the sponsor; A.C.M. had full access to all of the electrophysiologic data in this study and takes complete responsibility for the integrity of that data and the accuracy of the data analysis; R.G.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.W.P.H. is employed by GlaxoSmithKline. Received in original form May 10, 2004; accepted in final form October 4, 2004
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ABSTRACT
METHODS
-ethylbenzyl]-3-hydroxy-2-phenylquinoline-4-carboxamide), and SB 235375 ([-]-[S]-N- [
