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INTEGRATIVE |
1 Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, USA
2 Howard Florey Institute, University of Melbourne, Victoria, Australia 3010
3 The Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224, USA
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Abstract |
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(Received 3 January 2006;
accepted after revision 26 March 2006;
first published online 31 March 2006)
Corresponding author B. J. Canning: Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MA 21224, USA. Email: bjc{at}jhmi.edu
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Introduction |
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Action potentials in pre- and post-ganglionic sympathetic nerves potentially regulating airway smooth muscle tone have been recorded at rest and during airway stimulation (Widdicombe, 1966; Bachoo & Polosa, 1987; Habler et al. 1994; Shirai et al. 1995a,b). Reflex-mediated alterations in their activity have been attributed mostly to activation of vagal afferent nerves, primarily pulmonary stretch receptors (Barman & Gebber, 1976; Bachoo & Polosa, 1987; Yu et al. 1990; Seals et al. 1993; Habler et al. 1994; St Croix et al. 1999; Huang et al. 2000; Zhou et al. 2002). Spinal afferent nerves emanating from thoracic dorsal root ganglia (T1–T4) also innervate the airways and lungs (Kostreva et al. 1975, 1978; Saria et al. 1985; Kummer et al. 1992; Wang et al. 2003; Soukhova et al. 2003; Plato et al. 2006). Activation of these spinal afferent nerves can alter respiration and/or renal sympathetic nerve activity, but their role in regulating airway sympathetic nerves is unknown. In fact, no study has directly studied and quantified reflex regulation of airway smooth muscle by sympathetic nerves. Rather, the role of sympathetic nerves in regulating airway smooth muscle tone has been inferred from the effects of propranolol on bronchospasm. Mostly, however, the effects of ß-adrenoceptor antagonists on responsiveness of airways can be attributed to preventing the effects of hormonal catecholamines (Diamond, 1972; Colebatch & Engel, 1974; Underwood et al. 1997). It is likely that the difficulty with which the effects of neuronal catecholamines can be differentiated from those mediated by hormonal catecholamines in measures of whole lung mechanics has contributed to this gap in our understanding of airway neuronal control.
We have developed a preparation of the guinea-pig in which reflex responses of an isolated segment of the extrathoracic trachea can be monitored in situ (Mazzone & Canning, 2002c). Monitoring reflex effects in the trachea accurately predicts neuronal regulation of the airways as a whole but provide the added advantage that the pharmacology and specific neuronal pathways regulating these effects can be studied selectively and systematically. In the present study we describe reflex regulation of airway smooth muscle by sympathetic nerves.
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Methods |
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Immunohistochemistry of airway-identified dorsal root ganglia DRG neurones
Male Hartley guinea-pigs (200–250 g, Charles River, Wilmington, MA, USA) were anaesthetized with ketamine and xylazine (60 mg kg–1 and 10 mg kg–1, respectively, I.P.) and treated with atropine (0.05 mg kg–1, I.P.) to decrease bronchial mucus secretion and/or bronchospasm. The animal was placed head up on a 45 deg incline. The midcervical trachea was exposed and 400 µl of the tracer DiI (dissolved in 100% ethanol and diluted in sterile saline to a final concentration of 0.5 mg ml–1 in 1% ethanol) was instilled into the lumen using a 28.5-gauge needle. Animals were maintained in this position after tracer instillation and suture of the cut until they woke up. Ten to fourteen days later the guinea-pigs were deeply anaesthesized (pentobarbital, 100 mg kg–1, I.P.). When no heart beat or respiratory efforts were apparent, the chest was opened and the animal was transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS, 4°C) containing 6 U ml–1 heparin and 0.1% procaine. DRG were dissected and placed in 4% PFA for 2 h. After rinsing in PBS, DRG were cryoprotected in an 18% sucrose solution overnight at 4°C, subsequently frozen in O.C.T. medium (VWR, Bridgeport, NJ, USA) and sectioned (20 µm). DiI-labelled neurones were visualized by fluorescence microscopy (Olympus BX60), photographed using a digital camera and localized by their x–y coordinates on the slides. Tissue sections were then treated with blocking solution (10% goat serum, 1% bovine serum albumin and 0.5% Tween 20 in 0.1 M PBS) for 1 h at room temperature followed by incubation with primary antibodies against substance P (SP; 1: 100 dilution, Chemicon, Temecula, CA, USA) or neurofilament (NF; 1: 100 dilution, Sigma) at 4°C overnight. Thereafter the slides were incubated with fluorescence-labelled anti-primary antibodies (goat anti-mouse or rat IgG antibodies (1: 100 dilutions, Molecular Probes) for 2 h at room temperature. Antibodies were diluted in 0.1 M PBS containing 0.3% Triton and 1% bovine serum albumin. After secondary labelling the slides were coverslipped with antifade buffer (Molecular Probes). The airway-identified neurones were re-localized and examined for immunoreactivity for SP and NF as previously described (Mazzone & Canning, 2002b).
Electrophysiology
Dorsal root ganglia (DRG) neurones were retrogradely labelled with DiI as described above. Ten to fourteen days later, the animals were killed by asphyxiation in a chamber filled with 100% CO2 and exsanguinated. DRG (T1–T4) were quickly extracted from the animals, placed in 4°C Locke solution (composition (mM): 136 NaCl, 5·6 KCl, 14·3 NaHCO3, 1·2 NaH3PO4, 2·2 CaCl2, 1·2 MgCl2 and 10 dextrose, equilibrated continuously with 95% O2–5% CO2; pH 7·2–7·4), de-sheathed, cut into pieces and placed in Ca2+ and Mg2+-free Hanks' balanced salt solution (CMFH) (composition (mM): 138 NaCl, 5·0 KCl, 4·0 NaHCO3, 0·3 Na2HPO4, 0·3 KH2PO4, 5 dextrose and 0·03 Phenol Red). The ganglia were then incubated for 7 min in 10 ml CMFH containing 1 µg ml–1 papain (Boehringer Mannheim), which was activated by 0.2 mg ml–1 L-cysteine. After two washes in CMFH the tissue was incubated for 10 min in CMFH containing 2 mg ml–1 dispase (grade II, Boehringer Mannheim) and 1 mg ml–1 type 1A collagenase. Ganglia neurones were dissociated during this last incubation by trituration with a fire-polished Pasteur pipette. After two more washes in Leibovitz L-15 medium (Gibco) containing 10% (v/v) fetal bovine serum (FBS; JRH Biosciences, Lexena, KS, USA), the cells were resuspended in L-15–10% FBS and applied in 150 µl aliquots to poly D-lysine-coated coverslips in 2.5 mm culture plates. Neurones were allowed to settle and attach overnight at 37°C before use.
Within 9 h of plating on the coverslips, DiI-labelled DRG neurones were visualized using fluorescence microscopy for whole-cell patch-clamp recordings using an Axopatch 200B amplifier and pCLAMP8 software (Axon Instruments, Foster City, CA, USA). The resistance of the patch pipettes was 1–3 M
when they were filled with the following solution (mM): 140 KCl, 2 MgCl2, 1 CaCl2, 10 N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulphonic acid] (Hepes), 11 EGTA, 2 Mg-ATP, and 1 Li-GTP; pH 7.3 adjusted with KOH, 314 mosmol l–1. Coverslips were continuously superfused (6–7 ml min–1) during recording with a Locke solution (mM): 136 NaCl, 5.6 KCl, 1.2 NaH2PO4, 14.3 NaHCO3, 1.2 MgCl2, 2.2 CaCl2, and 10 dextrose, equilibrated with 95% O2–5%CO2 pH 7.3–7.5. The Locke solution was maintained at 33°C. Pipette voltage offset was neutralized prior to the formation of a gigaseal. Membrane resistance (Rm), series resistance (Rs), and membrane capacitance (Cm) were determined from current transients elicited by a 5 mV depolarizing step from a holding potential –60 mV, using the ‘Membrane Test’ application of pCLAMP8. Capacitance and 80%
Rs were compensated electronically. Criteria for cell inclusion in the study were as follows: Rs
10 M, Rm > 100 M, and stable recording with 80%
Rs compensation throughout the experiment. For testing the chemosensitivity of neurones to bath-applied capsaicin, cells were voltage-clamped to –60 mV and ionic currents were measured.
In vivo measurement of airway smooth muscle tone
Male Hartley guinea-pigs (300–400 g) were anaesthetized with urethane (1.48 ± 0.03 g kg–1, I.P.) and placed supine on a heated pad (see Fig. 1). A midline incision in the neck exposed the extrathoracic trachea, which was cannulated at its caudal-most end with a bent 15-gauge leur stub adaptor. After neuromuscular blockade with succinylcholine (2 mg kg–1
S.C.), the animal was mechanically ventilated (60 breaths min–1, 6 ml kg–1, 3 cmH2O positive end expiratory pressure). Depth of anaesthesia was repeatedly assessed prior and subsequent to blockade and supplemental anaesthetic administered as needed based on responses (withdrawal, cardiovascular) to sharp pinches to the limbs and skin. The ventilator was attached in series to an ultrasonic nebulizer and pulmonary inflation pressure was monitored using a pressure transducer attached to a side port of the tracheal cannula. Stainless steel dry fly hooks (Mustad, Auburn, NY, USA; size 14, 4.9 mm in width, 12 mm long) were placed between two to three cartilage rings in the cervical trachea on the lateral aspects of the trachea. One hook was tied to a fixed bar while the other was connected to an isometric force transducer (Grass Instruments, Quincy, MA, USA) to monitor tracheal tension (TT; grams force (mN) per tissue width (4.9 mm)). Baseline tension was set at 3–4 mN mm–1. The tracheal lumen was continuously superfused (20 ml min–1) with warmed (37°C) oxygenated Krebs buffer, introduced to the tracheal lumen via a small slit, two cartilage rings below the hooks. The buffer was collected at the larynx via gentle suction. The buffer (composition (mM): 118 NaCl, 5.4 KCl, 1 NaHPO4, 1.2 MgSO4, 1.9 CaCl2, 25 NaHCO3, 11.1 dextrose, pH 7.4) contained 3 µM indomethacin to prevent formation of neuromodulatory prostanoids and a mixture of neurokinin (NK) receptor antagonists (0.1 µM ZD6021 or 0.1 µM each CP99994, SR48968 and SB223412) to block the effects of peripherally released tachykinins. The 
-adrenoceptor antagonist phentolamine (1 µM) was also added to the superfusate to minimize prejunctional adrenoceptor-dependent effects and effects on the tracheal vasculature and/or airway smooth muscle.
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In initial experiments the optimal conditions for monitoring sympathetic nerve-mediated relaxations of the trachealis were determined. After a 10 min equilibration period, 1 µM atropine was added to the tracheal perfusate followed by bilateral vagotomy to eliminate any parasympathetic influences over tracheal tone. Thereafter, the trachealis was precontracted to 
50% of the maximum contraction by continuously superfusing 10–50 µM histamine into the tracheal lumen (precontraction is necessary to study any subsequently evoked relaxations). When the histamine contractions had stabilized, the cervical sympathetic trunks were electrically stimulated (5–10 V, 1–32 Hz, 1 ms pulse duration, 10 s trains) bilaterally to evoke relaxations. The adrenergic nature of these responses was confirmed by assessing the antagonistic effects of 2 µM propranolol on the evoked responses. For these pharmacological analyses, the nerves were stimulated at an optimal stimulation frequency (
16 Hz) at 5 min intervals before and during continuous perfusion of the trachea with propranolol (in control preparations, sympathetic nerve-mediated relaxations remain essentially unchanged (± 5%) in five consecutive stimuli at 5 min intervals; n
= 5). The effects of propranolol were assessed until two consecutive stimuli evoked identical responses. The results were expressed as a percentage blockade of the evoked relaxations. To determine the relative role of ß1- and ß2-adrenoceptors, the effects of the ß1- and ß2-adrenoceptor-selective antagonists practolol and ICI118551, respectively, on the sympathetic nerve-mediated relaxations were assessed. Relaxations were evoked by sympathetic nerve stimulation (16 Hz) before and during continuous perfusion with either 1 nM–0.1 µM ICI118551 or 0.01–3 µM practolol. Cumulatively increasing concentrations of the antagonists were administered until consecutive doses produced no further blockade. When the maximum effect of either compound was attained, the other antagonist was added at its optimal concentration to assess the effects of combined antagonism of ß1- and ß2-adrenoceptors. These results were expressed as a percentage blockade of the relaxant response.
At the end of each experiment, the H1 histamine receptor antagonist pyrilamine (1 µM) was added to the tracheal perfusate to induce a maximal reversal of the histamine contraction. Neurally evoked relaxations were expressed as a percentage of this maximum relaxation.
Once the optimal conditions for monitoring sympathetic nerve-mediated responses had been established, we studied reflex activation of the sympathetic nerves with aerosolized capsaicin. Capsaicin (0.1 M) was dissolved in 100% ethanol and diluted in sterile saline to 10–60 µM (median 30 µM). This range of concentrations of capsaicin is near threshold and/or submaximal for evoking airway responses (Thompson & Sheppard, 1988; Xiang et al. 1998). Two millilitres of the capsaicin solution was introduced in a nebulizer (Mystique, Airsep, Buffalo, NY, USA; particle size 5 µm); the nebulizer was turned on for 2–5 min (median 3.5 min) to induce sympathetically mediated tracheal relaxations. Capsaicin challenges with each concentration continued until pulmonary inflation pressure increased by 50% (secondary to the capsaicin-evoked axon reflex) or for 5 min with 100 µM challenge concentration when modest changes in pulmonary inflation pressure were noted (< 10% of preparations). Responses were monitored for 10 min or until a maximum effect was attained. The antagonistic effects of 3 µM propranolol added to the tracheal perfusate or intravenous administration of the ganglionic blocker hexamethonium (4 mg kg–1) on the evoked responses was assessed. To distinguish the effects of neuronal catecholamines from hormonal catecholamines in the capsaicin-induced reflexes, we assessed the effect of cutting both cervical sympathetic trunks and recurrent laryngeal nerves while leaving the tracheal vasculature intact. We also assessed the effects of adding 1 µM tetrodotoxin (TTX) to the tracheal perfusate on these relaxations (this would abolish transmitter release from sympathetic nerve terminals in the trachea while having no effect on the actions of circulating catecholamines). To evaluate the role of spinal afferents in these responses, bilateral dorsal rhizotomy from T1 to T4 was performed after upper thoracic laminectomy and opening of the dura. Control preparations consisted of a sham operation, with laminectomy and opening of the dura without cutting the dorsal roots. Finally, we assessed the effects of systemically administered tachykinin receptor antagonists on these responses. CP99994, SR48968 and SB223412 were administered simultaneously at 1 mg kg–1
I.V. prior to capsaicin challenge. Vehicle control experiments were carried out in parallel.
In another series of experiments, we assessed the effect of intravenous propranolol treatment on capsaicin and neurokinin A (NKA)-evoked increases in pulmonary inflation pressure. Concentration response curves for capsaicin (0.1–10 µg kg–1, I.V.) and NKA (0.1–10 nmol kg–1, I.V.) were constructed in the absence and presence of propranolol (1 mg kg–1, I.V.), given 10 min prior to capsaicin or NKA. Vehicle control experiments were carried out in parallel.
Drugs
ZD6021 (AstraZeneca, Wilmington, DE, USA), SR48968 and SB223412 (GlaxoSmithKline, King of Prussia, PA, USA), and CP99994 (Schering Plough, New Brunswick, NJ, USA) were gifts. All other drugs were purchased from Sigma (St Louis, MO, USA) unless specified. Stock solutions were made in distilled water and diluted in Krebs buffer except indomethacin and capsaicin (100% ethanol), and ZD6021, CP99994, SR48968 and SB223412 (100% DMSO). Drugs given intravenously or subcutaneously (1–50 mg ml–1) were dissolved in saline except: CP99994 and SR48968 were dissolved in DMSO (10 mg ml–1) and diluted (1 mg ml–1) in saline; SB223412 was dissolved in DMSO (10 mg ml–1) and diluted (1 mg ml–1) with 20% acid in saline.
Data analysis and statistics
All data are presented as mean ± S.E.M. Group means were compared by analysis of variance. When statistically significant differences amongst group means were detected by ANOVA, group means were compared using Scheffe's F test for unplanned comparisons. P values less than 0.05 are considered significant.
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Results |
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DiI delivered to the airways and lungs by intratracheal instillation in three guinea-pigs retrogradely labelled neurones in the right and left thoracic (T1–T4) dorsal root ganglia (DRG). Immunohistochemical analyses revealed that 72% (21 of 29 labelled neurones) of the airway projecting DRG neurones had immunoreactivity for substance P (SP; a marker for nociceptive-type, capsaicin-sensitive C-fibre afferent neurones; Szolcsanyi et al. 1988; Lawson et al. 1993) whereas only 14% (4 out of 29 neurones) were immunoreactive for neurofilament (NF; a marker for myelinated nerves; Lawson et al. 1993). No labelled neurones evaluated were dually labelled for SP and NF, and four labelled neurones were not labelled for either SP or NF (Fig. 2A). Whole-cell patch-clamp recordings revealed that the majority of airway projecting DRG neurones produced large (1.8 ± 0.43 nA, n = 22 neurones from 7 animals) inward currents to bath-applied 10 µM capsaicin (Fig. 2B). Together, these results imply that both A- and C-type DRG neurones innervate the airways (primarily tachykinin-containing C-fibres) and also show that capsaicin stimulates many airway afferent DRG neurones.
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Electrical stimulation (10–20 V, 1–32 Hz, 1 ms pulse duration, 10 s trains) of the cervical sympathetic trunks evoked frequency-dependent relaxations of the precontracted guinea-pig trachealis (Fig. 3). These relaxations were fast in onset and reversal, characteristic of adrenergic nerve-mediated relaxations of airway smooth muscle (Diamond & O'Donnell, 1980; Canning & Undem, 1993). At frequencies 16 Hz, the histamine-induced contractions were nearly completely reversed by the end of the 10 s trains. Coincident with the effects on the airways, cervical sympathetic nerve stimulation (16 Hz, 10 s) transiently increased heart rate and blood pressure by 10 ± 3% and 18 ± 2%, respectively (n
= 6). Cutting the cervical sympathetic trunks and stimulating either the rostral or caudal cut ends evoked relaxations that were abolished by the ganglionic blocker hexamethonium (4 mg kg–1
I.V.). Adding the ß-adrenoceptor antagonist propranolol to the tracheal perfusate also nearly abolished the relaxations evoked by sympathetic nerve stimulation. By contrast, neither ICI118551 (1 nM–0.1 µM) nor practolol (0.01–3 µM) substantially antagonized the electrically evoked relaxations. When added simultaneously, however, the ß1- (3 µM practolol) and ß2- (0.1 µM ICI118551) adrenoceptor antagonists mimicked the effects of the non-selective antagonist propranolol (Fig. 4).
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Based on the results of our electrophysiological analyses, we used capsaicin to evoke sympathetic nerve-mediated reflexes in the airways. As capsaicin also induces parasympathetic relaxant reflexes in the airways (Mazzone & Canning, 2002a,b,c), we cut the vagus nerves bilaterally, caudal to the nodose ganglia. Atropine was also added to the tracheal perfusate to prevent parasympathetic nerve-mediated contractions, and the trachealis was then precontracted with histamine. Under these conditions, capsaicin inhalation still evoked a slowly developing but pronounced fall in tracheal tension (Fig. 5). The average maximal relaxation induced by capsaicin inhalation was 63 ± 7% (n
= 13). Relaxations started within 2 min of initiating the capsaicin challenge and reached a maximum in 6 min (capsaicin challenges in the control preparations lasted 4.6 ± 0.8 min). The relaxations evoked were sustained for several minutes, long after terminating the capsaicin aerosol challenge and were reversed by propranolol. When propranolol was added prior to capsaicin challenge, the relaxations were attenuated (10 ± 9% of the maximum relaxation, n
= 7; Fig. 5B). Pretreatment with hexamethonium (4 mg kg–1
I.V.; n
= 3) or sympathetic denervation of the trachea (n
= 5) nearly abolished the reflex relaxations (–2 ± 4% and 6 ± 4% of the maximum relaxation, respectively; Table 1).
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Capsaicin inhalation invariably increased pulmonary inflation pressure (PIP) secondary to axon reflex-mediated, tachykinin-dependent bronchospasm (Thompson & Sheppard, 1988). In control preparations, the maximum increase in PIP averaged 78 ± 7% over baseline (n
= 10). The increases in PIP evoked by capsaicin typically preceded the relaxations measured in the trachealis and the magnitude of the tracheal relaxations was highly correlated (r2
= 0.91; P < 0.01) with the increases in PIP. It is thus possible that the sympathetic-adrenergic nerve-mediated reflex effects initiated by inhaled capsaicin occur secondary to a mechanical effect induced by the bronchospasm (or as a consequence of the bronchospasm). However, when bronchospasm (54 ± 23% increase in PIP) was evoked by inhalation of 10 µM NKA instead of capsaicin, little or no relaxant response was evoked (4 ± 3% of maximum relaxation; n
= 3). Second, the potency of NKA as a bronchoconstrictor when administered intravenously was potentiated by only 2-fold following pretreatment with propranolol (1 mg kg–1
I.V.), whereas propranolol increased the potency of I.V. capsaicin at evoking bronchospasm 10- to 30-fold (Fig. 6).
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Bilateral dorsal rhizotomy (T1–T4) prior to capsaicin inhalation essentially abolished the subsequently evoked reflex relaxation of the trachea (9 ± 5% of the maximum relaxation, n = 4; Fig. 8, Table 1). Sham operation in two animals did not prevent the subsequently evoked relaxation. Based on the effects of the dorsal rhizotomies and our immunohistochemical analyses, we subsequently evaluated the role of tachykinins on the reflex relaxations evoked. As mentioned above, NKA administered by inhalation or intravenously failed to mimic the effects of capsaicin challenge, so we assumed that any role of tachykinins in mediating this reflex are confined to actions in the spinal cord. To address this hypothesis, we treated animals with a mixture of NK1, NK2 and NK3 receptor antagonists (each at 1 mg kg–1 I.V.), prior to capsaicin application (as described in Methods, these antagonists were always present in the tracheal perfusate in all of the experiments carried out in this study). The neurokinin receptor antagonists nearly abolished the reflex relaxation of the trachea (3 ± 7% of the maximum relaxation, n = 5, Fig. 8, Table 1). The vehicles for these antagonists were without effect on the capsaicin-induced reflex relaxations (69 ± 20% of the maximum relaxation, n = 3).
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Discussion |
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Sympathetic innervation of airway smooth muscle
As has been well described in cats, dogs and guinea-pigs (Cabezas et al. 1971; Diamond & O'Donnell, 1980; Russell, 1980; Canning & Undem, 1993), sympathetic nerve stimulation evoked marked relaxations of the guinea-pig trachealis in situ. The frequency dependence and kinetics of the relaxations were identical to that previously described, and their sensitivity to propranolol confirmed the sympathetic and adrenergic nature of these responses. Comparable vasoconstrictor effects in the bronchial circulation have been described upon sympathetic nerve stimulation in cats and pigs (Hyman et al. 1990; Franco-Cereceda et al. 1995). These observations and many in vitro physiological and morphological studies suggest that sympathetic adrenergic innervation of the airways and lungs is common to all vertebrate species.
The sympathetic nerves innervating the trachea are derived from both the cervical as well as the thoracic (stellate) sympathetic ganglia (Kummer et al. 1992; B. J. Canning, unpublished observations). That stimulating either the rostral or caudal cut ends of the cervical sympathetic trunk evoked relaxations of the trachealis that were blocked by hexamethonium is consistent with this hypothesis. Innervation of the intrathoracic airways is primarily derived from the thoracic sympathetic ganglia. Immunohistochemical analyses and circumstantial evidence gathered in functional studies suggests that a subpopulation of the postganglionic thoracic sympathetic nerves innervating the airways are non-adrenergic, utilizing the peptide vasoactive intestinal peptide (VIP and related peptides) and/or the gaseous transmitter nitric oxide (NO, synthesized from arginine by the neuronal isoform of NO synthase) to relax airway smooth muscle (Bowden & Gibbins, 1992; Fischer et al. 1996; Matsumoto et al. 1997). In our pharmacological analyses, we found little evidence for a prominent non-adrenergic component of the sympathetic response in the trachea, as propranolol reduced the nerve-mediated relaxations (evoked electrically or reflexively) by an average of >95%. Comparable results have been reported previously (Diamond & O'Donnell, 1980; Canning & Undem, 1993).
The airway smooth muscle of guinea-pigs expresses both ß1- and ß2-adrenoceptors. Based on the effects of chemical sympathectomy with 6-hydroxydopamine or reserpine and the resulting supersensitivity to ß1- but not ß2-adrenoceptor selective agonists, it has been proposed that ß1-adrenoceptors on airway smooth muscle are ‘innervated’ and activated by neuronally released catecholamines, while the ß2-adrenoceptors are not innervated but activated by circulating catecholamines and inhaled ß2-adrenoceptor selective agonists used for treating pulmonary disease (Broadley et al. 1986; Grassby & Broadley, 1986). This hypothesis has not been directly tested, however, and can only be addressed using preparations such as ours in which the sympathetic nerves can be directly and selectively stimulated. We found that neither practolol nor ICI118551, when administered at concentrations selective for antagonizing ß1- and ß2-adrenoceptors, respectively, substantially blocked the sympathetic nerve-mediated relaxations of the trachealis (20–30% reduction). When administered simultaneously, however, the ß1- and ß2-adrenoceptor antagonists markedly reduced the sympathetic nerve-evoked relaxations. These data suggest that both ß1- and ß2-adrenoceptors mediate airway smooth muscle relaxation evoked by sympathetic-adrenergic nerve activation. Comparable results have been reported in studies of the sympathetic innervation of the pulmonary circulation in cats (Hyman et al. 1990).
Afferent nerves reflexively regulating airway sympathetic nerves
Retrograde neuronal tracing studies described here and previously reveal that spinal afferent nerves arising from thoracic DRG innervate the airways and lungs of several species including guinea-pigs (Saria et al. 1985; Kummer et al. 1992; Plato et al. 2006). Functional studies in rabbits, dogs and primates also suggest that spinal afferent nerves innervate the airways and lungs (Kostreva et al. 1975, 1978; Wang et al. 2003; Soukhova et al. 2003). We found that the majority of spinal afferent nerves innervating the airways and lungs express the neuropeptide substance P and were activated by capsaicin. Following vagotomy to prevent airway parasympathetic reflexes in the trachea (both contractions and relaxations; Mazzone & Canning, 2002a), capsaicin inhalation induced a marked relaxation of the trachealis. The effects of propranolol, TTX and sympathetic denervation of the trachea on this reflex provide conclusive evidence that sympathetic adrenergic nerves reflexively mediate these relaxations. The effects of dorsal rhizotomy suggest that the reflex initiated by capsaicin inhalation may be mediated by activation of the capsaicin-sensitive nerves innervating the airways and lungs.
We speculate that reflexes comparable to those we measured in the trachea regulate airway smooth muscle and vascular tone throughout the tracheobronchial tree. The effects of propranolol on the bronchoconstrictor response to intravenously administered capsaicin (10- to 30-fold increase in potency) are consistent with this notion. The data also suggest that the effects of capsaicin are mediated directly and are not dependent upon indirect effects (e.g. bronchospasm). Thus, NKA inhalation did not mimic the effects of inhaled capsaicin, and propranolol only modestly altered the response to intravenously administered NKA.
A small percentage of retrogradely labelled DRG neurones studied were capsaicin insensitive or expressed immunoreactivity for neurofilament, suggesting that a subpopulation of myelinated spinal afferent nerves innervate the airways and lungs. Their role in regulating airway autonomic nerve activity and their sensitivity to chemical and mechanical stimuli has not yet been assessed. We did, however, find evidence for vagal regulation of airway sympathetic nerves. Stimulating the rostral cut ends of the vagus nerves evoked pronounced relaxations of the trachealis. It is unclear what vagal afferent nerves were driving this response but it is likely that bronchopulmonary vagal afferent nerves play some role (Barman & Gebber, 1976; Bachoo & Polosa, 1987; Daly & Kirkman, 1988; Yu et al. 1990; Seals et al. 1993; Habler et al. 1994; St Croix et al. 1999; Huang et al. 2000; Zhou et al. 2002). Activation of bronchopulmonary afferent nerves can also initiate airway parasympathetic reflexes, both cholinergic contractions and non-adrenergic, non-cholinergic relaxations (Mazzone & Canning, 2002a). These coincident sympathetic and parasympathetic reflexes may act in concert or in parallel to maintain a stable airway caliber. Dysfunction or dysregulation of any of these components may lead to the airways obstruction associated with asthma and chronic obstructive pulmonary disease (see below).
Selective reflex regulation of airway sympathetic nerves and role in regulating airway caliber
The pronounced relaxations evoked reflexively by inhaled capsaicin in the vagotomized animals were prevented by propranolol and occurred independent of any marked changes in heart rate or blood pressure. These observations suggest that the spinal afferent nerves innervating the airways can selectively regulate airway sympathetic adrenergic nerve activity. This contrasts with vagal afferent nerves, which tonically influence basal sympathetic nerve activity and can reflexively modulate sympathetic nerves innervating multiple organs (Yu et al. 1990; Foreman, 1999; present study). This selective regulation of airway sympathetic nerves adds further evidence against historical notions regarding regulation of sympathetic nerve activity and its role in both vegetative and defensive reflex responses (Peterson et al. 1983; Morrison, 2001; Jänig & Habler, 2003).
Stimulating either the caudal or rostral cut ends of the cervical sympathetic trunks evoked hexamethonium-sensitive relaxations of the trachealis. This confirms previous studies indicating that at least a subpopulation of the preganglionic sympathetic nerves innervating the superior cervical ganglia also innervate neurones in the stellate ganglia (Lichtman et al. 1980). It is possible (but untested) that a single population of preganglionic neurones selectively innervates postganglionic neurones in both the stellate and superior cervical ganglia that project to the airways. Given that we observed reflex-mediated, sympathetic nerve-dependent airway smooth muscle relaxations either coincident with or independent of changes in heart rate or blood pressure, it is also possible that multiple and differentially regulated subpopulations of preganglionic sympathetic nerves regulate sympathetic outflow to the airways. Alternatively, airway afferent nerves may selectively regulate sympathetic outflow to the airways through modulatory actions in the spinal cord.
We found little evidence for the involvement of circulating catecholamines in mediating the response to inhaled capsaicin. Only when blood gases were compromised secondary to bronchospasm or asphyxia could a marked systemic effect be measured. However, there are many stimuli that initiate a surge in circulating catecholamines (e.g. histamine, allergen, asphyxia), so the role of adrenal regulation of airway caliber cannot be discounted (Diamond, 1972; Colebatch & Engel, 1974; Underwood et al. 1997). Circulating catecholamines may also tonically regulate airway smooth muscle tone and reactivity (Larson, 1985; Weinmann et al. 1985; Knox et al. 1992; Fontana et al. 2002; Mazzone & Canning, 2002c). The driving force behind this basal activation of ß-adrenoceptors in the airways is unclear.
The relative importance of sympathetic-adrenergic nerves in regulating airway tone is both species dependent and dependent upon the physiological conditions experienced by the animal (Canning & Fischer, 2001). In dogs, adrenergic nerves are the only functional relaxant nerves innervating the airways (Russell, 1980). However, in most other species including humans, non-cholinergic parasympathetic nerves subserve a primary role in mediating airway smooth muscle relaxation. In fact, although sympathetic-adrenergic innervation has been clearly demonstrated in human pulmonary arteries and circumstantial evidence suggests that adrenergic nerves also innervate the airway smooth muscle and glands of human airways and lungs (Gothert & Hentrich, 1985; Davis & Kannan, 1987; Pack et al. 1988; Martinez et al. 1995), the ß-adrenoceptor antagonist propranolol and/or high thoracic epidural anaesthesia has little or no effect on resting airway mechanics and no effect on airways responsiveness to constricting stimuli in healthy human subjects (Laitinen et al. 1976; Habib et al. 1979; Sterk et al. 1985; Groeben et al. 1994, 1995). Propranolol is also without effect on nerve-mediated responses evoked in vitro in human and non-human primate airway preparations (Richardson & Beland, 1976; Middendorf & Russell, 1980; Canning & Fischer, 2001). This is not due to insufficient ß-adrenoceptor expression by the airway smooth muscle, as ß2-receptor agonists have a profound bronchodilating effect in the airways of all human subjects, and potently and effectively relax isolated preparations of human airway smooth muscle from all patient populations (Goldie et al. 1990). Rather, the data suggest that the airways of healthy human subjects are either sparsely innervated and/or only modestly regulated by adrenergic nerves. Recruitment of these sympathetic reflexes in disease, perhaps to counteract exaggerated parasympathetic cholinergic tone, or to compensate for dysfunctional parasympathetic non-cholinergic nerves, may help regulate airway caliber (Sly et al. 1967; Grieco & Pierson, 1971; Molho et al. 1977; Larson, 1985; Sands et al. 1985; Ind et al. 1989; Noppen & Vincken, 1996; Heindl et al. 2001; Schilero et al. 2005).
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Baker
DG
&
Don
H (1987). Catecholamines abolish vagal but not acetylcholine tone in the intact cat trachea. J Appl Physiol
63, 2490–2498.
Baker DG & McDonald DM (1992). Distribution of catecholamine-containing nerves on blood vessels of the rat trachea. J Comp Neurol 325, 38–46.[CrossRef][Medline]
Barman
SM
&
Gebber
GL (1976). Basis for synchronization of sympathetic and phrenic nerve discharges. Am J Physiol
231, 1601–1607.
Bowden JJ & Gibbins IL (1992). Vasoactive intestinal peptide and neuropeptide Y coexist in non-noradrenergic sympathetic neurons to guinea pig trachea. J Auton Nerv Syst 38, 1–19.[CrossRef][Medline]
Broadley
KJ, Chess-Williams
RG
&
Grassby
PF (1986). A physiological basis for subclassifying ß-adrenoceptors examined by chemical sympathectomy of guinea-pigs. J Physiol
373, 367–378.
Broadstone RV, LeBlanc PH, Derksen FJ & Robinson NE (1991). In vitro responses of airway smooth muscle from horses with recurrent airway obstruction. Pulm Pharmacol 4, 191–202.[CrossRef][Medline]
Cabezas
GA, Graf
PD
&
Nadel
JA (1971). Sympathetic versus parasympathetic nervous regulation of airways in dogs. J Appl Physiol
31, 651–655.
Canning BJ & Fischer A (2001). Neural regulation of airway smooth muscle tone. Respir Physiol 125, 113–127.[CrossRef][Medline]
Canning
BJ
&
Undem
BJ (1993). Relaxant innervation of the guinea-pig trachealis: demonstration of capsaicin-sensitive and -insensitive vagal pathways. J Physiol
460, 719–739.
Colebatch
HJ
&
Engel
LA (1974). Constriction of the lung by histamine before and after adrenalectomy in cats. J Appl Physiol
37, 798–805.
Daly
MD
&
Kirkman
E (1988). Cardiovascular responses to stimulation of pulmonary C fibres in the cat: their modulation by changes in respiration. J Physiol
402, 43–63.
Davis C & Kannan MS (1987). Sympathetic innervation of human tracheal and bronchial smooth muscle. Respir Physiol 68, 53–61.[CrossRef][Medline]
Diamond
L (1972). Potentiation of bronchomotor responses by beta adrenergic antagonists. J Pharmacol Exp Ther
181, 434–445.
Diamond
L
&
O'Donnell
M (1980). A nonadrenergic vagal inhibitory pathway to feline airways. Science
208, 185–188.
Fischer A, Mayer B & Kummer W (1996). Nitric oxide synthase in vagal sensory and sympathetic neurons innervating the guinea-pig trachea. J Auton Nerv Syst 56, 157–160.[CrossRef][Medline]
Fontana
GA, Pantaleo
T, Lavorini
F, Bongianni
F, Mannelli
M, Bridge
PD
&
Pistolesi
M (2002). Handgrip-induced airway dilation in asthmatic patients with bronchoconstriction induced by MCh, inhalation. J Appl Physiol
93, 1723–1730.
Foreman RD (1999). Mechanisms of cardiac pain. Annu Rev Physiol 61, 143–167.[CrossRef][Medline]
Franco-Cereceda A, Matran R, Alving K & Lundberg JM (1995). Sympathetic vascular control of the laryngeo-tracheal, bronchial and pulmonary circulation in the pig: evidence for non-adrenergic mechanisms involving neuropeptide Y. Acta Physiol Scand 155, 193–204.[Medline]
Goldie RG, Paterson JW & Lulich KM (1990). Adrenoceptors in airway smooth muscle. Pharmacol Ther 48, 295–322.[CrossRef][Medline]
Gothert M & Hentrich F (1985). Identification of presynaptic ß2-adrenoceptors on the sympathetic nerve fibres of the human pulmonary artery. Br J Pharmacol 85, 933–941.[Medline]
Grassby
PF
&
Broadley
KJ (1986). Responses mediated via ß-1 adrenoceptors but not ß-2 adrenoceptors exhibit supersensitivity after chronic reserpine pretreatment. J Pharmacol Exp Ther
237, 950–958.
Grieco MH & Pierson RN Jr (1971). Mechanism of bronchoconstriction due to beta adrenergic blockade. Studies with practolol, propranolol, and atropine. J Allergy Clin Immunol 48, 143–152.[CrossRef][Medline]
Groeben H, Schwalen A, Irsfeld S, Lipfert P & Hopf HB (1995). Pulmonary sympathetic denervation does not increase airway resistance in patients with chronic obstructive pulmonary disease (COPD). Acta Anaesthesiol Scand 39, 523–526.[Medline]
Groeben H, Schwalen A, Irsfeld S, Tarnow J, Lipfert P & Hopf HB (1994). High thoracic epidural anesthesia does not alter airway resistance and attenuates the response to an inhalational provocation test in patients with bronchial hyperreactivity. Anesthesiology 81, 868–874.[Medline]
Habib
MP, Pare
PD
&
Engel
LA (1979). Variability of airway responses to inhaled histamine in normal subjects. J Appl Physiol
47, 51–58.
Habler HJ, Jänig W & Michaelis M (1994). Respiratory modulation in the activity of sympathetic neurones. Prog Neurobiol 43, 567–606.[CrossRef][Medline]
Heindl
S, Lehnert
M, Criee
CP, Hasenfuss
G
&
Andreas
S (2001). Marked sympathetic activation in patients with chronic respiratory failure. Am J Respir Crit Care Med
164, 597–601.
Huang
WX, Yu
Q
&
Cohen
MI (2000). Fast (3 Hz and 10 Hz) and slow (respiratory) rhythms in cervical sympathetic nerve and unit discharges of the cat. J Physiol
523, 459–477.
Hyman
AL, Lippton
HL
&
Kadowitz
PJ (1990). Analysis of pulmonary vascular responses in cats to sympathetic nerve stimulation under elevated tone conditions. Evidence that neuronally released norepinephrine acts on alpha 1-, alpha 2-, and beta 2-adrenoceptors. Circ Res
67, 862–870.
) and presence (•) of propranolol (2 mg kg–1I.V.). Pulmonary inflation pressure (PIP) was monitored to assess bronchospasm. The results are presented as the mean ±
S.E.M. of 4–8 experiments. *Maximum increase in PIP produced by either capsaicin or NKA was increased in the presence of propranolol relative to that evoked by the same doses of capsaicin or NKA in control animals (P < 0.05). Propranolol increased the potency of capsaicin by more than 10-fold (the dose of capsaicin increasing PIP by 100% (PD100) averaged 6.9 ± 1.0 and 0.6 ± 0.2 µg kg–1 in the absence and presence of propranolol, respectively; P < 0.01). By contrast, propranolol only modestly decreased the PD100 for NKA (6.1 ± 1.3 and 2.9 ± 0.5 nmol kg–1 in the absence and presence of propranolol, respectively; P < 0.05).
