Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

In the lungs, release of acetylcholine from the vagus nerves is under the local control of inhibitory muscarinic autoreceptors on the postganglionic nerves. Acetylcholine released from the vagus nerve stimulates both M₃ muscarinic receptors on airway smooth muscle, causing contraction and bronchoconstriction, and M₂ muscarinic receptors on the nerves, decreasing further release of acetylcholine (1). These neuronal M₂ receptors are present in most species studied, including humans (2). In the guinea pig, the function of these inhibitory receptors is markedly impaired after acute viral infection (3), acute ozone exposure (4), and antigen challenge of sensitized animals (5).

Loss of function of inhibitory M₂ receptors is characterized by airway hyperresponsiveness to electrical stimulation of the vagus nerves. Furthermore, airway hyperresponsiveness to histamine in antigen-challenged guinea pigs is due to increased vagally mediated reflex bronchoconstriction as a result of M₂ receptor dysfunction (6). Airway M₂ receptors are dysfunctional in some (7, 8), but not all (9), patients with asthma.

Glucocorticoids are commonly used in the treatment of asthma. Many mechanisms have been proposed to explain the beneficial effects of these medications (10). Most have focused on the anti-inflammatory properties of steroids. These medications may increase the expression of certain airway enzymes (11), increasing the degradation of proinflammatory or bronchoconstrictive mediators. Steroid treatment also increases -adrenoceptor density in the airways (12) and may decrease expression of neurokinin (13) and histamine (14) receptors.

In these studies, we investigated the effects of dexamethasone on vagally mediated bronchoconstriction and on airway M₂ muscarinic receptor function. We also studied the effects of dexamethasone on M₂ receptor expression and function in airway parasympathetic nerve cultures.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

In Vivo Studies

Animals. Female Dunkin-Hartley guinea pigs (300 to 350 g; supplied by Hilltop Animal Farms, Scottsdale, PA) were used. Protocols were approved by the Johns Hopkins University School of Hygiene and Public Health Animal Care and Use Committee.

Treatment of guinea pigs with dexamethasone. Dexamethasone (0.1 mg/kg) was administered by intraperitoneal injection daily for 2 d. In vivo physiologic studies were done 24 h after the final dose.

Anesthesia and measurement of pulmonary inflation pressure. Guinea pigs were anesthetized with urethane (1.5 g/kg, intraperitoneally). Heart rate and blood pressure were measured via a carotid artery cannula. Both vagus nerves were cut and the distal ends were placed on shielded electrodes immersed in a pool of mineral oil. The animal's body temperature was maintained at 37°C using a heating blanket. The animals were paralyzed with succinylcholine (10 µg/kg/min, intravenously) and ventilated via a tracheal cannula (tidal volume 1 ml/100 g body weight, 100 breaths/ min; Harvard Apparatus Co., South Natick, MA). Pulmonary inflation pressure (Ppi) was measured from a sidearm of the tracheal cannula.

Vagal stimulation and M₂ receptor function. To determine the response to electrical stimulation at various frequencies, the vagi were stimulated at 1-min intervals (1 to 25 Hz, 0.2 ms pulse duration, 10 V, 5 s pulse train). Bronchoconstriction was measured as an increase in Ppi.

To test M₂ receptor function, bronchoconstriction in response to stimulation of the vagus nerves was matched between animals at the beginning of each experiment by adjusting the voltage. Because the response of the neuronal M₂ receptors to acetylcholine released from the nerves is greater at high stimulus frequencies (1), the effects of antagonists are more readily apparent at the higher frequencies. Conversely, at low frequencies of stimulation exogenous agonists do not have to compete with endogenous acetylcholine, thus the effects of the exogenous agonists are more apparent. Because of this, experiments testing the function of the neuronal M₂ muscarinic receptor with the muscarinic antagonist gallamine were carried out using stimulation at 15 Hz, whereas those using the muscarinic agonist pilocarpine were carried out using stimulation at 2 Hz. For pilocarpine experiments, vagi were stimulated at 6 to 20 V, 0.2 mS pulse duration, 2 Hz, 22 s pulse train.

All animals were pretreated intravenously with guanethidine (10 mg/kg) to deplete noradrenaline and thus eliminate any involvement of the sympathetic nerves. When reproducible baseline responses to vagal stimulation had been obtained, pilocarpine (0.1 to 100 µg/kg, intravenously) was administered in cumulative fashion, and the effects on vagally mediated bronchoconstriction and bradycardia were recorded. Doses of pilocarpine greater than 30 µg/kg caused transient bronchoconstriction. Therefore, the effect of these doses of pilocarpine on vagally induced bronchoconstriction was measured after the Ppi had returned to baseline. Doses of pilocarpine greater than 100 µg/kg were not used because they caused sustained bronchoconstriction via stimulation of the M₃ muscarinic receptors on the airway smooth muscle. At the end of each experiment, vagally induced bronchoconstriction and bradycardia were abolished by atropine (1 mg/kg, intravenously) indicating that both of these responses were mediated via stimulation of muscarinic receptors.

The response of the airways to acetylcholine (which is degraded by cholinesterase) and methacholine (which is relatively cholinesterase-resistant [15]) was tested (1 to 10 µg/kg, intravenously). To determine the role of cholinesterase in determining the response to acetylcholine, animals were treated intravenously with physostigmine (250 µg/kg), and the subsequent response to acetylcholine was measured. To test the role of M₂ receptor dysfunction and cholinesterase in determining the response to nerve stimulation, animals were treated intravenously with either gallamine (30 mg/kg, to block M₂ receptors), physostigmine, or both gallamine and physostigmine, and the subsequent response to nerve stimulation was determined.

Lung lavage. At the end of each experiment, lungs were lavaged five times with 10-ml aliquots of warm phosphate-buffered saline (PBS). Cells were counted and differentials were obtained using cytospun slides stained with Diff-Quik (American Scientific Products, Stone Mountain, GA).

Drugs. Pilocarpine, succinylcholine, atropine, dexamethasone, gallamine, guanethidine, mouse recombinant interferon-, physostigmine, and urethane were purchased from Sigma (St. Louis, MO). Ketamine was purchased from Fort Dodge Animal Health (Fort Dodge, IA). Xylazine was purchased from Bayer (Shawnee Mission, KS). All drugs were dissolved and diluted in 0.9% NaCl.

Cell Culture Studies

Nerve cell culture. Parasympathetic nerve cells were cultured using a method modified from Burnstock and coworkers (16) as previously described (17). Briefly, guinea pig trachealis muscles, containing parasympathetic ganglia, were disaggregated using collagenase (0.2%) and plated on polystyrene. Nonadherent cells were replated on matrigel (Collaborative Biomedical Products, Bedford, MA) coated dishes. Cells were grown in serum-free medium (50% Ham's F12:50% Dulbecco's modified Eagle's medium, 20 µg/ml glutamine [BioWhittaker, Walkersville, MD], 500 µg/ml fatty acid free bovine serum albumin [Calbiochem, La Jolla, CA], 20 µg/ml rat transferrin, 10 µg/ml bovine insulin, 100 ng/ml, and 125 U/ml penicillin [GIBCO-BRL, Gaithersburg, MD]). Culture medium was supplemented with nerve growth factor (100 ng/ml), initially obtained from Quality Control Biochemicals (QCB) (Hopkinton, MA) and subsequently obtained from Sigma after the QCB nerve growth factor was discontinued. Cultures were incubated at 37°C in 5% CO₂ and were fed every 48 h. Twenty-four hours after plating, cytosine arabinoside (1.0 µM; Sigma) was added to the medium to inhibit growth of any dividing cells. Cells were used in experiments 7 d after plating.

Dexamethasone and interferon- treatment of cultured nerve cells. Cells were treated with either dexamethasone (1 µM), mouse recombinant interferon- (300 U/ml), or both dexamethasone and interferon-. This was left in until functional experiments were done or RNA was harvested. All studies of acetylcholine release, M₂ receptor function, and M₂ receptor gene expression were done 24 h after these treatments.

Testing acetylcholine release and M₂ receptor function. The parasympathetic nerve cells, grown in 96-well plates, were washed five times with 100 µl Krebs-Henseleit solution. The sixth sample was collected for measurement of acetylcholine. To determine frequency-dependent acetylcholine release, cells were then stimulated electrically using electrodes placed in the wells (0.5 to 5 Hz, 30 V, 0.2 ms pulse duration, 25 total pulses), and the acetylcholine released was measured as below. To determine M₂ receptor function, cells were stimulated (5 Hz, 30 V, 0.2 ms pulse duration, 5 s pulse train) in the absence and presence of atropine (10⁵ M) to block the M₂ receptors on the cells. After either set of experiments, the cells were depolarized with 3 M KCl for 5 min and another sample was collected to determine total acetylcholine content of the cells. Two assays were used for acetylcholine, a chemiluminescence assay and a radioimmunoassay, with very similar results.

Chemiluminescence assay. A chemiluminescence assay for acetylcholine was performed as previously described (17) using a modification of a method described by Israel and Lesbats (18). Each 10 µl sample was mixed with 0.5 ml of chemiluminescent reaction mixture made from 7.5 ml Kreb-Henseleit solution, 80 µl of 1,000 U/ml cholinesterase, 100 µl of 250 U/ml choline oxidase, 50 µl of 2 mg/ml horseradish peroxidase, and 100 µl of 1 mM luminol. Acetylcholine was measured in relative light units using a chemiluminometer (Wallac, Gaithersburg, MD) over 1 min. The increase in acetylcholine with electrical field stimulation was calculated as a percentage of the baseline. The total amount of acetylcholine in each well was calculated as the amount of acetylcholine from two 1-min washes, added to the acetylcholine in the field stimulated sample, and acetylcholine released by KCl. Each data point in the acetylcholine release studies represents the mean of five wells.

Radioimmunoassay. Acetylcholine was measured using a radioimmunoassay, as previously described (19). The reaction mixture was prepared by mixing 100 µl of rabbit antiserum to acetylcholine (CHG-40; 1:1,200 in 0.4% bovine gamma globulin); 100 µl 3H-labeled acetylcholine; 50 µl 0.01 M acetic acid; 180 µl 0.15 M Tris buffer (pH 7.4); and 10 µl of samples/standard. This antibody is highly specific for acetylcholine. As some of our samples contained atropine, we confirmed that atropine did not interfere with the acetylcholine assay. The reaction mixture was incubated overnight at 4°C. The acetylcholine-antiserum complex was precipitated with 500 µl saturated ammonium sulfate, washed with 50% ammonium sulfate, and dissolved in 500 µl of distilled water. The solution was then mixed with 6 ml of scintillation cocktail, and radioactivity was counted in a scintillation counter. The control curve was plotted using 0 to 300 pg/10 ml of acetylcholine (the lower limit of detection using this method is 0.3 pg/ml). The acetylcholine concentration in the test samples was estimated by comparing its radioactivity with the standard curve. Samples to be assayed via this method were collected with 0.05% diisopropylfluorophosphate to inhibit cholinesterase activity.

Competitive reverse transcriptase/polymerase chain reaction (RT-PCR) assay for M₂ receptor gene expression. An internal standard cellular RNA (cRNA) was generated as described previously (20) by excising 43 bp from a 568-bp RT-PCR product of guinea pig M₂ receptor messenger (mRNA) and transcribing this in vitro. To carry out the competitive RT-PCR assay, RNA was extracted from nerve cell cultures using the RNAzol B method and precipitated in isopropanol overnight. Contaminating genomic DNA was eliminated by treating with 2 U RNase-free DNase for 30 min at 37°C. Five hundred nanograms of RNA were added to internal standard cRNA (0.01 to 10 pg/sample) in the presence of Moloney murine leukemia virus reverse transcriptase (2.5 U/µl), deoxynucleotide triphosphates (dNTPs) (1 mM), RNase inhibitor (1 U/µl), and random hexamer primers (2.5 µM). Reverse transcription was carried out at 42°C for 20 min, after which the reverse transcriptase was inactivated by heating to 99°C for 5 min. The absence of contaminating genomic DNA was confirmed by carrying out control reactions in which the reverse transcriptase was omitted. Complementary DNA (cDNA) was amplified using AmpliTaq polymerase (1.5 U/50 µl) in the presence of 2 mM MgCl₂ and dNTPs (0.4 mM). The upstream primer was 5'-TCC TCT CTT TCA TCC TCT GG-3' and the downstream primer was GTG CCT GAG TCA CCT TTT TG-3'. These primers yield a product of 568 bases using guinea pig M₂ mRNA and 525 bases using the truncated internal standard cRNA. PCR was conducted for 40 cycles at 95°C for 45 s, 60°C for 45 s, and 72°C for 90 s. Final extension was at 72°C for 10 min. Products were run on agarose gels (4%) and stained with ethidium bromide (0.5 µg/ml). Amounts of product were determined by densitometry. The input amount of M₂ receptor mRNA was determined by interpolating to determine the amount of internal standard cRNA that yielded a ratio of M₂ mRNA product/cRNA product of 1 (20).

Immunostaining M₂ receptors. Nerve cells were fixed in methanol for 5 min at 20°C and incubated with 10% normal goat serum for 30 min. They were then incubated with primary antibody for M₂ receptors (rabbit anti-M₂ receptor, Research and Diagnostic Antibodies, 1:5,000 dilution) at 37°C for 3 h. The incubation time and the antibody concentration were optimized for minimal staining of M₂ receptors. The antigen was detected by using biotinylated antirabbit antibody (Vector Laboratories, Burlingame, CA) at a dilution of 1:100 for 30 min at room temperature. The slides were then incubated with avidin biotin complex (Vectastain) for 30 min, followed by chromogen AEC (Vector Laboratories) for 3 min. Nuclei were counterstained with 4% hematoxylin. To control for nonspecific binding of the antibody, one slide was incubated with 0.3% Triton-X in PBS instead of the primary antibody.

Analysis of data. In vivo responses to nerve stimulation and to acetylcholine, methacholine, and pilocarpine were analyzed using a repeated-measures analysis of variance. The frequency- response relationship for stimulated acetylcholine release was analyzed using repeated-measures analysis of variance. The effects of atropine on acetylcholine release in control and dexamethasone-treated cells were analyzed using a two-way analysis of variance with pairwise comparisons made using Student's t test with Bonferroni correction. The levels of M₂ receptor mRNA in control, dexamethasone-treated, and interferon-treated cells were compared using Student's t test with Bonferroni correction (21).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effects of Dexamethasone In Vivo

Baseline responses. Baseline values for heart rate (control, 335.6 ± 14.7 versus dexamethasone, 336.4 ± 10.6 beats/min), systolic blood pressure (control, 57.0 ± 2.9 versus dexamethasone, 54.0 ± 1.5 mm Hg), diastolic blood pressure (control, 31.0 ± 1.0 versus dexamethasone, 30.0 ± 1.1 mm Hg), and Ppi (control, 103 ± 6.8 versus dexamethasone, 114 ± 4.6 mm H₂O) were unaffected by dexamethasone treatment.

Effects of dexamethasone on vagally mediated bronchoconstriction in vivo. Electrical stimulation of both vagus nerves produced frequency-dependent bronchoconstriction (measured as an increase in Ppi), which rapidly reversed after stimulation was stopped. In dexamethasone-treated animals, the bronchoconstriction response to vagal stimulation was suppressed (Figure 1A). Vagally induced bronchoconstriction and bradycardia were completely blocked by atropine (1 mg/kg), indicating that they were mediated via the release of acetylcholine onto muscarinic receptors.

View larger version (21K): [in this window] [in a new window]   Figure 1.   (A) Dexamethasone decreases the response of guinea pig airways to electrical stimulation of the vagus nerves. Bilateral stimulation of vagi (10 V, 0.2 ms pulse duration, 1 to 25 Hz, 5 s pulse train) increases airway inflation pressure more in control animals (open squares) than in dexamethasone-treated animals (solid squares; n = 6, P < 0.001). Data are mean ± SE. (B) Dexamethasone potentiates the ability of pilocarpine to suppress vagally mediated bronchoconstriction. Data are expressed as the ratio of the response to vagal stimulation vagi (6 to 20 V, 0.2 ms pulse duration, 2 Hz, 22 s pulse train) after treatment with a given dose of pilocarpine to the response in the absence of pilocarpine. Thus a ratio of 1 would indicate no response to pilocarpine and a ratio of less than 1 indicates suppression of vagally mediated bronchoconstriction. Pilocarpine suppresses bronchoconstriction at lower doses in dexamethasone-treated animals (solid squares) than in control animals (closed squares; n = 5, P < 0.005). Data are mean ± SE.

Effects of dexamethasone on M₂ receptor function in vivo. In both control and dexamethasone-treated animals, pilocarpine suppressed the response to vagal stimulation, demonstrating a functional M₂ muscarinic receptor on the nerves (Figure 1B). In dexamethasone-treated animals, much lower doses of pilocarpine were required to suppress vagally mediated bronchoconstriction, demonstrating increased function of the M₂ receptors. Interestingly, the heart rate response to vagal stimulation, which is also mediated via M₂ receptors on the cardiac muscle, was unaffected by dexamethasone (P = 0.97).

Effects of dexamethasone on responses to intravenous acetylcholine and methacholine. Dexamethasone substantially decreased acetylcholine-induced bronchoconstriction (Figure 2A). Inhibiting cholinesterase activity by treating animals with physostigmine increased acetylcholine-induced bronchoconstriction more in dexamethasone-treated animals than in controls, so that the response to acetylcholine after physostigmine was the same in dexamethasone-treated and control animals. In contrast, the response to methacholine (which is relatively resistant to cholinesterase) was not affected by dexamethasone, demonstrating normal function of airway smooth muscle M₃ receptors (Figure 2B).

View larger version (21K): [in this window] [in a new window]   Figure 2.   (A) Dexamethasone decreases airway responsiveness to intravenous acetylcholine in vagotomized animals. Bronchoconstriction in dexamethasone-treated animals (solid squares; n = 11) is less than in control animals (open squares; n = 4, P < 0.03). Treating animals intravenously with physostigmine (250 µg/kg) to inhibit cholinesterase activity increases the response to acetylcholine to the same final level in dexamethasone-treated animals (solid circles; n = 4) and control animals (open circles; n = 4, P = 0.35). (B) Dexamethasone does not affect airway responsiveness to methacholine. Dexamethasone-treated animals (solid squares) have similar bronchoconstriction to control animals (open squares; n = 4, P = 0.49. Data are mean ± SE.

Effects of inhibiting cholinesterase and blocking M₂ receptors on the response to vagal stimulation. Blocking cholinesterase activity using physostigmine increased the response to vagal stimulation in both dexamethasone-treated and control animals (Figure 3). However, the response to vagal stimulation remained significantly suppressed in dexamethasone-treated animals that received physostigmine. Likewise, blocking M₂ receptors using gallamine (10 mg/kg, intravenously) increased the response to vagal stimulation in both dexamethasone-treated and control animals (Figure 4). Again, the response to vagal stimulation remained significantly suppressed in dexamethasone-treated animals that received gallamine. However, the combination of physostigmine and gallamine increased the response to vagal stimulation to the same final level in both dexamethasone-treated and control animals.

View larger version (27K): [in this window] [in a new window]   Figure 3.   Blocking cholinesterase (with physostigmine) or M2 receptors (with gallamine) each partially reverses dexamethasone-induced hyporesponsiveness to vagal stimulation. The combination of physostigmine and gallamine raises the response to vagal stimulation (15 Hz) to the same final level in dexamethasone-treated (solid bars) and control (open bars) animals. Tissues from control animals had significantly (P < 0.05) greater responses than tissues from dexamethasone-treated animals at baseline, after gallamine treatment, and after physostigmine treatment, but not after treatment with the combination of gallamine and physostigmine. In tissues from both control and dexamethasone-treated animals, treatment with gallamine, physostigmine, or the combination of gallamine and physostigmine significantly (P < 0.05) increased the response to electrical field stimulation. Data are mean ± SE.

View larger version (22K): [in this window] [in a new window]   Figure 4.   Release of acetylcholine from cultured airway parasympathetic neurons is suppressed by treatment with dexamethasone. Cells were stimulated electrically (1 V, 0.1 ms pulse duration, 0.5 to 6 Hz, 6 s pulse train). Data are mean ± SE (n = 6, P < 0.05.

Effects of dexamethasone on lung inflammatory cells. Total inflammatory cells in lung lavage fluid were not changed by dexamethasone treatment (control, 11.71 ± 1.63 × 10⁶ cells; dexamethasone 10.02 ± 0.98 × 10⁶ cells). Lymphocytes (control, 0.14 ± 0.05 × 10⁶ cells; dexamethasone 0.2 ± 0.07 × 10⁶ cells), eosinophils (control, 1.26 ± 0.25 × 10⁶ cells; dexamethasone 1.11 ± 0.33 × 10⁶ cells), neutrophils (control, 0.23 ± 0.1 × 10⁶ cells; dexamethasone 0.48 ± 0.08 × 10⁶ cells), and macrophages (control, 10.09 ± 1.5 × 10⁶ cells; dexamethasone 8.23 ± 0.86 × 10⁶ cells) were likewise not affected by dexamethasone.

Effects of Dexamethasone in Cultured Airway Parasympathetic Neurons

Release of acetylcholine in response to electrical field stimulation. The amount of acetylcholine released was greater in cells grown in culture medium containing nerve growth factor supplied by QCB (Figure 4) than in culture medium containing nerve growth factor supplied by Sigma (Figure 5). Total cellular acetylcholine content was greater in cells grown in the presence of QCB nerve growth factor (182 ± 9 pmol/culture) than in those grown in the presence of Sigma nerve growth factor (132 ± 8 pmol/culture; P = 0.0001).

View larger version (21K): [in this window] [in a new window]   Figure 5.   Atropine potentiates release of acetylcholine from cultured airway parasympathetic neurons more in dexamethasone treated cultures than in control cultures. Acetylcholine release was measured in the absence (open columns) and presence (shaded columns) of atropine. There is no difference in acetylcholine release between control and dexamethasone-treated cells when M2 receptors are blocked with atropine. Data are mean ± SE (n = 10 to 17). *Significantly different from control (no dexamethasone, no atropine); **Significantly different from dexamethasone alone.

Effects of dexamethasone on acetylcholine release by cultured airway parasympathetic neurons. Cells used in these experiments were all grown in medium that contained nerve growth factor supplied by QCB. Electrical field stimulation of cultured airway parasympathetic neurons increased release of acetylcholine in frequency-dependent fashion (Figure 4). Release of acetylcholine was substantially decreased in dexamethasone-treated cells. Dexamethasone did not change the total cellular acetylcholine content (control, 190 ± 15 pmol/culture; dexamethasone, 174 ± 11 pmol/culture; P = 0.37).

Effects of dexamethasone on M2 receptor function in cultured airway parasympathetic neurons. Cells used in these experiments were all grown in medium that contained nerve growth factor supplied by Sigma. Blocking M₂ receptors with atropine increased the release of acetylcholine in both dexamethasone-treated and control cultures (Figure 5). The degree of potentiation in dexamethasone-treated cells was greater than in control cells, so that the active release of acetylcholine from both groups was the same after M₂ receptors were blocked. Dexamethasone did not change the total cellular acetylcholine content (control, 129 ± 11 pmol/culture; dexamethasone, 134 ± 11 pmol/culture; P = 0.75).

Effects of dexamethasone on M₂ receptor expression in cultured airway parasympathetic neurons. Cells used in these experiments were grown in culture medium that contained nerve growth factor supplied by QCB in two experiments and by Sigma in two experiments. As there was no effect of the different nerve growth factors on M₂ receptor expression or on the effects of dexamethasone and interferon on M₂ receptor expression, the data from these experiments were pooled. M₂ receptor mRNA was increased 10-fold by dexamethasone (Figure 6). As we have previously shown (20), interferon- decreased M₂ receptor mRNA; this decrease was prevented by dexamethasone.

View larger version (106K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Figure 6.   Competitive RT-PCR assay for M2 receptor mRNA. (A) A representative gel. Numbers across the top of the gel represent the amount of internal standard cRNA, in picograms, added to each reaction. The size of the M2 receptor mRNA product is 568 bp and the size of the competing cRNA product is 525. (B) The means of four competitive RT-PCR experiments show that M2 receptor mRNA is increased tenfold in dexamethasone-treated cultures when compared with controls. Interferon- (IFN) decreases M2 receptor mRNA. The effect of interferon is reversed by dexamethasone (DEX). Data are mean ± SE (n = 4). *Significantly different from control.

M₂ receptor immunostaining. Immunostaining demonstrated M₂ receptor protein in both dexamethasone-treated and control cells. Staining intensity was increased in dexamethasone-treated cells (Figure 7). The increase in staining was most evident in the nerve cell processes.

View larger version (109K): [in this window] [in a new window]   Figure 7.   Immunostaining with antibody specific for the third transmembrane segment of the M2 muscarinic receptor demonstrates increased staining in dexamethasone-treated cultures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

These studies demonstrate that airway responsiveness to electrical stimulation of the vagus nerves is decreased by dexamethasone. The increased effects of pilocarpine and gallamine demonstrate that this decrease in response to vagal stimulation is due in part to increased function of the M₂ muscarinic receptors on the nerves.

Furthermore, airway responsiveness to acetylcholine is decreased by dexamethasone. As this hyporesponsiveness is no longer evident in the presence of physostigmine, it is likely that this reflects increased cholinesterase activity. The failure of dexamethasone to suppress the airway response to methacholine, which is substantially less susceptible to metabolism by acetylcholinesterase (15), further supports increased cholinesterase activity as the mechanism of dexamethasone-induced hyporesponsiveness to acetylcholine. The normal response to methacholine in dexamethasone-treated animals also demonstrates that M₃ receptors on the airway smooth muscle are unaffected.

Blocking either the M₂ receptor (using gallamine) or cholinesterase (using physostigmine) partially reversed dexamethasone-induced hyporesponsiveness to vagal stimulation. The combination of gallamine and physostigmine brought vagally induced bronchoconstriction to the same final level in both dexamethasone-treated and control animals. This suggests that dexamethasone-induced inhibition of the response to vagal stimulation is mediated by a combination of increased M₂ receptor function and increased cholinesterase activity. Interestingly, the heart rate response to vagal stimulation was not affected by dexamethasone, suggesting that the regulation of M₂ receptors on the heart muscle is controlled differently from the M₂ receptor on airway parasympathetic neurons.

Dexamethasone also decreased acetylcholine release and increased M₂ receptor function in cultured airway parasympathetic neurons. This increase corresponded to an increase in M₂ receptor mRNA and increased immunostaining with antibody specific for the M₂ receptor. There was no effect of dexamethasone on total acetylcholine content in the cells. Thus, dexamethasone increases expression and function of the M₂ receptor in vitro, and this increase in expression may be responsible for the increased M₂ receptor function in vivo.

Of note is the effect of different sources of nerve growth factor on the function of cultured airway parasympathetic neurons. Our earlier studies (Figure 4) were done in cells grown in medium that contained nerve growth factor supplied by QCB. These cells had significantly higher acetylcholine content and released fourfold more acetylcholine in response to electrical stimulation than did cells used in our later studies (Figure 5), which used cells grown in medium that contained nerve growth factor supplied by Sigma. This greater acetylcholine release allowed for a more dramatic suppression of acetylcholine release by dexamethasone (Figure 4) than was seen in subsequent studies (Figure 5). Despite this, the suppression of acetylcholine release by dexamethasone remained significant in the later studies, and the reversal of this effect in cells treated with atropine supports the hypothesis that dexamethasone suppresses acetylcholine release by increasing M₂ receptor expression and/or function.

Glucocorticoids are effective in the treatment of asthma. The main mechanism of action is generally thought to be suppression of airway inflammation. Some clinical studies have demonstrated improvement in airway hyperresponsiveness in patients with asthma after treatment with inhaled glucocorticoids (22), again postulated to be the result of anti-inflammatory effects. We have previously shown that dexamethasone prevents allergen-induced M₂ receptor dysfunction by inhibiting the recruitment of eosinophils to the airway nerves (23). Our current studies suggest that increased M₂ receptor function may also contribute to the beneficial effects of glucocorticoids.

Effects of glucocorticoids on muscarinic receptors have been mixed and may be tissue-specific. In airway smooth muscle, there is no effect on M₃ muscarinic receptors; however, expression of M₂ receptors on the airway smooth muscle, which decreases the response to -adrenergic stimulation, is decreased by glucocorticoid treatment (24). In contrast, total lung muscarinic receptor density, determined by radioligand binding, is increased by glucocorticoid treatment (12). This may include the M₂ receptors on the airway nerves. Likewise, in the hypothalamus, glucocorticoids increase muscarinic receptor density (25).

It is not possible, based on the current studies, to exclude an effect of dexamethasone on the signal transduction pathways activated by the M₂ receptor. Effects of steroids on G-proteins, including both Gi and G-proteins linked to potassium channels (both of which may be involved in M₂ receptor signal transduction), have been reported (26).

Glucocorticoids have variable effects of acetylcholine synthesis (29). A decrease in acetylcholine synthesis could account for the decreased response to vagal stimulation but would not produce the increased M₂ receptor function demonstrated in the pilocarpine studies. Measurement of acetylcholine content in the cultured airway parasympathetic neurons also demonstrated that this was not altered by dexamethasone treatment.

Thus, we have shown that dexamethasone decreases airway responsiveness to vagal stimulation via two mechanisms: increased M₂ receptor function and increased degradation of acetylcholine by cholinesterases. Studies in cultured airway parasympathetic neurons show that M₂ receptor mRNA expression is increased by dexamethasone and that this is associated with increased M₂ receptor protein and increased M₂ receptor function. Increased M₂ receptor function results in decreased acetylcholine release. At the same time, the acetylcholine that is released is degraded more rapidly via increased cholinesterases. These mechanisms may all contribute to the therapeutic effects of glucocorticoids by inhibiting reflex bronchoconstriction.