Introduction

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Tachykinins are a family of related neuropeptides including substance P (SP), neurokinin A (NKA), and neurokinin B (NKB), that preferentially interact with neurokinin NK₁, NK₂, and NK₃ receptors, respectively (1). In the airways, SP and NKA are present within sensory C-fibers, and are released by a number of physical and chemical stimuli (2, 3). The activation of sensory nerves may result in various inflammatory and reflex effects such as bronchoconstriction, plasma extravasation, mucus secretion, and cough (4). NKA is more potent than SP in causing contraction of human airway smooth muscle (5, 6), and NKA rather than SP inhalation causes bronchoconstriction in asthmatic patients (7, 8). NKA also induces airway hyperresponsiveness (AHR) in allergic guinea pigs and sheep (9). Studies with selective tachykinin antagonists have demonstrated an important role for NK₂ receptors in the airways. SR48968, a potent selective NK₂ receptor antagonist, blocks hyperpnea-induced as well as sodium metabisulfite-induced bronchoconstriction, inhibits citric acid-induced AHR, and suppresses cough induced by citric acid in guinea pigs (12). In human airways, NKA causes bronchoconstriction in normal subjects after inhalation of a neutral endopeptidase inhibitor, thiorphan, that prevents peptide degradation (16), and causes potent bronchoconstriction in asthmatic patients (17). Expression of messenger RNA (mRNA) for the NK₂ receptor is increased 4-fold in lung samples from asthmatic as compared with nonsmoking control subjects, although NK₁ receptor mRNA levels are similar in the two groups (18).

There are still concerns that regular or high doses of ₂-adrenoceptor agonists may contribute to exacerbations of asthma or mortality from the disease (19). Sustained use of ₂-adrenoceptor agonists is associated with loss of bronchoprotection against nonspecific bronchoconstrictors, such as methacholine and adenosine, and may result in a greater inflammatory response of the airways to allergens (20). However, the mechanisms of such adverse effects of ₂-adrenoceptor agonists are not fully understood. On the other hand, corticosteroids are more widely used, together with ₂-adrenoceptor agonists, in the treatment of asthma. We have previously shown that dexamethasone downregulates the expression of NK₂ receptors at both the mRNA and protein levels in bovine tracheal smooth muscle (21).

In the present study we examined whether a ₂-adrenoceptor agonist, fenoterol, alone or in combination with dexamethasone, affects expression of the NK₂ receptor, as well as the functional effects of fenoterol in response to NK₂ receptor stimulation in bovine tracheal smooth muscle, which provides sufficient tissues for molecular, binding, and functional studies.

	Materials and Methods

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Tissue Preparation

Fresh bovine tracheae were obtained from an abattoir and the smooth-muscle layer was dissected after stripping off epithelium, mucosa, and connective tissue. The smooth-muscle layer was cut into small pieces or strips in oxygenated Krebs-Henseleit solution ([in mM]: NaCl 118, KCl 5.9, MgSO₄ 1.2, CaCl₂ 2.5, NaH₂PO₄ 1.2, NaHCO₃ 25.5, and glucose 5.6). After washing, the pieces or strips of muscle were incubated in 4-(2-hydroxyethyl)-1-piperazine- N'-2-ethanesulfonic acid (Hepes)-modified Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamate, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C in a shaker incubator. For molecular and binding studies, pieces of bovine tracheal smooth muscle were placed in T-75 flasks and incubated in the absence or presence of 10⁹-10⁵ M fenoterol for the indicated periods. The tissues were then frozen and kept at 70°C for RNA extraction and membrane preparation. In some experiments, incubation of bovine tracheal smooth muscle with 10⁷ M fenoterol for 6 h was done in the presence of various concentrations of dexamethasone (10⁹-10⁷ M). To examine whether the effect of fenoterol was mediated by specific receptor subtypes, propranolol (a nonselective -adrenoceptor antagonist; 10⁷ M), ICI118551 (a selective ₂-adrenoceptor antagonist; 10⁷ M) or CGP20712A (a selective ₁-adrenoceptor antagonist; 10⁷ M) was added for 1 h before the addition of 10⁷ M fenoterol for a further 6 h. Preincubation of bovine tracheal smooth muscle with the protein synthesis inhibitor cycloheximide (10 µg/ml) for 1 h before the addition of 10⁷ M fenoterol and incubation for a further 6 h was done to assess whether new protein(s) synthesis was required for the upregulation of NK₂ receptors. Incubation of bovine tracheal smooth muscle with a direct adenylyl cyclase activator, forskolin (10⁵ M); with an analogue of nonhydrolyzable cyclic adenosine monophosphate (cAMP), 8-bromo-cAMP (10³ M); or with other cAMP-increasing agents such as prostaglandin E₂ (PGE₂) (10⁶ M) for 6 h was done to see whether the effect of fenoterol is mediated by an increase in cAMP. The half-life of messenger RNA (mRNA) for the NK₂ receptor was measured in control and fenoterol-treated tissues by incubation in the absence and presence of 10⁷ M fenoterol for 6 h before the addition of 5 µg/ml actinomycin D for various times (22).

For contraction experiments, smooth-muscle strips (~ 5 × 2 × 1 mm) were washed in sterile DMEM and then exposed to 10⁷ M fenoterol, or 10⁶ M dexamethasone alone and in combination, or to vehicle for 18 h (three or four smooth-muscle strips in 10 ml of the same medium used for the incubation of muscle pieces in a 25 cm² cell culture flask) at 37°C in a shaker incubator.

RNA Isolation

Total cellular RNA from bovine tracheal smooth muscle was isolated by acid guanidinium thiocyanate-phenol- chloroform extraction as described by Chomczynski and Sacchi (23). The PolyATtract mRNA isolation kit system IV (Promega, Southampton, UK) was used to prepare polyadenine-containing (poly[A]⁺) RNA according to the manufacturer's instructions. Samples of poly(A)⁺ RNA were size-fractionated on a 1% agarose/formaldehyde gel and blotted onto nylon membranes (Magna, Westborough, MA) through capillary action.

Northern Blot Analysis

Complementary DNA (cDNA) for the human NK₂ receptor (891-bp EcoRI/SacI fragment; kindly provided by Dr. J. E. Krause, Washington University, St. Louis, MO) and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (1.3-kb Pst1 fragment) were radiolabeled with a random primer labeling kit in the presence of [-³²P]deoxycytosine triphosphate ([-³²P]dCTP) (3,000 Ci/mmol; Amersham, Amersham, UK). The blot was prehybridized for 5 h in 50% formamide, 5× standard saline-citrate (SSC), 5× Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS), 10 mM NaH₂PO₄, and 100 mg/ml sonicated denatured salmon sperm DNA, and was then hybridized with ³²P- labeled cDNA probes for 12-16 h at 42°C. After hybridization, the blot was washed for 30 min at high stringency in 0.1× SSC/0.1% SDS at 55°C. The blot was exposed to Kodak OMAT XS film (Hemel Hempstead, UK) at 70°C with an intensifying screen for 1-3 d. The blot was hybridized first to a ³²P-labeled NK₂ receptor cDNA probe, and subsequently to a GAPDH cDNA probe after stripping. The autoradiograms were scanned with a laser densitometer (New Discovery Series; pdi, Huntington Station, NY) or with the Gel Documentation and Analysis System GDS8000 (UVP, Ltd., Cambridge, UK). The amount of NK₂ receptor mRNA was calculated by adding the densitometric areas of bands corresponding to the three different sizes of transcripts (see RESULTS), and was then quantified relative to the amount of GAPDH mRNA on the same filter.

Nuclear Run-On Transcription Assay

Nuclear run-on transcription assays were performed to determine whether fenoterol changed the transcription rate of the NK₂ receptor gene. Nuclei were isolated from frozen bovine tracheal smooth-muscle incubated with or without fenoterol (10⁷ M) for 6 h and stored at 70°C in Keller storage buffer (10 mM Tris-HCl, pH 7.5; 5 mM MgCl₂; 0.5 M Sorbitol; 2.5% Ficoll [MW = 400,000]; 0.008% spermidine; 1 mM dithiothreitol [DTT]; and 50% glycerol) at 5 × 10⁷ nuclei/100 µl. Each reaction (final volume = 0.4 ml) was conducted in the presence of 5 × 10⁷ isolated nuclei, 40 mM Tris-HCl (pH 8.3), 150 mM NH₄Cl, 7.5 mM MgCl₂, 0.625 mM adenosine triphosphate, 0.313 mM guanosine triphosphate, 0.313 mM cytosine triphosphate (Promega, Southampton, UK), 0.5 mCi [³²P]uridine triphosphate (800 Ci/mmol; DuPont-New England Nuclear, Hounslow, UK), and 120 units/ml recombinant ribonuclease (RNase) inhibitor. Transcription reactions were allowed to proceed for 30 min at 27°C before termination by the addition of 40 units of recombinant RNase inhibitor and 75 units of RQ-1 DNase (Promega). After DNase and proteinase K treatments, the radiolabeled RNA formed was purified by phenol-chloroform extraction and precipitated three times with ethanol in the presence of 1.33 M ammonium acetate. An equal number of counts from each sample was added to slot blots, with three different slots on the same blot. The blots consisted of 10 µg each of pGEM-3Z plasmid (as a control), and plasmids-containing inserts of human NK₂ receptor cDNA, or rat GAPDH cDNA immobilized on a nylon membrane. After hybridization for 72 h at 42°C, the blots were washed at a final stringency of 0.1× SSC and 0.1% SDS at 55°C, including a 30-min digestion with 1 mg/ml RNase A and 20 units/ml RNase T1 at 37°C to digest any single-stranded RNA not hybridized to DNA. After autoradiography, the film was scanned through laser densitometry and quantified by calculating the ratio of NK₂ receptor cDNA signal to GAPDH cDNA signal.

Radioligand Binding Assay

Frozen bovine tracheal smooth-muscle tissues were ground under liquid nitrogen, suspended in 10 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4) containing 0.32 M sucrose, and homogenized with a Polytron homogenizer (KINEMATICA AG, Lucerne, Switzerland) at a setting of 6 for 30-s bursts. The homogenate was centrifuged at 1,000 × g for 10 min at 4°C to remove debris, and the supernatant was then centrifuged at 40,000 × g for 20 min at 4°C. The resulting pellet was washed and recentrifuged at the same speed. The final pellet was resuspended in 50 mM Tris-HCl (pH 7.4). Protein concentration was determined by the method of Lowry and coworkers (24), using bovine serum albumin (BSA) as a standard.

The density and affinity of NK₂ receptors were assessed from saturation isotherms with the use of 0.5-10 nM [³H]SR48968 (25.7 Ci/mmol; DuPont NEN) in a final volume of 0.5 ml of assay buffer containing approximately 300-400 µg membrane protein in 50 mM Tris-HCl (pH 7.4), 0.4 mg/ml BSA, 3 mM MnCl₂, 0.04 mg/ml bacitracin, and 0.004 mg/ml chymostatin. Nonspecific binding was determined in the presence of 10⁶ M unlabeled SR48968. After incubation at 25°C for 30 min, bound and free radioactivity were separated through Whatman GF/C glass- fiber filters (Maidstone, UK), presoaked for at least 3 h in a buffer (pH 7.4) containing 50 mM Tris-HCl, 0.02% BSA, and 0.05% polyethylenimine. Filters were washed three times with 5 ml ice-cold buffer, using a Brandel cell harvester, and were then placed in vials with 4 ml of scintillation mixture (Filtron X; National Diagnostics, Hull, UK) and counted on a liquid scintillation counter (Model 2200 CA; Packard, Pangbourne, UK). Specific binding was determined by subtracting nonspecific binding from total binding, and usually constituted 71-88% of total binding. The maximal binding capacity (B_max) and dissociation constant (K_d) were analyzed with the computerized nonlinear regression program Ligand (25).

Contraction Measurements

After 18 h exposure to drugs or vehicle, smooth-muscle strips were washed twice and then mounted in 20-ml organ baths for isotonic recording under a 0.5-g preload in gassed Krebs-Henseleit buffer at 37°C. After two 30-min equilibration periods with a change of buffer in between, the strips were precontracted twice by cumulative administration of methacholine (10⁷-10⁵ M and 10⁷-10⁴ M, respectively), followed by washing periods of 60 min; between the two precontraction/washing periods, zero tone was established with 10⁷ M isoproterenol, followed immediately by a 15-min washing period. Subsequently, cumulative concentration-response curves to the specific NK₂ receptor agonist [-Ala⁸]-NKA(4-10) (PolyPeptide Laboratories GmbH; Wolfenbuettel, Germany) (26) were constructed, with concentrations ranging from 10¹⁰ M to 3 × 10⁶ M; experiments were performed in duplicate on six independent occasions, except for those involving dexamethasone plus fenoterol (n = 4).

Statistical Analysis

Data are shown as mean ± SEM. Groups of data were evaluated by analysis of variance (ANOVA). Data that appeared statistically significant were compared with paired or unpaired Student's t tests, with Bonferroni's correction for comparing the means of multiple groups. A value of P < 0.05 was considered significant.

Contractile responses induced by [-Ala⁸]-NKA(4-10) were expressed as a percentage of the response to 10⁴ M methacholine as assessed with the second precontraction curve for each individual smooth-muscle strip. Responses to 10⁴ M methacholine were not significantly affected by 18 h exposure to 10⁷ M fenoterol as measured in independent isometric contraction experiments. Differences between control and fenoterol-treated preparations were analyzed by ANOVA (contraction levels) and Wilcoxon's signed ranks test (contraction levels and log EC₅₀ values).

	Results

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Effect of Fenoterol on NK₂ Receptor mRNA

Poly(A)⁺ RNA isolated from both control and fenoterol-treated bovine tracheal smooth-muscle tissues gave rise to three hybridization bands with estimated sizes of the respective mRNAs of approximately 3.1, 2.7, and 2.3 kb (Figure 1A). All of these bands were in agreement with the previously reported sizes of NK₂ mRNAs (27). The time course of the induction of NK₂ receptor mRNA by fenoterol revealed that an increase in all three bands of NK₂ receptor mRNA could be detected after 4 h, and reached a plateau after 12 h (Figure 1). An increase in NK₂ receptor mRNA after 6 h could be detected at a concentration of fenoterol as low as 10⁹ M (Figure 2). Although the nonselective -adrenoceptor antagonist propranolol and selective ₁- and ₂-adrenoceptor antagonists CGP 20712A and ICI118551 respectively had no effect on the expression of NK₂ receptor mRNA, by themselves, propranolol or ICI118551 completely blocked the effect of fenoterol at 6 h, whereas CGP20712A had no effect (Figure 3), demonstrating that the effect of fenoterol on the NK₂ receptor was mediated entirely by ₂-adrenoceptors.

View larger version (45K): [in this window] [in a new window]   Figure 1.   Time course of fenoterol-induced NK2 receptor (NK2R) mRNA in bovine tracheal smooth muscle. (A) Northern blot analyses with cDNA for NK2 receptor and GAPDH after isolation of poly(A)+ RNA from bovine tracheal smooth muscle in absence (C) and presence of 107 M fenoterol (F) for indicated times. (B) Quantification of densitometric measurement as ratio of NK2 receptor mRNA (i.e., sum of all three bands) relative to GAPDH mRNA. Means ± SEM of four to six separate experiments are shown (**P < 0.01, ***P < 0.001 for difference from corresponding control value with Student's t test).

View larger version (31K): [in this window] [in a new window]   Figure 2.   Dose-response of fenoterol-induced NK2 receptor mRNA in bovine tracheal smooth muscle. (A) Northern blot analyses of isolated poly(A)+ RNA in the absence (C) or presence of increasing doses of fenoterol for 6 h. Lane 1: vehicle (control); lane 2: 109 M fenoterol; lane 3: 108 M; lane 4: 107 M; lane 5: 106 M; lane 6: 105 M. (B) Densitometric measurements of NK2 receptor mRNA. Means ± SEM of five separate experiments are shown (*P < 0.05, **P < 0.01, ***P < 0.001 for differences from control values with Student's t test).

View larger version (39K): [in this window] [in a new window]   Figure 3.   Effect of the nonselective and selective -antagonists propranolol (prop, 107 M), ICI118551 (ICI, 107 M) and CGP20712A (CGP, 107 M) on NK2 receptor mRNA in bovine tracheal smooth muscle. (A) Northern blot analysis of isolated poly(A)+ RNA in the absence of fenoterol (C, lane 1), 107 M fenoterol (F) alone for 6 h (lane 2), propranolol alone (lane 3), ICI118551 alone (lane 4), CGP20712A alone (lane 5), combination of propranolol and fenoterol (lane 6), ICI118551 and fenoterol (lane 7), and CGP20712A and fenoterol (lane 8). (B) Densitometric measurements of NK2 receptor mRNA. Means ± SEM of five separate experiments are shown (**P < 0.01, ***P < 0.001 for differences from control values with Student's t test).

The protein synthesis inhibitor cycloheximide also completely blocked the effect of fenoterol at 6 h on the expression of NK₂ receptor mRNA (Figure 4), indicating that new protein synthesis is required for upregulation of the receptor.

View larger version (29K): [in this window] [in a new window]   Figure 4.   Effect of the protein synthesis inhibitor cycloheximide (CHX, 10 µg/ml) on NK2 receptor mRNA in bovine tracheal smooth muscle. (A) Northern blot analysis of isolated poly(A)+ RNA in the absence of fenoterol (C, lane 1), 107 M fenoterol (F) alone for 6 h (lane 2), cycloheximide alone (lane 3), and the combination of fenoterol and cycloheximide (lane 4). (B) Densitometric measurements of NK2 receptor mRNA. Means ± SEM of four separate experiments are shown (***P < 0.001 for differences from control values with Student's t test).

Incubation of bovine tracheal smooth muscle with forskolin, a direct adenylyl cyclase activator; with 8-bromo-cAMP, an analogue of nonhydrolyzable cAMP; or with PGE₂ produced a significant increase in NK₂ receptor mRNA expression resembling the effect of fenoterol (Figure 5), suggesting that all these effects are mediated by an increase in cAMP.

View larger version (38K): [in this window] [in a new window]   Figure 5.   Effects of the direct adenylyl cyclase activator forskolin (Fors, 105 M), cAMP analog 8-bromo-cAMP (8b-cAMP, 103 M), and PGE2 (106 M) on NK2 receptor mRNA in bovine tracheal smooth muscle. (A) Northern blot analysis of isolated poly(A)+ RNA in the absence of any drug (C, lane 1), and in the presence of 107 M fenoterol (F, lane 2), forskolin (lane 3), 8-bromo-cAMP (lane 4), and PGE2 (lane 5) for 6 h. (B) Densitometric measurements of NK2 receptor mRNA. Means ± SEM of four separate experiments are shown (*P < 0.05; **P < 0.01, ***P < 0.001 for differences from control values with Student's t test).

A change in abundance of NK₂ receptor mRNA could result either from an alteration in the degradation rate or in the transcription rate of this mRNA. The stability of NK₂ receptor mRNA in the presence of the RNA polymerase inhibitor actinomycin D (5 µg/ml) was compared in control and in fenoterol-treated bovine tracheal smooth-muscle tissues (Figure 6). The half-life of NK₂ receptor mRNA was approximately 5 h, but no degradation occurred after preteatment with fenoterol over the same period. These results suggest that the increase in NK₂ receptor mRNA in the presence of fenoterol is at least partly due to the increased stability of NK₂ receptor mRNA.

View larger version (11K): [in this window] [in a new window]   Figure 6.   Effect of fenoterol on stability of NK2 receptor mRNA in bovine tracheal smooth muscle. Actinomycin D (5 µg/ml) was added at 0 min to bovine tracheal smooth muscle pretreated for 6 h with vehicle (open circles) or 107 M fenoterol (closed circles) for the times indicated. Means ± SEM of four separate experiments are shown.

The rate of transcription of newly synthesized NK₂ receptor mRNA calculated as a ratio to that of GAPDH mRNA was increased by 25% after treatment with fenoterol as compared with the control value (Figure 7).

View larger version (20K): [in this window] [in a new window]   Figure 7.   Effect of fenoterol on transcription of NK2 receptor gene in bovine tracheal smooth muscle as assessed with nuclear run-on assay in the absence (control) and presence of 107 M fenoterol for 6 h. (A) Representative autoradiogram showing quantification of GAPDH (1); pGEM-3Z (2); and NK2 receptor (3). (B) Densitometric measurement showing transcription rate of NK2 receptor gene as the ratio to GAPDH in control and fenoterol-treated tissues. Means ± SEM of three separate experiments are shown (*P < 0.05 for differences from control values with Student's t test).

The expression of NK₂ receptor mRNA after incubation with fenoterol in the presence of various concentrations of dexamethasone was also examined. Suppression by dexamethasone of the fenoterol-induced increase in NK₂ receptor mRNA in all three bands could be detected at a dexamethasone concentration as low as 10⁹ M. The further suppressive effect of dexamethasone in the presence of fenoterol could also be detected through levels of NK₂ receptor mRNA that were below the control level at higher dexamethasone concentrations (Figure 8), in accord with our previous findings (21).

View larger version (26K): [in this window] [in a new window]   Figure 8.   Dose-response effect of dexamethasone (dex) on fenoterol-induced NK2 receptor mRNA in bovine tracheal smooth muscle. (A) Northern blot analyses of isolated poly(A)+ RNA in the absence (C) and presence of fenoterol (F) and presence of increasing doses of dexamethasone plus fenoterol for 6 h. Lane 1: vehicle (control); lane 2: 107 M fenoterol (F) alone; lane 3: 109 M dexamethasone plus fenoterol; lane 4: 108 M dexamethasone plus fenoterol; lane 5: 107 M dexamethasone plus fenoterol. (B) Densitometric measurements of NK2 receptor mRNA. Means ± SEM of three separate experiments are shown (**P < 0.01, ***P < 0.001 for differences from control values with Student's t test).

Effect of Fenoterol on NK₂ Receptor Binding

The [³H]SR48968 binding assay showed a significant increase in the number of binding sites of fenoterol after 12 h (B_max = 157 ± 14 fmol/mg protein versus 208 ± 10 fmol/mg protein for vehicle and fenoterol treatment, respectively; P < 0.05, n = 4; Figure 9), and after 18 h (B_max = 155 ± 20 fmol/mg protein versus 255 ± 27 fmol/mg protein for vehicle and fenoterol treatment, respectively; P < 0.01, n = 4; Figure 9), with no significant change at 4 h. The K_d values did not change significantly in vehicle and fenoterol-treated tissues (0.91 ± 0.21 nM versus 0.89 ± 0.22 nM and 0.95 ± 0.31 nM versus 1.11 ± 0.29 nM at 12 h and 18 h, respectively).

View larger version (13K): [in this window] [in a new window]   Figure 9.   Time-course effect of fenoterol on number of NK2 receptors as determined through binding of [3H]SR48968 in bovine tracheal smooth muscle incubated for various time points without drug (open columns) or with 107 M fenoterol (closed columns). Means ± SEM of four separate experiments are shown (*P < 0.05, **P < 0.01 for differences from control values with Student's t test).

Effect of Fenoterol on [-Ala⁸]-NKA(4-10)- Induced Contraction

The specific NK₂ receptor agonist [-Ala⁸]-NKA(4-10) induced concentration-dependent contractions in control bovine tracheal smooth-muscle strips at concentrations of 3 × 10⁸ M and greater; at 3 × 10⁶ M, contraction amounted to 77 ± 5% (mean ± SEM of six experiments) of the methacholine-induced maximum. Half-maximal contraction was obtained at a log concentration (log EC₅₀) of 6.64 ± 0.08. In contrast to the control preparations, contractions in fenoterol-exposed strips began at a concentration of 10⁸ M of agonist, reaching 86 ± 5% at 3 × 10⁶ M (P < 0.05 or less as compared with the control value at all agonist concentrations from 3 × 10⁸ M and greater); the log EC₅₀ value was increased to 6.92 ± 0.13 (P < 0.01 compared with control) (Figure 10).

View larger version (18K): [in this window] [in a new window]   Figure 10.   Concentration-response curves for the specific NK2 receptor agonist [-Ala8]-NKA(4-10) in bovine tracheal smooth-muscle strips incubated for 18 h without drug (closed circles), or with 107 M fenoterol (closed squares), 106 M dexamethasone (closed triangles), or dexamethasone plus fenoterol (closed inverted triangles). Contraction is presented as percentage of the response to 100 µM methacholine in the second precontraction curve. Means ± SEM of four to six experiments, each performed in duplicate, are shown.

Preparations that had been exposed to dexamethasone alone contracted at [-Ala⁸]-NKA(4-10) concentrations of 3 × 10⁸ M and more, as in the control preparations, but reaching somewhat lower levels of contraction at concentrations of 10⁷ M and higher. However these differences in contraction levels, which reached 68 ± 7% at 3 × 10⁶ M [-Ala⁸]-NKA(4-10), did not reach statistical significance, and neither did log EC₅₀ (6.46 ± 0.06) change significantly after dexamethasone (P < 0.10 compared with control). Dexamethasone did, however, completely prevent the fenoterol-induced increase in NK₂ receptor-mediated smooth-muscle contraction. Thus, after combined exposure of bovine tracheal smooth-muscle strips to dexamethasone and fenoterol, the concentration-response curve was almost superimposable on that obtained after dexamethasone alone, reaching 72 ± 9% (mean ± SEM of four experiments) at 3 × 10⁶ M [-Ala⁸]-NKA(4-10); half-maximal contraction was obtained at a log EC₅₀ of 6.48 ± 0.09 (P < 0.02 compared with fenoterol alone).

	Discussion

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We demonstrated a time- and dose-dependent influence of fenoterol treatment on bovine tracheal smooth-muscle NK₂ receptors at both the mRNA and protein levels, and the mechanisms for this effect. The fenoterol-induced increase in expression of the gene for the NK₂ receptor was accompanied by an increase in the functional sensitivity of bovine tracheal smooth muscle to an NK₂ receptor agonist. We have provided the first description of the molecular and functional regulation of the NK₂ receptor by a ₂-adrenoceptor agonist. Furthermore, we have shown that a corticosteroid prevents the ₂-adrenoceptor agonist- induced changes in NK₂ receptor expression.

NK₂ receptor mRNA bands with estimated sizes of approximately 3.1, 2.7, and 2.3 kb were expressed in both control and fenoterol-treated bovine tracheal smooth muscle. Tsuchida and coworkers (27) reported the presence of three similar hybridized bands, which were due to differences in the lengths at the extreme 5' sequences of the 5'-untranslated regions, in rat adrenal gland and eye tissues. We consider all three bands to be specific, since we used a washing at the highest stringency (i.e., 0.1 × SSC/ 0.1% SDS at 60°C for two periods of 30 min each). Interestingly, all three bands were regulated identically in each independent experiment. It is not known whether these three bands of mRNA are translated into a functionally active receptor, nor is the role of these mRNA species currently understood.

Treatment with fenoterol led to an increase in NK₂ receptor mRNA within 4 h, which reached a plateau after 12 h, but a significant increase in NK₂ receptor protein was observed after 12 h. The increase in NK₂ receptor mRNA clearly occurred before the increase in NK₂ receptor number. Hence, the increase in NK₂ receptor density that we observed is likely to be due to increased mRNA levels, leading to increased translation of receptor mRNA and expression of receptor at the cell surface. The fenoterol- induced upregulation of NK₂ receptor mRNA was mediated by ₂-adrenoceptors, since both the nonselective -adrenoceptor antagonist propranolol and the selective ₂-adrenoceptor antagonist ICI118551 completely blocked the effect of 10⁷ M fenoterol at 6 h, whereas the selective ₁-adrenoceptor antagonist CGP20712A had no effect. In accord with these findings, our contraction experiments clearly showed that ₂-adrenoceptor-mediated upregulation of NK₂ receptor number (and steady-state mRNA levels) had a functional effect, since the contractile response to a selective NK₂ receptor agonist began at lower concentrations of this agonist and reached a higher level of contraction at any given concentration of the agonist. Fenoterol-induced upregulation of NK₂ receptor mRNA was prominent at fenoterol concentrations (10⁹-10⁷ M) similar to those required for the relaxation of precontracted guinea pig and human airway smooth muscle (28, 29). This increased responsiveness may have important consequences in terms of the use by asthmatic patients of ₂-adrenoceptor agonists as bronchodilators or bronchoprotectors. In support of this, NK₂ receptor mRNA expression in the lungs of asthmatic patients was found to be significantly increased in comparison with that in the lungs of nonsmoking controls (18). Interestingly, all of the asthmatic patients were taking ₂-adrenoceptor agonists by inhaler, either alone or in combination with an inhaled corticosteroid.

Dexamethasone blocked fenoterol-induced upregulation of NK₂ receptor mRNA dose-dependently to a level below the control level, in accord with our previous finding that dexamethasone alone decreased NK₂ receptor expression in bovine tracheal smooth muscle (21). We found that dexamethasone decreased transcription of the gene for the NK₂ receptor, which presumably compensates for the increased transcription and mRNA stability induced by fenoterol. This is similar to the protective effect of corticosteroid in downregulating ₂-adrenoceptor expression induced by prolonged -adrenoceptor agonist exposure in rat lung (30). In the contractile responses of bovine tracheal smooth muscle, there was a tendency for the NK₂ receptor-mediated contraction curve to shift to the right after treatment with dexamethasone alone, although this did not reach statistical significance. This may well be the result of NK₂ receptor number decreasing only moderately after such treatment (~ 19% reduction), as shown in our previous study (21). More important, however, was that dexamethasone treatment completely prevented fenoterol-induced increases in the contractile potency of NK₂ receptors. This finding agrees well with the results of our mRNA studies, in which dexamethasone kept mRNA levels below the control level, even in the presence of 10⁷ M fenoterol. It therefore appears that a corticosteroid has a greater influence than a ₂-adrenoceptor agonist on NK₂ receptor expression.

In addition, the effect of fenoterol on the expression of NK₂ receptor mRNA at 6 h was blocked by the protein synthesis inhibitor cycloheximide, suggesting that the synthesis of a new protein(s) is required for the induction on NK₂ receptor mRNA. The nature of this protein is unclear. In the combination treatment with dexamethasone and fenoterol, dexamethasone might also have inhibited the synthesis of this protein, and this remains to be investigated. Incubation of bovine tracheal smooth muscle with the direct adenylyl cyclase activator forskolin, with a nonhydrolyzable analogue of cAMP (8-bromo-cAMP), or with another cAMP-increasing agent (PGE₂), produced a significant increase in NK₂ receptor mRNA expression. Thus, the fenoterol-induced increase in NK₂ receptor mRNA is likely to be mediated by an increase in cAMP through the activation of ₂-adrenoceptors.

The NK₂ receptor mRNA upregulation observed in our study may be due to an increased transcription rate as well as an increased stability of mRNA in response to an increase in cAMP. We found an increase in NK₂ receptor mRNA half-life after fenoterol treatment, which may partly explain the receptor upregulation. -Adrenoceptor agonists have previously been shown to have effects on mRNA stability, as in the case of fibroblast growth factor-2 mRNA in rat cortical astrocytes (31), c-fos mRNA levels in rat brain (32), and m₂ muscarinic acetylcholine receptor mRNA levels in chicken heart cells (33). The last-named effect was blocked by Rp-cAMP, an inhibitor of cAMP-dependent protein kinase, indicating that cAMP mediated this effect. The rate of transcription of newly synthesized NK₂ receptor mRNA as assessed with a nuclear run-on assay was also increased, by 25%, after fenoterol treatment for 6 h as compared with the control value, suggesting that both an increase in NK₂ receptor mRNA stability and an increased rate of transcription of the NK₂ receptor gene are likely to account for the upregulation of NK₂ receptors.

The tachykinin-induced contraction of human bronchus in vitro is mediated solely by NK₂ receptors (6). Selective NK₂ receptor antagonists block hyperpnea-induced bronchoconstriction in guinea pigs (12) and reduce allergen- induced bronchoconstriction in guinea pigs (9, 10). These results suggest that NKA, acting via NK₂ receptors, may play a role in the AHR that is an important feature of asthma. Although we are not aware of any previous studies addressing NK₂ receptor-mediated contraction in bovine tracheal smooth muscle as such, it appears that the sensitivity of the response, with the NK₂ receptor agonist used, is very similar to that in human bronchus (pD₂ = 6.9; E_max  70% of acetylcholine) (5, 34). Therefore, bovine trachea appears to be a reasonable model for human airway smooth muscle in this respect.

₂-Adrenoceptor agonists are the most effective bronchodilators currently available for the treatment of asthma. However, there is still some concern that excessive use of ₂-adrenoceptor agonists in some patients may contribute to asthma morbidity or mortality (19). The regular use of inhaled ₂-adrenoceptor agonists such as fenoterol has been associated with an increased risk of death or near death from asthma (35, 36). If the increase in NK₂ receptor expression and response as observed in the present study also occurs in human airway smooth muscle in vivo, it might contribute to the adverse effects suggested to occur with the regular or sustained use of ₂-adrenoceptor agonists.

In summary, we have shown that ₂-adrenoceptor agonist treatment increases NK₂ receptor mRNA expression and receptor number, as well as airway smooth-muscle contractile responses to an NK₂ receptor agonist, through a decrease in the rate of mRNA degradation and an increase in the rate of gene transcription. The combined use of a steroid and ₂-adrenoceptor agonist not only prevents the ₂-adrenoceptor agonist-induced increase in NK₂ receptor mRNA and in the contractile response, but also exerts a stronger inhibitory effect by reducing the steady-state level of NK₂ receptor mRNA in airway smooth muscle. Future studies will focus on identifying the factors responsible for ₂-adrenoceptor agonist-induced NK₂ receptor gene transcription, and on delineating the mechanism(s) involved in the interaction between steroid and ₂-adrenoceptor agonist. The findings in such studies may have implications for both the pathophysiology and treatment of asthma.