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Published ahead of print on September 15, 2005, doi:10.1165/rcmb.2005-0177OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 574-581, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0177OC

Effect of Cigarette Smoke Exposure In Vivo on Bronchial Smooth Muscle Contractility In Vitro in Rats

Yoshihiko Chiba, Masahiko Murata, Hiroko Ushikubo, Yuji Yoshikawa, Akiyoshi Saitoh, Hiroyasu Sakai, Junzo Kamei and Miwa Misawa

Department of Pharmacology and Department of Pathophysiology and Therapeutics, School of Pharmacy, Hoshi University, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Yoshihiko Chiba, Ph.D., Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. E-mail: chiba{at}hoshi.ac.jp


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Cigarette smoking is a risk factor for the development of airway hyperresponsiveness and chronic obstructive pulmonary disease. Little is known concerning the effect of cigarette smoking on the contractility of airway smooth muscle. The current study was performed to determine the responsiveness of bronchial smooth muscles isolated from rats that were subacutely exposed to mainstream cigarette smoke in vivo. Male Wistar rats were exposed to diluted mainstream cigarette smoke for 2 h/d every day for 2 wk. Twenty-four hours after the last cigarette smoke exposure, a marked airway inflammation (i.e., increases in numbers of neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid and peribronchial tissues) was observed. In these subacutely cigarette smoke-exposed animals, the responsiveness of isolated intact (nonpermeabilized) bronchial smooth muscle to acetylcholine, but not to high K+-depolarization, was significantly augmented when compared with the air-exposed control group. In {alpha}-toxin–permeabilized bronchial smooth muscle strips, the acetylcholine-induced Ca2+ sensitization of contraction was significantly augmented in rats exposed to cigarette smoke, although the contraction induced by Ca2+ was control level. Immunoblot analyses revealed an increased expression of RhoA protein in the bronchial smooth muscle of rats that were exposed to cigarette smoke. Taken together, these findings suggest that the augmented agonist-induced, RhoA-mediated Ca2+ sensitization may be responsible for the enhanced bronchial smooth muscle contraction induced by cigarette smoking, which has relevance to airway hyperresponsiveness in patients with chronic obstructive pulmonary disease.

Key Words: airway hyperresponsiveness • bronchial smooth muscle • Ca2+ sensitization • chronic obstructive pulmonary disease • cigarette smoking


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD), which is one of the most important causes of morbidity and mortality in the world. Cigarette smoking induces an inflammatory response in the airways that might play a key role in the pathogenesis of COPD. Furthermore, multicenter clinical trials (Lung Health Study) showed that current smokers with functional evidence of early COPD have airway hyperresponsiveness (1, 2). Similarly, a dose-dependent effect of cigarette smoking on airway responsiveness was reported (3). The latter study supports the concept that cigarette smoke has a primary effect on airway responsiveness.

Bronchodilators are the mainstay of current management for patients with COPD. Anticholinergic bronchodilators, such as tiotropium bromide, have become standard care in the control of COPD (4, 5). There is evidence that the cholinergic tone of the airways may be increased in patients with COPD (6, 7). Some of the chemical and oxidizing pollutants generated by cigarette smoking affect airway smooth muscle contractility directly (8, 9). Therefore, it is possible that one of the factors that contribute to the exaggerated airway narrowing in patients with COPD may be an abnormality of the nature of airway smooth muscle. Rapid relief from airway limitation in patients with COPD by bronchodilators may also suggest an involvement of augmented airway smooth muscle contraction in the airway obstruction. Experimental evidence has been reported that chronic exposure to cigarette smoke augments the in vivo responsiveness of airways to cholinergic agonists in rats (10), guinea pigs (11), and mice (12). However, little is known concerning the effect of cigarette smoking in vivo on the contractility of airway smooth muscle in vitro. In the present study, the responsiveness of bronchial smooth muscles isolated from the rats that were subacutely exposed to mainstream cigarette smoke was compared with that from control animals.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals and Cigarette Smoke Exposure
Male Wistar rats (6 wk of age, specific pathogen–free, 170–190 g; Charles River Japan, Kanagawa, Japan) were used. All experiments were approved by the Animal Care Committee at the Hoshi University (Tokyo, Japan).

Rats were randomly divided into two groups to be exposed to mainstream cigarette smoke (CS group) or room air (control group). In the CS group, animals were exposed to diluted mainstream cigarette smoke for 2 h/d everyday for 2 wk by using an automated smoking machine (Model INH06-CIGR01; Medical Interface Project Station, Osaka, Japan). Each awake rat was held in an exposure chamber that was connected to the smoking machine. A puff of mainstream cigarette smoke (35 ml) generated from hi-lite cigarettes (a total of 1.4 mg nicotine and 17 mg tar/cigarette) (Japan Tobacco, Tokyo, Japan) was diluted with 280 ml of room air and delivered to the chamber. Each cigarette was puffed 40 times with suction volume of 600 ml/min.

Determination of Inflammatory Cells in Bronchoalveolar Lavage Fluids
Twenty-four hours after the last cigarette smoke or room air exposure, bronchoalveolar lavage fluid (BALF) was collected by the method previously described (16–18) with minor modification. The rats were killed by exsanguination from abdominal aorta under chloral hydrate (400 mg/kg, intraperitoneally) anesthesia, the chest of each animal was opened, and a 20-gauge blunt needle was tied into the proximal trachea. BALF was obtained by intratracheal instillation of 1 ml/100 g body weight of PBS (pH 7.5, room temperature) into the lung while it was kept within the thoracic cavity. The lavage was reinfused into the lung twice before final collection. BAL cells were isolated by centrifugation at 500 x g. The resultant pellet was resuspended in 500 µl of 10% formaldehyde and incubated for 10 min. The cells were washed with PBS and resuspended in 500 µl of PBS. An aliquot of BAL cell suspension was used for cell counts with a hemocytometer. A 10-µl aliquot of cell suspension was mounted on silane-coated glass slides and air-dried. Cell types were identified and counted by differential staining microscopy with Diff-Quik (Baxter Healthcare, Miami, FL) under 400x magnification. Eosinophils were defined as cells that had positive staining (red ~ pink granules) with polymorphonuclei. Inflammatory cell populations were determined by randomly counting 100 cells in duplicate and multiplying the total cell counts per milliliter of BALF by the percentages of each cell type.

Histology
Airways below the main bronchi were fixed in 10% formaldehyde and embedded in Paraplast Plus paraffin. Sections (4 µm) were obtained from blocks and mounted on silane-coated glass slides, deparaffinized with xylene and graded ethanol, and processed for hematoxylin and eosin staining.

Functional Study for Intact Bronchial Smooth Muscles
To determine whether the cigarette smoke exposure in vivo affects the bronchial smooth muscle responsiveness in vitro, the isometric contraction of the circular smooth muscle of the main bronchus was measured as described previously (19, 20). In brief, 24 h after the last cigarette smoke or room air exposure, the rats were killed by exsanguinations from the abdominal aorta under chloral hydrate anesthesia (400 mg/kg, intraperitoneally). The airway tissues below the larynx to lungs were immediately removed. A 4-mm length (3 mm diameter) of the left main bronchus was isolated (8–9 cartilages), and the resultant tissue ring preparation was suspended in an organ bath at a resting tension of 1 g. The organ bath contained modified Krebs-Henseleit solution with the following composition (mM): NaCl 118.0, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25.0, KH2PO4 1.2, and glucose 10.0 (pH 7.4). The isometric contraction of the circular smooth muscle was measured with a force-displacement transducer (TB-612T; Nihon Kohden, Tokyo, Japan). During an equilibration period, the tissues were washed three or four times at 15- to 20-min intervals and equilibrated slowly to a baseline tension of 1 g. After the equilibration period, the concentration-response curve to acetylcholine (ACh) (10–7–10–3 M in final concentration) was constructed cumulatively. A higher concentration of ACh was successively added after attainment of a plateau response to the previous concentration. In another series of experiments, isotonic K+ solution (10–90 mM in final concentration) was cumulatively administered in the presence of atropine and indomethacin (both 10–6 M) to determine the bronchial smooth muscle responsiveness to high K+ depolarization.

Functional Study for {alpha}-Toxin–Permeabilized Bronchial Smooth Muscles
To determine the change in ACh-induced Ca2+ sensitization of the bronchial smooth muscle contraction in the CS group, the {alpha}-toxin–permeabilized smooth muscles of intrapulmonary bronchi were prepared as described previously (13, 15) with minor modification. Briefly, 24 h after the last cigarette smoke or room air exposure, the third branch of intrapulmonary bronchi was isolated, carefully cleaned of lung parenchyma and adhering connective tissue, and cut into ring strips (~ 0.2 mm width, 0.5 mm diameter). The epithelium was removed by gently rubbing with keen-edged tweezers (13, 15). The ring strips were then permeabilized by a 30-min treatment with 83.3 µg/ml {alpha}-toxin (Sigma, St. Louis, MO) in the presence of Ca2+ ionophore A23187 (10 µM; Sigma) at room temperature in relaxing solution. Relaxing solution contained 20 mM Pipes, 7.1 mM Mg2+-dimethanesulfonate, 108 mM K+-methanesulfonate, 2 mM EGTA, 5.875 mM Na2ATP, 2 mM creatine phosphate, 4 U/mL creatine phosphokinase, 1 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and 1 µg/ml E-64 (pH 6.8) containing 10 µM A23187. Free Ca2+ concentration was changed by adding an appropriate amount of CaCl2 (13). The permeabilized muscle strip was suspended in a 400-µl organ bath at room temperature. The contractile force developed was measured by an isometric transducer (T7–8-240; Orientec, Tokyo, Japan) under a resting tension of 50 mg. To determine the involvement of RhoA in the ACh-induced myofilament Ca2+ sensitization, the {alpha}-toxin–permeabilized muscle strips were treated with Clostridium botulinum C3 exoenzyme (1 µg/ml; Calbiochem-Novabiochem, La Jolla, CA) in the presence of 100 µM NAD for 20 min at room temperature.

Western Blot Analyses
Protein samples of bronchial tissues were prepared as previously described (13, 15). The airway tissues below the main bronchi to lungs were removed and immediately soaked in ice-cold, oxygenated Krebs-Henseleit solution. The airways were carefully cleaned of adhering connective tissues, blood vessels, and lung parenchyma under a stereomicroscopy. The epithelium was removed as much as possible by gently rubbing with keen-edged tweezers (13, 15). The bronchial tissue (containing the main and intrapulmonary bronchi) segments were quickly frozen with liquid nitrogen, and the tissue was crushed to pieces by Cryopress (CP-100W; Microtec, Co. Ltd., Chiba, Japan) (15 s x 3). The tissue powder was homogenized in ice-cold tris(hydroxymethyl) aminomethane (Tris) (10 mM; pH 7.5) buffer containing 5 mM MgCl2, 2 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 1% Triton X-100, and 1% sodium cholate. The tissue homogenate was centrifuged (3,000 x g at 4°C for 15 min), and the resultant supernatant was stored at –85°C until use.

To determine the level of RhoA protein in bronchial smooth muscles, the samples (10 µg of total protein per lane) were subjected to 15% SDS-PAGE, and the proteins were electrophoretically transferred to a poly(vinylidene fluoride) membrane. After blocking with 3% gelatin, the poly(vinylidene fluoride) membrane was incubated with polyclonal rabbit anti-RhoA antibody (1:3,000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2,500 dilution) (Amersham Biosciences, Piscataway, NJ), detected by an enhanced chemiluminescent system (Amersham Biosciences), and analyzed by a densitometry system. Detection of house-keeping gene was performed on the same membrane by using monoclonal mouse anti–{beta}-actin antibody (1:3,000 dilution) (Santa Cruz Biotechnology) to confirm the same amount of proteins loaded. The levels of each protein were expressed as percentage of control. The mean value of the control group was set at 100%.

Statistical Analyses
Data were expressed as the mean with SE. Statistical significance was determined by unpaired Student's t test or two-way ANOVA with post hoc Bonferroni/Dunn (StatView for Macintosh ver. 5.0; SAS Insitute, Cary, NC). A value of P < 0.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Airway Inflammation after the 2-wk Cigarette Smoke Exposure
To determine the changes in airway biology after subacute exposure to mainstream cigarette smoke, differential cell count in BALF and histologic examinations were performed. Total cell counts in the BALF of rats in the CS group were significantly increased as compared with those of the control group (Figure 1). When the differential cell count was performed under Diff-Quik staining, BALF of rats in the CS group had significantly increased numbers of neutrophils (0.57 ± 0.21 x 106/ml of BALF; n = 5, P < 0.05) and lymphocytes (0.42 ± 0.08 x 106/ml of BALF; n = 5, P < 0.01) as compared with the control group (0.00 ± 0.00 and 0.05 ± 0.03 x 106/ml of BALF, respectively; n = 4). A marked increase in macrophages was found, although no statistical significance was observed (Figure 1). Histologic examinations revealed a marked inflammation of the lungs (Figure 2) and main bronchi (Figure 3) in the CS group. A remarkable infiltration of inflammatory cells into the subepithelial and smooth muscle layers was found. Unlike the BAL cell components, many neutrophils were found under higher magnification (1,000x) with oil-immersion (data not shown). Although detachment of epithelium was not observed, an epithelial hyperplasia was found, especially in the intrapulmonary bronchi (Figures 2C and 2D).



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  		Figure 1. Effect of repeated exposure to mainstream cigarette smoke (CS) on cell counts in bronchoalveolar lavage fluid (BALF) in rats. Differential cell count was performed under Diff-Quik staining to identify eosinophils (Eos), neutrophils (Neu), lymphocytes (Lym), and macrophages (Mac). Each column represents the mean ± SEM from four (Control) and five (CS) experiments. The numbers of neutrophils and lymphocytes in the BALF were significantly increased after the 2-wk exposure to cigarette smoke. *P < 0.05 and **P < 0.01 versus control by unpaired Student's t test.

 





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  		Figure 2. Histologic change of lungs after the 2-wk cigarette smoke exposure in rats. Sections (4 µm) of formalin-fixed tissues of control (A and B) and CS animals (C and D) were stained with hematoxylin and eosin before examination by light microscopy. Original magnification: A and C, x100; B and D, x400. Many inflammatory cells were infiltrated into the periphery of intrapulmonary bronchi in the CS group (C and D).

 





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  		Figure 3. Histologic change of the left main bronchus after the 2-wk cigarette smoke exposure in rats. Sections (4 µm) of formalin-fixed tissues of control (A and B) and CS animals (C and D) were stained with hematoxylin and eosin before examination by light microscopy. Original magnification: A and C, x100; B and D, x400. Inflammatory cells were infiltrated into the smooth muscle layer in the CS rats (C and D).

 
Bronchial Smooth Muscle Hyperresponsiveness after the2-wk Cigarette Smoke Exposure
To determine the effect of subacute exposure to mainstream cigarette smoke on bronchial smooth muscle contractility, the smooth muscle responsiveness to stimulation of plasma membrane receptors (e.g., muscarinic cholinoceptors) and nonreceptor-mediated stimulation (e.g., isotonic high K+-depolarization) was determined. ACh stimulation caused concentration-dependent contractile responses in the control and CS groups (Figure 4, left panel). The contraction induced by ACh was inhibited by atropine (10–6 M; data not shown) completely and by 4-diphenylacetoxy N-methylpiperidine, an M3 receptor antagonist, with high affinity (21), indicating an involvement of M3 subtype of receptors in the ACh-induced contraction of rat bronchial smooth muscle. The ACh-induced contraction of bronchial smooth muscles isolated from rats in the CS group was significantly augmented as compared with that from the control animals (P < 0.05 by two-way ANOVA with post hoc Bonferroni/Dunn) (Figure 4, left panel). Although no change in the pD2 value (–logarithm of 50% effective ACh concentration [M], calculated by individual concentration-response curve) was observed between groups (5.05 ± 0.06 in the control and 4.92 ± 0.08 in the CS group), the maximal contraction was significantly increased in the CS group (1.63 ± 0.09 g) when compared with the control group (1.20 ± 0.08 g; P < 0.01). No significant difference in the contractile response induced by high K+-depolarization was observed between groups (Figure 4, right panel).



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  		Figure 4. Effect of repeated exposure to mainstream cigarette smoke (CS) on bronchial smooth muscle responsiveness to acetylcholine (ACh) (left panel) and isotonic high K+ depolarization (right panel) in rats. Intact smooth muscle preparations (~ 4 mm length, 3 mm diameter) were isolated from the left main bronchi of the 2-wk CS (closed circles) or control animals (open circles). Each point represents the mean ± SEM from 15 animals. The bronchial smooth muscle responsiveness to ACh (left panel), but not to high K+-depolarization (right panel), was significantly augmented in the CS group (*P < 0.05 by two-way ANOVA with post hoc Bonferroni/Dunn).

 
We determined the effects of acute (1 d) and chronic (4 wk) exposure to mainstream cigarette smoke on bronchial smooth muscle responsiveness to ACh. Acute exposure had no effect on ACh responsiveness. Moreover, further augmentation of ACh responsiveness in the chronically (4 wk) exposed animals was not observed when compared with the subacutely (2 wk) exposed group (data not shown).

Augmented ACh-Induced Ca2+ Sensitization of Bronchial Smooth Muscle Contraction after the 2-wk Cigarette Smoke Exposure
The results obtained by the intact smooth muscle study described previously suggest that intracellular signaling pathway(s) mediated by an activation of heterotrimeric G-protein–coupled receptor(s) might be augmented in bronchial smooth muscle of rats in the CS group. Recent studies have demonstrated an augmented RhoA/Rho-kinase–mediated Ca2+ sensitization associated with the increased contractility of airway smooth muscle in animal models of allergic bronchial asthma (13–15). These findings remind us of the augmented agonist-induced Ca2+ sensitization of bronchial smooth muscle contraction in rats in the CS group. To test this hypothesis, the permeabilized bronchial smooth muscle study was conducted. In all muscle strips treated with 83.3 µg/ml {alpha}-toxin for 30 min, the application of free Ca2+ (pCa = 6.5, 6.3, 6.0, 5.5, and 5.0) induced a concentration-dependent contractile response (Figure 5, lower panel), indicating successful permeabilization. In the {alpha}-toxin–permeabilized intrapulmonary bronchial smooth muscles, no significant difference in the responsiveness of Ca2+ was observed between groups (Figure 5, lower panel). When the Ca2+ concentration was clamped at pCa = 6.3, the application of ACh in the presence of 50 µM GTP caused a further contraction (i.e., ACh-induced Ca2+ sensitization) in the control and CS groups (Figure 5, upper panel). The ACh-induced Ca2+ sensitization was blocked by treatment with a muscarinic receptor antagonist atropine (10–6 M, data not shown) and a RhoA inactivator C3 exoenzyme (1 µg/ml for 20 min) (Figure 6). The ACh-induced Ca2+ sensitization of bronchial smooth muscle contraction was significantly augmented in the CS group when compared with the control group (P < 0.01 by two-way ANOVA with post hoc Bonferroni/Dunn) (Figure 5, upper panel).



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  		Figure 5. Effect of repeated exposure to mainstream cigarette smoke (CS) on acetylcholine (ACh)-induced Ca2+ sensitization of bronchial smooth muscle contraction in rats. {alpha}-Toxin–permeabilized smooth muscles of intrapulmonary bronchi (~ 0.2-mm width, < 0.5-mm diameter) were prepared from the 2-wk CS (closed circles) or control animals (open circles). The ACh-induced Ca2+ sensitization (upper panel) was measured in the presence of pCa (–log concentration of Ca2+) 6.3 and 50 µM GTP. The contraction mediated by Ca2+ was measured in the same muscle strips (lower panel). Each point represents the mean ± SEM from six animals. The ACh-induced Ca2+ sensitization of bronchial smooth muscle contraction was significantly augmented in the CS group (upper panel; **P < 0.01 by two-way ANOVA with post hoc Bonferroni/Dunn), whereas the Ca2+-mediated contraction was the control level (lower panel).

 


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  		Figure 6. Effect of a selective RhoA inactivator, Clostridium botulinum C3 exoenzyme, on acetylcholine (ACh)-induced Ca2+ sensitization of the {alpha}-toxin–permeabilized muscle strip obtained from a CS rat. After the permeabilization, Ca2+-induced contractile response in the absence and presence of ACh and GTP were observed as indicated (before C3). The strip was incubated with C3 exoenzyme (see MATERIALS AND METHODS), and the contractile response was re-estimated (after C3). Traces are representative of three animals. The ACh-induced Ca2+ sensitization of contraction was blocked by treatment with C3 exoenzyme.

 
Changes in the Expression Levels of Contractile Proteins after the 2-wk Cigarette Smoke Exposure
In animal models of allergic asthma, it has been suggested that the augmented agonist-induced Ca2+ sensitization of airway smooth muscle contraction might be mediated by an increased expression of RhoA protein (13–15). In the present study, the effect of cigarette smoke exposure on the expression level of RhoA protein in bronchial smooth muscle was investigated. The RhoA expression in bronchial smooth muscle was significantly increased after the 2-wk cigarette smoke exposure (Figure 7). No significant change in the contents of CPI-17 (protein kinase C-potentiated protein phosphatase-1 inhibitory protein of 17 kD), an alternative protein that is reportedly involved in the agonist-induced Ca2+ sensitization (22, 23), or myosin light chain was observed between groups (Figures 8 and 9).



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  		Figure 7. Effect of repeated exposure to mainstream cigarette smoke (CS) on the expression of RhoA protein in rat bronchial smooth muscle. Upper panel shows the typical immunoblots of {beta}-actin (45 kD) and RhoA (21 kD) in the control (left) or CS animals (right). The relative densities of the RhoA band are summarized in the lower panel. Each column represents the mean ± SEM from five animals. The levels of each protein were expressed as percentage of control: the mean value of the five control animals was set at 100%. The RhoA expression in bronchial smooth muscles of the 2-wk CS rats was significantly increased as compared with the control group. *P< 0.05 versus control by unpaired Student's t test.

 


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  		Figure 8. Effect of repeated exposure to mainstream cigarette smoke (CS) on the expression of CPI-17, an inhibitor of smooth muscle myosin light chain phosphatase, in rat bronchial smooth muscle. Upper panel shows the typical immunoblots of {beta}-actin (45 kD) and CPI-17 (17 kD) in the control (left) or CS animals (right). The relative densities of the CPI-17 band are summarized in the lower panel. Each column represents the mean ± SEM from five animals. The levels of each protein were expressed as percentage of control; the mean value of the five control animals was set at 100%. No significant difference in the CPI-17 expression was observed between groups.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This study demonstrated that the 2-wk exposure of mainstream cigarette smoke to rats caused neutrophilic airway inflammation, which is commonly observed in patients with COPD (24–27). The bronchial smooth muscle responsiveness to ACh, but not to high K+-depolarization, was significantly augmented in the CS group when compared with the control group. Moreover, the ACh-induced Ca2+ sensitization of bronchial smooth muscle contraction was significantly augmented in the CS group, although the contraction induced by Ca2+ was control level. This is the first study, to our knowledge, that demonstrates an augmented Ca2+ sensitization of bronchial smooth muscle contraction induced by subacute cigarette smoke exposure.

It has been suggested that cigarette smoking is a risk factor for the development of airway hyperresponsiveness and COPD (1–3). Experimental evidence has demonstrated an augmented in vivo responsiveness of airways after cigarette smoke exposure (10–12). Although many investigators have made an effort to clarify the mechanism(s) of cigarette smoke–induced airway hyperresponsiveness, the underlying mechanism is unclear. In rats, Xu and colleagues (10) found that exposure to mainstream cigarette smoke resulted in an in vivo airway hyperresponsiveness to methacholine. In their study, however, there was no evidence that the hyperresponsiveness was related to changes in elastic recoil pressure of airways, to airway–parenchymal interdependence, or to airway inflammation (10), all of which have been reported to affect airway responsiveness in vivo (28–31). On the other hand, some of the chemical and oxidizing pollutants generated by cigarette smoking affect airway smooth muscle contractility directly (8, 9). We hypothesized that airway hyperresponsiveness induced by cigarette smoke exposure might result from an alteration in airway smooth muscle contractility.

Typically, smooth muscle contraction is mediated mainly by an increase in cytosolic Ca2+ via the activation of plasma membrane Ca2+ channels and/or Ca2+ release from the sarcoplasmic reticulum. The increased cytosolic Ca2+ forms the 4Ca2+-calmodulin-myosin light chain (MLC) kinase complex and activates MLC kinase. The activated MLC kinase phosphorylates the 20 kD MLC, leading to smooth muscle contraction (22, 23). In the present study, no significant change in the contraction of intact (nonpermeabilized) bronchial smooth muscle induced by high K+ depolarization was observed between the control and CS groups (Figure 4, right panel). In the CS group, the Ca2+-mediated contraction of {alpha}-toxin–permeabilized muscle strips was also within the control level (Figure 5, lower panel). Moreover, no change in the expression level of smooth-muscle MLC was demonstrated between groups (Figure 9). Therefore, it is unlikely that the function of voltage-dependent Ca2+ channels on plasma membrane and the contraction mediated by Ca2+ might change after the 2-wk cigarette smoke exposure. Furthermore, not only ACh-induced (Figure 4) but also endothelin-1–induced (data not shown) contraction was augmented in the bronchial smooth muscles of rats in the CS group, indicating that intracellular signal transduction rather than specific plasma membrane receptors might change after the cigarette smoke exposure.



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  		Figure 9. Effect of repeated exposure to mainstream cigarette smoke (CS) on the expression of myosin light chain (MLC) in rat bronchial smooth muscle. Upper panel shows the typical immunoblots of {beta}-actin (45 kD) and MLC (20 kD) in the control (left) or CS animals (right). The relative densities of the MLC band are summarized in the lower panel. Each column represents the mean ± SEM from five animals. The levels of each protein were expressed as percentage of control; the mean value of the five control animals was set at 100%. No significant difference in the MLC expression was observed between groups.

 
Airway smooth muscle remodeling has been suggested as one of the factor that enhances its contractility in allergic bronchial asthma (32, 33). Gosens and colleagues (33) reported that the enhanced contraction was associated with an increase in smooth muscle myosin expression in lungs. It has also been reported that cigarette smoke exposure to tracheal explants directly induces airway remodeling in rats (34, 35). However, the current study showed that the contraction of intact (nonpermeabilized) bronchial smooth muscles induced by high K+-depolarization was not changed in the 2-wk CS group (Figure 4). The subacute cigarette smoke exposure also had no effect on the contraction of permeabilized muscle strips induced by Ca2+ (Figure 5). Moreover, no significant difference in the expression levels of MLC was observed between groups (Figure 9). Thus, severe airway smooth muscle remodeling may not be induced by the 2-wk in vivo exposure in rats.

In addition to the Ca2+-dependent contraction of smooth muscle, the MLC phosphorylation is regulated by MLC phosphatase, Ca2+ independently, and thus further contraction occurs when smooth muscle is stimulated by agonists (termed agonist-induced Ca2+ sensitization) (22, 23). The agonist-induced Ca2+ sensitization of smooth muscle contraction has been demonstrated in various types of smooth muscle, including airways (13, 15, 36). Recent studies have suggested a participation of a monomeric GTP-binding protein, RhoA, and its downstream target, Rho-kinase, in the agonist-induced Ca2+ sensitization (22, 23). An involvement of CPI-17 (17-kD protein kinase C-potentiated inhibitory protein for heterotrimeric MLC phosphatase) has also been suggested in the Ca2+ sensitizing effect (22, 37). There is increasing evidence that augmentation of the Ca2+ sensitization of smooth muscle contraction might be involved in several human diseases, such as hypertension (38–40) and coronary (41–43) and cerebral vasospasms (44, 45). In the respiratory tract, an augmentation of agonist-induced Ca2+ sensitization with increased expression of RhoA and CPI-17 proteins has been reported in bronchial smooth muscle of a rat model of allergic asthma, which has bronchial smooth muscle hyperresponsiveness (13, 46). Similarly, an enhanced RhoA/Rho-kinase–mediated contraction of airway smooth muscle has been demonstrated in guinea pig (14) and murine (15) models of allergic asthma. The cigarette smoke exposure in vivo caused a hyperresponsiveness of the isolated bronchial smooth muscle (Figure 4), an augmented agonist-induced Ca2+ sensitization (Figure 5), and an increased expression of RhoA protein (Figure 7). It seems likely that the augmented agonist-induced, RhoA-mediated Ca2+ sensitization of bronchial smooth muscle contraction is responsible for the airway hyperresponsiveness induced by subacute cigarette smoke exposure.

Although the underlying mechanism(s) responsible for the cigarette smoke–induced hyperresponsiveness and upregulation of RhoA in bronchial smooth muscle is not clear, there is experimental evidence that reactive oxygen species (ROS) might have the ability to induce upregulation of RhoA by enhancing its transcription in nonsmooth muscle cells (47). ROS are produced by inflammatory cells, such as neutrophils (48), which were significantly increased in BALF (Figure 1) and markedly increased in peribronchial tissues (Figures 2 and 3) after the subacute cigarette smoke exposure. Cigarette smoke is also a potent source of ROS (48). On the other hand, cigarette smoke contains potent respiratory irritants, such as acrolein, an unsaturated aliphatic aldehyde. It has been reported that inhalation of acrolein at a concentration of 1 ppm induced airway hyperresponsiveness to ACh in guinea pigs (49). Ben-Jebria and colleagues (50) reported that acrolein inhalation in vivo caused hyperresponsiveness of isolated tracheal smooth muscle to cholinergic agonists but not to high K+ depolarization in ferrets. The latter report may partially support our results.

In addition to their contractile ability, airway smooth muscle cells display proinflammatory activities when stimulated. For instance, human airway smooth muscle cells produce a neutrophil chemotactic factor, IL-8, by stimulation with IL-1{beta}, TNF-{alpha}, and TGF-{beta} (51–53). Recent study also suggests that cigarette smoke may directly cause the release of IL-8 from human airway smooth muscle cells (54). On the other hand, RhoA-mediated production of IL-8 was reported in human colonic epithelial cells (55). Although it is not known whether smooth muscle cells have the same mechanism, the increased expression of RhoA may augment the release of IL-8, resulting in a further neutrophilia and a deterioration of the disease. Further studies are needed on this matter.

In conclusion, subacute exposure to cigarette smoke caused an augmented ACh-induced Ca2+ sensitization of contraction with an upregulation of RhoA in bronchial smooth muscle, resulting in an enhanced contractile response, which might be a major cause of airway hyperresponsiveness in patients with COPD.


   Acknowledgments
 
The authors thank Kimiko Wada, Miyuuji Komatsu, and Ayako Takahashi for their technical assistance.


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2005-0177OC on September 15, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form May 10, 2005

Accepted in final form August 25, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Tashkin DP, Altose MD, Bleecker ER, Connett JE, Kanner RE, Lee WW, Wise R. The lung health study: airway responsiveness to inhaled methacholine in smokers with mild to moderate airflow limitation. The Lung Health Study Research Group. Am Rev Respir Dis 1992;145:301–310.[Medline]
  2. Tashkin DP, Altose MD, Connett JE, Kanner RE, Lee WW, Wise RA. Methacholine reactivity predicts changes in lung function over time in smokers with early chronic obstructive pulmonary disease. The Lung Health Study Research Group. Am J Respir Crit Care Med 1996;153:1802–1811.[Abstract]
  3. Tashkin DP, Simmons MS, Chang P, Liu H, Coulson AH. Effects of smoked substance abuse on nonspecific airway hyperresponsiveness. Am Rev Respir Dis 1993;147:97–103.[Medline]
  4. Barnes PJ. The role of anticholinergics in chronic obstructive pulmonary disease. Am J Med 2004;117:24S–32S.
  5. Gross NJ. Tiotropium bromide. Chest 2004;126:1946–1953.[Abstract/Free Full Text]
  6. Gross NJ, Co E, Skorodin MS. Cholinergic bronchomotor tone in COPD: estimates of its amount in comparison with that in normal subjects. Chest 1989;96:984–987.[Abstract/Free Full Text]
  7. Nisar M, Earis JE, Pearson MG, Calverley PM. Acute bronchodilator trials in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992;146:555–559.[Medline]
  8. Ben-Jebria A, Marthan R, Rossetti M, Savineau JP, Ultman JS. Effect of in vitro exposure to acrolein on carbachol responses in rat trachealis muscle. Respir Physiol 1993;93:111–123.[CrossRef][Medline]
  9. Marthan R, Roux E, Savineau JP. Human bronchial smooth muscle responsiveness after in vitro exposure to oxidizing pollutants. Cell Biol Toxicol 1996;12:245–249.[CrossRef][Medline]
  10. Xu LJ, Dandurand RJ, Lei M, Eidelman DH. Airway hyperresponsiveness in cigarette smoke-exposed rats. Lung 1993;171:95–107.[CrossRef][Medline]
  11. Wu ZX, Lee LY. Airway hyperresponsiveness induced by chronic exposure to cigarette smoke in guinea pigs: role of tachykinins. J Appl Physiol 1999;87:1621–1628.[Abstract/Free Full Text]
  12. Barrett EG, Wilder JA, March TH, Espindola T, Bice DE. Cigarette smoke-induced airway hyperresponsiveness is not dependent on elevated immunoglobulin and eosinophilic inflammation in a mouse model of allergic airway disease. Am J Respir Crit Care Med 2002;165:1410–1418.[Abstract/Free Full Text]
  13. Chiba Y, Takada Y, Miyamoto S, MitsuiSaito M, Karaki H, Misawa M. Augmented acetylcholine-induced, Rho-mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats. Br J Pharmacol 1999;127:597–600.[CrossRef][Medline]
  14. Schaafsma D, Gosens R, Bos IS, Meurs H, Zaagsma J, Nelemans SA. Allergic sensitization enhances the contribution of Rho-kinase to airway smooth muscle contraction. Br J Pharmacol 2004;143:477–484.[CrossRef][Medline]
  15. Chiba Y, Ueno A, Shinozaki K, Takeyama H, Nakazawa S, Sakai H, Misawa M. Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice. Respir Res 2005;6:4.[CrossRef][Medline]
  16. Chiba Y, Arimoto T, Yoshikawa T, Misawa M. Elevated nitric oxide synthase activity concurrent with antigen-induced airway hyperresponsiveness in rats. Exp Lung Res 2000;26:535–549.[CrossRef][Medline]
  17. Chiba Y, Kusakabe T, Kimura S. Decreased expression of uteroglobin-related protein 1 in inflamed mouse airways is mediated by IL-9. Am J Physiol Lung Cell Mol Physiol 2004;287:L1193–L1198.[Abstract/Free Full Text]
  18. Chiba Y, Srisodsai A, Supavilai P, Kimura S. Interleukin-5 reduces the expression of uteroglobin-related protein (UGRP) 1 gene in allergic airway inflammation. Immunol Lett 2005;97:123–129.[CrossRef][Medline]
  19. Chiba Y, Misawa M. Inhibition of neutral endopeptidase increases airway responsiveness to ACh in nonsensitized normal rats. J Appl Physiol 1995;78:394–402.[Abstract/Free Full Text]
  20. Chiba Y, Sakai H, Misawa M. Possible involvement of Gi3 protein in augmented contraction of bronchial smooth muscle from antigen-induced airway hyperresponsive rats. Biochem Pharmacol 2001;61:921– 924.[CrossRef][Medline]
  21. Chiba Y, Sakai H, Misawa M. Characterization of muscarinic receptors in rat bronchial smooth muscle in vitro. Res Commun Mol Pathol Pharmacol 1998;102:205–208.[Medline]
  22. Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 2003;83:1325–1358.[Abstract/Free Full Text]
  23. Chiba Y, Misawa M. The role of RhoA-mediated Ca2+ sensitization of bronchial smooth muscle contraction in airway hyperresponsiveness. J Smooth Muscle Res 2004;40:155–167.[CrossRef][Medline]
  24. Saetta M. Airway inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;160:S17–S20.[Abstract/Free Full Text]
  25. Maestrelli P, Saetta M, Mapp CE, Fabbri LM. Remodeling in response to infection and injury: airway inflammation and hypersecretion of mucus in smoking subjects with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001;164:S76–S80.[Abstract/Free Full Text]
  26. Qiu Y, Zhu J, Bandi V, Atmar RL, Hattotuwa K, Guntupalli KK, Jeffery PK. Biopsy neutrophilia, neutrophil chemokine and receptor gene expression in severe exacerbations of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;168:968–975.[Abstract/Free Full Text]
  27. O'Donnell RA, Peebles C, Ward JA, Daraker A, Angco G, Broberg P, Pierrou S, Lund J, Holgate ST, Davies DE, et al. Relationship between peripheral airway dysfunction, airway obstruction, and neutrophilic inflammation in COPD. Thorax 2004;59:837–842.[Abstract/Free Full Text]
  28. Boucher RC, Johnson J, Inoue S, Hulbert W, Hogg JC. The effect of cigarette smoke on the permeability of guinea pig airways. Lab Invest 1980;43:94–100.[Medline]
  29. Hulbert WM, McLean T, Hogg JC. The effect of acute airway inflammation on bronchial reactivity in guinea pigs. Am Rev Respir Dis 1985;132:7–11.[Medline]
  30. James AL, Dirks P, Ohtaka H, Schellenberg RR, Hogg JC. Airway responsiveness to intravenous and inhaled acetylcholine in the guinea pig after cigarette smoke exposure. Am Rev Respir Dis 1987;136:1158–1162.[Medline]
  31. Bellofiore S, Eidelman DH, Macklem PT, Martin JG. Effects of elastase-induced emphysema on airway responsiveness to methacholine in rats. J Appl Physiol 1989;66:606–612.[Abstract/Free Full Text]
  32. Fernandes DJ, Mitchell RW, Lakser O, Dowell M, Stewart AG, Solway J. Do inflammatory mediators influence the contribution of airway smooth muscle contraction to airway hyperresponsiveness in asthma? J Appl Physiol 2003;95:844–853.[Abstract/Free Full Text]
  33. Gosens R, Bos IS, Zaagsma J, Meurs H. Protective effects of tiotropium bromide in the progression of airway smooth muscle remodeling. Am J Respir Crit Care Med 2005;171:1096–1102.[Abstract/Free Full Text]
  34. Wang RD, Tai H, Xie C, Wang X, Wright JL, Churg A. Cigarette smoke produces airway wall remodeling in rat tracheal explants. Am J Respir Crit Care Med 2003;168:1232–1236.[Abstract/Free Full Text]
  35. Wang RD, Wright JL, Churg A. Transforming growth factor-{beta}1 drives airway remodeling in cigarette smoke–exposed tracheal explants. Am J Respir Cell Mol Biol 2005;33:387–393.[Abstract/Free Full Text]
  36. Ozaki H, Kwon SC, Tajimi M, Karaki H. Changes in cytosolic Ca2+ and contraction induced by various stimulants and relaxants in canine tracheal smooth muscle. Pflugers Arch 1990;416:351–359.[CrossRef][Medline]
  37. Woodsome TP, Eto M, Everett A, Brautigan DL, Kitazawa T. Expression of CPI-17 and myosin phosphatase correlates with Ca2+ sensitivity of protein kinase C-induced contraction in rabbit smooth muscle. J Physiol 2001;535:553–564.[Abstract/Free Full Text]
  38. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 1997;389:990–994.[CrossRef][Medline]
  39. Mukai Y, Shimokawa H, Matoba T, Kandabashi T, Satoh S, Hiroki J, Kaibuchi K, Takeshita A. Involvement of Rho-kinase in hypertensive vascular disease: a novel therapeutic target in hypertension. FASEB J 2001;15:1062–1064.[Free Full Text]
  40. Seko T, Ito M, Kureishi Y, Okamoto R, Moriki N, Onishi K, Isaka N, Hartshorne DJ, Nakano T. Activation of RhoA and inhibition of myosin phosphatase as important components in hypertension in vascular smooth muscle. Circ Res 2003;92:411–418.[Abstract/Free Full Text]
  41. Shimokawa H, Seto M, Katsumata N, Amano M, Kozai T, Yamawaki T, Kuwata K, Kandabashi T, Egashira K, Ikegaki I, et al. Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm. Cardiovasc Res 1999;43:1029–1039.[Abstract/Free Full Text]
  42. Kandabashi T, Shimokawa H, Miyata K, Kunihiro I, Kawano Y, Fukata Y, Higo T, Egashira K, Takahashi S, Kaibuchi K, et al. Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1beta. Circulation 2000;101:1319–1323.[Abstract/Free Full Text]
  43. Hiroki J, Shimokawa H, Higashi M, Morikawa K, Kandabashi T, Kawamura N, Kubota T, Ichiki T, Amano M, Kaibuchi K, et al. Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells. J Mol Cell Cardiol 2004;37:537–546.[CrossRef][Medline]
  44. Sato M, Tani E, Fujikawa H, Kaibuchi K. Involvement of Rho-kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm. Circ Res 2000;87:195–200.[Abstract/Free Full Text]
  45. Chrissobolis S, Sobey CG. Evidence that Rho-kinase activity contributes to cerebral vascular tone in vivo and is enhanced during chronic hypertension: comparison with protein kinase C. Circ Res 2001;88:774–779.[Abstract/Free Full Text]
  46. Sakai H, Chiba Y, Hirano T, Misawa M. Possible involvement of CPI-17 in augmented bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats. Mol Pharmacol 2005;68:145–151.[Abstract/Free Full Text]
  47. Turcotte S, Desrosiers RR, Beliveau R. HIF-1{alpha} mRNA and protein upregulation involves Rho GTPase expression during hypoxia in renal cell carcinoma. J Cell Sci 2003;116:2247–2260.[Abstract/Free Full Text]
  48. Barnes PJ. Mediators of chronic obstructive pulmonary disease. Pharmacol Rev 2004;56:515–548.[Abstract/Free Full Text]
  49. Leikauf GD, Leming LM, O'Donnell JR, Doupnik CA. Bronchial responsiveness and inflammation in guinea pigs exposed to acrolein. J Appl Physiol 1989;66:171–178.[Abstract/Free Full Text]
  50. Ben-Jebria A, Crozet Y, Eskew ML, Rudeen BL, Ultman JS. Acrolein-induced smooth muscle hyperresponsiveness and eicosanoid release in excised ferret tracheae. Toxicol Appl Pharmacol 1995;135:35–44.[CrossRef][Medline]
  51. John M, Au BT, Jose PJ, Lim S, Saunders M, Barnes PJ, Mitchell JA, Belvisi MG, Chung KF. Expression and release of interleukin-8 by human airway smooth muscle cells: inhibition by Th-2 cytokines and corticosteroids. Am J Respir Cell Mol Biol 1998;18:84–90.[Abstract/Free Full Text]
  52. Fong CY, Pang L, Holland E, Knox AJ. TGF-{beta}1 stimulates IL-8 release, COX-2 expression, and PGE2 release in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2000;279:L201–L207.[Abstract/Free Full Text]
  53. Chung KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J 2000;15:961–968.[Abstract]
  54. Oltmanns U, Chung KF, Walters M, John M, Mitchell JA. Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle. Respir Res 2005;6:74.[CrossRef][Medline]
  55. Zhao D, Kuhnt-Moore S, Zeng H, Wu JS, Moyer MP, Pothoulakis C. Neurotensin stimulates IL-8 expression in human colonic epithelial cells through Rho GTPase-mediated NF-kappa B pathways. Am J Physiol Cell Physiol 2003;284:C1397–C1404.[Abstract/Free Full Text]



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