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J Appl Physiol 97: 2154-2159, 2004. First published August 20, 2004; doi:10.1152/japplphysiol.00752.2003
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Involvement of p42/44 MAPK and RhoA protein in augmentation of ACh-induced bronchial smooth muscle contraction by TNF-{alpha} in rats

Hiroyasu Sakai, Shinobu Otogoto, Yoshihiko Chiba, Kazuho Abe, and Miwa Misawa

Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo 142-8501, Japan

Submitted 22 July 2003 ; accepted in final form 23 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Bronchial asthma is characterized by chronic inflammation of airway tissues and nonspecific airway hyperresponsiveness (AHR), but the underlying mechanisms of AHR have yet to be elucidated. Recently, tumor necrosis factor-{alpha} (TNF-{alpha}) has been identified as a proinflammatory cytokine that might be important in the hyperresponsiveness of airway tissue. We have investigated the effects of SB-203580 (a p38 MAPK inhibitor), U-0126 (an inhibitor of p42/44 MAPK activation), and cycloheximide (an inhibitor of protein synthesis) on TNF-{alpha}-augmented ACh-induced bronchial smooth muscle contraction. We have also investigated the phosphorylation of p42/44 MAPK and upregulation of RhoA protein by TNF-{alpha}. Treatment of rat bronchial smooth muscles with TNF-{alpha} (300 and 1,000 ng/ml for 24 h) resulted in a significant upward shift in the concentration-response curve to ACh, but not to high K+, compared with control tissues. The effect of TNF-{alpha} was completely blocked by pretreatment with U-0126 or cycloheximide, but not with SB-203580. Immunoblotting demonstrated that p42/44 MAPK was phosphorylated and RhoA protein was increased in bronchial tissue by TNF-{alpha}. Furthermore, the TNF-{alpha}-induced upregulation of RhoA protein was abolished by U-0126 pretreatment. In conclusion, we suggest that TNF-{alpha} might be one of the important mediators involved in the pathogenesis of augmented bronchial smooth muscle contractility in AHR. For the first time, we have demonstrated that augmentation of ACh-induced contractile response evoked by TNF-{alpha} was mediated by synthesis of protein, such as RhoA, through activation of p42/44, but not p38 MAPK, in rat bronchial smooth muscle.

acetylcholine asthma

AIRWAY HYPERRESPONSIVENESS (AHR) and inflammation leading to increased airway resistance are characteristic features of asthma (6). The importance of AHR in the pathogenesis of asthma has been suggested by its correlation with the severity of this disease (19). Therefore, the underlying mechanisms of AHR should be determined for the therapy of asthma.

The inflammation of asthmatic lungs is characterized by infiltration of airway walls with eosinophils, mast cells, and lymphocytes. Activation of these cells results in the release of a plethora of inflammatory mediators that individually or in concert induce changes in the airway wall geometry and produce the symptoms of the disease. On the other hand, there is increasing evidence that tumor necrosis factor-{alpha} (TNF-{alpha}), one of the proinflammatory cytokines released from these inflammatory cells, is directly linked to the airway inflammation and hyperresponsiveness observed in asthma (27). TNF-{alpha} is elevated in the sputa and bronchoalveolar lavage fluid (BALF) of patients with bronchial asthma (7, 29). In mouse (20) and guinea pig (32) models of lung inflammation, increased levels of TNF-{alpha} have been detected in the BALF of sensitized animals after challenge with antigen. In addition, in vivo pretreatment of rat and human airways with aerosolized TNF-{alpha} produced an enhanced increase in airway resistance similar to that observed in asthma, when the airways were challenged with endogenous agonists (17, 31). Pharmacological evidence has also pointed toward an important role of TNF-{alpha} in AHR. TNF-{alpha} receptor (TNFR) fusion protein, which can potently block endogenous TNF-{alpha}, was effective in reducing the enhanced airway reactivity and inflammatory cell infiltration into the airways in sensitized guinea pigs and Brown Norway rats in vivo (25). Moreover, incubation of airway smooth muscle cells with TNF-{alpha} can increase agonist-stimulated intracellular Ca2+ release (5). These observations suggest that TNF-{alpha} may be one of the primary components responsible for the bronchial smooth muscle hyperresponsiveness observed in asthma. Indeed, TNF-{alpha} has been shown to augment muscarinic receptor agonist-induced airway smooth muscle contractions in humans (28) and mice (2).

We previously demonstrated in vivo and in vitro hyperresponsiveness in rats that were actively sensitized and repeatedly challenged with aerosolized antigen (9, 10). We reported that the muscarinic receptor density of bronchial tissues in an AHR rat model with repeated antigen challenge was within the normal range (10). Moreover, no significant difference in the ACh-induced increase in cytosolic Ca2+ concentration of the main bronchial smooth muscle was observed between control and AHR rats (12). These findings strongly suggest that the mechanisms responsible for the augmented ACh-induced contraction of the main bronchial smooth muscle might exist in postmuscarinic receptor signaling, including an augmented Ca2+ sensitization. In the previous study, the level of RhoA, an important protein that mediates Ca2+ sensitization (24) and ACh-induced Ca2+ sensitization, was significantly increased in a bronchial preparation of repeatedly antigen-challenged rats compared with control rats. The Ca2+ sensitization was abolished by pretreatment with the Rho inhibitor, C3 toxin (13). It is therefore possible that increased RhoA seems to enhance the Ca2+-sensitizing signal, resulting in augmentation of the ACh-induced contractile response in a rat model of AHR.

Mitogen-activated protein kinases (MAPKs), a family of serine/threonine kinases, consist of at least three distinct members: extracellular signal-regulated kinase (also called p42/44 MAPK), p38 MAPK, and c-Jun NH2-terminal kinase (15). MAPKs regulate a variety of cellular responses, including inflammation, cell cycle progression, proliferation, differentiation, and protein synthesis (20). Recently, p38 and p42/44 MAPKs were reported to be activated by proinflammatory cytokines such as TNF-{alpha} (14). However, whether MAPKs are involved in the TNF-{alpha}-induced AHR is unclear. The effects of inhibitors of p38 MAPK (MEK) and protein synthesis on TNF-{alpha}-augmented ACh-induced bronchial smooth muscle contraction were therefore investigated. Furthermore, we investigated phosphorylation of p42/44 MAPK and upregulation of RhoA protein by TNF-{alpha}, and the effect of an MEK inhibitor on TNF-{alpha}-induced upregulation of RhoA protein was elucidated by immunoblot analysis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   Male Wistar rats (6 wk of age, specific pathogen-free, 170–190 g; Charles River Japan) were housed for appropriate time intervals in the Animal Center of Hoshi University after their arrival. Constant temperature (22 ± 1°C) and humidity (55 ± 10%) were maintained, with a fixed 12:12-h light-dark cycle and free access to food and water. Experiments were done according to the guiding principles for the care and use of laboratory animals approved by the Animal Care Committee of Hoshi University (Tokyo, Japan).

Functional study.   The animals were killed by exsanguination from the abdominal aorta under anesthesia with chloral hydrate (400 mg/kg ip). An ~4-mm length of the left main bronchus was isolated by a method described elsewhere (9). After treatment with 10 µM U-0126 [a selective inhibitor of p42/44 MAPK (MEK) activation], 30 µM SB-203580 (a selective p38 MAPK inhibitor), 10 µM cycloheximide (an inhibitor of protein synthesis), or 0.1% dimethyl sulfoxide (vehicle) for 1 h, the bronchial strips were incubated with 100, 300, or 1,000 ng/ml TNF-{alpha} or its vehicle in Krebs-Henseleit solution (in mM: 118.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 KH2PO4, and 10.0 glucose, pH 7.4) at room temperature for 24 h. The bronchial rings were mounted on two L-shaped stainless steel prongs in a 5-ml organ bath, which contained modified Krebs-Henseleit solution. The buffer solution was maintained at 37°C and oxygenated with 95% O2-5% CO2. One prong was connected to a force displacement transducer (model TB-612T, Nihon Kohden) for continuous recording of isometric tension. The other prong was connected to a displacement device, which allowed adjustment of the distance between the two parallel prongs. The resting tension was set by a transducer equipped with a manipulator. Briefly, just after the tissue was placed in an organ bath, tension (~1.5 g) was loaded onto the tissue. During an equilibration period in the organ bath, the tissue was washed three to four times at 15- to 20-min intervals and equilibrated gradually to a baseline tension of 1.0 g. In some cases, a further equilibration period was required for tissue stabilization. At 15 min after the last wash, higher concentrations of ACh were successively added after a plateau response to the previous concentration was attained. After measurement of responsiveness to ACh, the bronchial smooth muscle was also depolarized with isotonic high-K+ solution prepared by isosmotic replacement of NaCl by KCl in the presence of 10–6 M atropine and 10–6 M indomethacin.

Immunoblot analysis.   To quantify phosphorylation of p42/44 MAPK, Western blot was performed with homogenates of the main and intrapulmonary bronchi. p42/44 MAPK is activated by growth and neurotrophic factors through phosphorylation of Thr202 and Tyr204 by the immediately upstream MAPK kinase (MEK). Briefly, the samples (10 µg of total protein per lane) were subjected to 12% SDS-PAGE, and the proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. After the membrane was blocked with 3% gelatin, it was incubated with primary antibody [polyclonal rabbit anti-p42/44 MAPK or polyclonal rabbit anti-phospho-p42/44 MAPK (phosphospecific antisera), 1:5,000 or 1:2,500 dilution, respectively; Cell Signaling Technology]. Then the membrane was incubated with horseradish peroxidase-conjugated sheep anti-rabbit IgG (1:2,500 dilution; Sigma) and detected by an enhanced chemiluminescent (ECL) system (Amersham). The relative phosphorylation of p42/44 MAPK was obtained from the ratio of the intensity of phospho- to total p42/44 MAPK labeling on the blot.

In the RhoA, {alpha}-actin, and myosin light chain (MLC) expression study, the samples were subjected to 15% SDS-PAGE. Proteins were then electrophoretically transferred to PVDF membranes. After they were blocked, the membranes were incubated with the primary antibodies. Rabbit anti-RhoA (1:2,000 dilution; Santa Cruz Biotechnology), mouse anti-{alpha}-smooth muscle actin (1: 1,000; Sigma), rabbit anti-MLC (1:1,000; Santa Cruz Biotechnology), and mouse anti-GAPDH (1:3,000 dilution; Chemicon) were used as the primary antibodies. Then the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000 dilution; Amersham) and goat anti-mouse IgG (1:5,000 dilution; Amersham) and detected by an ECL system. The ratio of corresponding RhoA to GAPDH in each lane was calculated as an index of RhoA, {alpha}-actin, and MLC levels. We used GAPDH as an internal control, because this protein is also constitutively expressed in most tissues and is the most widely accepted internal control in the molecular biology literature.

In the study of ACh-induced MLC phosphorylation, the bronchial preparations were incubated with 300 ng/ml TNF-{alpha} or its vehicle in Krebs-Henseleit solution at room temperature for 24 h. The resulting preparations were stimulated by 10–3 M ACh for 10 min. Then the samples were homogenized with tissue protein extraction reagent (T-PER, Pierce). After the samples (20 µg) were subjected to 15% SDS-PAGE, Western blot was performed. The membranes were incubated with the primary antibodies. Goat anti-phospho-MLC (Thr18/Ser19, 1:250 dilution; Santa Cruz Biotechnology) and rabbit anti-MLC (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Then the membranes were incubated with horseradish peroxidase-conjugated donkey anti-goat IgG (1:5,000 dilution; Santa Cruz Biotechnology) and goat anti-rabbit IgG (1:5,000 dilution; Amersham) and detected by an ECL system. The ratio of corresponding phospho-MLC to MLC was calculated as an index of phospho-MLC.

Statistical analyses.   Values are means ± SE. Statistical significance of difference was determined by two-way ANOVA or Bonferroni/Dunn's post test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Functional study.   Figure 1 shows the effects of 24 h of incubation with TNF-{alpha} on bronchial smooth muscle responsiveness. Compared with vehicle-incubated control muscles, the ACh responsiveness in TNF-{alpha}-treated groups was significantly augmented in a TNF-{alpha} concentration-dependent manner (Fig. 1A): the Emax tension (the maximal tension induced by 10–3 M ACh) of the groups treated with 300 and 1,000 ng/ml TNF-{alpha} [1.69 ± 0.09 g (P < 0.01) and 1.76 ± 0.08 g (P < 0.001), respectively] was significantly greater than that of the vehicle-treated group (1.16 ± 0.09 g). The bronchial smooth muscle contraction induced by U-46619, a thromboxane A2 mimic, was also augmented by TNF-{alpha} (300 ng/ml) treatment (data not shown), indicating that the effect of TNF-{alpha} is not specific for muscarinic receptor signaling. On the other hand, no significant difference in the response to isotonic high K+ (10, 30, and 60 mM) was observed among the groups (Fig. 1B).



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  		Fig. 1. ACh (A) and high K+ (B) concentration-response curves for rat bronchial ring after treatment with tumor necrosis factor-{alpha} (TNF-{alpha}; 100, 300, and 1,000 ng/ml) or its vehicle for 24 h. Values are means ± SE from 6 experiments. In TNF-{alpha}-treated group, ACh responsiveness of main bronchial smooth muscles was significantly [P < 0.05 (by ANOVA) for 300 ng/ml and P < 0.01 (by ANOVA) for 1,000 ng/ml] augmented compared with vehicle-treated group. *P < 0.05; **P < 0.01; ***P < 0.001, 1,000 ng/ml TNF-{alpha} vs. vehicle. #P < 0.05; ##P < 0.01, 300 ng/ml TNF-{alpha} vs. vehicle.

 
Figure 2 shows the effect of 30 µM SB-203580, a p38 MAPK inhibitor, on bronchial smooth muscle responsiveness after 24 h of incubation with TNF-{alpha} (300 ng/ml). Compared with the TNF-{alpha}-treated control group, the TNF-{alpha}-augmented ACh responsiveness was not significantly different from that in the SB-203580-treated groups. However, 10 µM U-0126, an inhibitor of p42/44 MAPK activation, significantly inhibited the TNF-{alpha}-augmented ACh responsiveness. Cycloheximide (10 µM), an inhibitor of protein synthesis, also significantly inhibited the TNF-{alpha}-augmented ACh responsiveness.



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  		Fig. 2. Effects of SB-203580, U-0126, and cycloheximide (CHX) on augmentation of ACh-induced contractile response evoked by TNF-{alpha}. Rat bronchial ring preparations preincubated with SB-203580 (30 µM), U-0126 (10 µM), cycloheximide (10 µM), or vehicle for 1 h were treated with TNF-{alpha} (300 ng/ml for 24 h). After 24-h incubation period, bronchial smooth muscle responsiveness to ACh was measured isometrically. Values are means ± SE from 4–8 experiments. Effect of TNF-{alpha} was significantly inhibited by pretreatment with U-0126 (P < 0.05, by ANOVA) and cyclohexamide (P < 0.05, by ANOVA), but not with SB-203580. *P < 0.05; **P < 0.01, U-0126 + TNF-{alpha} vs. vehicle + TNF-{alpha}. #P < 0.05; ##P < 0.01, CHX + TNF-{alpha} vs. vehicle + TNF-{alpha}.

 
Western blot analysis.   Using phosphospecific antisera, we studied whether TNF-{alpha} treatment led to activation of the p42/44 MAPK pathway in the rat bronchial preparation. Activation of the p42/44 MAPK pathway occurred in a time-dependent manner. Phosphorylated p42 and p44 MAPKs were significantly increased by 20 min of treatment with TNF-{alpha} (Fig. 3).



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  		Fig. 3. Phosphorylation of p42/44 MAPK by TNF-{alpha} in rat main and intrapulmonary bronchial preparations. Preparations were stimulated with TNF-{alpha} (300 ng/ml) for 10 and 20 min. A: representative blots of samples subjected to 10% SDS-PAGE and incubated with anti-phospho-p42/44 MAPK (top) and anti-p42/44 MAPK antibodies (bottom). B: relative densities of phosphorylated p42/44 MAPK in TNF-{alpha}-treated or vehicle-treated (control) bronchial preparation. Values are means ± SE from 4–5 different animals. *Significantly different from control, P < 0.05.

 
In the present study, anti-RhoA antibody was used to detect RhoA protein in the TNF-{alpha}- and vehicle-treated bronchial preparations. Representative RhoA and GAPDH immunoblots of the TNF-{alpha}-treated and control preparations are shown in Fig. 4A. Immunoblotting with the antibody against RhoA showed a single 21-kDa band, indicating the existence of RhoA proteins in the rat bronchi. The ratios of corresponding RhoA to GAPDH were calculated and are represented in Fig. 4B. Expression of RhoA protein in the TNF-{alpha}-treated bronchial preparation was significantly increased compared with that in the vehicle control preparation (P < 0.05). Figure 4 also shows the effects of U-0126 on the TNF-{alpha}-induced augmented expression of RhoA protein. U-0126 significantly inhibited the RhoA expression induced by TNF-{alpha} (Fig. 4B). No significant difference in the expression of actin and MLC was observed between groups (Fig. 5).



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  		Fig. 4. Effect of U-0126 on TNF-{alpha}-induced upregulation of RhoA protein. A: typical immunoblots of RhoA and GAPDH in control, vehicle + TNF-{alpha} (300 ng/ml for 24 h), and U-0126 (10 mM) + TNF-{alpha}-treated bronchial preparations. B: relative density of RhoA to GAPDH (RhoA/GAPDH) in bronchial preparation. Values are means ± SE from 4–6 different animals. *Significantly different from TNF-{alpha} + vehicle, P < 0.05. #Significantly different from control, P < 0.05.

 


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  		Fig. 5. A: typical immunoblots of myosin light chain (MLC), {alpha}-actin, and GAPDH in TNF-{alpha}-treated (300 ng/ml for 24 h) or vehicle-treated bronchial smooth muscle. B: relative densities of MLC and {alpha}-actin to GAPDH in TNF-{alpha}- and vehicle-treated bronchial smooth muscles. Values are means ± SE from 4 different animals. No significant difference in expression of MLC and {alpha}-actin was observed between groups.

 
Figure 6 shows the effect of TNF-{alpha} pretreatment on the ACh-induced phosphorylation of MLC in rat bronchial preparations. When analyzed by Western blot, MLC phosphorylation was represented by a distinct single band, as in a previous report (32). Treatment with ACh induced a significant increase in MLC phosphorylation in control preparations (no TNF-{alpha}), i.e., ACh-induced MLC phosphorylation. The ACh-induced MLC phosphorylation was significantly augmented by TNF-{alpha} pretreatment. However, no significant difference in the phosphorylation of MLC was observed between TNF-{alpha}- and vehicle-treated groups.



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  		Fig. 6. A: typical immunoblots of ACh (10–3 M)-induced phosphorylation of MLC (p-MLC) and MLC in TNF-{alpha}-treated (300 ng/ml for 24 h) or vehicle-treated bronchial smooth muscle. B: relative density of p-MLC to MLC in TNF-{alpha}-treated and vehicle-treated bronchial smooth muscles. Values are means ± SE from 4 different animals. MLC was significantly phosphorylated by ACh. *Significantly different from control + ACh, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In the present study, treatment of rat bronchial smooth muscle with TNF-{alpha} (300 and 1,000 ng/ml for 24 h) resulted in a significant upward shift in the concentration-response curve to ACh, but not to high K+, compared with control tissues. Thus we used 300 ng/ml TNF-{alpha} in most experiments. The augmented ACh responsiveness to TNF-{alpha} was completely blocked by pretreatment with U-0126 or cycloheximide, but not with SB-203580. To study whether TNF-{alpha} activates p42/44 MAPK in a bronchial preparation, phosphorylated p42/44 MAPK was determined by immunoblotting, and TNF-{alpha} was found to induce p42/44 MAPK phosphorylation in bronchial tissue. TNF-{alpha} significantly increased the expression of RhoA protein in the bronchus. U-0126 inhibited the expression of RhoA enhanced by TNF-{alpha} treatment in the bronchus.

TNF-{alpha} has been implicated in the pathophysiology of proinflammatory disorders, including bronchial asthma. Elevated levels of TNF-{alpha} have been detected in BALF from asthmatic patients (7). Amrani et al. (4) showed that exposure of human airway smooth muscle cells to TNF-{alpha} for 24 h potentiates the increase in cytosolic free Ca2+ induced by contractile agonists such as carbachol and bradykinin. However, in the present study, TNF-{alpha} potentiated the ACh-, but not high K+-, induced contraction of rat bronchial smooth muscle. Accordingly, TNF-{alpha} may increase bronchial smooth muscle contractilities through an agonist receptor such as the muscarinic receptor, but not through a Ca2+ channel, such as the voltage-dependent Ca2+ channel.

We previously demonstrated that the increased expression of RhoA protein by allergic stimulation seems to enhance the Ca2+-sensitizing signal, resulting in augmentation of the ACh-induced contractile response in antigen-challenged AHR rats (13). We have confirmed that TNF-{alpha} treatment augments ACh-induced bronchial smooth muscle contraction; ACh was previously reported to be one of the activators of RhoA in bronchial smooth muscle (11). We have demonstrated that TNF-{alpha} augmented the expression of RhoA protein in a bronchial preparation. Thus TNF-{alpha} produced by allergic stimulation might increase the agonist-induced Ca2+-sensitizing signal via enhanced expression of RhoA protein.

The mechanisms underlying the TNF-{alpha}-induced increase in bronchial smooth muscle contractilities have not been fully elucidated. TNF-{alpha} initiates its pleiotropic action by binding to two types of receptors, designated p55 (TNFR1) and p75 (TNFR2) according to their apparent molecular mass. These receptors are coexpressed on the surface of mast cells (30). Although TNFR1 and TNFR2 were found to be coexpressed on airway smooth muscle cells (3, 5), the majority of TNF-{alpha} effects on airway smooth muscle is mediated by TNFR1 (2). TNFR1 was shown to regulate the TNF-{alpha}-induced expression of adhesion molecules (3). However, the receptor type implicated in the TNF-{alpha}-induced increase in bronchial smooth muscle contractility and in the expression of RhoA protein has not been identified.

A number of studies have demonstrated the involvement of MAPKs in modulating airway smooth muscle cell proliferation. However, it is unclear whether MAPKs participate in the agonist-induced bronchial smooth muscle contractilities augmented by TNF-{alpha}. Although the transcription factor(s) involved in RhoA expression is unknown, it has been suggested that TNF-{alpha} modulates gene transcription via activation of MAPK, i.e., phosphorylation of MAPK (1). It has also been reported that phosphorylation of MAPK occurs immediately after TNF-{alpha} stimulation (21). So, in the present study, the phosphorylation level of MAPK was measured 10–20 min after TNF-{alpha} stimulation to determine whether MAPK is also activated by TNF-{alpha} in rat bronchial smooth muscle. Although Amrani et al. (1) demonstrated that TNF-{alpha} activated p38 MAPK in airway smooth muscle cells, we showed that SB-203580 did not prevent the TNF-{alpha}-induced potentiation of the ACh-induced contraction of rat bronchial smooth muscle. However, we have demonstrated that TNF-{alpha} activates p42/44 MAPK in rat bronchus and that inhibition of p42/44 MAPK activation by U-0126 completely blocked the TNF-{alpha}-induced potentiation of the ACh-induced contraction of rat bronchial smooth muscle. Furthermore, the augmented ACh-induced bronchial smooth muscle contraction by TNF-{alpha} was abolished by cycloheximide, an inhibitor of protein synthesis. These findings suggest that augmentation of the ACh-induced bronchial smooth muscle contraction by TNF-{alpha} may involve the protein synthesis process, including RhoA protein through the p42/44 MAPK pathway.

In the present study, TNF-{alpha} augmented the ACh-induced maximal contraction with an increased expression of RhoA in bronchial smooth muscle, whereas no significant increase was observed in the contractile proteins (Fig. 5). This result might be consistent with that of our previous study in which the maximal contraction induced by ACh was augmented concurrently with an overexpression of RhoA protein in bronchial smooth muscles of antigen-induced AHR rats (13), whereas no change was observed in high-K+ depolarization-induced contraction (26). By using the rat model of AHR, we also demonstrated that the maximal response of ACh-induced Ca2+ sensitization was significantly augmented and completely abolished in the presence of the RhoA inhibitor, C3 exoenzyme (13). Indeed, in the present study, we demonstrated that bronchial smooth muscle contraction induced by U-46619 was also augmented by TNF-{alpha} treatment. ACh-induced phosphorylation of MLC was augmented by pretreatment with TNF-{alpha}. Thus TNF-{alpha} may augment agonist-induced maximum contraction. Moreover, the increased expression of RhoA protein might affect the maximal contraction induced by contractile agonists.

The delayed increase in protein expression induced by TNF-{alpha} may be initiated by the early phosphorylation of MAPK (1, 21, 22). For instance, Chen et al. (8) reported that the TNF-{alpha}-induced increase in monocyte chemoattractant protein type 1 (MCP-1), which was observed ~24 h after TNF-{alpha} treatment, is mediated via the activation of MAPK and transcriptional factors such as AP-1 and NF-{kappa}B in vascular smooth muscle cells. They also demonstrated that TNF-{alpha}-induced phosphorylation of MAPK occurs within 30 min. MAPK phosphorylation and MCP-1 overexpression induced by TNF-{alpha} was abolished by PD-98059, an inhibitor of p42/44 MAPK activator. So, in our present study, MAPK phosphorylation was measured 10 and 20 min after treatment with TNF-{alpha} to identify an initial event for the TNF-{alpha}-induced increase in RhoA expression.

In conclusion, we suggest that TNF-{alpha} might be one of the important mediators involved in the pathogenesis of the augmented bronchial smooth muscle contractility in antigen-induced AHR and that augmentation of the ACh-induced contractile response by TNF-{alpha} is mediated by synthesis of protein(s) such as RhoA through activation of p42/44 MAPK, but not p38 MAPK, in rat bronchial smooth muscle.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported in part by Ministry of Education, Culture, Sports, Science, and Technology of Japan Grant-in-Aid for Scientific Research 13670101.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Yuri Sakai, Mayu Hirahara, and Reiko Maki for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Chiba, Dept. of Pharmacology, School of Pharmacy, Hoshi Univ., 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan (E-mail: chiba{at}hoshi.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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