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.

While nonspecific airway hyperresponsiveness (AHR) is a central feature of allergic bronchial asthma, the mechanism underlying the developmont of AHR is not clearly understood. We have previously demonstrated in vitro hyperresponsiveness of bronchial smooth muscle to acetylcholine (ACh) in rats that were actively sensitized and repeatedly challenged with aerosolized antigen. It has also been demonstrated that the AChinduced, RhoA-mediated Ca 2+ sensitization is markedly augmented concomitantly with an increased expression and activation of RhoA protein in the bronchial smooth muscle of the antigen-treated rats. In the present study, we have investigated whether TNF-α, a proinflammatory cytokine which is involved in bronchial asthma, causes upregulation of RhoA mRNA and protein in the rat bronchus. Treatment of rat bronchial smooth muscle preparations with TNF-α (300 ng/ml for 24 hr) significantly shifted the concentrationresponse curve to ACh upwards, but did not alter the response to high K + , when compared to that of control tissues. Levels of RhoA mRNA and protein in the TNF-α-treated bronchus were significantly greater than those in the control group. In conclusion, it is suggested that the augmentation of the ACh-induced contractile response evoked by TNF-α might be mediated by an upregulation of RhoA in rat bronchial smooth muscle.