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Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo, Japan
Received June 28, 2004; accepted April 5, 2005
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Abstract |
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), which is regulated not only by the Ca2+/calmodulin-dependent MLC kinase-mediated pathway but also by a Ca2+-independent mechanism (Ca2+ sensitization) (Somlyo and Somlyo, 1994). Multiple second messengers/signaling pathways, including the RhoA/ROCK (Rho-associated coiled-coil–forming protein kinase) (Kimura et al., 1996; Kureishi et al., 1997; Uehata et al., 1997) and protein kinase C (PKC) (Walsh et al., 1994; Jensen et al., 1996) pathways, have reportedly been linked to the Ca2+ sensitization mechanisms. ROCK and PKC have been proposed to mediate the inhibition of myosin light-chain phosphatase (MLCP) in response to various agonists (Somlyo and Somlyo, 2000).
CPI-17 (named PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa), which is activated by PKC and acts on an MLCP-specific target, was isolated from pig aorta smooth muscle extracts (Eto et al., 1995). Expression of CPI-17 is highly restricted to smooth muscle tissues (Woodsome et al., 2001). Phosphorylation of Thr38 in CPI-17 converts it to a potent MLCP inhibitor with an IC50 of 
5 nM (Eto et al., 1995, 1997). Phospho-CPI-17 enhances myosin phosphorylation and contraction of permeabilized arterial smooth muscle (Li et al., 1998). Permeabilization of femoral artery strips using Triton X-100 depletes endogenous CPI-17 with loss of the contractile response to phorbol ester. The PKC-induced contraction of permeabilized artery was reconstituted by addition of recombinant CPI-17 (Kitazawa et al., 1999). Furthermore, the expression pattern of CPI-17 among six different smooth muscle tissues correlates with their extent of PKC-induced contraction, implying that CPI-17 is key to the PKC-mediated Ca2+ sensitization (Woodsome et al., 2001). Assays with purified kinases showed that Thr38 of CPI-17 can be phosphorylated by multiple kinases such as PKC, ROCK, protein kinase N, and Zip-like kinase (Eto et al., 1995; Hamaguchi et al., 2000; Koyama et al., 2000; MacDonald et al., 2001).
Airway hyper-responsiveness (AHR) associated with heightened airway resistance and inflammation is the characteristic feature of asthma (Bousquet, 2000). The importance of AHR in the cause of bronchial asthma was suggested by the correlation with the severity of the illness (Lotvall et al., 1998). Therefore, understanding of the fundamental mechanism of AHR is important to determine medical treatment for asthma.
Our previous studies found both in vivo and in vitro hyper-responsiveness to ACh and other spasmogens in rats that were actively sensitized and repeatedly challenged with aerosolized antigen (Chiba and Misawa, 1993; Misawa and Chiba, 1993, 1995). In this animal model of AHR, the muscarinic receptor density of bronchial tissues was within the normal range (Chiba and Misawa, 1995). Furthermore, no significant difference in the ACh-induced increase in cytosolic Ca2+ concentration of the main bronchial smooth muscle was observed between the control and AHR rats (Chiba et al., 1999a). These findings strongly suggest that the mechanisms responsible for the augmented ACh-induced contraction of the main bronchial smooth muscle might exist in after receptor signaling, including augmented Ca2+ sensitization. Indeed, Ca2+ sensitization in bronchial preparation of rats repeatedly challenged with antigen was significantly enhanced compared with that of control rats (Chiba et al., 1999b). However, the mechanism of augmented Ca2+ sensitization to contractile agonists in bronchial smooth muscle from AHR rats remains to be solved in detail. The aim of the present study was to explore the role of CPI-17 in hyper-responsiveness of bronchial smooth muscle in antigen-induced AHR rats.
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Materials and Methods |
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Sensitization and Antigenic Challenge. Rats were sensitized and repeatedly challenged with 2,4-dinitrophenylated Ascaris suum antigen by a method described previously (Chiba et al., 1999a,b). Our previous and current studies (Fig. 1) revealed that the sensitization procedure alone had no effect on the ACh responsiveness of the bronchial muscle and muscarinic receptors property in rats (Chiba and Misawa, 1995). Therefore, in the present study, age-matched nonsensitized normal rats were used as control.
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) and stored at –85°C until use. cDNAs were prepared from the total RNA (0.5 µg) by using a Takara RNA PCR kit (ver. 2.1; Takara, Tokyo, Japan) in a total volume of 20 µl of reaction buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM MgCl2, 1 mM dNTP mixture, 1 U/ml RNase inhibitor, 2.5 µM concentration of random 9-mers, and 0.25 U/ml avian myeloblastosis virus reverse transcriptase. The reaction mixture was incubated for 10 min at 30°C followed by 60 min at 42°C to initiate the synthesis of the cDNAs. Reverse transcriptase was inactivated at 99°C for 5 min. Then, the RT reaction mixture (10 µl) was added to 0.5 µl of 0.1 mM forward primer, 0.5 µl of 0.1 mM reverse primer, 4 µl of 10x amplification buffer (100 mM Tris-HCl, pH 8.3, and 0.5 M KCl), 3 µl of 25 mM MgCl2, 31.8 µl of H2O, and 0.25 µl of 5 U/ml Taq polymerase. The PCR primers for rat CPI-17 used were 5'-GCGAGTCACCGTCAAATACGAC-3' (sense) and 5'-TCCTCTGTGGGATTCAGGCAAGC-3' (antisense). The PCR primers for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used were 5'-CCATCACTGCCACTCAGAAGAC-3' (sense) and 5'-TACTCCTTGGAGGCCATGTAGG-3' (antisense), which were designed from published sequences (NM_110403 and XM_342145). The thermal cycle profile used in the present study was 1) denaturing for 30 s at 95°C, 2) annealing primers for 30 s at 60°C, and 3) extending the primers for 60 s at 72°C. The PCR amplifications were performed for 25, 30, and 35 cycles for CPI-17 and 20, 25, and 30 cycles for GAPDH. A portion (10 µl) of the PCR mixture was subjected to electrophoresis on 2% agarose gel and visualized by densitometer (Atto Densitograph; Atto Co., Tokyo, Japan). The ratios of the corresponding CPI-17 (30 cycles)/GAPDH (20 cycles) were calculated as indices of CPI-17 mRNA levels.
Protein Extraction. Membrane and cytosolic fractions of bronchial tissue were prepared by a method described previously (Chiba et al., 1999b) with minor modifications. In brief, the airway tissues below the main bronchi were removed and immediately soaked in ice-cold, oxygenated Krebs-Henseleit solution. They were carefully cleaned of adhering connective tissues, blood vessels, and lung parenchyma under stereomicroscopy. Then, the bronchial tissue was equilibrated in oxygenated Krebs-Henseleit solution (37°C) for 60 min with 10-min washout intervals. After the equilibration period, the tissue segments were stimulated by an indicated concentration of ACh (10–5–10–3 M) for 20 min. In some experiments, the bronchial preparations were pretreated with Y-27632 (ROCK inhibitor; 10–6 M) or calphostin C (PKC inhibitor; 10–6 M) to determine the role(s) of ROCK and/or PKC on the ACh-induced phosphorylation and translocation of CPI-17 and MLC phosphorylation. The concentration of Y-27632 (10–6 M) and calphostin C (10–6 M) used had no effect on Ca2+-induced contraction of bronchial smooth muscle (Chiba et al., 2001; Sakai et al., 2005). Thus, 10–6 M concentrations of Y-27632 and calphostin C were used in the present study. The reaction was stopped by quickly freezing with liquid nitrogen, and the tissue was then homogenized in 1 ml of ice-cold homogenization buffer with the following composition: 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 µg/ml leupeptin, and 20 µg/ml aprotinin. The homogenate was used to quantify the expression of CPI-17. The tissue homogenate was centrifuged (105,000g, 4°C for 30 min), and the supernatant was collected as the cytosolic fraction. The pellet was resuspended in 3 ml of homogenization buffer and recentrifuged (105,000g, 4°C for 30 min). The resultant pellet was resuspended in 2 ml of ice-cold homogenization buffer and used as the membrane fraction. These preparations were stored at –80°C until use.
Western Blot Analyses. To quantify the expression, translocation and phosphorylation of CPI-17 proteins, immunoblotting was performed as described previously (Chiba et al., 1999b). In brief, the samples (10 µg of protein per lane) were subjected to 15% SDS-PAGE. Proteins were then electrophoretically transferred for 4 h onto PVDF membranes (Hybond-ECL; Amersham Biosciences, Little Chalfont, UK) in ice-cold transfer buffer (20% methanol containing 25 mM Tris and 192 mM glycine). After repeated washing with Tris buffer (20 mM Tris and 500 mM NaCl, pH 7.5) containing 0.1% (v/v) Tween 20 (TTBS), the PVDF membranes were incubated with blocking buffer (3% gelatin in TTBS) for 1.5 h at room temperature. The PVDF membranes were then incubated with primary antibody, polyclonal goat anti-CPI-17 (1:1500 dilution; Santa Cruz Biotechnology) or polyclonal goat anti-Thr38 phospho-CPI-17 (1:5000 dilution; Santa Cruz Biotechnology) in antibody buffer (1% gelatin in TTBS) for 12 h at room temperature. The PVDF membranes were then washed five times (each for 15 min) with TTBS. They were incubated with horseradish peroxidase-conjugated anti-goat IgG (Amersham) for 1.5 h at room temperature, and then washed five times with TTBS. The blots were detected with an enhanced chemiluminescent method (ECL System; Amersham) and quantified by densitometry (Atto Densitograph Software ver. 4.0). To normalize the CPI-17 contents to an internal control protein, 
-actin, immunoblotting was also performed on the same gel by using monoclonal mouse anti--actin N-terminal (Sigma-Aldrich, St. Louis, MO) and goat anti-mouse IgG (Amersham Biosciences). The ratios of corresponding phosphorylated CPI-17/-actin and CPI-17/-actin in each lane were calculated as indices of phosphorylated and total CPI-17 protein levels. The membrane/total CPI-17 in each animal sample was calculated according to the formula (membrane CPI-17/-actin)/[(membrane CPI-17/-actin) + (cytosolic CPI-17/-actin)]. In the ACh-induced MLC phosphorylation study, The bronchial preparation were stimulated by ACh 10–3 M for 10 min. Then, the samples were homogenized with T-PER tissue protein extraction reagent (Pierce, Rockford, IL). After the samples (20 µg) were subjected to 15% SDS-polyacrylamide gel electrophoresis, Western blot was performed. The membranes were incubated with the primary antibodies. The primary antibodies used were goat anti-p-MLC (Thr18/Ser19; 1:250 dilution; Santa Cruz Biotechnology, Inc.) or rabbit anti-myosin light chain (1:1000; Santa Cruz Biotechnology, Inc.). Then, the membranes were incubated with horseradish peroxidase-conjugated donkey anti-goat IgG (1:5000 dilution; Santa Cruz Biotechnology, Inc.) and goat anti-rabbit IgG (1:5000 dilution; Amersham Bisociences), detected by an ECL system. The ratio of corresponding p-MLC/MLC was calculated as an index of p-MLC.
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Results |
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To determine the ACh (10–5–10–3 M)-induced phosphorylation of CPI-17 in bronchial smooth muscle of the rat, immunoblottings were performed by using phospho-[Thr38]-specific antibody. The ACh-induced phosphorylation of CPI-17 was increased in a concentration-dependent manner in both groups (Fig. 3). It is noteworthy that the ACh-induced CPI-17 phosphorylation at Thr38 was significantly augmented in bronchus from rats repeatedly challenged with antigen.
As shown in Fig. 4A, CPI-17 protein was expressed in both the membrane and cytosolic fractions of bronchial smooth muscles at resting state (no ACh stimulation). No significant difference in the ratio of membrane to total CPI-17 at resting state was observed between the control (0.216 ± 0.048) and repeated antigen challenge (0.172 ± 0.029) groups. The CPI-17 contents in the membrane fractions were significantly increased by ACh (10–5–10–3 M) stimulation in a concentration-dependent manner, although the ratio of cytosolic to total CPI-17 was significantly decreased in each group (data not shown) (i.e., ACh-induced translocation of CPI-17 to plasma membrane). As shown in Fig. 4B, the ACh-induced translocation of CPI-17 was significantly augmented in repeated antigen challenge group compared with the control group.
MLC phosphorylation was represented a distinct single band. Treatment of ACh (10–3 M) induced a significant increase in MLC phosphorylation between groups; that is, ACh induced MLC phosphorylation. The ACh-induced MLC phosphorylation was significantly augmented in the repeated antigen challenge group. However, no significant difference in the phosphorylation of MLC of basement was observed between groups (Fig. 5).
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Discussion |
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). Furthermore, isolated smooth muscle of the bronchus from the AHR rat had a hyper-responsiveness (Misawa and Chiba, 1993; Chiba and Misawa, 1995). The augmented contractile responsiveness of bronchi to ACh obtained from AHR rats in the present study was concordant with our previous results. It has been demonstrated, with the use of permeabilized bronchial smooth muscles, that ACh-induced Ca2+ sensitization was inhibited by C3 exoenzyme in normal bronchial smooth muscle (Chiba et al., 1999b). Moreover, the contraction of permeabilized muscle, but not the increase in [Ca2+]i, induced by ACh was much enhanced in bronchial smooth muscle of the airway hyper-responsive rats (Chiba et al., 1999b). Thus, the increased smooth muscle contractility is proposed to be related to augmented agonist-induced Ca2+ sensitization of myofilaments in AHR rats. At least two pathways might be responsible for the increased Ca2+ sensitization: the RhoA/ROCK and PKC (Hori and Karaki, 1998). Our previous studies demonstrated that enhancement of the ACh-induced Ca2+ sensitization in hyper-responsive muscle was effectively inhibited by C3 exoenzyme, a RhoA inhibitor, in -escin–permeabilized rat bronchial smooth muscle. The augmented contraction of the intact (nonpermeabilized) bronchial smooth muscle in response to ACh was also inhibited by Y-27632, a ROCK inhibitor (Chiba et al., 2001). The increased smooth muscle contractility was therefore suggested to be related to augmented, agonist-induced, RhoA-mediated Ca2+ sensitization of myofilaments. It is possible that the increased expression of RhoA in the bronchial smooth muscle causes an enhancement of RhoA-mediated Ca2+ sensitization, resulting in the augmented contraction at the AHR state. On the other hand, CPI-17, a phosphorylation-dependent inhibitory protein of myosin phosphatase, has been suggested to be the downstream effector of PKC (Kitazawa et al., 1999; Woodsome et al., 2001), because PKC phosphorylates and activates CPI-17. In the present study, we observed that the levels of CPI-17 mRNA and protein were significantly increased in bronchial smooth muscle from AHR rats. In addition, the ACh-induced phosphorylation of CPI-17 was significantly increased in bronchial smooth muscle from AHR rats. It is interesting that the ACh-induced phosphorylation and translocation of CPI-17 was inhibited by the pretreatment of Y-27632 or calphostin C in bronchial tissue of rats repeatedly challenged with antigen. Moreover, pretreament with Y-27632 or calphostin C also inhibited the ACh-induced phosphorylation of MLC. The inhibitory effects of Y-27632 and PKC inhibitor on agonist-induced phosphorylation of CPI-17 has also been demonstrated in rabbit femoral arterial smooth muscle (Kitazawa et al., 2000). We therefore propose here that Ca2+ sensitization mediated not only by RhoA/ROCK and PKC/CPI-17 but also by cross-talk of RhoA/ROCK and CPI-17 pathways may play important roles in the ACh-induced bronchial smooth muscle contraction and phosphorylation of MLC. In the state of antigen-induced AHR, these Ca2+ sensitization pathways may be activated much more intensely than in the non-AHR state.
We here investigated the ACh-induced membrane associated CPI-17. The translocation of CPI-17 was observed by ACh-stimulation in rat bronchial smooth muscle. In the antigen-induced AHR rats, the translocation of CPI-17 was much more augmented. Taggart et al. (1999) showed that receptor agonist stimulation of uterine smooth muscle cell causes a redistribution of RhoA, ROCK, and PKC-
from the cytosol to the cell periphery. Furthermore, myosin phosphatase (MYPT1) has also been shown to translocate to membrane in vascular smooth muscle cells treated with prostaglandin F2 (Shin et al., 2002). It is thus possible that the phosphorylation and translocation to plasma membrane of CPI-17 have important roles in agonist-induced smooth muscle contraction and Ca2+ sensitization with relevance of RhoA, ROCK, and PKC.
In conclusion, we have for the first time suggested that enhancement of the Ca2+-sensitizing effect mediated by markedly up-regulated expression and increased activity of CPI-17 might contribute to the augmented contractility of airway smooth muscle at the antigen-induced AHR.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: MLC, myosin light chain; PKC, protein kinase C; ROCK, Rho-associated coiled-coil–forming protein kinase; MLCP, myosin light chain phosphatase; CPI-17, PKC-potentiated inhibitory protein for heterotrimeric MLCP phosphatase of 17 kDa; AHR, airway hyper-responsiveness; PCR, polymerase chain reaction; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Y-27632, N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride; TTBS, Tris buffer containing Tween 20; PVDF, polyvinylidene difluoride; ACh, acetylcholine.
Address correspondence to: Hiroyasu Sakai, Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. E-mail: sakai{at}hoshi.ac.jp
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