Involvement of RhoA-mediated Ca²⁺ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice

Male BALB/c mice (6-week old, specific pathogen-free; Charles River Japan, Inc., Kanagawa, Japan) were used. All experiments were approved by the Animal Care Committee at the Hoshi University (Tokyo, Japan). Preparation of a murine model of allergic bronchial asthma, which has in vivo airway hyperresponsiveness (AHR), was performed as described by Kato et al. [20]. In brief, mice were actively sensitized by intraperitoneal injections of 8 μg ovalbumin (OA; Seikagaku Co., Tokyo, Japan) with 2 mg Imject Alum (Pierce Biotechnology, Inc., Rockfold, IL, USA) on day 0 and day 5. The sensitized mice were challenged with aerosolized OA-saline solution (5 mg/ml) for 30 min on days 12, 16 and 20. A control group of mice received the same immunization procedure but inhaled saline aerosol instead of OA challenge. The aerosol was generated with an ultrasonic nebulizer (Nihon Kohden, Tokyo, Japan) and introduced to a Plexiglas chamber box (130 × 200 mm, 100 mm height) in which the mice were placed.

Twenty-four h after the last antigen challenge, the mice were sacrificed by exsanguination from abdominal aorta under urethane (1.6 g/kg, i.p.) anesthesia. Then the airway tissues under the larynx to lungs were immediately removed. About 3 mm length of the left main bronchus (about 0.5 mm diameter) was isolated and epithelium was removed by gently rubbing with keen-edged tweezers [21]. The resultant tissue ring preparation was then suspended in a 5 ml-organ bath by two stainless-steel wires (0.2 mm diameter) passed through the lumen. For all tissues, one end was fixed to the bottom of the organ bath while the other was connected to a force-displacement transducer (TB-612T, Nihon Kohden) for the measurement of isometric force. A resting tension of 0.5 g was applied. The buffer solution 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. The buffer solution was maintained at 37°C and oxygenated with 95% O₂-5% CO₂. The BSM responsiveness to exogenously applied Ca²⁺ in acetylcholine (ACh)-stimulated or high K⁺-depolarized muscle was determined as previously [22]. In brief, after an equilibration period, the organ bath solution was replaced with Ca²⁺-free solution containing 10^-6 M nicardipine with the following composition (mM); NaCl 122.4, KCl 4.7, MgSO₄ 1.2, NaHCO₃ 25.0, KH₂PO₄ 1.2, glucose 10.0 and EGTA 0.05. Fifteen min later, 1 mM ACh was added and, after attainment of a plateau (almost baseline level) response to ACh, a cumulative concentration-response curve for Ca²⁺ (0.1–6.0 mM) was made. A higher concentration of Ca²⁺ was added after the response to the previous concentration reached a plateau. In another series of experiments, bronchial smooth muscles were depolarized with 60 mM K⁺, instead of ACh, in the presence of 10^-6 M atropine and in the absence of nicardipine in the Ca²⁺-free solution. All these functional studies were performed in the presence of 10^-6 M indomethacin. The concentration of indomethacin had no effect both on baseline tension and on the ACh- and high K⁺-induced constrictions of BSMs (data not shown).

To determine the change in Ca²⁺ sensitization of BSM contraction, permeabilized BSMs were prepared as described previously [21] with minor modification. In brief, 24 h after the last antigen challenge, the left main bronchus was isolated as described above and cut into ring strips (about 200 μm width, 500 μm diameter). The epithelium was removed by gently rubbing with keen-edged tweezers. The ring strips were then permeabilized by a 30-min treatment with 83.3 μg/ml α-toxin (Sigma, St. Louis, MO, USA) 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 (FCCP) 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. The apparent binding constant of EGTA for Ca²⁺ was considered to be 10⁶ M^-1 [23]. The permeabilized muscle strip was then 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 α-toxin-permeabilized muscle strips were treated with Clostridium botulinum C3 exoenzyme (10 μg/ml; Calbiochem-Novabiochem Corp., La Jolla, CA) in the presence of 100 μM NAD for 20 min at room temperature.

Protein samples of BSMs were prepared as previously [21]. In breif, 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 [21]. Then the bronchial tissue (containing the main and intrapulmonary bronchi) segments were quickly frozen with liquid nitrogen, and the tissue was crushed to pieces by CryopressTM (CP-100W; Microtec, Co. Ltd., Chiba, Japan: 15 sec × 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 then centrifuged (3,000 g, 4°C for 15 min) and the resultant supernatant was stored at -85°C until use. To determine the level of RhoA protein in BSMs, the samples (10 μg of total protein per lane) were subjected to 15% SDS-PAGE and the proteins were then electrophoretically transferred to a PVDF membrane. After blocking with 3% gelatin, the PVDF membrane was incubated with polyclonal rabbit anti-RhoA antibody (1:3,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Then the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2,500 dilution; Amersham Biosciences, Co., Piscataway, NJ, USA), detected by an enhanced chemiluminescent system (Amersham Biosciences, Co.) and analyzed by a densitometry system. Thereafter, the primary and secondary antibodies were stripped and the membrane was reprobed by using monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:3,000 dilution; Chemicon International, Inc., Temecula, CA, USA) to confirm the same amount of proteins loaded.

The active form of RhoA, GTP-bound RhoA, in BSMs was measured by RhoA pull down assay. In brief, bronchial tissues containing the main and intrapulmonary bronchi were isolated as described above. The isolated bronchial tissues were equilibrated in oxygenated Krebs-Henseleit solution at 37°C for 1 hr. After the equilibration period, the tissues were stimulated by ACh (10^-3 M for 10 min) and were quickly frozen with liquid nitrogen. The tissues were then lysed in lysis buffer with the following composition (mM); HEPES 25.0 (pH 7.5), NaCl 150, IGEPAL CA-630 1%, MgCl₂ 10.0, EDTA 1.0, glycerol 10%, NaF 25.0, sodium orthovanadate 1.0 and peptidase inhibitors. Active RhoA in tissue lysates (200 μg protein) was precipitated with 25 μg GST-tagged Rho binding domain (amino acids residues 7–89 of mouse rhotekin; Upstate, Lake Placid, NY, USA), which was expressed in Escherichia coli and bound to glutathione-agarose beads. The precipitates were washed three times in lysis buffer, and after adding the SDS loading buffer and boiling for 5 min, the bound proteins were resolved in 15% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with anti-RhoA antibody as described above.

Phosphorylated proteins were detected by using the fluorescent Pro-Q-Diamond dye (Molecular Probes, Eugene, OR, USA), which can directly detect phosphate groups attached to tyrosine, serine or threonine residues in gels [24]. In brief, bronchial tissue lysates (50 μg protein) with SDS loading buffer prepared as described above were resolved in 10 – 20% gradient polyacrylamid gels (Atto Co., Tokyo, Japan). Proteins were fluorescently stained by fixing the gels in 50% methanol and 10% acetic acid for 1 h. The gels were washed with deionised water for 20 min, stained with Pro-Q-Diamond for 1.5 h and destained by three washes in 4% acetonitrile in 50 mM sodium acetate, pH 4.0, for 2 h. Gels were scanned with a fluorimager, a Typhoon 9410 laser scanner (Amersham Biosciences, Co.), with excitation at 532 nm and a 580 nm band pass emission filter for Pro-Q-diamond dye detection. Phosphorylated proteins were quantified densitometrically with the ImageQuant software (Amersham Biosciences, Co.). After scanning, the gels were washed with deionised water for 30 min and incubated in 0.7% glycine-0.2% SDS in 0.3% Tris buffer for 15 min. The proteins were then electrophoretically transferred to a PVDF membrane and immunoblottings for myosin phosphatase target subunit 1 (MYPT1; polyclonal goat anti-MYPT1 antibody; 1:1000; Santa Cruz Biotechnology, Inc.), GAPDH and myosin light chain (MLC; polyclonal rabbit anti-MLC2 antibody; 1:3000; Santa Cruz Biotechnology, Inc) were performed as described above.

Under Ca²⁺-free condition (in the presence of 10^-6 M nicardipine and 0.05 mM EGTA), ACh (10^-3 M) generated a transient phasic contraction in all BSM preparations used. The generated tension of BSM from the repeatedly OA-challenged mice (69 ± 12 mg, N = 6) was significantly greater than that from the sensitized control animals (20 ± 12 mg, N = 6; P < 0.05). The concentration of nicardipine used in the present study completely blocked high K⁺ (10–90 mM)-induced BSM contraction in Ca²⁺ (2.5 mM) containing normal Krebs-Henseleit solution (data not shown), indicating that voltage-dependent Ca²⁺ channels were completely blocked in this condition.

The tension returned to baseline level within 5 min after the ACh application, and then the contraction induced by cumulatively administered Ca²⁺ was measured. Figure 1A shows the concentration-response curves to Ca²⁺ of murine BSMs that were preincubated with nicardipine (10^-6 M) and ACh (10^-3 M) under Ca²⁺-free (0.05 mM EGTA) condition. Addition of Ca²⁺ induced a concentration-dependent BSM contraction in both the sensitized control and OA-challenged groups. The contractile response to Ca²⁺ of the ACh-stimulated BSMs from the repeatedly OA-challenged mice was markedly augmented as compared to that from the sensitized control animals. By contrast, no significant difference in the response to Ca²⁺ of BSMs depolarized with 60 mM K⁺ (in the absence of nicardipine and presence of 10^-6 M atropine) was observed between groups (Fig. 1B). Likewise, the ACh (10^-7–10^-3 M) concentration-response curve determined in normal Krebs-Henseleit solution (2.5 mM Ca²⁺) was significantly shifted upward in BSMs from the OA-challenged mice as compared with that from the sensitized control animals, whereas no significant difference in the contractile response induced by isotonic high K⁺ (10–90 mM) was observed between groups (data not shown).

The BSM contractility was also determined by using α-toxin-permeabilized BSM preparations. In all BSM preparations treated with a-toxin, application of free Ca²⁺ (pCa = 6.5, 6.3, 6.0, 5.5 and 5.0) induced a concentration-dependent reproducible contractile response, indicating successful permeabilization. In the α-toxin-permeabilized BSM, no significant difference in the Ca²⁺ responsiveness or the maximal contractile response induced by pCa 5.0 (Emax) was observed between the sensitized control (pEC₅₀[Ca²⁺ (M)] = 5.67 ± 0.04, Emax = 26.7 ± 1.2 mg; N = 6) and repeatedly OA-challenged (pEC₅₀[Ca²⁺ (M)] = 5.78 ± 0.15, Emax = 22.8 ± 5.9 mg; N = 6) groups. In both groups, when the Ca²⁺ concentration was clamped at pCa 6.0, application of ACh (10^-5–10^-3 M) in the presence of GTP (10^-4 M) caused a further contraction, i.e., ACh-induced Ca²⁺ sensitization, in an ACh concentration-dependent manner (Fig. 2). The ACh-induced Ca²⁺ sensitization was significantly greater in the repeatedly OA-challenged group (Fig. 2B).

To determine an involvement of RhoA protein in the ACh-induced Ca²⁺ sensitization, the effect of pretreatment with C3 exoenzyme on the contractile response of the α-toxin-permeabilized BMS was also investigated. The C3 treatment alone had no significant effect on the Ca²⁺ responsiveness of α-toxin-permeabilized BSMs in any groups (data not shown). However, the ACh (10^-3 M, in the presence of 10^-4 M GTP)-induced Ca²⁺ sensitizing effect was inhibited by treatment with C3 in both the sensitized control and OA-challenged groups (Fig. 3). Interestingly, the remaining C3-insensitive component of the ACh-induced Ca²⁺ sensitization was the same level between groups, whereas the Ca²⁺ sensitization before treatment with C3 was significantly greater in BSMs of the OA-challenged mice (Fig. 3B). These findings indicate that the C3-sensitive Ca²⁺ sensitization, probably mediated by RhoA [25, 26], might be augmented in BSMs of the OA-challenged AHR mice.

The expression of RhoA protein in BSM homogenates was assessed by using immunoblotting. As shown in Fig. 4A, immunoblotting with the antibody against RhoA gave a single 21 kD band, indicating the expression of RhoA protein in murine BSM. The level of RhoA protein in samples of the OA-challenged mice was significantly increased as compared with that of the sensitized control animals. Moreover, the GTP-bound active form of RhoA in ACh-stimulated BSMs was markedly increased in the OA-challenged mice (Fig. 5).

Figure 6 shows the levels of total and phosphorylated MLCs in BSMs determined by immunoblotting and Pro-Q Diamond dye staining, respectively. Immunoblotting with the antibody against MLC protein revealed a single 20 kD band, which contains both phosphorylated and non-phosphorylated MLC proteins (total MLC). The levels of total MLC were the same between groups (Fig. 6, middle panel). In the Pro-Q Diamond dye-stained gels, there were several positive bands, i.e., phosphorylated proteins [24], in the ACh-stimulated BSM samples. Among them, a 20 kD band corresponding to MLC was distinctly found (Fig. 6, bottom panel). The ACh-induced phosphorylation of MLC in BSMs of OA-challenged mice was markedly augmented as compared with those of control animals. A Pro-Q Diamond dye-positive 140 kD band probably corresponding to MYPT1, i.e., phosphorylated MYPT1, was also found in the ACh-stimulated BSM samples and was increased in the OA-challenged group (data not shown).