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ABSTRACT: Increasing evidence suggests that protein kinase C (PKC) is involved in the Ca(2+) sensitization of various smooth muscle contractions. However, the exact role of PKC on bronchial smooth muscle (BSM) contraction is still unclear. In the present study, to determine the role of PKC activation in the BSM contraction, the effects of phorbol 12,13-dibutyrate (PDBu) on BSM tone were examined in the absence and presence of contractile stimulation. Although PDBu had no effect on the basal tone, the contraction induced by acetylcholine, high K(+) depolarization or Ca(2+) ionophore A23187 was significantly augmented by PDBu. Western blot analyses also revealed that the increase in the level of phosphorylated myosin light chain (MLC) induced by high K(+) depolarization was significantly augmented by PDBu treatment. Interestingly, neither high K(+) depolarization alone nor PDBu alone caused CPI-17 phosphorylation, but a significant phosphorylation of CPI-17 was observed when BSMs were co-stimulated by high K(+) and PDBu. Thus, a certain level of intracellular Ca(2+) might be needed both for an activation of CPI-17 and an induction of contraction induced by PDBu in mouse BSMs.
Respiratory Physiology & Neurobiology 09/2010; 173(2):120-4. · 2.24 Impact Factor
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ABSTRACT: Statins have been proposed as a novel treatment of respiratory diseases. To determine the beneficial effects of statins on allergic bronchial asthma, the effect of systemic treatment with lovastatin on antigen-induced airway inflammation was investigated.
Male BALB/c mice were used.
Mice were sensitized and repeatedly challenged with ovalbumin (OA) antigen to induce asthmatic response. Animals were also treated with lovastatin (4 mg/kg/day, i.p.) once a day prior to and during the antigen inhalation period.
Inflammatory cell counts and levels of interleukin (IL)-4, IL-13, eotaxin, thymus and activation-regulated chemokine and leukotriene B(4) (LTB(4)) in bronchoalveolar lavage (BAL) fluids were measured.
Significant increases in eosinophils and levels of the T helper 2 cytokines, chemokines and LTB(4) in BAL fluids in association with the increments of total and OA-specific immunoglobulin E (IgE) in sera were observed in the repeatedly antigen-challenged mice. The airway eosinophilia was ameliorated by lovastatin, whereas it had no significant effect on the levels of these inflammatory mediators or IgE.
Lovastatin may be beneficial for the treatment of allergic inflammatory diseases in the airways, such as allergic bronchial asthma.
Agents and Actions 06/2009; 58(7):363-9. · 1.59 Impact Factor
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ABSTRACT: To determine a change in airway smooth muscle contractility in a murine model of allergic asthma, the responsiveness of airway smooth muscles isolated from ovalbumin (OA)-sensitized and -challenged mice was compared with that from control animals.
Actively sensitized mice were repeatedly challenged by ovalbumin (OA) antigen inhalation. Twenty-four h after the last antigen challenge, tracheal and bronchial smooth muscle responsiveness to acetylcholine (ACh) and endothelin-1 (ET-1) were measured. Airway microvascular leakage and histochemistry were also determined as indices of airway inflammation.
Both the ACh and ET-1 responsiveness of bronchial, but not tracheal, smooth muscles were significantly augmented in OA-challenged mice, whereas no significant change in the expression levels of M2, M3 and ETB receptors was observed. The Evans blue dye extravasation in the main bronchial, but not tracheal, tissue of OA-challenged mice was significantly increased as compared with that of sensitized control animals. A marked inflammatory cells infiltration was also observed in bronchial but not tracheal tissues of OA-challenged mice.
Repeated antigen challenge to sensitized mice caused a hyperresponsiveness of bronchial, but not tracheal, smooth muscle accompanied with bronchial tissue inflammation.
Inflammation Research 12/2004; 53(11):636-42. · 2.11 Impact Factor
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ABSTRACT: The present study compared the effects of repeated antigen exposure on the development of hyperresponsiveness and the expression of RhoA in the main bronchial and lower tracheal smooth muscles of sensitized
Actively sensitized rats were repeatedly challenged by antigen inhalation. Twenty-four hours after the final antigen challenge the isometrical contractions of the bronchial and tracheal smooth muscles were measured. Immunoblottings were also performed using bronchial and tracheal homogenates and the density ratios of RhoA/beta-actin were calculated to quantify the levels of RhoA.
Acetylcholine-induced contraction of bronchial, but not tracheal, smooth muscle of antigen-treated rats was significantly augmented as compared with that of control rats, indicating that airway hyperresponsiveness appeared by antigen challenge in bronchial smooth muscle. RhoA expression in bronchial, but not tracheal, smooth muscle was significantly increased in the antigen-treated animals.
The increased expression of RhoA is suggested to have an important role in developing hyperresponsiveness of bronchial smooth muscle.
Inflammation Research 12/2001; 50(11):577-80. · 2.11 Impact Factor
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ABSTRACT: In the present study, the effects of a selective Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor, Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] on acetylcholine-induced contraction and Ca(2+) sensitization of rat bronchial smooth muscle were examined. Intact and beta-escin-permeabilized muscles of the third branch of intrapulmonary bronchi were used. In intact muscles, Y-27632 (10(-6)-10(-4) M) concentration-dependently inhibited acetylcholine-induced contractile responses. In acetylcholine (10(-3) M)-precontracted intact muscles, the maximal relaxation (about 50% inhibition of contraction) was obtained by a concentration of 10(-4) M Y-27632, which had no effect on the resting tone. In beta-escin-permeabilized muscles, addition of acetylcholine (10(-5)-10(-3) M) plus GTP (100 microM) induced a further contraction, i.e., Ca(2+) sensitization at a constant Ca(2+) concentration of pCa=6.0. The acetylcholine-induced Ca(2+) sensitization was completely blocked in the presence of 10(-4) M Y-27632, whereas the Ca(2+)-induced contraction itself was not affected by Y-27632. Immunoblot study revealed the expression of ROCK-I and ROCK-II proteins in the intrapulmonary bronchi of rats. These findings suggest that Y-27632 dilates acetylcholine-mediated contraction of rat bronchial smooth muscle by inhibiting RhoA/ROCK-mediated Ca(2+) sensitization.
European Journal of Pharmacology 10/2001; 427(1):77-82. · 2.52 Impact Factor
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ABSTRACT: Acetylcholine (ACh)-induced translocation of RhoA in bronchial smooth muscle of repeatedly antigen-challenged rats that have a marked airway hyperresponsiveness (AHR) was examined. ACh induced time- and concentration-dependent translocation of RhoA to the plasma membrane, indicating an activation of RhoA in bronchial smooth muscle. The level of ACh-induced RhoA translocation was further increased markedly in the AHR group as compared to that in the control group. It is suggested that the augmented activation of RhoA observed in the hyperresponsive bronchial smooth muscle might be responsible for the enhanced ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction associated with AHR.
British Journal of Pharmacology 08/2001; 133(6):886-90. · 4.41 Impact Factor
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ABSTRACT: To obtain information on the activation pathway of the monomeric G protein, RhoA, in bronchial smooth muscle, the expression of G alpha12 and G alpha13 in bronchial smooth muscle of the rat was determined. The levels of these G proteins were also compared between antigen-induced airway hyperresponsive and normal control groups.
Actively sensitized rats were repeatedly challenged by antigen inhalation. Twenty-four hours after the final antigen challenge, membrane preparations of bronchial smooth muscles were prepared. Immunoblottings were performed, and the density ratios of G alpha12/beta-actin and G alpha13/beta-actin were calculated to quantify the levels of these G-protein alpha subunits.
Both G alpha12 and G alpha13 proteins were expressed in rat bronchial smooth muscle. The levels of bronchial G alpha12 and G alpha13 proteins in the repeatedly antigen challenged rats were significantly increased as compared with those in control animals; the magnitude of upregulation in the airway-hyperresponsive group was 89% and 68% in the control group, respectively.
G alpha12 and G alpha13 proteins were expressed in rat bronchial smooth muscle. Considering the probable involvement of G12 and G13 proteins in Ca2+ sensitization through Rho protein, the augmented expression of such G proteins after repeated antigen challenge may be responsible for the hyperresponsiveness of bronchial smooth muscle contraction in rats.
Inflammation Research 07/2001; 50(6):333-6. · 2.11 Impact Factor
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ABSTRACT: To investigate a possible involvement of pertussis toxin (PTX)-sensitive heterotrimeric G proteins in the pathogenesis of airway hyperresponsiveness, the effect of PTX treatment on the augmented contractile response to acetylcholine (ACh) in bronchial smooth muscle of antigen-induced airway hyperresponsive rats was determined. In bronchial smooth muscle of airway hyperresponsive rats that were actively sensitized and repeatedly challenged with 2,4-dinitrophenylated Ascaris suum antigen, ACh-induced contractions were markedly augmented. The augmented contractile responses in the airway hyperresponsive group were significantly inhibited after treatment with PTX (1 microg/mL for 6 hr, 37 degrees ), whereas only a slight attenuation was observed in the normal control group. The level of G(alpha)i3 (measured by immunoblotting), but not other alpha-subunits of G(i/o) family proteins, in bronchial smooth muscle of the airway hyperresponsive rats was significantly increased as compared with that of control animals. It is concluded that PTX-sensitive muscarinic contractile responses of bronchial smooth muscle might be augmented upon antigen-induced airway hyperresponsiveness in rats, probably due to an up-regulation of G(alpha)i3 protein of bronchial smooth muscle.
Biochemical Pharmacology 05/2001; 61(7):921-4. · 4.70 Impact Factor
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ABSTRACT: Smooth muscle responsiveness of intrapulmonary small bronchi obtained from repeatedly antigen-challenged rats was compared with that from control animals to determine whether smooth muscle contractility of peripheral airways is augmented by such repeated challenge. In intact (non-permeabilized) smooth muscles of intrapulmonary bronchi, the acetylcholine (ACh)-induced contractile response was significantly augmented in the repeated challenge group, although 60-mM K+-induced contraction was within the normal level. In beta-escin-permeabilized muscles, no significant difference between groups was observed in the Ca2+-induced contractile responses. Thus, augmented ACh-induced contraction of intact intrapulmonary small bronchial smooth muscle might be, at least in part, due to an enhanced ACh-mediated Ca2+-sensitizing signal.
The Japanese Journal of Pharmacology 11/2000; 84(2):221-4.
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ABSTRACT: Nonspecific airway hyperresponsiveness (AHR) is a common feature of allergic bronchial asthmatics, but the underlying mechanism (s) of AHR have yet to be elucidated. The importance of AHR in the pathogenesis of asthma has been suggested by its relevance to the severity of this disease. There is thus a need to understand the underlying mechanisms of AHR for the sake of asthma therapy. In the present minireview, we discussed the involvement of the augmented agonist-induced Ca2+ sensitization of airway smooth muscle contraction in the pathogenesis of AHR. Treatment with acetylcholine (ACh) of a beta-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced a stronger contractile force even when the Ca2+ concentration was clamped at 1 microM. The ACh-induced Ca2+ sensitization of myofilaments was found to be significantly greater in antigen-induced airway hyperresponsive rats than in control rats. The ACh-induced Ca2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of the Rho family proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was demonstrated to be significantly increased in the airway hyperresponsive rats. Thus, the increased airway smooth muscle contractility observed in asthmatics may be related to the augmented agonist-induced, Rho-mediated Ca2+ sensitization of myofilaments.
Folia Pharmacologica Japonica 10/1999; 114(3):185-90.
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ABSTRACT: Treatment with acetylcholine (ACh) of a beta-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced force when the Ca2+ concentration was clamped at 1 microM. The ACh-induced Ca2+ sensitization of myofilaments was significantly greater in antigen-induced airway hyperresponsive rats than in control rats. The ACh-induced Ca2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of Rho family of proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was significantly increased in the airway hyperresponsive rats. Thus, increased airway smooth muscle contractility observed in asthmatics may be related to augmented agonist-induced, Rho-mediated Ca2+ sensitization of myofilaments.
British Journal of Pharmacology 07/1999; 127(3):597-600. · 4.41 Impact Factor
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ABSTRACT: To investigate the alteration in acetylcholine (ACh)-induced increase in Ca2+ sensitization of bronchial smooth muscle contraction concurrent with the airway hyperresponsiveness (AHR), the ACh-induced increases in cytosolic Ca2+ ([Ca2+]) level and contractile response were simultaneously determined by using Fura-2 loaded bronchial smooth muscle. The left main bronchi were isolated from AHR rats which were sensitized and repeatedly challenged with DNP-Ascaris antigen. The tissue ring preparations were incubated in loading solution containing 10 microM Fura-2AM for 3 hr at room temperature. Then the isometrical contraction and [Ca2+]i (F340/F380) were monitored. Although the ACh (10(-3) M)-induced contractile response in AHR group (322 +/- 60 % of 60 mM K+ induced contraction) was significantly greater than that in control animals (173 +/- 15 %, p<0.05), the ACh (10(-3) M)-induced increase in [Ca2+]i was without significant difference between the two groups (128 +/- 15 and 171 +/- 29% of 60 mM K+ -induced increase in [Ca2+]i, respectively). These findings suggest that an augmentation of ACh-induced Ca2+ sensitization may occur in bronchial smooth muscle of the rats with antigen-induced AHR.
Research communications in molecular pathology and pharmacology 01/1999; 106(1-2):77-85.
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ABSTRACT: This study was undertaken to assess the important muscarinic receptor subtype in acetylcholine (ACh)-induced rat bronchial smooth muscle contraction. Ring smooth muscle strips of the left main bronchus were used. Isometrical contraction was measured in response to ACh in cumulative concentrations (10(-7)-10(-3) M) with and without preincubations with the muscarinic receptor antagonists, pirenzepine (an M1 antagonist), methoctramine (an M2 antagonist), and 4-diphenylacetoxy N-methylpiperidine (4-DAMP; an M1/M3 antagonist). Preincubation with these antagonists resulted in concentration-dependent rightward shifts of the concentration-response curves to ACh. pA2 values (means+/-sem) were 8.80+/-0.10 for 4-DAMP, 7.03+/-0.06 for pirenzepine and 5.91+/-0.36 for methoctramine, indicating that the most important muscarinic receptor mediating ACh-induced contraction of rat bronchial smooth muscle is of the M3 type.
Research communications in molecular pathology and pharmacology 12/1998; 102(2):205-8.
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ABSTRACT: Nonspecific airway hyperresponsiveness (AHR) is a common feature of allergic bronchial asthmatics, but the underlying mechanisms of AHR have yet to be elucidated. The importance of AHR in the pathogenesis of asthma has been suggested by its relevance to the severity of this disease. There is thus a need to understand the underlying mechanisms of AHR for the sake of asthma therapy. In allergic asthmatics, airway smooth muscles (ASMs) obtained from in vivo hyperresponsive patients have in vitro hyperresponsiveness to cholinergic agonists. It is therefore possible that the mechanisms responsible for the AHR exist, at least in part, on the ASM site. Although ASM is known to contract in response to acetylcholine via muscarinic M3 receptors and this contractile response is augmented during AHR, no alteration in muscarinic receptor density in ASM has been demonstrated in various AHR models. It is thus likely that augmented intracellular signaling might be a possible reason for the AHR. In fact, recent investigations demonstrated increases in the levels of GTP binding protein, Ca2+ mobilization and inositol 1,4,5-trisphosphate generation and so on in hyperresponsive ASM.
Folia Pharmacologica Japonica 05/1998; 111(4):249-56.
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ABSTRACT: The present study investigated the changes in NMDA receptor subunit proteins in diazepam-withdrawn rat cerebral cortex, using Western blotting analysis. The protein levels of the NR1 and NR2B, but not NR2A, subunits were significantly increased in diazepam-withdrawn rats compared to those in control rats. Therefore, an increase in the NR1 and NR2B subunit proteins may be responsible for both the previously observed upregulation of [3H]dizocilpine binding in the cerebral cortex and the appearance of diazepam withdrawal signs.
European Journal of Pharmacology 01/1998; 341(2-3):R1-2. · 2.52 Impact Factor
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ABSTRACT: 1. The electrical field stimulation (EFS)-induced bronchoconstriction in vitro in rats challenged by DNP-Ascaris antigen was significantly greater than that in normal rats. 2. Morphine inhibited the EFS-induced bronchoconstriction in normal rats. Whereas the inhibition of EFS-induced bronchoconstriction by the opioid was little, if any, in the DNP-Ascaris-challenged rats. 3. These findings suggest that dysfunction of presynaptic inhibitory modulation through the opioid receptor may take place in the airways of DNP-Ascaris-challenged rats.
General Pharmacology 05/1996; 27(3):441-4.
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ABSTRACT: The antagonist and agonist binding sites of muscarinic receptors were investigated by using membrane preparations of airways from nonsensitized normal control, sensitized control and repeatedly antigen challenged rats. The in vitro bronchial responsiveness to ACh was markedly increased in repeatedly antigen challenged group but not in sensitized control group. No significant difference was observed in receptor density and antagonist affinity among these three groups. The affinity of ACh for high-affinity agonist binding sites of repeatedly antigen challenged group was much greater than those in the other groups; the affinity significantly reduced in the presence of GTP gamma S. We concluded that enhanced G protein level might be involved in inducing airway hyperresponsiveness in rats.
Comparative biochemistry and physiology. Part C, Pharmacology, toxicology & endocrinology 08/1995; 111(3):351-7.
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ABSTRACT: The origin of Ca2+ contributing to the enhancement of acetylcholine-induced bronchial smooth muscle constriction in airway hyperresponsiveness induced by antigen challenge was investigated. Under Ca(2+)-free (concomitant with 10(-6) M nicardipine) conditions, the contractile responses of bronchial rings to 1 mM acetylcholine were significantly greater in rings from rats with hyperresponsive airways (0.15 +/- 0.04 g) than those of rings from normal rats (0.02 +/- 0.004 g; P < 0.05). The cumulatively administered Ca2+ induced a markedly greater bronchoconstriction in rings from rats with hyperresponsive airways in Ca(2+)-free solution when muscles were pretreated with 1 mM acetylcholine (in the presence of 10(-6) M nicardipine) than in rings from normal rats, whereas no significant difference in Ca(2+)-induced bronchoconstriction was observed between the two groups when muscles were pretreated with 60 mM K+ (in the presence of 10(-6) M atropine). These findings suggest that enhancement of the availability of Ca2+ released from intracellular stores and/or influxed through receptor-operated Ca2+ channels in airway smooth muscles might be involved in the airway hyperresponsiveness to acetylcholine in rats.
European Journal of Pharmacology 05/1995; 278(1):79-82. · 2.52 Impact Factor
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ABSTRACT: The effects of sensory neuropeptides on the airway responsiveness to acetylcholine (ACh) were investigated in normal nonsensitized rats. The airway responsiveness to inhaled ACh was significantly increased after treatment with neurokinin A (NKA; 0.001%) or substance P (SP; 0.01%) aerosol in the presence of the neutral endopeptidase (NEP) inhibitor. NKA had a more potent effect than SP. Interestingly, the intravenous treatment with NEP inhibitor alone also induced airway hyperresponsiveness (AHR) to inhaled ACh. This AHR was significantly attenuated by pretreatment with a nonselective NK-receptor antagonist, [D-Pro2,D-Trp7,9]SP, systemic capsaicin, or bilateral cervical vagotomy, indicating that decreased NEP activity results in accumulation of endogenous sensory neuropeptide(s) and enhancement of vagal reflex to cause AHR. The airway responsiveness to ACh of isolated left main bronchus was also increased after treatment with 10(-6) M NKA, but not SP, together with 10(-6) M phosphoramidon. This in vitro AHR to ACh induced by phosphoramidon plus NKA was significantly attenuated by pretreatment with 10(-6) M tetrodotoxin. These findings suggest that overaccumulated sensory neuropeptides, especially NKA, may enhance the probability of transmitter release, probably via NK2 receptors, and that the enhanced transmitter release might be involved in AHR in rats.
Journal of Applied Physiology 03/1995; 78(2):394-402. · 3.75 Impact Factor
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ABSTRACT: The effects of ozagrel, a thromboxane A2 (TXA2) synthase inhibitor, and CV-3988, a platelet activating factor (PAF) antagonist, was investigated on the repeatedly antigenic challenge-induced airway hyperresponsiveness (AHR) in rats. Rats were actively sensitized with DNP-Ascaris antigen and received 3 inhalations of antigen (challenges) or saline (sensitized control) every 48 hr. These animals were also pretreated with ozagrel (100 mg/kg, p.o., 30 min before), CV-3988 (3 mg/kg, i.v., 5 min before) or respective vehicle (water and saline, respectively) before each inhalation of antigen or saline. The in vivo airway responsiveness to cumulatively inhaled acetylcholine (ACh; 0.001-0.03%, each for 3 min) was measured 24 hr after the last inhalation of antigen or saline under anesthesia. A marked AHR was observed after repeated antigenic challenge when compared with the sensitized control group (5.5-9.5 times in order). This AHR was significantly, but partly, attenuated by pretreatment with ozagrel although this treatment alone had no effect on the airway responsiveness to inhaled ACh in sensitized control animals. On the other hand, CV-3988 had no inhibitory effect on this AHR. These findings suggest that TXA2, but not PAF, is one of the most important mediators participating in the pathogenesis of the antigen-induced AHR in rats.
Research communications in chemical pathology and pharmacology 07/1994; 84(3):341-9.
