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Fiscal Year 1999 : ¥100,000 (Direct Cost : ¥100,000)
Fiscal Year 1998 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1997 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Although we tested the effect of supernatants obtained from human lung mast cell-, macrophage-, or eosinophil-culture medium on contractility of human broncial smooth muscle (SM), viability of the reparations always run down, Ricombinat IL-1β, TNFα, or both did not show remakable changes in CaィイD12+ィエD1 sensitivity of permeabilized airway SM. CaィイD12+ィエD1 sensitivity of permeabilized airway SM from TNFα transgenic mice either sensitized or non-sensitized with ovalbumin did not increase. Therefore, we extended the experiments to clarify the mechanism of airway SM contraction.
Initial phase of airway SM contration : IPィイD23ィエD2 but neither cyclic ADP-ribose nor nicotinate adenine dinucleotide phosphate plays a key role in carbachol (CCh)-initiated contraction, and IPィイD23ィエD2 utilizes single compartment of the caffeine/ryanodine-sensitive stored CaィイD12+ィエD1 in airway SM, such as rabbit tracheal and human bronchial SM.
Sustained phase of airway SM contration : Both rabbit tracheal and huma
n bronchial SM expressed RhoA p21, Rho-associated coiled coil forming protein kinase (ROCK) I, and ROCK II. Y-27632, a ROCK inhibitor, completely reversed CCh-induced contraction of intact and α-toxin-permeabilized airway SM, indicating that Rho/ROCK-mediated CaィイD12+ィエD1 sensitization plays a central role in the sustained phase of airway SM contraction.
To clarify the relative importance of ROCK and protein kinase C (PKC), we investigated the effects of Y-27632 and GF 109203X (a PKC inhibitor) on GTPγS-induced contraction. Although a Y-27632-insensitive component was retained during the early phase of the GTPγS response, Y-27632 and GF 109203X in combination abolished the GTPγS response. GTPγS caused only a small contraction in the absence of CaィイD12+ィエD1. Wortmannin, a myosin light chain kinase (MLCK) inhibitor, completely inhibited CaィイD12+ィエD1-induced contraction. ATP-triggered contraction of the strip which had been treated with calyculin A, a phosphatase inhibitor, in rigor solutions was markedly slowed by wortmannin but not by Y-27632. GTPγS, but not PDBu, contracted the β-escin-permeabilized trachea in the absence of CaィイD12+ィエD1, but the presence of CaィイD12+ィエD1-independent MLCK. We conclude that although ROCK does not directly phosphorylate MLCィイD220ィエD2 in situ, ROCK plays a primary role in G-protein-mediated CaィイD12+ィエD1 sensitization through inhibition of myosin phosphatase. PKC partially contributes to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway SM.
ROCK inhibition in vivo : Y-27632 inhalation inhibited acetylcholine or antigen-induced increasein lung resisistance of guinea-pig in vivo.
Hence, we demonstrated the mechanism of airway SM contraction at the molecular levels ; importance of IPィイD23ィエD2-mediated and Rho/ROCK-mediated signaling. However, we have to study further to establish an asthma model of airway SM in vitro. Less