Therapy for bronchial asthma by regulating a small G protein as a targeting molecule

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This project was designed to determine whether Rho, a small G protein, is useful to therapy for bronchial asthma as a targeting molecule. Since Rho, and it's effector enzyme, Rho-kinase, have functional relevance for Ca^<2+> sensitization, cell proliferation, cell migration, and cytoskeleton, the Rho/Rho-kinase pathway may related to eosinophil infiltration, airway hyperresponsiveness, β-adrenergic desensitization, and airway remodeling which are pathophysiogy of bronchial asthma. Isometric tension and concentrations of intracellular Ca^<2+> [Ca^<2+>]_i were simultaneously measured using fura-2 loaded tracheal smooth muscle in guinea pigs. When isoproterenol, a β-adrenergic receptor agonist, was cumulatively applied to the tissues precontracted with 1 μM methacholine, isoproterenol caused an inhibition of contraction induced by methacholine with reducing [Ca^<2+>]_i in a concentration-dependent manner. However, a reduction in tension was greater than that in [Ca^<2+>]_i in the inhibito … More ry action of isoproterenol, indicating that β-adrenergic action antagonizes not only Ca^<2+> mobilization but also Ca^<2+> sensitization. Loss of the latter process leads to dysfunction of β-adrenergic receptors. Hydrogen peroxide, an indicator of oxidative stress, generated force with elevating [Ca^<2+>]_i. However, Y-27632, an inhibitor of Rho-kinase, inhibited contraction by hydrogen peroxide without reducing [Ca^<2+>]_i, indicating that airflow limitation by oxidative stress is associated with activation of Rho-kinase. After exposure to sphingosine 1-phosphate (S1P), a bioactive lysophospholipid released from inflammatory cells, methacholine-induced contraction was markedly enhanced without elevating [Ca^<2+>]_i, and Y-27632 antagonized the augmented responsiveness to methacholine by pretreatment with S1P. Airway hyperresponsiveness is mediated by Rho-induced Ca^<2+> sensitization via inactivation of myosin phosphatase. In cultured human bronchial smooth muscle cells, cell proliferation and DNA synsethsis induced by FBS was inhibited by simvastatin, a HMG-CoA reductase, and Y-27632. Rho/Rho-kinase activity regulated by the mevalonate pathways in cholesterol synthesis contributes to cell proliferation related to airway remodeling. In sensitize mice, antigen challenges caused eosinophil infiltration to airways and an increase in the lung resistance in response to methacholine. Administration of Y-27632 and fasudil, another inhibitor of Rho-kinase, inhibited eosinophilia and augmented lung resistance by antigens challenges. The Rho/Rho-kinase pathway is involved in the airway inflammation by eosinophil recruitment.In conclusion, inhibiting the Rho/Rho-kinase pathway may have therapeutic potential for bronchial asthma. Less