beta-escin and Bronchial-Hyperreactivity

The mechanical properties and Ca2+ sensitivity of an organ-culture model derived from guinea pig airways have been examined. The cultured explants develop airway hyperresponsiveness to pharmacological agonists after 3-day culture, when compared with fresh and ovalbumin-sensitized tissues. The reactivity of cultured explants is dependent on the presence of the epithelium. They are also sensitive to glucocorticosteroid pretreatments, which neutralize the TNFalpha antibody and NF-kappaB inhibitor. Hence, specific immunostaining of NF-kappaB subunits (p65 and p50) was increased in the nuclear extract of cultured explants. In beta-escin-permeabilized preparations, step-increases in pCa revealed enhanced Ca2+ sensitivity of the contractile apparatus in cultured explants, which was prevented by epithelium removal. Pretreatments of cultured explants with neutralizing TNFalpha antibody and NF-kappaB inhibitor consistently reduced their Ca2+ sensitivity. These findings suggest that AHR developed in this organ culture model may be triggered by an inflammatory process mediated by the TNFalpha and NF-kappaB transcription factor, which results in an increased sensitivity to [Ca2+]i.

Topics: Animals; Anti-Inflammatory Agents; Antibodies, Blocking; Antigens; Blotting, Western; Bronchi; Bronchial Hyperreactivity; Calcium; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Escin; Female; Glucocorticoids; Guinea Pigs; Isometric Contraction; Male; Microscopy, Fluorescence; NF-kappa B; Organ Culture Techniques; Ovalbumin; Subcellular Fractions; Tumor Necrosis Factor-alpha

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.

Topics: Acetylcholine; Actin Cytoskeleton; Animals; Antigens; Bronchi; Bronchial Hyperreactivity; Calcium; Escin; GTP-Binding Proteins; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Rats; Rats, Wistar