Asthma is characterized by bronchial inflammation, airway smooth muscle hyperplasia, and enhanced non-specific airway contractility. Based on emerging evidence in support of an association between smooth muscle hyperplasia and enhanced agonist-mediated smooth muscle contraction, two interrelated hypotheses are raised: I: That inflammatory cell mediators which produce bronchonstriction are also growth inductive to airway smooth muscle; and II: That induction of airway smooth muscle hyperplasia is associated with altered agonist-mediated airway smooth muscle contractility. In addressing these hypotheses, proliferative responses, agonist-mediated contractile responses, and mechanisms regulating cell- surface receptor expression and binding will be examined in cultured rabbit airway smooth muscle (ASM) cells. A: To assess the interrelationship between inflammatory cell mediators, ASM growth, and altered ASM contractility, we will investigate: 1) whether the bronchoactive mediators: histamine, leukotrienes C4 and D4, prostaglandin D2, platelet activating factor, eosinophil-derived major basic protein substance P, and the endothelial cell-derived peptide, endothelin, each everts a proliferative effect in cultured ASM cells coupled to altered expression of the protooncogenes, c-myc and c-fos; and 2) whether proliferating ASM cells depict altered agonist-mediated contractile responsiveness, given by changes in intracellular free-calcium and contractile cell size distribution. The latter will be determine by fluorescence measurement of cells loaded with the calcium-sensitive dye, fura-2 and analysis of ASM cell size distribution curves using the Coulter counter technique, respectively. B: To assess mechanisms underlying altered contractile reponsiveness in hyperplastic ASM cells, we will investigate: 1) whether proliferating ASM cells exhibit changes in the receptor densities and ligand-binding affinities of the muscarinic-cholinergic and beta-adrenergic receptors; and 2) whether ASM cell growth-associated changes in muscarinic-cholinergic and beta-adrenergic receptor binding care growth- associated changes in muscarinic-cholinergic and beta-adrenergic receptor binding are coupled to modulation in the quantities of mRNA coded for these receptors and their rates of gene transcription. The latter will be assessed by Northern blot analysis and nuclear run-off studies, respectively. collectively, the proposed studies should provide new mechanistic insights into the interrelationship between inflammatory cell mediators, airway smooth muscle hyperplasia, and altered airway contractility which underlies the pathogenesis of asthma.
