C-AMP & CONTRACTILE REGULATION IN AIRWAY SMOOTH MUSCLE

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In asthma therapy a major goal is to achieve reversal of airway smooth muscle spasm. The most commonly used bronchodilators, beta adrenergic agonists and the methylxanthines, are thought to act via the cyclic AMP pathway. The ability of isoproterenol to relax contracted airway smooth muscle differs depending on the particular agonist used to contract the tissue. A working hypothesis is that certain agents (methacholine) produce contractions resistant to isoproterenol because they directly inhibit the cyclic AMP pathway. Other contractile agents (serotonin, histamine) are more easily reversed by isoproterenol and we will test the hypothesis that these do not inhibit the cyclic AMP pathway. We will contract isolated airway smooth muscle strips with methacholine, seratonin, histamine and potassium and compare the relative ability of these four agents to inhibit isoproterenol-induced relaxation, cyclic AMP increases and protein kinase activation. We will use canine trachealis because many comparable preparations can be collected from one donor and because the cellular homogeneity of samples permits valid correlation of mechanical events with changes in biochemical composition. The beta receptor density in cervical trachealis is greater than that in thoracic trachialis. We will perform experiments on both regions in order to evaluate the influence that natural variation in beta receptor density has on the functional antagonism between contractile and relaxant agonists. A major theory of asthma proposes that the molecular basis of asthma is a deficiency in the beta receptor cyclase system, and the reduced beta receptor density in thoracic trachealis provides an in vitro model of such a phenomenon. Experiments will be done to test the ability of methacholine, serotonin, histamine and potassium to resist relaxation, cyclic AMP increases and protein kinase activation induced by prostaglandin E2, Forskolin, cyclic AMP analogues, methylxanthines and non-cyclic AMP-mediated relaxants such as nitroprusside. These relaxants activate the cyclic AMP pathway at sites progressively farther along in the causal sequence. These results should facilitate localization of the site of action of methacholine since a relaxant which activates at a subsequent site should by-pass the methacholine inhibited site and readily relax the contraction. After the basic behavior of the system and mechanical-biochemical correlations have been worked out in the canine trachealis, experiments will be done to extend these principles to more peripheral airways.