Airways obstruction is the sine qua non of asthma. Although excess mucus secretions contribute to this phenomenon, the primary cause is a decrease in airway caliber due to airway smooth muscle contraction. An increase in cellular cyclic AMP levels has been associated with the relaxation of airway smooth muscle. There are, at present, two mutually non-exclusive hypotheses for cyclic AMP-mediated relaxation. One hypothesis asserts that relaxation is mediated through the phosphorylation of myosin light chain kinase (MLCK) by cyclic AMP-dependent protein kinase; the other asserts that relaxation results from a decrease in intracellular calcium following a rise in cyclic AMP levels. We propose to use a multidisciplinary approach to determine if either, or both, of these mechanisms contribute to cyclic AMP-mediated relaxation. First, the role of cyclic AMP-mediated phosphorylation of MLCK in canine tracheal smooth muscle (canine TSM) relaxation will be studied by determining (a) the temporal relationship between MLCK phosphorylation and relaxation, (b) the sites that are phosphorylated and (c) the effect of phosphorylation on MLCK activity. Second, the interaction between MLCK phosphorylation and cytoplasmic calcium during relaxation of skinned and intact canine TSM will be studied. In these experiments, the cytoplasmic calcium concentration will be manipulated and antibodies that inhibit MLCK phosphorylation will be used to study how these processes affect each other. Finally, changes in cell calcium in response to pharmacological stimulation of intact canine TSM will be quantitated. The Ca2+ measurements will then be correlated with changes in cyclic AMP levels, MLCK phosphorylation, myosin dephosphorylation and relaxation of canine TSM. These experiments utilize physiological, pharmacological, biochemical and immunological methods. Such an approach should provide insights into the roles of two important cellular messengers (calcium and cyclic AMP) and two important enzymes (myosin and myosin light chain kinase) in regulating the contractile properties of airway smooth muscle. These studies should also indicate avenues for pharmacological manipulation of airway caliber.
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