Exaggerated airway narrowing is a central feature of asthma. We have found that mice lacking a specific protein (an integrin) only in smooth muscle have exaggerated airway narrowing. In this proposal we will determine how this integrin normally prevents airway narrowing, a process that could be perturbed in diseases such as asthma.
The ¿9¿1 integrin is highly expressed in airway smooth muscle. Mice we have generated lacking this integrin only in smooth muscle cells have marked in vivo airway hyperresponsiveness and lung slices from these mice have increased airway narrowing. We have previously shown that the ¿9 subunit cytoplasmic domain directly binds the enzyme spermine/spermidine acetyltransferase (SSAT), the rate limiting step in catabolism of higher order polyamines, and that this association and polyamine catabolism are important modulators of ¿9¿1 function. Pharmacologic stabilization of SSAT also augments ¿9¿1-dependent prevention of airway smooth muscle contraction. In the current application we will systematically evaluate the effects of ¿9¿1 on responses of airway smooth muscle to multiple contractile agonists and to isoproterenol-induced relaxation using 4 parallel experimental systems (in vivo AHR, tracheal ring contraction, airway narrowing in lung slices and shortening of airway smooth muscle cells). We will utilize a variety of mutant and chimeric constructs of ¿9 and SSAT, in vivo and in vitro studies with SSAT knockout mice, catalytically inactive SSAT mutants and genetic and pharmacologic inhibitors to thoroughly examine what contribution interaction of ¿9 with SSAT makes to this response. Force generatioin in smooth muscle depends on calcium-dependent actin-myosin cross-bridging and parallel actin polymerization, and we will systematically evaluate the effects of ¿9¿1 on each of these pathways. Because the two major cytosolic effects of higher order polyamines are prevention of potassium efflux through Kir channels and activation of the lipid kinase, PIP5K1¿, we will pay special attention to the roles of Kir channels and PIP5K1¿ in this process.The proposed studies will test the overall hypothesis that ligated ¿9¿1 normally serves as a brake on airway narowing by concentrating SSAT, catabolizing polyamines and thus inhibiting potassium efflux and/or PIP2 production, resulting in reduced calcium oscillations, decreased actin-myosin cross-bridging and/or impaired actin polymerization. Abnormalities in this pathway, either acquired or genetic, could contribute to diseases such as asthma that are characterized by enhanced airway narrowing.