?-adrenergic receptors (?AR) stimulate cAMP production via activation of Gs and adenylyl cyclase (AC) activity to induce smooth muscle relaxation. Muscarinic acetylcholine receptors (mAChR) activate both Gq and Gi to cause a two-pronged contraction: M3 receptor-mediated elevation of intracellular Ca2+ and M2 receptor-mediated inhibition of AC activity. ?AR mediate relaxation of airway smooth muscle via both cAMP-dependent and -independent mechanisms but in gastrointestinal smooth muscle ?AR induce relaxation primarily via cAMP-dependent pathways. As a result of this differing dependence upon cAMP, Gi-coupled M2 receptors play a large role in contraction of gastrointestinal smooth muscle but not in airway smooth muscle. Airway smooth muscle overexpressing AC6 display enhanced ?AR-mediated cAMP formation and relaxation but not increased basal cAMP levels or response to activation of prostaglandin EP2 receptors. This selective effect of AC6 overexpression appears due to compartmentation of the exogenous AC6 with ?AR in caveolae and lipid rafts and the exclusion of EP2 receptors from these microdomains. Because other isoforms of AC localize differently than AC6, this project will examine the native AC isoform expression in airway and ileal smooth muscle then will assess the effects of expressing lipid raft and non-raft localized AC isoforms on biochemical signaling and regulation of contractile tone in these two tissues. The central hypothesis is that the localization of receptors and AC's in lipid rafts of smooth muscle determines the signaling pathways used by receptors to regulate contractile tone and that this localization differs between smooth muscle-containing tissues. The goal is to understand the cellular compartmentation of receptors (focusing primarily, but not exclusively, on BAR and mAChR) and AC's in ileal and airway smooth muscle and to determine the functional importance of this compartmentation in regulation of smooth muscle tone in both normal and asthmatic airways. It may be possible to enhance AC expression or function to selectively regulate smooth muscle function in asthma or gastrointestinal disease. ? ?
