RT Journal Article SR Electronic T1 Human bronchial smooth muscle cells express adenylyl cyclase isoforms 2, 4 and 6 in distinct membrane microdomains JF Journal of Pharmacology and Experimental Therapeutics JO J Pharmacol Exp Ther FD American Society for Pharmacology and Experimental Therapeutics SP jpet.110.177923 DO 10.1124/jpet.110.177923 A1 Amy S. Bogard A1 Congfeng Xu A1 Rennolds Ostrom YR 2011 UL http://jpet.aspetjournals.org/content/early/2011/01/12/jpet.110.177923.abstract AB Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, as β-adrenergic receptor (βAR) agonists stimulate AC activity and increase airway diameter. We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC). RT-PCR and immunoblot analyses detected expression of AC2, AC4 and AC6. Forskolin-stimulated AC activity in membranes from hBSMC displayed Ca2+-inhibited and Gβγ-stimulated AC activity, consistent with expression of AC6, AC2 and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca2+ but unaltered by Gβγ, while butaprost-stimulated AC activity was stimulated by Gβγ but unaffected by Ca2+ addition. Using sucrose density centrifugation to isolate lipid raft fractions, we found that only AC6 localized in lipid raft fractions while AC2 and AC4 localized in non-raft fractions. Immunoisolation of caveolae using caveolin-1 antibodies yielded Ca2+-inhibited AC activity (consistent with AC6 expression) while the non-precipitated material displayed Gβγ-stimulated AC activity (consistent with expression of AC2 and/or AC4). Overexpressing AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to PGE2 or butaprost. β2AR, but not EP2 or EP4 receptors, co-localized with AC5/6 in lipid raft fractions. Thus, particular GPCR's couple to discreet AC isoforms based, in part, upon their co-localization in membrane microdomains. These different cAMP signaling compartments in airway smooth muscle cells are responsive to different hormones and neurotransmitters and can be regulated by different coincident signals such as Ca2+ and Gβγ.