Human Bronchial Smooth Muscle Cells Express Adenylyl Cyclase Isoforms 2, 4, and 6 in Distinct Membrane Microdomains

Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because β-adrenergic receptor (AR) agonists stimulate AC activity and increase airway diameter. We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC). Reverse transcriptase-polymerase chain reaction 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, whereas 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, whereas AC2 and AC4 localized in nonraft fractions. Immunoisolation of caveolae using caveolin-1 antibodies yielded Ca2+-inhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed Gβγ-stimulated AC activity (consistent with expression of AC2 and/or AC4). Overexpression of AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to prostaglandin E2 or butaprost. β2AR, but not prostanoid EP2 or EP4 receptors, colocalized with AC5/6 in lipid raft fractions. Thus, particular G protein-coupled receptors couple to discreet AC isoforms based, in part, on their colocalization 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.