Abstract

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 G_βγ-stimulated AC activity, consistent with expression of AC6, AC2, and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca²⁺ but unaltered by G_βγ, whereas butaprost-stimulated AC activity was stimulated by G_βγ 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 G_βγ-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 G_βγ.