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

Abstract

Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because ␤-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 Ca 2ϩ -inhibited and G ␤␥ -stimulated AC activity, consistent with expression of AC6, AC2, and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca 2ϩ but unaltered by G ␤␥ , whereas butaprost-stimulated AC activity was stimulated by G ␤␥ but unaffected by Ca 2ϩ 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 Ca 2ϩ -inhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed G ␤␥ -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 E 2 or butaprost. ␤ 2 AR, but not prostanoid EP 2 or EP 4 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 Ca 2ϩ and G ␤␥ .

Adenylyl Cyclase 2 Selectively Couples to E Prostanoid Type 2 Receptors, Whereas Adenylyl Cyclase 3 Is Not Receptor-Regulated in Airway Smooth Muscle

“…1). Consistent with findings from other cell types, AC3 and AC5/6 localize to lipid rafts, whereas AC2 localized to nonraft membranes (Ostrom et al, 2000Ostrom and Insel, 2004;Bogard et al, 2011).…”

“…Our data show that mBSMCs express mRNA for AC1, AC3, and AC5 along with low levels of AC4. We could detect expression only of AC3 and AC5/6 protein, although immunological approaches to define ACs can be problematic (Bogard et al, 2011). This AC expression profile differs significantly from human airway smooth muscle cells, which express AC2, AC4, and AC6 (Bogard et al, 2011).…”

“…AC isoform expression was assessed by RT-PCR using the primer pairs described previously (Bogard et al, 2011). Total RNA was extracted from mBSMCs grown to 60 to 70% confluence by using a RNeasy RNA isolation kit (QIAGEN, Valencia, CA).…”

“…Incubating cells with AC2 or AC3 adenoviruses also led to detectible AC expression, with AC2 in nonraft fractions and AC3 in lipid raft fractions. Commercially available AC isoform antibodies yield multiple bands from mBSMCs and many other cell types (Liu et al, 2008;Bogard et al, 2011). Genuine immunoreactive bands were confirmed by comparing blots from control and AC-overexpressing cells (Supplemental Fig.…”

“…Lipid rafts are microdomains of the plasma membrane that are biochemically similar to caveolae but morphologically indistinguishable from the rest of the plasma membrane (Hooper, 1999;Simons and Toomre, 2000;Galbiati et al, 2001). Data from several cell types indicate that AC6 selectively couples to ␤AR and not prostanoid EP receptors because of colocalization of AC6 and ␤AR in lipid rafts (Ostrom et al, 2001;Liu et al, 2008;Bogard et al, 2011). Nonraft localized EP receptors activate cAMP production, but it is unknown which AC isoforms they couple to for regulating cell function.…”