Adenylyl cyclase 2 selectively couples to E prostanoid type 2 receptors, whereas adenylyl cyclase 3 is not receptor-regulated in airway smooth muscle

Abstract

Adenylyl cyclases (ACs) are important regulators of airway smooth muscle function, because β-adrenergic receptor (βAR) agonists stimulate AC activity and cAMP production. We have previously shown in a number of cell types that AC6 selectively couples to βAR and these proteins are coexpressed in lipid rafts. We overexpressed AC2, AC3, and AC6 in mouse bronchial smooth muscle cells (mBSMCs) and human embryonic kidney (HEK)-293 cells by using recombinant adenoviruses and assessed their localization and regulation by various G protein-coupled receptors (GPCRs). AC3 and AC6 were expressed primarily in caveolin-rich fractions, whereas AC2 expression was excluded from these domains. AC6 expression enhanced cAMP production in response to isoproterenol but did not increase responses to butaprost, reflecting the colocalization of AC6 with β(2)AR but not E prostanoid type 2 receptor (EP(2)R) in lipid raft fractions. AC2 expression enhanced butaprost-stimulated cAMP production but had no effect on the β(2)AR-mediated response. AC3 did not couple to any GPCR tested. Forskolin-induced arborization of mBSMCs was assessed as a functional readout of cAMP signaling. Arborization was enhanced by overexpression of AC6 and AC3, but AC2 had no effect. GPCR-stimulated arborization mirrored the selective coupling observed for cAMP production. With the addition of the phosphodiesterase 4 (PDE4) inhibitor rolipram AC2 accelerated forskolin-stimulated arborization. Thus, AC2 selectively couples to EP(2)R, but signals from this complex are limited by PDE4 activity. AC3 does not seem to couple to GPCR in either mBSMCs or HEK-293 cells, so it probably exists in a distinct signaling domain in these cells.

Fig. 1.

A, AC isoform mRNA expression in mBSMCs was measured by RT-PCR. AC isoform-specific primer pairs were used with 35 cycles of RT-PCR (see Materials and Methods). Representative images of three experiments are shown. Arrows indicate expected sizes of PCR product. Molecular weight markers (MW) are shown in the first lane. β-Actin primers were used with no reverse transcriptase (noRT) or cDNA (β-actin) templates. B, cAMP accumulation in whole cells pretreated with IBMX was measured (see Materials and Methods) in response to various agonists known to stimulate AC activity in other cells. Data are presented as mean ± S.E.M. (n = 3–4). *, p < 0.05; **, p > 0.01 by paired t test compared with basal (no drug). Fsk, forskolin; Iso, isoproterenol; Buta, butaprost; Bera, beraprost;NECA, adenosine-5′N-ethylcarboxamide; CGRP, calcitonin gene-related peptide.

Fig. 2.

Immunoblot analysis of fractions from lipid raft isolation from mBSMCs. Cells were fractionated by using a nondetergent method and separated by sucrose density centrifugation (see Materials and Methods). Gradients were collected in 10 0.5-ml fractions and analyzed for appropriate separation of marker proteins (data not shown). Fractions were separated by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting by using the indicated primary antibody. In some studies, cells were incubated with recombinant adenoviruses expressing AC2, AC3, or AC6 for 24 h. Shown are regions of the gels at the approximate molecular mass of the expected immunoreactive band. Images shown are representative of three to five experiments. Cav, caveolin.

Fig. 3.

Colocalization of AC isoforms and caveolin-1 in mBSMCs. Cells were blocked with 1% bovine serum albumin and then incubated with rabbit anti-AC2, -AC3, or -AC6, or mouse anticaveolin-1 antibodies at 37°C for 1 h. After washing, the cells were incubated with Alexa 594-conjugated goat anti-mouse IgG and Alexa 488-conjugated goat-anti-rabbit IgG for 1 h. In some studies, cells were incubated with recombinant adenoviruses expressing AC2, AC3, or AC6 for 24 h. Images shown are representative of four to five experiments. Degree of overlap calculated from the merged images is shown in Table 1.

Fig. 4.

cAMP accumulation in mBSMC or HEK-293 cells expressing AC2, AC3, or AC6. mBSMCs (A, B, and D) or HEK-293 cells (C) were incubated with recombinant adenoviruses expressing either lacZ (control), AC2, AC3, or AC6, then cAMP production was measured (see Materials and Methods) A, mBSMCs were stimulated with a maximal concentration of forskolin (Fsk) in the presence of IBMX. *, p < 0.05; **, p > 0.01 by paired t test compared with basal. #, p < 0.05 by paired t test compared with lacZ condition. B, mBSMCs were treated with 0.1 μM Fsk, 0.1 μM isoproterenol, 0.3 μM butaprost, or 0.3 μM beraprost in the presence of IBMX. Data are presented as the percentage increase in cAMP levels when an AC was expressed over the lacZ condition, with the dashed line indicating no increase over lacZ. *, p < 0.05; **, p > 0.01 by paired t test compared with lacZ. C, HEK-293 cells were treated with vehicle, 10 μM Fsk, 1 μM Iso, or 1 μM PGE₂. Data are presented as the fold increase in basal cAMP levels (vehicle-treated lacZ cells), with the dashed line indicating no increase over basal. *, p < 0.05; **, p > 0.01 by paired t test compared with lacZ. D, mBSMCs treated with a maximal concentration of Fsk or vehicle without the inclusion of a PDE inhibitor. *, p < 0.05; **, p > 0.01 by paired t test compared with lacZ. #, p < 0.05 by paired t test compared with basal. ns, not significant. All data are presented as mean ± S.E.M. (n = 3–5).

Fig. 5.

Arborization of mBSMC after overexpression of either AC2, AC3, or AC6. Cells were incubated with the indicated drugs, and time-lapse photography was used to monitor cell shape changes for 40 min after addition of the indicated drug in the absence (A) or presence of the PDE4-specific inhibitor rolipram (1 μM; B). The pixel density of three to four cells in each image was calculated, and the rate of increase in pixel density over time was used to calculate arborization rate. The percentage of change in arborization rate versus lacZ cells was calculated, and data are presented as the mean ± S.E.M. of three experiments (9–12 observations). *, p < 0.05 by paired t test compared with lacZ. Representative images of the cell shape change are shown at the top.

Fig. 6.

Schematic diagram of GPCR/AC localization and signaling in mBSMCs. β₂AR and prostacyclin receptors (IPR) primarily signal via AC6 localized in lipid raft microdomains. AC3, when overexpressed, localizes in these same domains but does not seem to couple to any GPCR. Signals emanating from this domain mediate arborization of the cells. In nonraft membranes, prostanoid EP₂ receptors localize with AC2, but cAMP signals from these locales do not seem to regulate the arborization response. PDE4 is expressed in this location and its activity seems responsible for limiting cAMP in this compartment, because treatment of cells with rolipram uncovers an EP₂R-AC2 stimulation of arborization. Buta, butaprost; Bera, beraprost; MB, membrane.