Adenylyl cyclase type 6 overexpression selectively enhances beta-adrenergic and prostacyclin receptor-mediated inhibition of cardiac fibroblast function because of colocalization in lipid rafts

Abstract

Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Fibroblasts are activated by factors such as transforming growth factor beta and inhibited by agents that elevate 3',5'-cyclic adenosine monophosphate (cAMP) levels. cAMP signal generation and response is known to be compartmentalized in many cell types in part through the colocalization of receptors and specific adenylyl cyclase isoforms in lipid rafts and caveolae. The present study sought to define the localization of key G protein-coupled receptors with adenylyl cyclase type 6 (AC6) in lipid rafts of rat cardiac fibroblasts and to determine if this colocalization was functionally relevant. We found that cardiac fibroblasts produce cAMP in response to agonists for beta-adrenergic (isoproterenol), prostaglandin EP2 (butaprost), adenosine (adenosine-5'-N-ethylcarboxamide, NECA), and prostacyclin (beraprost) receptors. Overexpression of AC6 increased cAMP production stimulated by isoproterenol and beraprost but not by butaprost or NECA. A key function of fibroblasts is the production of collagen. Isoproterenol- and beraprostmediated inhibition of collagen synthesis was also enhanced by AC6 overexpression, while inhibition by butaprost and NECA were unaltered. Lipid raft fractions from cardiac fibroblasts contain the preponderance of beta-adrenergic receptors and AC6 but exclude EP2 receptors. While we could not determine the localization of native prostacyclin receptors, we were able to determine that epitope-tagged prostanoid IP receptors (IPR) expressed in COS7 cells did localize, in part, in lipid raft fractions. These findings indicate that IP receptors are expressed in lipid rafts and can activate raft-localized AC isoforms. AC6 is completely compartmentized in lipid raft domains where it is activated solely by coresident G protein-coupled receptors to regulate cardiac fibroblast function.

Fig. 1

cAMP production stimulated by certain GPCR is enhanced by overexpression of AC6. cAMP production was measured in cells incubated with either LacZ- or AC6-expressing recombinant adenovirus for 24 h. Cells were incubated with the indicated drugs along with a phosphodiesterase inhibitor for 10 min at 37°C before cells were lysed and cAMP content assayed by EIA (see “Materials and methods”). Each bar represents the mean±SEM of at least three experiments. Asterisk denotes p<0.05, double asterisk denotes p<0.01 as compared to the same condition in LacZ cells, and number sign denotes p<0.05 as compared to basal by ANOVA

Fig. 2

Localization of GPCRs and ACs in lipid raft fractions from rat cardiac fibroblasts. Expression of caveolin-1, AC3, AC5/6, β₂AR, and EP₂R was assessed by SDS-PAGE and immunoblot analysis of buoyant (cav) and “heavy” (non-cav) membrane fractions and whole-cell lysates (WCL) from cardiac fibroblasts isolated using a nondetergent method followed by sucrose density centrifugation. Fractions from cells overexpressing AC6 (using a recombinant adenovirus) were also analyzed for AC5/6 immunoreactivity. Cav samples were pooled from the fractions at the 5–35% sucrose interface while the non-cav samples were pooled from the fractions in the 45% sucrose layer. Equal volumes of each fraction were loaded to represent the relative proportions of protein as they exist in intact cells. Right panels show full-length immunoblots probed with AC3 and AC5/6 antibodies. Images are representative of at least three experiments

Fig. 3

βAR and IPR but not EP₂R stimulate AC activity in immunoisolated caveolae. Caveolin-1 antibodies were used to immunoprecipitate caveolar fractions isolated by Triton-X100 insolubility. Precipitates and supernatants were assayed for AC activity as described in “Materials and methods.” Caveolin-1 immunoprecipitates contained the bulk of caveolin-1 and AC5/6 immunoreactivity while the supernatants contained very little caveolin-1 and less AC5/6 (top panels). Forskolin (Fsk), isoproterenol (Iso), and beraprost-stimulated AC activities in the IPs were enhanced by AC6 overexpression while butaprost-stimulated AC activity was unaffected (bottom panel). Data are expressed as the fold increase induced by AC6 overexpression (raw data is shown in Table 2). Asterisk denotes p<0.05, and double asterisk denotes p<0.01 as compared to 1 (no increase over control) by ANOVA

Fig. 4

HA-IPR are detected in lipid raft fractions and intracellular stores in COS-7 cells. a cAMP production was measured using [³H]adenine labeling in COS-7 cells transiently transfected with an HA-tagged IPR construct. Responses to forskolin or various concentrations of beraprost were compared in cells treated with recombinant adenovirus expressing either LacZ (control) or AC6. Each bar or point represents the mean±SEM of at least three experiments. Asterisk denotes p<0.05, double asterisk denotes p<0.01 as compared to the same condition in LacZ cells, and number sign denotes p<0.05 as compared to basal by ANOVA. b COS7 cells transiently transfected with HA-IPR were fractionated using a nondetergent method and analyzed by SDS-PAGE and immunoblotting (left panels) or fixed and visualized by immunofluorescent microscopy (right panels). Much HA-IPR is detected in intracellular stores and “heavy” fractions, but a portion is detected in the plasma membrane and buoyant lipid raft fractions coexpressing caveolin-1. Images are representative of at least three experiments

Fig. 5

cAMP-elevating agents inhibit collagen synthesis in rat cardiac fibroblasts. [³H]Proline incorporation stimulated by 2.5% FBS was measured in the absence or presence of increasing concentrations of forskolin (a), isoproterenol (b), beraprost (c), or butaprost (d). Cells were serum deprived for 24 h and then treated with the indicated drug for 20 min before addition of 2.5% FBS for another 48 h (see “Materials and methods”). Each bar or point represents the mean±SEM of at least three experiments. Asterisk denotes p<0.05 as compared to 2.5% FBS, and number sign denotes p<0.05 as compared to 0.25% FBS (basal) by ANOVA

Fig. 6

AC6 overexpression enhances the inhibition of collagen synthesis mediated by βAR and IPR but not EP₂R or adenosine receptors. [³H]Proline incorporation stimulated by 2.5% FBS was measured in the absence or presence of forskolin, isoproterenol, beraprost, butaprost, or NECA. Cells were serum deprived for 24 h and then treated with the indicated drug for 20 min before addition of 2.5% FBS for another 48 h (see “Materials and methods”). Data are expressed as the total incorporated proline counts (a) or as the percent inhibition of the FBS-stimulated proline incorporation (b, forskolin inhibits greater than 100% in the AC6 conditions because it induces a level of incorporation that is less than the 0% FBS condition). Each bar represents the mean±SEM of at least three experiments. Asterisk denotes p<0.05 as compared to the LacZ condition, and number sign denotes p<0.05 as compared to 2.5% FBS (0% inhibition) by ANOVA