Angiotensin II Enhances Adenylyl Cyclase Signaling via Ca2+/Calmodulin

Abstract

Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between G q and G s signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by ␤-adrenergic receptors (␤-AR). The potentiation of ␤-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of G q . Ionomycin and thapsigargin increased intracellular Ca 2؉ levels and potentiated isoproterenoland forskolin-stimulated cAMP production, whereas chelation of Ca 2؉ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or G␤␥ did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca 2؉ -activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased ␤AR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that ␤AR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca 2؉ -dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of crosstalk between G q and G s signaling pathways in cardiac fibroblasts.

Inhibition of cardiac myofibroblast formation and collagen synthesis by activation and overexpression of adenylyl cyclase

“…Assays were terminated by aspiration of media and addition of 250 l of ice-cold trichloroacetic acid (7.5%) to each well. RIA was used to quantitate cAMP (14,17); results were normalized to the amount of protein per sample, as determined by a colorimetric protein assay (Bio-Rad).…”

“…We used an adenoviral vector to enhance expression in CF of AC6, an AC isoform expressed natively at relatively high levels (14). Basal cAMP levels did not differ between LacZ-and AC6-overexpressing CF (Fig.…”

“…G protein-coupled receptor (GPCR) agonists that signal through G s to activate AC and stimulate cAMP production can inhibit collagen synthesis (13)(14)(15). Forskolin and GPCR agonists that stimulate AC were recently shown to inhibit serum or TGF-␤-stimulated ␣-SMA expression and collagen formation by human lung fibroblasts (16,17).…”

Focal Adhesions in (Myo)fibroblasts Scaffold Adenylyl Cyclase with Phosphorylated Caveolin

“…AC5/6 Localizes with cav-1 throughout the Cell Membrane and with Phospho-cav-1 at FA Sites-AC5/6 is predominantly detected in buoyant/caveolar membrane fractions, but a portion of AC5/6 localizes in heavy/non-caveolar membrane fractions in cardiac cells (31,32). Immunohistochemical analysis and quantification of AC5/6 and phospho-cav-1 staining of CF revealed that, in addition to being distributed with cav-1 throughout the plasma membrane, 17 Ϯ 4% of AC 5/6 colocalizes with phospho-cav-1 and 57 Ϯ 12% of phospho-cav-1 colocalizes with AC 5/6 at FA sites ( Fig.…”

The Cytosolic Domains of Ca2+-sensitive Adenylyl Cyclases Dictate Their Targeting to Plasma Membrane Lipid Rafts

“…AC1 and AC8 are activated by Ca 2ϩ in a calmodulin-dependent manner, whereas AC5 and AC6 are inhibited by Ca 2ϩ , independently of calmodulin (6). Accumulating evidence from heterologous expression studies is beginning to suggest that Ca 2ϩ -sensitive ACs (specifically AC5, AC6, and AC8) are enriched in lipid rafts, whereas Ca 2ϩ -insensitive ACs (AC2, AC4, and AC7) (4,7,8) are excluded from rafts and reside in the bulk PM. This differential localization may be directly related to specific properties of AC regulation, since the residence of Ca 2ϩ -regulatable ACs in lipid rafts is crucial for their responsiveness to physiological elevations in cytosolic Ca 2ϩ .…”