Angiotensin II Enhances Adenylyl Cyclase Signaling via Ca 2 / Calmodulin

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 Gq and Gs 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 Gq. Ionomycin and thapsigargin increased intracellular Ca levels and potentiated isoproterenoland forskolin-stimulated cAMP production, whereas chelation of Ca with 1,2-bis(2-aminophenoxy)ethaneN,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 -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 ARregulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca -dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of crosstalk between Gq and Gs signaling pathways in cardiac fibroblasts.

involves an altered balance of synthesis and degradation of extracellular matrix (ECM) 1 by cardiac fibroblasts and can lead to abnormal accumulation of ECM in the interstitial space (i.e. fibrosis) (1,2). Several G protein-coupled receptors (GPCR) that signal through G q have been implicated in the pathogenesis of cardiac hypertrophy and failure (3,4). Conversely, GPCRs that signal via G s may inhibit collagen deposition by cardiac fibroblasts (5).
Two major pro-fibrotic signals in the heart are cytokines, such as transforming growth factor-␤, and the peptide hormone ANG II, both of which increase collagen synthesis by cardiac fibroblasts (6,7). ANG II also inhibits matrix metalloproteinase expression by cardiac fibroblasts, thereby attenuating degradation of ECM proteins (8). An anti-fibrotic role for cAMP is supported by evidence that adenosine and prostacyclin inhibit cardiac fibroblast proliferation and collagen synthesis through activation of A 2b and prostanoid receptors, respectively, which each couple via G s , to enhanced cAMP production (9,10). ␤-Adrenergic receptors have been shown to stimulate fibroblast proliferation via epidermal growth factor receptor transactivation (11) but their role in regulating collagen synthesis in cardiac fibroblasts is poorly documented.
Adenylyl cyclase (AC) catalyzes the synthesis of cAMP and its expression limits the ability of a cardiac cell to maximally produce this second messenger (12,13). Nine different transmembrane AC isoforms exist, each with different amino acid sequence, tissue and chromosomal distribution, and regulation (14). Differences in regulation include stimulation or inhibition by G␤␥, Ca 2ϩ , and various protein kinases. AC5 and AC6, which are the predominant isoforms expressed in cardiac myocytes (15), represent a subfamily of ACs that are related in structure and regulation. These isoforms are inhibited by protein kinase A, Ca 2ϩ , G i , G␤␥, and nitric oxide (14,16,17). By contrast, AC1, AC3, and AC8 are stimulated by Ca 2ϩ /calmodulin (yet AC3 can also be inhibited by calmodulin kinase-II) (14,18,19). AC2 and AC4 are activated by G␤␥ and AC2 and AC7 can be activated by phosphorylation by protein kinase A and/or PKC (14). Thus, the AC isoform expression in a cell determines the interaction between cAMP production and other signal transduction cascades.
Meszaros et al. (20) have recently described a signaling "cross-talk" between two key GPCR signal transduction pathways in cardiac fibroblasts. Isoproterenol (ISO), a ␤-adrenergic receptor (␤AR) agonist, activates G s and stimulates cAMP production 10-fold over basal levels. ANG II activates G q -coupled angiotensin receptors that, by themselves, do not alter cAMP production in cardiac fibroblasts, but in combination with ISO potentiate the ␤AR response, resulting in a 2-fold potentiation of ISO-stimulated cAMP production. The goal of the present study was to identify the molecular mechanism and physiological consequence of the cross-talk between these two signaling pathways in cardiac fibroblasts. The current data indicate that G q -mediated elevation of intracellular Ca 2ϩ induced by ANG II potentiates G s -stimulated cAMP formation, probably via stimulation of AC3. Moreover, we show that the potentiation is functionally relevant: combined treatment of cardiac fibroblasts with ANG II and ISO inhibits ANG II-promoted collagen synthesis more so than ISO alone. Thus, we identify an endogenous signaling pathway by which intracellular Ca 2ϩ enhances cAMP production and limits collagen synthesis, and perhaps fibrosis, in the heart.

EXPERIMENTAL PROCEDURES
Materials-Cell culture reagents were obtained from Invitrogen. Primary antibodies to AC isoforms and all secondary antibodies were obtained from Santa Cruz Biotechnology. Antibodies to caveolin were from BD Pharmingen. ANG II, UTP, Fura-2/AM, forskolin, and BAPTA/AM were obtained from Calbiochem. All other drugs and reagents were of reagent grade and obtained from Sigma.
Preparation and Culture of Adult Rat Cardiac Fibroblasts-Cardiac fibroblasts were prepared from adult male 250 -300-g Sprague-Dawley rat hearts. Following rapid excision of the hearts, the ventricles were isolated, minced, pooled, and placed in a collagenase/pancreatin digestion solution. After sequential digestions, the fibroblasts were pelleted and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin, streptomycin, fungizone, and 10% fetal bovine serum (FBS, Gemini Bio-Products). After a 30-min period of attachment to uncoated culture plates, cells that were weakly attached or unattached were rinsed free and discarded. After 2-3 days, confluent cultures were amplified by trypsinization and seeding onto new dishes. For signaling assays, only early passage (Յ3) cells grown to 80 -90% confluency were used. The purity of these cultures was greater than 95% cardiac fibroblasts as determined by positive staining for vimentin and negative staining for smooth muscle actin and von Willebrand factor, as previously described (21).
Adenoviral Gene Transfer to Cardiac Fibroblasts-Wild-type G q was cloned into the PACCMVpLpA shuttle vector for production of adenovirus, as previously described (3). We found that a titer of 200 virus particles per cell was optimal for G q expression without cytotoxicity, as determined by trypan blue exclusion and morphological examination with phase-contrast light microscopy. We used 1000 viral particles per cell of an adenovirus construct to maximally overexpress the murine adenylyl cyclase type 6 (AC6) gene, produced as described previously (22). Cardiac fibroblasts were infected with the appropriate virus and incubated at 37°C 18-24 h in serum-free DMEM prior to the assays. Preliminary experiments using LacZ expression and ␤-galactosidase staining indicated that the efficiency of the adenoviral construct to increase gene expression was 40% (data not shown).
Quantitation of cAMP Production-Cardiac fibroblasts cultured on 24-well plates were washed three times with 37°C serum-free and NaHCO 3 -free DMEM supplemented with 20 mM HEPES, pH 7.4. Cells were equilibrated for 30 min, then assayed for cAMP accumulation by incubation with drugs of interest in the presence of 0.2 mM isobutylmethylxanthine for 10 min. When antagonists or inhibitors were used, these agents were equilibrated with cells for 15 min before addition of agonists. Assay medium was aspirated and 250 l of ice-cold trichloroacetic acid (7.5% w/v) was immediately added to each well to terminate reactions. Trichloroacetic acid extracts were assayed for cAMP content by either Direct ELISA kit (Assay Designs) or radioimmunoassay as described previously (22). Data were corrected for the amount of total acid-precipitable protein per well. In other studies, AC activity was measured in cardiac fibroblast membranes as previously described (23). Briefly, membranes were prepared by rinsing cells twice in ice-cold phosphate-buffered saline then scraping cells into hypotonic homogenizing buffer (30 mM Na-HEPES, 5 mM MgCl 2 , 1 mM EGTA, 2 mM dithiothreitol, pH 7.5) and homogenizing with 20 strokes in a Dounce homogenizer. Homogenate was spun at 300 ϫ g for 5 min at 4°C. Supernatant was then transferred to a clean centrifuge tube and spun at 5,000 ϫ g for 10 min. Pellet was suspended in membrane buffer (30 mM Na-HEPES, 5 mM MgCl 2 , 2 mM dithiothreitol, pH 7.5) to attain ϳ1 mg/ml total protein concentration. Assay was conducted by adding 30 l of membranes into tubes containing assay buffer (30 mM Na-HEPES, 100 mM NaCl, 1 mM EGTA, 10 mM MgCl 2 , 1 mM isobutylmethylxanthine, 1 mM ATP, 10 mM phosphocreatine, 5 M GTP, 60 units/ml creatine phosphokinase, and 0.1% bovine serum albumin, pH 7.5) and drugs of interest. The mixture was incubated for 15 min at 30°C and reactions were stopped by boiling for 5 min. cAMP content of each tube was assayed for cAMP content by radioimmunoassay.
Intracellular Ca 2ϩ Measurements-Fibroblasts (0.25 ϫ 10 5 cells) were plated on 22-mm glass coverslips. The cells were washed once in phosphate-buffered saline and incubated in 1 ml of DMEM containing 1 M Fura-2/AM at 37°C for 30 min. Cells were then washed once with DMEM and placed in a 37°C chamber containing 1.5 ml of HEPESbuffered saline (HBS: 130 mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM MgCl 2 , 1.0 mM CaCl 2 , 25 mM HEPES, pH 7.4), such that groups of 5-8 cells could be viewed using an inverted Olympus IX-70 microscope. Spectrofluorometric measurements were collected using Delta Scan System spectofluorometer (Photon Technology), where the field was excited at 380 and 340 nm and the emission ratio was collected at 511 nm. Agonists were administered from ϫ1000 stocks to maintain a constant volume of 1.5 ml.
Reverse Transcriptase-PCR to Identify AC Isoforms-Total RNA was extracted from cardiac fibroblasts grown to 80 -90% confluency on 15-cm plates using Trizol reagent (Invitrogen). A DNase reaction was performed to eliminate DNA contaminants and the RNA was reverse transcribed using Superscript II (Invitrogen) and poly-T priming. Individual isoform-selective primer pairs were used to amplify each isoform of AC. Primers were based on rat or murine sequences as previously described (24). PCR reactions with each primer pair were performed on cDNA template, genomic DNA (positive control), and minus RT (negative control) template using 35 cycles and an annealing temperature of 56°C. Sequence analysis was used to confirm the identity of all PCR products.
Purification of Caveolin-enriched Membrane Fractions-Cardiac fibroblasts were fractionated using a detergent-free method adapted from Song et al. (25), and described previously (22). The faint lightscattering band resulting from sucrose density centrifugation was collected from the 5-35% sucrose interface. The bottom 4 ml of the gradient (45% sucrose) was collected as non-caveolar membranes.
Immunoblot Analysis-Individual fractions or whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis (Nu-PAGE, Invitrogen). Equal volumes of fractions were loaded so that each lane represents similar proportions from the cells, resulting in ϳ10-fold lower amounts of protein loaded in the caveolin-enriched membrane fraction lanes. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting and probed with primary antibody (see "Materials"). Bound primary antibodies were visualized by chemiluminescence. Images of immunoblot analyses and RT-PCR are representative of at least 3 separate experiments. In some experiments, the optical density of bands was calculated using a digital imaging system and LabWorks software (UVP Bioimaging Systems) and reported as arbitrary optical density units.

Assay of Collagenase-sensitive [ 3 H]Proline Incorporation-[ 3 H
]Proline incorporation by cardiac fibroblasts was measured according to modified methods of Guarda et al. (26). Briefly, cells were transferred to 12-well plates then were serum starved in 0% FBS for 24 h. Proline incorporation was then assayed by adding 1 Ci/well of [ 3 H]proline (PerkinElmer Life Sciences) along with, where indicated, drugs of interest and 2.5% FBS for 24 h. Cells were removed by trypsinization and protein precipitated overnight with 20% trichloroacetic acid. Protein was pelleted by centrifugation and washed 3 times with 1.0 ml of 5% trichloroacetic acid ϩ 0.01% proline. Pellets were dissolved with 0.2 M NaOH and the solutions titrated to neutral pH with 0.2 M HCl. Collagenase (2 mg/ml: Worthington Biochemical Corp.) in Tris/CaCl 2 /N-ethymaleimide buffer was added to each tube and samples were allowed to incubate for 1 h at 37°C. Samples were then placed on ice and proteins were precipitated with 10% trichloroacetic acid for 1 h. Samples were centrifuged at 14,000 rpm for 10 min and the collagenase-sensitive [ 3 H]proline in the supernatant was determined by liquid scintillation counting.
Analysis of Data-Statistical comparisons (t tests and one-way analysis of variance) and graphics were performed using Graph Pad Prism 3.0 (GraphPad Software).

Cross-talk between ANG II and ␤AR Signaling Pathways Is
Mediated by Phospholipase C and G q -Meszaros et al. (20) have described that ANG II, perhaps acting via G q , potentiates ␤AR and G s -mediated cAMP production in cardiac fibroblasts. In the first series of experiments we assessed whether crosstalk between G q and G s was dependent upon G q -promoted phospholipase C (PLC) activity (promoted by G q activation). We incubated cardiac fibroblasts with the specific PLC inhibitor, U73122, prior to hormonal stimulation with ANG II (100 nM) or UTP (30 M) in combination with ISO (1 M, Fig. 1). UTP provides a second class of agonist to assess cross-talk because it is an efficacious activator of the G q /PLC/inositol trisphosphate pathway in these cells (20). Neither ANG II nor UTP alone caused altered cAMP accumulation. At the concentration used in these experiments, ISO routinely produces a 10 -12-fold increase in cAMP levels in cardiac fibroblasts (20). ANG II and UTP potentiated the response to ISO by 1.8 Ϯ 0.1 and 2.0 Ϯ 0.1-fold (relative to ISO alone), respectively. Whereas U73122 alone did not affect basal cAMP accumulation or the ISO response, this inhibitor (at a concentration that reduced stimulated phosphoinositide hydrolysis 75-80%) completely eliminated the potentiation of cAMP accumulation by ANG II and UTP. These data suggest that G q -linked activation of PLC is required for the effect of G q potentiation of G s -AC activity.
To assess whether activation of G q might mediate the effects of ANG II on ␤AR signaling, we used adenoviral-mediated gene transfer of G q to increase its expression in cardiac fibroblasts (Fig. 1B). Increased expression of G q enhanced inositol phosphate accumulation by both ANG II (1.6-fold increase over control) and UTP (2.6-fold increase over control) and significantly enhanced the ANG II potentiation of ISO-stimulated cAMP (3.9 Ϯ 0.5-fold over ISO alone) compared with control cells incubated with the null (PACCMVpLpA) virus (2.5 Ϯ 0.3-fold over ISO alone). Thus, results in Fig. 1 are consistent with the conclusion that activation of GPCRs that activate G q and PLC enhance ␤AR/G s -mediated cAMP formation.
To determine whether signaling by G␤␥ subunits generated by activation of G q mediates the observed cross-talk, we used an adenovirus to express the C-terminal peptide of GRK 2 (␤ARK 1 ). This peptide (␤ARKct) binds to free G␤␥ subunits, and inhibits both G␤␥ signaling and GRK 2 activation (27). We exposed cardiac fibroblasts to either ␤ARKct or null virus and measured cAMP accumulation stimulated by ISO with and without ANG II. Expression of ␤ARKct did not attenuate ANG II-induced potentiation of ISO-stimulated cAMP accumulation in cardiac fibroblasts (null virus: ISO 10.8 Ϯ 0.2-fold over basal, ANG II ϩ ISO 19.4 Ϯ 2.2; ␤ARKct virus: ISO 9.2 Ϯ 0.5, ANG II ϩ ISO 20.8 Ϯ 0.6) but did induce a 4-fold increase in ISO potency, consistent with its action blocking GRK 2 activation (27).
Another possible pathway by which ANG II could effect cAMP production is via receptor tyrosine kinase signaling. However, cross-talk was not inhibited in cells treated with  Fig. 1 indicate that ANG II enhances ISO-mediated cAMP production via the action of G q and PLC rather than G␤␥ or tyrosine kinase-associated signal- ing, an important distinction given that ANG II activates both G protein and tyrosine kinase pathways in cardiac fibroblasts (28,29).
Requirement for Elevation of Intracellular Ca 2ϩ but Not PKC-Because G q activation by ANG II elevates intracellular Ca 2ϩ levels and activates PKC in cardiac fibroblasts (29), we examined the role of both responses as potential mechanisms by which ANG II influences cross-talk with G s . We utilized the Ca 2ϩ ionophore, ionomycin, to elevate intracellular Ca 2ϩ levels in a concentration-dependent manner ( Fig. 2A) that synergistically enhanced ISO-induced cAMP production (Fig. 2B) by 2.7 Ϯ 0.4 to 4.0 Ϯ 0.9-fold compared with ISO alone (indicated by the dashed line) using 10 and 100 nM ionomycin, respectively. Ionomycin alone did not alter basal cAMP production. Conversely, pharmacological inhibition of PKC by GF109203X (10 M) failed to inhibit the ANG II-induced potentiation of the ISO response (ANG II ϩ ISO 2.0 Ϯ 0.1-fold over ISO alone, plus GF109203X 1.9 Ϯ 0.1) but did reduce UTP-stimulated phosphoextracellular signal regulated kinase immunoreactivity (basal, 36 Ϯ 6.1 OD; 100 M UTP, 1037 Ϯ 41; UTP ϩ GF109203X, 180 Ϯ 14.4). Similar negative results were seen with the PKC inhibitors calphostin C and staurosporine (data not shown). Thus, G q /G s cross-talk appears to be mediated by the effect of ANG II to elevate intracellular Ca 2ϩ and not activation of PKC.
ANG II and Ionomycin Potentiate Forskolin-induced cAMP Production-To test whether the synergistic effects of ANG II on ISO-stimulated cAMP formation might occur via an effect distal to ␤AR, we utilized forskolin, which directly activates AC (albeit with some dependence upon G s ). Forskolin (1 M) alone stimulated cAMP production by 8 -10-fold over basal levels, a level of response similar to that seen with 0.1-1 M ISO. ANG II potentiated forskolin-induced cAMP levels (over forskolin alone) in a concentration-dependent manner in the range tested (1-100 nM), reaching statistical significance at 10 and 100 nM (1.6 and 2-fold over forskolin alone, respectively, Fig.  3A). Likewise, ionomycin (100 nM) significantly potentiated forskolin-induced cAMP levels (from 8.3 Ϯ 1.3-fold alone to 25.5 Ϯ 7.4-fold in combination, relative to controls, Fig. 3B). These results indicate that the effect of ANG II is distal to the receptor and that ionomycin can mimic this effect by elevating intracellular Ca 2ϩ .
Effects of Ca 2ϩ Store Release and Ca 2ϩ Buffering on G q -G s Cross-talk-We next sought to determine whether G q -G s crosstalk might occur via G q -promoted Ca 2ϩ release from intracellular stores and if we could inhibit the cross-talk by buffering intracellular Ca 2ϩ . Thapsigargin, which inhibits the sarcoendoplasmic reticular Ca 2ϩ -ATPase pump, elevated intracellular Ca 2ϩ levels in a concentration-dependent manner (Fig. 4A). Thapsigargin at a concentration of 100 nM was maximally efficacious in stimulating rapid Ca 2ϩ release; this concentration also synergistically enhanced forskolin-induced cAMP production ( Fig. 4B) with slight but statistically insignificant effects on basal cAMP production. We obtained similar results when these experiments were performed in the absence of extracellular Ca 2ϩ (data not shown), indicating that Ca 2ϩ release from internal stores was sufficient to observe the enhancement in cAMP formation. These results, together with those from the studies with ionomycin, indicate that potentiation of the cAMP signal can occur from elevation of intracellular Ca 2ϩ , either by storage release or influx promoted by a Ca 2ϩ ionophore. Conversely, preincubation with 1 M BAPTA/AM for 30 min prior to agonist stimulation prevented ANG II-induced Ca 2ϩ transients (Fig. 4C) and blocked the synergistic effects of ANG II on forskolin-mediated cAMP production (Fig. 4D). Thus, elevation of intracellular Ca 2ϩ is both necessary and sufficient for potentiation of cAMP production by ANG II. The results seen with forskolin indicate that Ca 2ϩ works at the level of G s /AC rather than ␤AR.
AC Isoform Expression in Cardiac Fibroblasts-Because the identity of AC isoforms expressed in cardiac fibroblasts could result in specific signaling characteristics, we sought to define the AC isoforms expressed in these cells and in particular to assess isoforms that are regulated by Ca 2ϩ . RT-PCR analysis using isoform-specific primers revealed that cardiac fibroblasts express mRNA for AC2, AC3, AC4, AC5, AC6, AC7, and AC8 (Fig. 5A). Each of the primer pairs amplified appropriate genomic DNA sequence but did not yield PCR products when RNA (no reverse transcriptase) was used as template (data not shown). PCR reactions using primers for AC1 and AC9 yielded products that were not of the expected size or sequence. We also conducted immunoblot analysis to detect expression of AC proteins. Because AC immunoreactivity in cardiac myocytes is enriched in buoyant, caveolin-rich fractions in a manner that improves immunological detection (22,30), we fractionated cardiac fibroblasts to isolate caveolin-rich fractions and performed immunoblot analyses. As shown in Fig. 5B, caveolin-1 immunoreactivity was detected in buoyant fractions (caveolinrich fraction) and was excluded from non-buoyant fractions. Immunoreactivity for AC3 and AC5/6 (the antibody used does not distinguish between AC5 and AC6) was detected primarily in caveolin-enriched membrane fractions, whereas immunoreactivity of AC2, AC4, and AC7 was detected only in non-caveolin-enriched membrane fractions (Fig. 5B). No immunoreac-

FIG. 5. Cardiac fibroblasts express multiple AC isoforms with different subcellular distributions.
A, RT-PCR analysis was performed using AC isoform-specific primer pairs (see "Experimental Procedures") and cDNA, mRNA (no reverse transcriptase negative control, not shown), or genomic DNA (positive control, not shown). Arrows indicate the size of the expected PCR product from each primer pair. Image is representative of three experiments. B, expression of AC isoforms was assessed by immunoblot analysis of caveolin-rich (cav) and non-caveolin rich (non-cav) membrane fractions from cardiac fibroblasts (see "Experimental Procedures"). Each fraction was separated by SDS-PAGE, transferred to membrane, and probed with antibodies specific for AC1, AC2, AC3, AC4, AC5/6, AC7, AC8, AC9, and caveolin 1. Nonspecific immunoreactive bands are partially evident at the top of images for AC5/6 and AC8. Each image is representative of at least three experiments. tivity was detected for AC8 or AC9 (Fig. 5B). Thus, cardiac fibroblasts express numerous AC isoforms but the isoforms appear to be differentially localized in caveolin-rich membrane microdomains.
AC Isoform Specificity of G q -G s Cross-talk-We hypothesized that expression of a Ca 2ϩ -calmodulin-stimulable isoform of AC was essential for the cross-talk we observe. Thus, we pretreated cardiac fibroblasts with a specific calmodulin inhibitor (10 M calmidazolium) and found that it significantly attenuated cross-talk induced by ANG II plus ISO or by ionomycin plus forskolin (Fig. 6, A and B, respectively). We also measured Ca 2ϩ and forskolin-stimulated AC activity in membrane preparations from cardiac fibroblasts with and without exogenously added calmodulin; 3 M free Ca 2ϩ in the presence of forskolin (10 M) did not stimulate AC activity unless calmodulin (0.1 M) was added to the reaction (Fig. 6C). This Ca 2ϩ -stimulable AC activity was inhibited by the inclusion of a calmodulin inhibitor (1 M fluphenazine, which did not alter basal or forskolin-stimulated AC activity). These results indicate that Ca 2ϩ -calmodulin activation of AC is a plausible mechanism for the effect of ANG II and ionomycin to enhance cAMP production by ␤AR.
We next probed whether it was possible to attenuate crosstalk between ANG II and cAMP production by altering the balance of Ca 2ϩ -stimulated versus Ca 2ϩ -insensitive AC isoforms by using an adenovirus to overexpress an isoform of AC that is not Ca 2ϩ -stimulable, AC6 (14). Incubation of cardiac fibroblasts with the AC6 adenovirus for 18 h resulted in a 2-fold increase in maximal forskolin-stimulated cAMP production (Fig. 6D). Based on evidence that the extent of AC overexpression is proportional to the increase in maximal forskolin response (13), the conditions used here approximately doubled the total cellular content of AC and generated cardiac fibroblasts expressing predominantly AC6. Immunoblot analyses indicated an appreciable increase in detectible AC5/6 protein in caveolin-enriched membrane fractions (Fig. 6D, inset). ANG II FIG. 6. Calmidazolium and overexpression of AC6 inhibit cross-talk. A, cardiac fibroblasts were pretreated with the calmodulin inhibitor calmidazolium (CDZ) for 30 min prior to reactions. The potentiation effect produced by the combination of ANG II and ISO was significantly blocked in the presence of CDZ, but did not have a significant effect on ISO alone. Data are mean Ϯ S.E. expressed as fold-change over control. *, statistically significant differences versus ANG II ϩ ISO (*, p Ͻ 0.05) by Student's t test. B, cells were pretreated with CDZ for 30 min prior to reactions. CDZ significantly inhibited cAMP potentiation induced by IN and FSK, but did not have a significant effect on FSK alone. Data are mean Ϯ S.E. expressed as fold-change over control. *, statistically significant differences versus IN ϩ FSK (*, p Ͻ 0.05) by Student's t test. C, AC activity was measured in cardiac fibroblast membranes with or without calmodulin (0.1 M) and with or without fluphenazine (1 M). Data are expressed as -fold over basal AC activity. #, statistically significant difference (p Ͻ 0.05) versus Fsk alone; **, statistically significant difference (p Ͻ 0.01) versus control by Student's t test. D, production of cAMP in response to forskolin or forskolin plus ANG II was measured in control and AC6-overexpressing cardiac fibroblasts. ANG II potentiation of FSK was significant (p Ͻ 0.01) by Student's t test) in control cells but not significant (p ϭ 0.14) in AC6 overexpressing cells. Immunoblot analysis indicated an appreciable increase in AC5/6 protein in caveolin-enriched membrane (Cav) fractions of cardiac fibroblasts incubated with AC6 adenovirus (inset). potentiation of forskolin-stimulated cAMP was unchanged by AC6 overexpression in terms of absolute amount of cAMP stimulated over forskolin alone (14.2 Ϯ 1.4 pmol of cAMP/mg of protein in control cells versus 17.7 Ϯ 8.8 in AC6 cells), but was significantly inhibited in terms of the fractional effect (p ϭ 0.14, Fig. 6D). Thus, the potentiative effect of ANG II appeared not to extend to the overexpressed AC6. The inability of ANG II to potentiate forskolin-stimulated cAMP production to the same degree in AC6-overexpressing cells was not related to a limited capacity of cardiac fibroblasts to synthesize cAMP in this latter condition, as combined treatment of the cells with ISO and forskolin-stimulated cAMP levels to 27.6 Ϯ 3.48 pmol/mg protein, a level nearly 3-fold higher than the combination of forskolin and ANG II. Thus, cross-talk is decreased in cells manipulated to express increased levels of AC6, an AC isoform not activated by Ca 2ϩ . Taken together, the data in Figs. 1-6 implicate the central role of both Ca 2ϩ influx and a Ca 2ϩcalmodulin-stimulable AC isoform in mediating the observed G q -G s cross-talk.
Role of Cross-talk on Collagen Synthesis-To investigate the functional relevance of the cross-talk between ANG II signaling and the cAMP pathway, we assessed the synthesis of collagen by cardiac fibroblasts. Increased cellular cAMP levels can inhibit cardiac fibroblast-mediated production of collagen (31). Thus, we tested whether simultaneous incubation with cAMPelevating agents and ANG II would inhibit collagen synthesis to a greater extent than incubation with cAMP-elevating agents alone. FBS-stimulated collagenase-sensitive [ 3 H]proline incorporation was measured in cardiac fibroblasts as an index of collagen formation in the absence and presence of ANG II and in the absence and presence of ISO. 2.5% FBS stimulated collagen synthesis 2.6 Ϯ 0.2-fold over basal levels. ANG II alone increased collagen synthesis (data not shown) but in the presence of 2.5% FBS induced no significant increase over FBS alone (Fig. 7A). ISO (100 M) inhibited FBS-stimulated collagen synthesis (31 Ϯ 9% inhibition, Fig. 7, A and B). However, in the presence of ANG II, ISO inhibited proline incorporation 48 Ϯ 8%, an effect that was significantly different (p Ͻ 0.01) from serum-stimulated levels (Fig. 7, A and B). Moreover, the ANG II enhancement of the ISO effect was reduced to nonsignificant levels by chelation of Ca 2ϩ (with 0.1 M BAPTA/AM) or inhibition of calmodulin (with 0.1 M calmidazolium). Thus, the data support the conclusion that the enhanced ␤AR-mediated cAMP production that ANG II promotes via elevation of Ca 2ϩ and signaling via calmodulin can blunt collagen synthesis. Taken together, the data suggest that coincidental G s and G q signals interact to modulate cardiac fibroblast signaling and function, and thereby promote a G s -mediated inhibition of collagen synthesis. DISCUSSION The focus of the present work was to define the mechanism of G q -G s cross-talk, which we previously described in adult rat cardiac fibroblasts (20). The striking potentiation of ␤AR signaling in the presence of ANG II might occur via several different mechanisms. ANG II activates AT 1 receptors in cardiac fibroblasts, thereby activating both G q -coupled and tyrosine kinase signaling pathways (28,29). Several pieces of evidence indicate that this cross-talk is mediated by activation of G q : 1) at least 2 other G q -coupled receptors (P2Y and bradykinin) are capable of inducing similar potentiation (Fig. 1A and Ref. 20); 2) blockade of PLC eliminates potentiation of ISO response by either ANG II or UTP (Fig. 1A); 3) overexpression of G q enhances cross-talk (Fig. 1B); 4) chelation of a key second messenger of the G q pathway, intracellular Ca 2ϩ , results in attenuation of cross-talk; and 5) pharmacological agents that directly increase cytosolic Ca 2ϩ concentrations (ionomycin, thapsigargin) also potentiate cAMP accumulation (Figs. 2 and 3). These data, along with evidence that exclusion of extracellular Ca 2ϩ does not effect cross-talk (data not shown), indicate that ANG II-induced potentiation of ␤AR signaling is triggered by G q activation and Ca 2ϩ release from internal stores and that Ca 2ϩ influx is not required for enhanced cAMP production. Consistent with this conclusion, pharmacological inhibition of the other major pathway activated by G q , PKC, had no effect on cross-talk.
Our studies with forskolin allowed us to test the target at which Ca 2ϩ acts to increase ␤AR signaling. As a direct activator of AC, albeit requiring G s activation for full effect (32), forskolin-stimulated cAMP formation was also activated by coincident signals that elevate intracellular Ca 2ϩ (e.g. ANG II, ionomycin, or thapsigargin, Figs. 3 and 4). These results led us to focus on the role of AC in G q -G s cross-talk. We were surprised to discover that cardiac fibroblasts express mRNA for seven of the nine transmembrane AC isoforms (Fig. 5). This expression of multiple isoforms contrasts with results from cardiac myocytes, which appear to express predominantly 2 isoforms, AC5 and AC6 (15). Of the isoforms expressed in cardiac fibroblasts, the protein for AC3 appears to be predominantly expressed, particularly in caveolin-rich fractions (Fig.  5B).
Given its level of expression and action as a Ca 2ϩ -stimulable isoform, AC3 is likely critical for the cross-talk that we observe in cardiac fibroblasts. The other AC isoforms capable of being activated by Ca 2ϩ , AC1 and AC8, were not detected by immunoblot analysis (although AC8 mRNA was detected in PCR studies). Previous data using heterologously expressed protein indicate that AC3 stimulation by Ca 2ϩ is dependent upon coincidental activation of AC by either G s or forskolin (18), results consistent with what we observe in cardiac fibroblasts. An important role for a Ca 2ϩ -stimulable AC isoform in the observed cross-talk is inferred by data from vascular smooth muscle (33) and directly supported by our studies overexpressing AC6. By increasing AC6 expression, we altered the balance of AC isoform expression in favor of an isoform not stimulated by Ca 2ϩ and found that the G q -G s cross-talk was fractionally reduced. Taken together, these data provide a clear-cut example of AC isoform expression imparting distinct signaling characteristics and regulation of cell function. The membrane microdomains in which expression of AC isoforms occurs can influence cell responses (22,34). Caveolae are a subset of lipid rafts, plasma membrane microdomains enriched in sphingolipid and cholesterol, and are the site of enrichment of many membrane-associated signaling molecules (35). We found that among numerous AC isoforms expressed in cardiac fibroblasts, only AC3 and AC5/6 localize in buoyant, caveolin-rich domains. These data are the first of which we are aware that show that native AC isoforms localize differently with respect to caveolae/lipid rafts. Smith et al. (36) recently reported that AC7 and AC8 heterologously expressed in HEK-293 cells localized differently with respect to caveolin-rich domains (AC8 in caveolar fractions, AC7 in non-caveolar fractions). The reason for the difference between the data from Smith et al. (36) and the present studies (we observe AC8 in non-caveolar fractions, Fig. 5) is unclear, but localization of GPCR and AC isoforms in caveolar fractions can be highly cell-specific and can differ between exogenously and endogenously expressed protein (24). More work is needed to understand the functional importance, if any, of the observed pattern of AC isoform expression in different plasma membrane microdomains of these cells.
Our studies of collagen synthesis (Fig. 7) suggest the physiological importance of G q -G s cross-talk. Agents that elevate cellular cAMP attenuate collagen synthesis by cardiac fibroblasts (5). In our studies, ISO alone (in the absence of a phosphodiesterase inhibitor) could significantly inhibit collagen synthesis, and the addition of ANG II, which produced a 2-fold increase in ISO-stimulated cAMP accumulation, led to a greater reduction in collagen synthesis. These results appear to conflict with observations that ␤AR antagonists can reduce fibrosis in heart failure (37,38). However, the beneficial effects of ␤AR blockade are likely dominated by improvements in cardiac contractility (which would lead to decreased levels of renin-angiotensin and possibly other hormones) and myocyte survival, which in turn influence ECM formation and turnover as well as fibroblast proliferation. Our findings are consistent with data on certain other G s -linked agonist action on cardiac fibroblasts (31,39) and with a previous report that the ␤AR antagonist, propranolol, inhibits collagen deposition in pulmonary fibroblasts, cells not subjected to the mechanical forces of a contractile organ (40). In addition, our studies examine fibroblasts over a short period (24 h) and in isolation, removed from circulating catecholamines and the mechanical forces of cardiac contraction.
In conclusion, the current results demonstrate that ANG II enhances ␤AR signaling via activation of G q and elevated intracellular Ca 2ϩ . We propose that a Ca 2ϩ -stimulable isoform of AC, likely AC3, is the regulatory site of this Ca 2ϩ /calmodulindependent potentiation and acts as an integrator of these signaling pathways. Moreover, cross-talk between the G q and G s pathways (i.e. enhanced cAMP production) appears to have functional consequences for regulation of the ECM in myocardium, acting to limit collagen synthesis.