Angiotensin II Enhances Adenylyl Cyclase Signaling via Ca2+/Calmodulin
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m212659200
ISSN1083-351X
AutoresRennolds S. Ostrom, Jennifer E. Naugle, Miki Hase, Caroline Gregorian, James S. Swaney, Paul A. Insel, Laurence L. Brunton, J. Gary Meszaros,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoCardiac 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 β-adrenergic receptors (β-AR). The potentiation of β-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ 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 Gβγ did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-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 βAR-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 βAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts. 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 β-adrenergic receptors (β-AR). The potentiation of β-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ 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 Gβγ did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-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 βAR-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 βAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts. Cardiac hypertrophy is associated with increased cardiac mass, a gradual decline in contractile function and eventual heart failure. The remodeling associated with these changes involves an altered balance of synthesis and degradation of extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; GPCR, G protein-coupled receptors; ANG II, angiotensin II; AC, adenylyl cyclase; PKC, protein kinase C; ISO, isoproterenol; βAR, β-adrenergic receptors; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RT, reverse transcriptase; PLC, phospholipase C. by cardiac fibroblasts and can lead to abnormal accumulation of ECM in the interstitial space (i.e. fibrosis) (1Eghbali M. Weber K.T. Mol. Cell. Biochem. 1990; 96: 1-14Google Scholar, 2Weber K.T. Sun Y. Tyagi S.C. Cleutjens J.P. J. Mol. Cell Cardiol. 1994; 26: 279-292Google Scholar). Several G protein-coupled receptors (GPCR) that signal through Gq have been implicated in the pathogenesis of cardiac hypertrophy and failure (3Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn 2nd, G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Google Scholar, 4Ramirez M.T. Sah V.P. Zhao X.L. Hunter J.J. Chien K.R. Brown J.H. J. Biol. Chem. 1997; 272: 14057-14061Google Scholar). Conversely, GPCRs that signal via Gs may inhibit collagen deposition by cardiac fibroblasts (5Dubey R.K. Gillespie D.G. Jackson E.K. Hypertension. 1998; 31: 943-948Google Scholar). Two major pro-fibrotic signals in the heart are cytokines, such as transforming growth factor-β, and the peptide hormone ANG II, both of which increase collagen synthesis by cardiac fibroblasts (6Weber K.T. Circulation. 1997; 96: 4065-4082Google Scholar, 7Lee A.A. Dillmann W.H. McCulloch A.D. Villarreal F.J. J. Mol. Cell. Cardiol. 1995; 27: 2347-2357Google Scholar). ANG II also inhibits matrix metalloproteinase expression by cardiac fibroblasts, thereby attenuating degradation of ECM proteins (8Brilla C.G. Rupp H. Funck R. Maisch B. Eur. Heart J. 1995; 16: 107-109Google Scholar). An anti-fibrotic role for cAMP is supported by evidence that adenosine and prostacyclin inhibit cardiac fibroblast proliferation and collagen synthesis through activation of A2b and prostanoid receptors, respectively, which each couple via Gs, to enhanced cAMP production (9Dubey R.K. Gillespie D.G. Zacharia L.C. Mi Z. Jackson E.K. Hypertension. 2001; 37: 716-721Google Scholar, 10Yu H. Gallagher A.M. Garfin P.M. Printz M.P. Hypertension. 1997; 30: 1047-1053Google Scholar). β-Adrenergic receptors have been shown to stimulate fibroblast proliferation via epidermal growth factor receptor transactivation (11Kim J. Eckhart A.D. Eguchi S. Koch W.J. J. Biol. Chem. 2002; 277: 32116-32123Google Scholar) 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 (12Post S.R. Hilal-Dandan R. Urasawa K. Brunton L.L. Insel P.A. Biochem. J. 1995; 311: 75-80Google Scholar, 13Gao M. Ping P. Post S. Insel P.A. Tang R. Hammond H.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1038-1043Google Scholar). Nine different transmembrane AC isoforms exist, each with different amino acid sequence, tissue and chromosomal distribution, and regulation (14Hanoune J. Defer N. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 145-174Google Scholar). Differences in regulation include stimulation or inhibition by Gβγ, Ca2+, and various protein kinases. AC5 and AC6, which are the predominant isoforms expressed in cardiac myocytes (15Ishikawa Y. Sorota S. Kiuchi K. Shannon R.P. Komamura K. Katsushika S. Vatner D.E. Vatner S.F. Homcy C.J. J. Clin. Invest. 1994; 93: 2224-2229Google Scholar), represent a subfamily of ACs that are related in structure and regulation. These isoforms are inhibited by protein kinase A, Ca2+, Gi, Gβγ, and nitric oxide (14Hanoune J. Defer N. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 145-174Google Scholar, 16McVey M. Hill J. Howlett A. Klein C. J. Biol. Chem. 1999; 274: 18887-18892Google Scholar, 17Hill J. Howlett A. Klein C. Cell Signal. 2000; 12: 233-237Google Scholar). By contrast, AC1, AC3, and AC8 are stimulated by Ca2+/calmodulin (yet AC3 can also be inhibited by calmodulin kinase-II) (14Hanoune J. Defer N. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 145-174Google Scholar, 18Choi E.J. Xia Z. Storm D.R. Biochemistry. 1992; 31: 6492-6498Google Scholar, 19Wei J. Wayman G. Storm D.R. J. Biol. Chem. 1996; 271: 24231-24235Google Scholar). AC2 and AC4 are activated by Gβγ and AC2 and AC7 can be activated by phosphorylation by protein kinase A and/or PKC (14Hanoune J. Defer N. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 145-174Google Scholar). Thus, the AC isoform expression in a cell determines the interaction between cAMP production and other signal transduction cascades. Meszaros et al. (20Meszaros J.G. Gonzalez A.M. Endo-Mochizuki Y. Villegas S. Villarreal F.J. Brunton L.L. Am. J. Physiol. 2000; 278: C154-C162Google Scholar) have recently described a signaling "cross-talk" between two key GPCR signal transduction pathways in cardiac fibroblasts. Isoproterenol (ISO), a β-adrenergic receptor (βAR) agonist, activates Gs and stimulates cAMP production 10-fold over basal levels. ANG II activates Gq-coupled angiotensin receptors that, by themselves, do not alter cAMP production in cardiac fibroblasts, but in combination with ISO potentiate the βAR 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 Gq-mediated elevation of intracellular Ca2+ induced by ANG II potentiates Gs-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 Ca2+ enhances cAMP production and limits collagen synthesis, and perhaps fibrosis, in the heart. 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 (≤3) 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 (21Gustafsson A.B. Brunton L.L. Mol. Pharmacol. 2000; 58: 1470-1478Google Scholar). Adenoviral Gene Transfer to Cardiac Fibroblasts—Wild-type Gq was cloned into the PACCMVpLpA shuttle vector for production of adenovirus, as previously described (3Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn 2nd, G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Google Scholar). We found that a titer of 200 virus particles per cell was optimal for Gq 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 (22Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079Google Scholar). 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 β-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 NaHCO3-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 μl 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 (22Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079Google Scholar). 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 (23Ostrom R.S. Gregorian C. Drenan R.M. Xiang Y. Regan J.W. Insel P.A. J. Biol. Chem. 2001; 276: 42063-42069Google Scholar). 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 MgCl2, 1 mm EGTA, 2 mm dithiothreitol, pH 7.5) and homogenizing with 20 strokes in a Dounce homogenizer. Homogenate was spun at 300 × g for 5 min at 4 °C. Supernatant was then transferred to a clean centrifuge tube and spun at 5,000 × g for 10 min. Pellet was suspended in membrane buffer (30 mm Na-HEPES, 5 mm MgCl2, 2 mm dithiothreitol, pH 7.5) to attain ∼1 mg/ml total protein concentration. Assay was conducted by adding 30 μl of membranes into tubes containing assay buffer (30 mm Na-HEPES, 100 mm NaCl, 1 mm EGTA, 10 mm MgCl2, 1 mm isobutylmethylxanthine, 1 mm ATP, 10 mm phosphocreatine, 5 μm 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 Ca2+ Measurements—Fibroblasts (0.25 × 105 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 μm 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 HEPES-buffered saline (HBS: 130 mm NaCl, 5 mm KCl, 10 mm glucose, 1 mm MgCl2, 1.0 mm CaCl2, 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 ×1000 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 (24Ostrom R.S. Liu X. Head B.P. Gregorian C. Seasholtz T.M. Insel P.A. Mol. Pharmacol. 2002; 62: 983-992Google Scholar). 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. (25Song S.K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Google Scholar), and described previously (22Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079Google Scholar). The faint light-scattering 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 ∼10-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 [3H]Proline Incorporation—[3H]Proline incorporation by cardiac fibroblasts was measured according to modified methods of Guarda et al. (26Guarda E. Katwa L.C. Myers P.R. Tyagi S.C. Weber K.T. Cardiovasc. Res. 1993; 27: 2130-2134Google Scholar). 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 μCi/well of [3H]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 + 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/CaCl2/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 [3H]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 βAR Signaling Pathways Is Mediated by Phospholipase C and Gq—Meszaros et al. (20Meszaros J.G. Gonzalez A.M. Endo-Mochizuki Y. Villegas S. Villarreal F.J. Brunton L.L. Am. J. Physiol. 2000; 278: C154-C162Google Scholar) have described that ANG II, perhaps acting via Gq, potentiates βAR and Gs-mediated cAMP production in cardiac fibroblasts. In the first series of experiments we assessed whether cross-talk between Gq and Gs was dependent upon Gq-promoted phospholipase C (PLC) activity (promoted by Gq activation). We incubated cardiac fibroblasts with the specific PLC inhibitor, U73122, prior to hormonal stimulation with ANG II (100 nm)or UTP (30 μm) in combination with ISO (1 μm, Fig. 1). UTP provides a second class of agonist to assess cross-talk because it is an efficacious activator of the Gq/PLC/inositol trisphosphate pathway in these cells (20Meszaros J.G. Gonzalez A.M. Endo-Mochizuki Y. Villegas S. Villarreal F.J. Brunton L.L. Am. J. Physiol. 2000; 278: C154-C162Google Scholar). 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 (20Meszaros J.G. Gonzalez A.M. Endo-Mochizuki Y. Villegas S. Villarreal F.J. Brunton L.L. Am. J. Physiol. 2000; 278: C154-C162Google Scholar). ANG II and UTP potentiated the response to ISO by 1.8 ± 0.1 and 2.0 ± 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 Gq-linked activation of PLC is required for the effect of Gq potentiation of Gs-AC activity. To assess whether activation of Gq might mediate the effects of ANG II on βAR signaling, we used adenoviral-mediated gene transfer of Gq to increase its expression in cardiac fibroblasts (Fig. 1B). Increased expression of Gq 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 ± 0.5-fold over ISO alone) compared with control cells incubated with the null (PACCMVpLpA) virus (2.5 ± 0.3-fold over ISO alone). Thus, results in Fig. 1 are consistent with the conclusion that activation of GPCRs that activate Gq and PLC enhance βAR/Gs-mediated cAMP formation. To determine whether signaling by Gβγ subunits generated by activation of Gq mediates the observed cross-talk, we used an adenovirus to express the C-terminal peptide of GRK2 (βARK1). This peptide (βARKct) binds to free Gβγ subunits, and inhibits both Gβγ signaling and GRK2 activation (27Drazner M.H. Peppel K.C. Dyer S. Grant A.O. Koch W.J. Lefkowitz R.J. J. Clin. Invest. 1997; 99: 288-296Google Scholar). We exposed cardiac fibroblasts to either βARKct or null virus and measured cAMP accumulation stimulated by ISO with and without ANG II. Expression of βARKct did not attenuate ANG II-induced potentiation of ISO-stimulated cAMP accumulation in cardiac fibroblasts (null virus: ISO 10.8 ± 0.2-fold over basal, ANG II + ISO 19.4 ± 2.2; βARKct virus: ISO 9.2 ± 0.5, ANG II + ISO 20.8 ± 0.6) but did induce a 4-fold increase in ISO potency, consistent with its action blocking GRK2 activation (27Drazner M.H. Peppel K.C. Dyer S. Grant A.O. Koch W.J. Lefkowitz R.J. J. Clin. Invest. 1997; 99: 288-296Google Scholar). 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 AG1478 (10 μm), a specific tyrosine kinase inhibitor (ANG II + ISO, 2.0 ± 0.1-fold over ISO alone; ANG II + ISO + AG1478 2.0 ± 0.1) or with PP2 (1 μm), a selective Src inhibitor (data not shown). These findings and those of Fig. 1 indicate that ANG II enhances ISO-mediated cAMP production via the action of Gq and PLC rather than Gβγ or tyrosine kinase-associated signaling, an important distinction given that ANG II activates both G protein and tyrosine kinase pathways in cardiac fibroblasts (28Dostal D.E. Booz G.W. Baker K.M. Mol. Cell. Biochem. 1996; 157: 15-21Google Scholar, 29Hou M. Pantev E. Moller S. Erlinge D. Edvinsson L. Acta Physiol. Scand. 2000; 168: 301-309Google Scholar). Requirement for Elevation of Intracellular Ca2+ but Not PKC—Because Gq activation by ANG II elevates intracellular Ca2+ levels and activates PKC in cardiac fibroblasts (29Hou M. Pantev E. Moller S. Erlinge D. Edvinsson L. Acta Physiol. Scand. 2000; 168: 301-309Google Scholar), we examined the role of both responses as potential mechanisms by which ANG II influences cross-talk with Gs. We utilized the Ca2+ ionophore, ionomycin, to elevate intracellular Ca2+ levels in a concentration-dependent manner (Fig. 2A) that synergistically enhanced ISO-induced cAMP production (Fig. 2B) by 2.7 ± 0.4 to 4.0 ± 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 μm) failed to inhibit the ANG II-induced potentiation of the ISO response (ANG II + ISO 2.0 ± 0.1-fold over ISO alone, plus GF109203X 1.9 ± 0.1) but did reduce UTP-stimulated phosphoextracellular signal regulated kinase immunoreactivity (basal, 36 ± 6.1 OD; 100 μm UTP, 1037 ± 41; UTP + GF109203X, 180 ± 14.4). Similar negative results were seen with the PKC inhibitors calphostin C and staurosporine (data not shown). Thus, Gq/Gs cross-talk appears to be mediated by the effect of ANG II to elevate intracellular Ca2+ 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 βAR, we utilized forskolin, which directly activates AC (albeit with some dependence upon Gs). Forskolin (1 μm) alone stimulated cAMP production by 8–10-fold over basal levels, a level of response similar to that seen with 0.1–1 μm 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 ± 1.3-fold alone to 25.5 ± 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 Ca2+. Effects of Ca2+ Store Release and Ca2+ Buffering on Gq-GsCross-talk—We next sought to determine whether Gq-Gs cross-talk might occur via Gq-promoted Ca2+ release from intracellular stores and if we could inhibit the cross-talk by buffering intracellular Ca2+. Thapsigargin, which inhibits the sarcoendoplasmic reticular Ca2+-ATPase pump, elevated intracellular Ca2+ levels in a concentration-dependent manner (Fig. 4A). Thapsigargin at a concentration of 100 nm was maximally efficacious in stimulating rapid Ca2+ 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 Ca2+ (data not shown), indicating that Ca2+ 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 Ca2+, either by storage release or influx promoted by a Ca2+ ionophore. Conversely, preincubation with 1 μm BAPTA/AM for 30 min prior to agonist stimulation prevented ANG II-induced Ca2+ transients (Fig. 4C) and blocked the synergistic effects of ANG II on forskolin-mediated cAMP production (Fig. 4D). Thus, elevation of intracellular Ca2+ is both necessary and sufficient for potentiation of cAMP production by ANG II. The results seen with forskolin indicate that Ca2+ works at the level of Gs/AC rather than βAR. 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 Ca2+. 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 (22Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079Google Scholar, 30Schwencke C. Okumura S. Yamamoto M. Geng Y.J. Ishikawa Y. J. Cell. Biochem. 1999; 75: 64-72Google Scholar), 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 (caveolin-rich 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 immunore-activity of AC2, AC4, and AC7 was detected only in non-caveolin-enriched membrane fractions (Fig. 5B). No immunoreactivity 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 Gq-GsCross-talk—We hypothesized that expression of a Ca2+-calmodulin-stimulable isoform of AC was ess
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