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Nitric Oxide Inhibition of Adenylyl Cyclase Type 6 Activity Is Dependent upon Lipid Rafts and Caveolin Signaling Complexes

2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês

10.1074/jbc.m313440200

1083-351X

Rennolds S. Ostrom, Richard A. Bundey, Paul A. Insel,

Metabolism, Diabetes, and Cancer

Several cell types, including cardiac myocytes and vascular endothelial cells, produce nitric oxide (NO) via both constitutive and inducible isoforms of NO synthase. NO attenuates cardiac contractility and contributes to contractile dysfunction in heart failure, although the precise molecular mechanisms for these effects are poorly defined. Adenylyl cyclase (AC) isoforms type 5 and 6, which are preferentially expressed in cardiac myocytes, may be inhibited via a direct nitrosylation by NO. Because endothelial NO synthase (eNOS and NOS3), β-adrenergic (βAR) receptors, and AC6 all can localize in lipid raft/caveolin-rich microdomains, we sought to understand the role of lipid rafts in organizing components of βAR-Gs-AC signal transduction together with eNOS. Using neonatal rat cardiac myocytes, we found that disruption of lipid rafts with β-cyclodextrin inhibited forskolin-stimulated AC activity and cAMP production, eliminated caveolin-3-eNOS interaction, and increased NO production. βAR- and Gs-mediated activation of AC activity were inhibited by β-cyclodextrin treatment, but prostanoid receptor-stimulated AC activity, which appears to occur outside caveolin-rich microdomains, was unaffected unless eNOS was overexpressed and lipid rafts were disrupted. An NO donor, SNAP, inhibited basal and forskolin-stimulated cAMP production in both native cardiac myocytes and cardiac myocytes and pulmonary artery endothelial cells engineered to overexpress AC6. These effects of SNAP were independent of guanylyl cyclase activity and were mimicked by overexpression of eNOS. The juxtaposition of eNOS with βAR and AC types 5 and 6 results in selective regulation of βAR by eNOS activity in lipid raft domains over other Gs-coupled receptors localized in nonraft domains. Thus co-localization of multiple signaling components in lipid rafts provides key spatial regulation of AC activity. Several cell types, including cardiac myocytes and vascular endothelial cells, produce nitric oxide (NO) via both constitutive and inducible isoforms of NO synthase. NO attenuates cardiac contractility and contributes to contractile dysfunction in heart failure, although the precise molecular mechanisms for these effects are poorly defined. Adenylyl cyclase (AC) isoforms type 5 and 6, which are preferentially expressed in cardiac myocytes, may be inhibited via a direct nitrosylation by NO. Because endothelial NO synthase (eNOS and NOS3), β-adrenergic (βAR) receptors, and AC6 all can localize in lipid raft/caveolin-rich microdomains, we sought to understand the role of lipid rafts in organizing components of βAR-Gs-AC signal transduction together with eNOS. Using neonatal rat cardiac myocytes, we found that disruption of lipid rafts with β-cyclodextrin inhibited forskolin-stimulated AC activity and cAMP production, eliminated caveolin-3-eNOS interaction, and increased NO production. βAR- and Gs-mediated activation of AC activity were inhibited by β-cyclodextrin treatment, but prostanoid receptor-stimulated AC activity, which appears to occur outside caveolin-rich microdomains, was unaffected unless eNOS was overexpressed and lipid rafts were disrupted. An NO donor, SNAP, inhibited basal and forskolin-stimulated cAMP production in both native cardiac myocytes and cardiac myocytes and pulmonary artery endothelial cells engineered to overexpress AC6. These effects of SNAP were independent of guanylyl cyclase activity and were mimicked by overexpression of eNOS. The juxtaposition of eNOS with βAR and AC types 5 and 6 results in selective regulation of βAR by eNOS activity in lipid raft domains over other Gs-coupled receptors localized in nonraft domains. Thus co-localization of multiple signaling components in lipid rafts provides key spatial regulation of AC activity. Seven membrane-spanning G protein-coupled receptors (GPCR) 1The abbreviations used are: GPCR, G protein-coupled receptor(s); AC, adenylyl cyclase(s); βAR, β-adrenergic receptor; βCD, β-cyclodextrin; cAMP, cyclic AMP; eNOS, endothelial nitric-oxide synthase; EP2R, prostanoid EP2 receptor; l-NMMA, N-monomethyl-l-arginine; NO, nitric oxide; NOS, nitric-oxide synthase; PDE, phosphodiesterase; PGE2, prostaglandin E2, SNAP, (±)-S-nitroso-N-acetylpenicillamine; MES, 2-(N-morpholino)ethanesulfonic acid; CPAE, calf pulmonary artery endothelial cell(s). signal via heterotrimeric G-proteins that regulate effector molecules that generate second messengers. Numerous GPCR, including β-adrenergic (βAR) and certain prostanoid receptors, couple to Gs to stimulate adenylyl cyclase (AC) activity and the generation of cyclic AMP (cAMP). In cardiac myocytes, increased cAMP levels change several aspects of cardiac function to enhance, for example, rate and force of contraction and the rate of relaxation. cAMP action primarily occurs via activation of protein kinase A, which alters intracellular Ca2+ dynamics and contractile function by phosphorylating calcium channels, troponin I and phospholamban (1Rapundalo S.T. Solaro R.J. Kranias E.G. Circ. Res. 1989; 64: 104-111Crossref PubMed Scopus (121) Google Scholar, 2Hartzell H.C. Mery P.F. Fischmeister R. Szabo G. Nature. 1991; 351: 573-576Crossref PubMed Scopus (195) Google Scholar). βAR, activated by catecholamines, is the predominant Gs-coupled GPCR in cardiac myocytes, but other receptors are also capable of regulating AC activity in these cells (3Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar). We and others have demonstrated that β1AR are efficiently coupled to activation of AC in cardiac myocytes due to their high degree of co-localization in a membrane microdomain composed of lipid rafts or caveolae, where AC is preferentially expressed (3Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar, 4Ostrom R.S. Gregorian C. Drenan R.M. Xiang Y. Regan J.W. Insel P.A. J. Biol. Chem. 2001; 276: 42063-42069Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 5Schwencke C. Yamamoto M. Okumura S. Toya Y. Kim S.J. Ishikawa Y. Mol. Endocrinol. 1999; 13: 1061-1070PubMed Google Scholar, 6Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). β2AR and prostanoid EP2R and EP4R couple with lower efficiency to AC due to either a transient localization in lipid rafts (β2AR) or exclusion from these microdomains (EP2/4R) (4Ostrom R.S. Gregorian C. Drenan R.M. Xiang Y. Regan J.W. Insel P.A. J. Biol. Chem. 2001; 276: 42063-42069Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Thus, co-localization of components in a signal transduction cascade appears to be a critical determinant of signaling efficiency by receptors that stimulate AC. Caveolae, detectable as plasma membrane invaginations enriched in the protein caveolin, are considered a subset of lipid rafts, membrane regions that are enriched in sphingolipids and cholesterol. The distinct lipid environment in lipid rafts and caveolae favors retention of certain plasma membrane proteins, creating a unique signaling microdomain (7Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, 8Anderson R. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1739) Google Scholar, 9Razani B. Woodman S.E. Lisanti M.P. Pharmacol. Rev. 2002; 54: 431-467Crossref PubMed Scopus (855) Google Scholar, 10Pike L.J. J. Lipid Res. 2003; 44: 655-667Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar, 11Foster L.J. De Hoog C.L. Mann M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5813-5818Crossref PubMed Scopus (735) Google Scholar). The fact that only certain GPCR, AC, and portions of cellular Gs reside in this microdomain challenges the traditional concept that membrane-associated signaling proteins are randomly distributed in the plasma membrane and interact via diffusion (9Razani B. Woodman S.E. Lisanti M.P. Pharmacol. Rev. 2002; 54: 431-467Crossref PubMed Scopus (855) Google Scholar). Instead, many proteins involved in GPCR signal transduction are apparently restricted to lipid raft domains, perhaps in preformed signaling complexes that facilitate rapid and specific signal transduction (12Ostrom R.S. Post S.R. Insel P.A. J. Pharmacol. Exp. Ther. 2000; 294: 407-412PubMed Google Scholar, 13Steinberg S.F. Brunton L.L. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 751-773Crossref PubMed Scopus (321) Google Scholar) and provide the close interaction needed for cross-talk between molecules of other signal transduction pathways (9Razani B. Woodman S.E. Lisanti M.P. Pharmacol. Rev. 2002; 54: 431-467Crossref PubMed Scopus (855) Google Scholar, 14Fagan K.A. Smith K.E. Cooper D.M. J. Biol. Chem. 2000; 275: 26530-26537Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 15Shaul P.W. Annu. Rev. Physiol. 2002; 64: 749-774Crossref PubMed Scopus (480) Google Scholar). One signaling molecule highly enriched in lipid rafts is endothelial nitric-oxide synthase (eNOS, NOS3) (16Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). NO is important for cardiovascular physiology (17Kelly R.A. Balligand J.L. Smith T.W. Circ. Res. 1996; 79: 363-380Crossref PubMed Scopus (634) Google Scholar, 18Feron O. Saldana F. Michel J.B. Michel T. J. Biol. Chem. 1998; 273: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), but the role of NO in regulation of cardiac contractility has been controversial (19Stoyanovsky D. Murphy T. Anno P.R. Kim Y.M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (232) Google Scholar, 20Petroff M.G. Kim S.H. Pepe S. Dessy C. Marban E. Balligand J.L. Sollott S.J. Nat. Cell Biol. 2001; 3: 867-873Crossref PubMed Scopus (270) Google Scholar). NO appears to have mixed effects; at certain concentrations and certain subcellular locations, NO enhances Ca2+ transients by activating ryanodine receptor Ca2+ release channels, whereas at other concentrations and locations NO can inhibit βAR-induced cardiac inotropy (21Hare J.M. Loh E. Creager M.A. Colucci W.S. Circulation. 1995; 92: 2198-2203Crossref PubMed Scopus (200) Google Scholar, 22Hare J.M. Givertz M.M. Creager M.A. Colucci W.S. Circulation. 1998; 97: 161-166Crossref PubMed Scopus (149) Google Scholar, 23Ziolo M.T. Katoh H. Bers D.M. Am. J. Physiol. 2001; 281: H2295-H2303Crossref PubMed Google Scholar). The effect of NO on βAR signaling is specific to eNOS relative to other NOS isoforms, perhaps because of the spatial co-localization of this isoform with βAR and AC in caveolae (24Barouch L.A. Harrison R.W. Skaf M.W. Rosas G.O. Cappola T.P. Kobeissi Z.A. Hobai I.A. Lemmon C.A. Burnett A.L. O'Rourke B. Rodriguez E.R. Huang P.L. Lima J.A. Berkowitz D.E. Hare J.M. Nature. 2002; 416: 337-339Crossref PubMed Google Scholar). NO regulation of βAR-mediated inotropy may contribute to blunted function in failing hearts perhaps because of an increased number of caveolae (25Hare J.M. Lofthouse R.A. Juang G.J. Colman L. Ricker K.M. Kim B. Senzaki H. Cao S. Tunin R.S. Kass D.A. Circ. Res. 2000; 86: 1085-1092Crossref PubMed Scopus (107) Google Scholar). The precise mechanism by which NO attenuates βAR signaling is not well characterized, but it has been suggested that NO can regulate AC types 5 and 6, the predominant AC isoforms expressed in the heart, via S-nitrosylation (26McVey M. Hill J. Howlett A. Klein C. J. Biol. Chem. 1999; 274: 18887-18892Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 27Hill J. Howlett A. Klein C. Cell Signal. 2000; 12: 233-237Crossref PubMed Scopus (42) Google Scholar). The present study was designed to examine the role of lipid rafts/caveolin-rich domains in GPCR-Gs-AC signaling and to assess if NO is an important regulator of cAMP production stimulated by GPCR. By using an NO donor and by overexpressing eNOS and AC6 in cardiac myocytes and vascular endothelial cells, we conclude that NO inhibits AC activity, attenuating signaling via GPCR that are co-localized with eNOS and AC in lipid rafts. Thus, NO selectively regulates βAR-stimulated cAMP production by inhibiting raft-localized AC6. In contrast, AC activity stimulated by GPCR not localized in lipid raft domains is unaffected by NO or raft disruption unless eNOS is overexpressed and lipid rafts are disrupted. The data thus emphasize the key role of lipid rafts in maintaining the association between caveolin and eNOS so as to facilitate regulation of βAR-Gs-AC signaling. Materials—Adenovirus expressing the murine AC6 cDNA was generated as described previously (28Gao M. Ping P. Post S. Insel P.A. Tang R. Hammond H.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1038-1043Crossref PubMed Scopus (109) Google Scholar). Adenovirus expressing the bovine eNOS cDNA was kindly donated by Dr. Donald Heistad and the Vector Development Core at the University of Iowa (29Ooboshi H. Chu Y. Rios C.D. Faraci F.M. Davidson B.L. Heistad D.D. Am. J. Physiol. 1997; 273: H265-H270PubMed Google Scholar). Primary antibodies for β1AR, β2AR, and AC5/6 and all secondary antibodies were obtained from Santa Cruz Biotechnology. Primary antibodies for caveolin-3 and eNOS were obtained from BD PharMingen. Primary antibody for EP2R was obtained from Cayman Chemical. Radiolabeled chemicals were obtained from PerkinElmer Life Sciences. Forskolin, N-monomethyl-l-arginine (l-NMMA), and SNAP were obtained from Calbiochem. All other chemicals and reagents were obtained from Sigma. Measurement of cAMP Production—Neonatal rat ventricular myocytes were prepared and maintained as described previously (3Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar). One day after plating, cells were incubated with indicated adenoviral construct(s) for 20 h (10–100 multiplicity of infection/cell), following which cells were washed extensively and allowed to equilibrate for 24 h. Myocytes were washed three times with serum-free and NaHCO3-free Dulbecco's modified Eagle's medium supplemented with 20 mm HEPES, pH 7.4 (DMEH). In some assays, cells were treated with 2% 2-hydroxypropyl-β-cyclodextrin (βCD) for 1 h at 37 °C to disrupt lipid rafts and washed with DMEH. Other cells were treated with βCD for 1 h, washed with DMEH, and then treated with βCD-cholesterol complexes (10 μg/ml cholesterol-βCD in a 1:6 molar ratio; Sigma catalog no. C4951) to deliver cholesterol back to the cells (30Rong J.X. Shapiro M. Trogan E. Fisher E.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13531-13536Crossref PubMed Scopus (380) Google Scholar). Cells were equilibrated in DMEH for 30 min and then assayed for cAMP accumulation by adding drugs of interest in the presence of a cyclic nucleotide phosphodiesterase (PDE) inhibitor, 0.2 mm isobutylmethylxanthine for 10 min. When inhibitors were used, these agents were incubated with cells for 5 min before the 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 radioimmunoassay, as previously described (4Ostrom R.S. Gregorian C. Drenan R.M. Xiang Y. Regan J.W. Insel P.A. J. Biol. Chem. 2001; 276: 42063-42069Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Calf pulmonary artery endothelial cells (CPAE; ATCC number CCL-209) were cultured in minimum essential medium containing Earle's salt (Invitrogen) and 20% fetal bovine serum (Omega Scientific) at 37 °C in 95% humidified air, 5% CO2. Cells were grown in 75-cm2 flasks and passed once a week with a split ratio of 1:5. cAMP accumulation was measured as described above on passage 7–15 CPAE seeded in 24-well plates. Measurement of NOS and AC Activity—AC and NOS activities were measured in membranes prepared from neonatal rat ventricular myocytes. Cells were rinsed twice in ice-cold PBS and then scraped into hypotonic homogenizing buffer (30 mm Na-HEPES, 5 mm MgCl2, 1 mm EGTA, 2 mm dithiothreitol, pH 7.5) and homogenized with 20 strokes of a Dounce homogenizer. The homogenate was centrifuged at 300 × g for 5 min at 4 °C; the supernatant was then transferred to a centrifuge tube and centrifuged at 5,000 × g for 10 min. The pellet was suspended in buffer (30 mm Na-HEPES, 5 mm MgCl2, 2 mm dithiothreitol, pH 7.5) to attain ∼1 mg/ml total protein concentration before being added into tubes containing drug and either AC 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 unit/ml creatine phosphokinase, and 0.1% bovine serum albumin, pH 7.5.) or NOS assay buffer (Stratagene). AC activity assays were terminated after 15 min by boiling, and cAMP content was determined by radioimmunoassay. NOS activity was assayed by measuring the conversion of [3H]arginine to [3H]citrulline using the NOSdetect™ assay kit (Stratagene). Membrane Fractionation—Neonatal rat ventricular myocytes were fractionated using a detergent-free method adapted from Song et al. (31Song S.K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar) and described previously (4Ostrom R.S. Gregorian C. Drenan R.M. Xiang Y. Regan J.W. Insel P.A. J. Biol. Chem. 2001; 276: 42063-42069Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Briefly, cells were homogenized in 500 mm sodium carbonate plus mammalian protease inhibitor mixture (Sigma catalog no. P-8340) with 20 strokes in a Dounce homogenizer, three 10-s bursts in a tissue grinder, and then three 20-s bursts with a sonicator. The homogenate was brought to 45% sucrose by the addition of 90% sucrose in 25 mm MES, 150 mm NaCl, pH 6.5, and loaded in an ultracentrifuge tube. A discontinuous sucrose gradient was layered on top of the sample by placing 4 ml of 35% sucrose and then 4 ml of 5% sucrose. The gradient was centrifuged at 33,000 rpm on a SW41Ti rotor (Beckman Instruments) for 16–20 h at 4 °C. Fractions were collected in 1-ml aliquots from the top of the gradient. Immunoprecipitation—Caveolin-3 was immunoprecipitated from isolated lipid raft fractions and from whole cell lysates using a protein A- or protein G-agarose method. For precipitations from lipid raft fractions, we pooled fractions 4 and 5 from the gradient after membrane fractionation and treated half the sample with 2% βCD and the other half with vehicle for 2 h on ice. For precipitations from whole cells, 10-cm plates of either control or βCD-treated neonatal rat ventricular myocytes were washed twice with cold PBS, scraped in 1 ml of lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Igepal CA-630 plus mammalian protease inhibitor mixture; Sigma catalog no. P-8340) and homogenized on ice with 20 strokes in a Dounce homogenizer. Both types of samples were precleared with protein A-agarose or protein G-agarose (Roche Applied Science) and then incubated with primary antibody for 1–3 h on a rocking platform at 4 °C. Antibody conjugates were precipitated by incubating with protein A/G-agarose overnight on a rocking platform at 4 °C and centrifuging at 13,000 × g for 5 min. Protein A/G-agarose pellets were then washed once in lysis buffer followed by three washes each in wash buffer 2 (50 mm Tris-HCl, pH 7.5, 500 mm NaCl, 0.2% Igepal CA-630) and wash buffer 3 (10 mm Tris-HCl, pH 7.5, 0.2% Igepal CA-630). Pellets and immunoprecipitation supernatants were suspended in sample buffer containing 20% β-mercaptoethanol and heated at 70 °C for 10 min. Proteins in both the immunoprecipitates and the supernatants were analyzed by immunoblot analysis. Immunoblot Analysis—Proteins were separated by SDS-PAGE as described previously (32Ostrom R.S. Liu X. Head B.P. Gregorian C. Seasholtz T.M. Insel P.A. Mol. Pharmacol. 2002; 62: 983-992Crossref PubMed Scopus (124) Google Scholar). Briefly, samples were loaded into polyacrylamide gels (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Millipore Corp.) by electroblotting. Membranes were blocked in 3% phosphate-buffered saline/milk, incubated with primary antibody (see "Materials") followed by secondary antibody with conjugated horseradish peroxidase (Santa Cruz Biotechnology). Blots were visualized using chemiluminescence and a digital imaging system (UVP, Inc.). All bands shown migrated at the expected size, as determined by comparison with molecular weight standards (Santa Cruz Biotechnology). The amount of protein per fraction was determined using a dye-binding protein assay (Bio-Rad). Data Analysis and Statistics—Data are presented as the mean ± S.E. of at least three separate experiments or as representative images of at least three separate experiments. Statistical comparisons (t tests and one-way analysis of variance), nonlinear regression analysis, and graphics were performed using Graph Pad Prism 4.0 (GraphPad Software). For analysis of concentration-response curves, individual experiments were fitted by nonlinear regression, and paired t tests were performed comparing EC50 values and maximum responses between treatment conditions. NO as a Regulator of cAMP Production in Cardiac Myocytes and Endothelial Cells—To determine if NO is a regulator of cardiac myocyte cAMP production, we used a NO donor, (±)-S-nitroso-N-acetylpenicillamine (SNAP) and measured both basal and forskolin-stimulated cAMP production. Inclusion of SNAP (10 μm) significantly reduced basal and forskolin-stimulated cAMP production in both control cardiac myocytes and myocytes incubated with an adenovirus to overexpress AC6 (Fig. 1A). The inhibition by SNAP of forskolin-stimulated cAMP was greater in AC6-overexpressing cells in terms of absolute amounts of cAMP (197 ± 36 pmol of cAMP in control cells and 525 ± 93 pmol of cAMP in AC6-overexpressing cells) but similar in proportional terms (38 ± 5.3% inhibition in control cells, 38 ± 2.2% inhibition in AC6-overexpressing cells). We also investigated whether a NOS inhibitor, l-NMMA, could release a tonic inhibition of cAMP production. Inclusion of 1 mm l-NMMA did not alter basal or forskolin-stimulated cAMP production in control cardiac myocytes but significantly increased forskolin-stimulated cAMP production in AC6-overexpressing cells (Fig. 1A). These data imply that increases in NO can blunt formation of cAMP in neonatal cardiac myocytes but that NO derived from endogenous NOS activity does not tonically inhibit cAMP production in these cells unless adenylyl cyclase is expressed at greater than ambient levels. In cardiac fibroblasts, NO production can act via soluble guanylyl cyclase to induce the expression of a phosphodiesterase, PDE2, which inhibits cAMP accumulation stimulated by forskolin or isoproterenol (33Gustafsson A.B. Brunton L.L. Am. J. Physiol. Cell Physiol. 2002; 283: C463-C471Crossref PubMed Scopus (44) Google Scholar). Therefore, we tested a 5-fold higher concentration of isobutylmethylxanthine (1 mm), the nonspecific PDE inhibitor used in our other assays, a PDE2-specific inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, and a guanylyl cyclase inhibitor, 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one, in measurements of AC activity in membranes from cardiac myocytes to determine if a similar mechanism exists in these cells. We found that neither high levels of isobutylmethylxanthine nor inclusion of erythro-9-(2-hydroxy-3-nonyl)adenine or 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one altered the ability of SNAP to inhibit forskolin-stimulated AC activity (Fig. 1B). These data are consistent with the idea that NO inhibits activity of AC5 and AC6 independent of guanylyl cyclase activation or induction of a cyclic nucleotide phosphodiesterase. We hypothesized that direct NO regulation of AC activity would be evident in other cell types that expressed eNOS and AC6. To test this idea, we used CPAE and measured the effects of an NO donor on cAMP production. By contrast with the results that we observed in cardiac myocytes, SNAP (10 μm) did not reduce basal or forskolin-stimulated cAMP production in CPAE cells (Fig. 2). However, SNAP did reduce forskolin-stimulated cAMP levels in CPAE cells engineered to overexpress AC6. Although l-NMMA (1 mm) did not alter basal or forskolin-stimulated cAMP production in control CPAE cells, l-NMMA significantly (p < 0.05 by paired t test) increased forskolin-stimulated cAMP production in AC6-overexpressing CPAE cells. Thus, CPAE cells only demonstrate NO-inhibited cAMP production when engineered to overexpress AC6, a result consistent with the idea that eNOS-derived NO preferentially inhibits activity of AC6 (26McVey M. Hill J. Howlett A. Klein C. J. Biol. Chem. 1999; 274: 18887-18892Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 27Hill J. Howlett A. Klein C. Cell Signal. 2000; 12: 233-237Crossref PubMed Scopus (42) Google Scholar). The Role of Intact Lipid Rafts in GPCR-Gs-AC Signal Transduction—Previous reports indicate that eNOS is highly localized in lipid rafts and caveolae in endothelial cells and cardiac myocytes and that its activity is inhibited by binding to the caveolin scaffold (18Feron O. Saldana F. Michel J.B. Michel T. J. Biol. Chem. 1998; 273: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Therefore, we hypothesized that intact lipid rafts or caveolae retain eNOS in the inactive state, thereby perhaps minimizing its role in regulating cAMP production under normal conditions (Fig. 1B). To test this hypothesis, we disrupted lipid rafts by treating cells with 2% βCD, a cholesterol binding agent, for 1 h and then measured cAMP production. These conditions lead to the disruption of morphologically identified caveolae (34Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1910) Google Scholar) and to the loss of caveolin-3, eNOS, and AC5/6 immunoreactivity from buoyant fractions of a discontinuous sucrose gradient (Fig. 3) (6Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). We overexpressed AC6 in these studies in order to enhance the absolute extent of NO inhibition of cAMP accumulation (as shown in Fig. 1). βCD treatment inhibited forskolin- and isoproterenol-stimulated cAMP production but did not inhibit PGE2-stimulated cAMP production (Fig. 4A), consistent with the co-localization of βAR and AC6 in lipid raft/caveolin-rich domains and the exclusion of PGE2 receptors from those domains (3Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar, 6Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). Adding cholesterol back to the cells by treating with βCD-cholesterol complexes following βCD treatment largely reversed these effects, indicating the specificity and reversibility of βCD treatment. To investigate the impact of βCD treatment in more detail, we measured cAMP production in response to multiple concentrations of forskolin, isoproterenol, and PGE2. βCD treatment caused a 5.8 ± 0.3-fold rightward shift (p < 0.01) in the forskolin concentration-response curve with no significant change in the maximal response (Fig. 4B). Although inclusion of l-NMMA (1 mm) did not alter the forskolin response in untreated cells (Fig. 1B), l-NMMA partially reversed the rightward shift induced by βCD treatment (a 2.3 ± 0.3-fold leftward shift as compared with the βCD response, p < 0.05) (Fig. 4B). NOS activity was increased in membranes prepared from cardiac myocytes treated with βCD as compared with those prepared from vehicle-treated cells (Fig. 4A, inset). Therefore, lipid raft disruption appears to inhibit forskolin-stimulated cAMP production due, in part, to an increase in NOS activity. By contrast with the impact of βCD treatment on forskolin response, we found different effects on the concentration-response curves for isoproterenol and PGE2 (Fig. 4, C and D), the receptors for which show different patterns for localization in lipid raft, caveolin-rich domains (Fig. 3) (4Ostrom R.S. Gregorian C. Drenan R.M. Xiang Y. Regan J.W. Insel P.A. J. Biol. Chem. 2001; 276: 42063-42069Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). βCD treatment caused no shift in the isoproterenol concentration-response curve and reduced the maximal response by 37 ± 8% as compared with untreated cells (p < 0.05; Fig. 4C). Inclusion of the NOS inhibitor caused a leftward shift of the response curve (1.5 ± 0.2-fold as compared with control, 1.6 ± 0.4-fold versus βCD treated alone, both p < 0.05) but did not alter the βCD-induced reduction in maximal response. Thus, βCD treatment has qualitatively different effects on βAR-stimulated versus forskolin-stimulated cAMP production; for βAR, but not forskolin, we found an inhibition of maximal response, whereas for both types of agonists, we observe a NOS-dependent component that decreases potency (to a greater extent in the case of forskolin). In contrast to effects on forskolin- and isoproterenol-mediated responses, PGE2 potency and maximal response were not statistically altered by βCD treatment and/or inclusion of l-NMMA (Fig. 4D). The minimal effect of disruption of lipid rafts on cAMP production stimulated by prostanoid receptors presumably results from exclusion of these receptors from those microdomains (4Ostrom R.S. Gregorian C. Drenan R.M. Xiang Y. Regan J.W. Insel P.A. J. Biol. Chem. 2001; 276: 42063-42