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

• Published in: The Journal of biological chemistry 2004-05, Vol.279 (19), p.19846-19853
• Main Authors: Ostrom, Rennolds S, Bundey, Richard A, Insel, Paul A
• Format: Article
• Language: English
• Subjects: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases - metabolism; Animals; Animals, Newborn; beta-Cyclodextrins; Cattle; Caveolin 1; Caveolin 3; Caveolins - chemistry; Caveolins - metabolism; Cell Membrane - metabolism; Cells, Cultured; Cyclic AMP - metabolism; Cyclodextrins - chemistry; Dose-Response Relationship, Drug; Immunoblotting; Membrane Microdomains - chemistry; Membrane Microdomains - metabolism; Mice; Models, Biological; Myocardial Contraction; Myocardium - enzymology; Nitric Oxide - metabolism; Nitric Oxide Synthase - chemistry; Penicillamine - analogs & derivatives; Penicillamine - pharmacology; Precipitin Tests; Protein Binding; Protein Isoforms; Protein Structure, Tertiary; Rats; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M313440200

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-G s -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 G s -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 G s -coupled receptors localized in nonraft domains. Thus co-localization of multiple signaling components in lipid rafts provides key spatial regulation of AC activity.