Nitric oxide inhibition of adenylyl cyclase type 6 activity is dependent upon lipid rafts and caveolin signaling complexes
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Rennolds S Ostrom1, Richard A Bundey, Paul A Insel
1Department of Pharmacology, and Vascular Biology Center of Excellence, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Abstract:
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), beta-adrenergic (betaAR) 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 betaAR-G(s)-AC signal transduction together with eNOS. Using neonatal rat cardiac myocytes, we found that disruption of lipid rafts with beta-cyclodextrin inhibited forskolin-stimulated AC activity and cAMP production, eliminated caveolin-3-eNOS interaction, and increased NO production. betaAR- and G(s)-mediated activation of AC activity were inhibited by beta-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 betaAR and AC types 5 and 6 results in selective regulation of betaAR 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.
Insights
Nitric oxide (NO) regulates cardiac function by inhibiting adenylyl cyclase (AC) within lipid rafts. This spatial organization controls beta-adrenergic receptor signaling and cardiac contractility.
Area of Science:
- Cardiovascular Physiology
- Molecular Cell Biology
- Biochemistry
Background:
- Nitric oxide (NO) is produced by NO synthase (NOS) in cardiac cells and influences cardiac contractility.
- The precise mechanisms by which NO affects heart function, particularly contractile dysfunction in heart failure, are not fully understood.
- Adenylyl cyclase (AC) isoforms 5 and 6, prevalent in cardiac myocytes, are potential targets for NO-mediated inhibition.
Purpose of the Study:
- To investigate the role of lipid rafts in organizing signaling molecules involved in beta-adrenergic receptor (betaAR)-G(s)-AC signal transduction and endothelial NO synthase (eNOS).
- To elucidate how lipid raft disruption affects NO production and AC activity in cardiac myocytes.
- To understand the spatial regulation of AC activity by eNOS within specific cellular microdomains.
Main Methods:
- Neonatal rat cardiac myocytes were used to study signaling pathways.
- Lipid rafts were disrupted using beta-cyclodextrin.
- AC activity, cAMP production, and protein interactions (caveolin-3 and eNOS) were measured.
- NO production was assessed, and experiments utilized NO donors (SNAP) and genetic manipulation (eNOS overexpression).
Main Results:
- Disruption of lipid rafts inhibited forskolin-stimulated AC activity and cAMP production.
- Beta-cyclodextrin treatment reduced caveolin-3 and eNOS interaction, increased NO production, and inhibited betaAR- and G(s)-mediated AC activation.
- Prostanoid receptor-stimulated AC activity was unaffected in native cells but altered upon eNOS overexpression and lipid raft disruption.
- NO donors and eNOS overexpression reduced cAMP production, indicating NO's inhibitory effect on AC.
Conclusions:
- Lipid rafts play a crucial role in organizing betaAR, G(s), AC isoforms, and eNOS, thereby spatially regulating betaAR signaling.
- eNOS activity within lipid rafts selectively modulates betaAR signaling compared to other G(s)-coupled receptors in non-raft domains.
- The co-localization of signaling components within lipid rafts provides critical spatial control over AC activity and cellular function.
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