| Abstract: |
Caveolae harbor different serpentine receptors, intracellular components of signaling cascades, and certain enzymes, including endothelial nitric oxide synthase (eNOS). The regulation of eNOS activity by Ca2+/calmodulin and caveolin has been described. We have previously demonstrated that nitric oxide (NO) can modulate signaling initiated via receptors localized to caveolae. In the present study, we show that NO donors induced an increase in the monomeric form of this scaffolding protein in cultured endothelial cells, the effect mimicked by 8-bromo cGMP. Proximity imaging of endothelial cells transfected with the thermotolerant green fluorescent protein-caveolin-1 construct demonstrated that sodium nitroprusside resulted in the increased fluorescence ratio of 410:470 nm, consistent with the distancing of fluorescently tagged caveolin-1. Pulse labeling of endothelial cells with cholera toxin B subunit indicated that sodium nitroprusside reversibly decreased its binding. Signaling via G protein-coupled receptors resident to caveolae was inhibited by pretreatment with NO donor. The data demonstrate that NO modulation of cell signaling is accomplished in part by regulating the state of caveolin-1 oligomerization. NO-induced attenuation of signaling involves reversible dissociation of caveolin scaffold, thus providing both spatial and temporal modulation of signal transduction. |
| Keywords: |
signal transduction; controlled study; human cell; animals; cells, cultured; fluorescence; green fluorescent protein; calcium; luminescent proteins; enzyme regulation; recombinant fusion proteins; vascular endothelium; endothelium, vascular; enzyme inhibitors; green fluorescent proteins; rats; oxidative stress; receptor; g protein coupled receptor; centrifugation, density gradient; vasodilator agents; oligomerization; dissociation; nitric oxide synthase; cell labeling; muscle, smooth, vascular; nitric oxide; nitric oxide synthase type iii; calcium ion; calmodulin; caveolae; caveolin 1; nitric oxide donors; endothelin-1; nitroprusside sodium; humans; human; priority journal; article; caveolins; cholera toxin; cytosolic calcium concentration; 8 bromo cyclic gmp; cholera toxin b subunit; serpentine; ion-selective electrodes; nitroprusside
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