In:
American Journal of Physiology-Heart and Circulatory Physiology, American Physiological Society, Vol. 301, No. 3 ( 2011-09), p. H903-H911
Abstract:
In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. In the absence of the requisite eNOS cofactor tetrahydrobiopterin (BH 4 ), NADPH oxidation is uncoupled from NO generation, leading to the production of superoxide. Although this phenomenon is apparent with purified enzyme, cellular studies suggest that formation of the BH 4 oxidation product, dihydrobiopterin, is the molecular trigger for eNOS uncoupling rather than BH 4 depletion alone. In the current study, we investigated the effects of both BH 4 depletion and oxidation on eNOS-derived superoxide production in endothelial cells in an attempt to elucidate the molecular mechanisms regulating eNOS oxidase activity. Results demonstrated that pharmacological depletion of endothelial BH 4 does not result in eNOS oxidase activity, whereas BH 4 oxidation gave rise to significant eNOS-oxidase activity. These findings suggest that the endothelium possesses regulatory mechanisms, which prevent eNOS oxidase activity from pterin-free eNOS. Using a combination of gene silencing and pharmacological approaches, we demonstrate that eNOS-caveolin-1 association is increased under conditions of reduced pterin bioavailability and that this sequestration serves to suppress eNOS uncoupling. Using small interfering RNA approaches, we demonstrate that caveolin-1 gene silencing increases eNOS oxidase activity to 85% of that observed under conditions of BH 4 oxidation. Moreover, when caveolin-1 silencing was combined with a pharmacological inhibitor of AKT, BH 4 depletion increased eNOS-derived superoxide to 165% of that observed with BH 4 oxidation. This study identifies a critical role of caveolin-1 in the regulation of eNOS uncoupling and provides new insight into the mechanisms through which disease-associated changes in caveolin-1 expression may contribute to endothelial dysfunction.
Type of Medium:
Online Resource
ISSN:
0363-6135
,
1522-1539
DOI:
10.1152/ajpheart.00936.2010
Language:
English
Publisher:
American Physiological Society
Publication Date:
2011
detail.hit.zdb_id:
1477308-9
SSG:
12
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