The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study

2002 ◽  
Vol 362 (3) ◽  
pp. 733-739 ◽  
Author(s):  
Jeannette VÁSQUEZ-VIVAR ◽  
Pavel MARTÁSEK ◽  
Jennifer WHITSETT ◽  
Joy JOSEPH ◽  
Balaraman KALYANARAMAN
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Spin Trapping ◽  
No Formation ◽  
Superoxide Formation ◽  
Oxide Synthase ◽  
Epr Spin Trapping ◽  
Endothelial Nitric Oxide ◽  
Superoxide Release

Augmentation of superoxide levels has been linked to impaired relaxation in hypertension, diabetes and hypercholesterolaemia. Purified endothelial nitric oxide synthase (eNOS) generates superoxide under limited availability of 5,6,7,8-tetrahydrobiopterin (BH4). Thus alterations in endothelial BH4 levels have been postulated to stimulate superoxide production from eNOS. This possibility was examined by determining the concentration-dependent effects of BH4, and its analogues, on superoxide formation by eNOS. Superoxide was quantified by EPR spin trapping, which is the only available technique to quantify superoxide from eNOS. Using 5-ethoxycarbonyl-5-methyl-pyrroline N-oxide, we show that only fully reduced BH4 diminished superoxide release from eNOS, with efficiency BH4>6-methyl-BH4>5-methyl-BH4. In contrast, partially oxidized BH4 analogues, 7,8-dihydrobiopterin (7,8-BH2) and sepiapterin had no effect. Neither l-arginine nor NG-nitro-l-arginine methyl ester (l-NAME) abolished superoxide formation. Together, BH4 and l-arginine stimulated ˙NO production at maximal rates of 148nmol/min per mg of protein. These results indicate that BH4 acts as a ‘redox switch’, decreasing superoxide release and enhancing ˙NO formation. This role was verified by adding 7,8-BH2 or sepiapterin to fully active eNOS. Both 7,8-BH2 and sepiapterin enhanced superoxide release while inhibiting ˙NO formation. Collectively, these results indicate that the ratio between oxidized and reduced BH4 metabolites tightly regulates superoxide formation from eNOS. The pathological significance of this scenario is discussed.

Endothelial dysfunction does not require loss of endothelial nitric oxide synthase

2000 ◽  
Vol 278 (6) ◽  
pp. H2020-H2027 ◽  
Author(s):  
Roberto R. Giraldez ◽  
Alexander Panda ◽  
Jay L. Zweier
Keyword(s):  
Nitric Oxide ◽  
Endothelial Dysfunction ◽  
Nitric Oxide Synthase ◽  
Immunoelectron Microscopy ◽  
Endothelial Nitric Oxide Synthase ◽  
Western Blots ◽  
No Formation ◽  
Triton X ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Whereas altered nitric oxide (NO ⋅) formation from endothelial nitric oxide synthase (NOS) causes impaired vascular reactivity in a number of cardiovascular diseases, questions remain regarding how endothelial injury results in impaired NO ⋅ formation. It is unknown if loss of NOS expression or activity is required or if other factors are involved. Detergent treatment has been used to induce endothelial dysfunction. Therefore, NOS and NO ⋅ synthesis were characterized in a rat heart model of endothelial injury and dysfunction induced by the detergent Triton X-100. Cardiac NO ⋅ formation was directly measured by electron paramagnetic resonance spectroscopy. NOS activity was determined by thel-[14C]arginine conversion assay. Western blots and immunohistology were applied to define the amounts of NOS present in heart tissue before and after Triton treatment. Immunoelectron microscopy was performed to assess intracellular NOS distribution. A short bolus of Triton X-100, 0.25%, abolished responses to histamine and calcium ionophore while preserving response to nitroprusside. Complete blockade of NO ⋅ generation occurred after Triton treatment, but NOS activity assayed with addition of exogenous substrate and cofactors was unchanged, and identical 135-kDa NOS bands were seen on Western blots, indicating that NOS was not removed from the heart or structurally damaged by Triton. Immunohistochemistry showed no change in NOS localization after Triton treatment, and immunoelectron microscopy revealed similar NOS distribution in the plasma membrane and intracellular membranes. These results demonstrate that the endothelial dysfunction was due to decreased NO ⋅ synthesis but was not caused by loss or denaturation of NOS. Thus endothelial dysfunction due to mild endothelial membrane injury may occur in the presence of active NOS and is triggered by loss of NOS substrates or cofactors.

Localization of endothelial nitric oxide synthase in the normal and failing human atrial myocardium

1996 ◽  
Vol 54 ◽  
pp. 786-787
Author(s):  
Chi-Ming Wei ◽  
Margarita Bracamonte ◽  
Shi-Wen Jiang ◽  
Richard C. Daly ◽  
Christopher G.A. McGregor ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Cardiac Tissues ◽  
Mammalian Tissues ◽  
End Stage Heart Failure ◽  
End Stage ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Nitric oxide (NO) is a potent endothelium-derived relaxing factor which also may modulate cardiomyocyte inotropism and growth via increasing cGMP. While endothelial nitric oxide synthase (eNOS) isoforms have been detected in non-human mammalian tissues, expression and localization of eNOS in the normal and failing human myocardium are poorly defined. Therefore, the present study was designed to investigate eNOS in human cardiac tissues in the presence and absence of congestive heart failure (CHF).Normal and failing atrial tissue were obtained from six cardiac donors and six end-stage heart failure patients undergoing primary cardiac transplantation. ENOS protein expression and localization was investigated utilizing Western blot analysis and immunohistochemical staining with the polyclonal rabbit antibody to eNOS (Transduction Laboratories, Lexington, Kentucky).

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