Abstract 12532: Increasing Dihydrobiopterin Causes Dysfunction of Endothelial Nitric Oxide Synthase in Rats in vivo
Background: Vascular content of dihydrobiopterin (BH2), an oxidized form of tetrahydrobiopterin (BH4), has been reported to be elevated in arteriosclerotic/atherosclerotic vascular lesions where eNOS is dysfunctional. However, no study has ever addressed the potential role of BH2 in the regulation of eNOS activity in vivo. Thus, in this study, we tested our hypothesis that an increasing BH2 concentration causes eNOS dysfunction in rats.
Methods and Results: Male Wistar rats were treated with either saline, or methotrexate (MTX) which is a specific dihydrofolate reductase inhibitor that blocks intracellular conversion of BH2 to BH4, or MTX plus sepiapterin (SEP) which is a BH2 precursor (n=8-10). Combined treatment with MTX and SEP did not significantly change aortic BH4 levels as compared with control (saline) treatment (79±20 vs. 73±6 pmol/g). However, the MTX/SEP treatment significantly and markedly increased aortic BH2 levels compared with the control treatment (291±29 vs. 33±6 pmol/g, P<0.01), and this was associated with a significant increase in basal blood pressure (BP) levels (142±4 vs. 128±4 mmHg, P<0.05). On the other hand, treatment with MTX alone did not significantly affect aortic levels of BH2 (80±9) or BH4 (59±9) or basal BP levels (137±8). Notably, as compared with the control treatment, the MTX/SEP treatment, but not with the MTX treatment, significantly impaired vasodilator and depressor responses induced by intravenous bolus injection of acetylcholine (ACh), a physiological eNOS activator, and also aggravated ACh-induced endothelium-dependent relaxations in isolated aortas (all P<0.05), without affecting endothelium-independent relaxations to sodium nitroprusside, a NO donor. Importantly, the MTX/SEP treatment, but not the MTX treatment, significantly enhanced aortic superoxide production (14.7±1.3 vs. 10.3±0.6 RLU/mg, P<0.05), and this increase was abolished in the presence of L-NAME, a NOS inhibitor (P<0.05), suggesting the involvement of eNOS uncoupling.
Conclusions: These results demonstrate for the first time that increasing BH2 causes eNOS dysfunction in rats in vivo in the absence of BH4 deficiency, providing a novel insight into the regulatory mechanism of eNOS activity.
- © 2011 by American Heart Association, Inc.