Abstract 13894: Dihydrobiopterin Causes Endothelial Nitric Oxide Synthase Dysfunction in Rats in vivo
Background: Vascular levels of dihydrobiopterin (BH2), an oxidized form of tetrahydrobiopterin (BH4), have been reported to be elevated in arteriosclerotic lesions where endothelial nitric oxide synthase (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, 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). Treatment with MTX alone did not significantly affect aortic BH4 (59±9 vs. 73±6 pmol/g) or BH2 (80±9 vs. 33±6 pmol/g) levels or basal blood pressure (BP) (137±8 vs. 128±4 mmHg) as compared with saline treatment (control). Treatment with MTX/SEP also did not significantly change aortic BH4 levels (79±20). However, MTX/SEP treatment significantly and markedly increased aortic BH2 levels by 8.7 times (291±29, P<0.01), and this was associated with a significant increase in basal BP (142±4, P<0.05). Notably, MTX/SEP treatment, but not with MTX treatment, significantly impaired vasodilator and depressor responses induced by intravenous bolus injection of acetylcholine (ACh), a physiological eNOS activator, and aggravated ACh-induced endothelium-dependent relaxations in isolated aortas (all P<0.05), without affecting endothelium-independent relaxations to sodium nitroprusside, a NO donor. Western blot analysis showed that MTX/SEP treatment did not significantly alter eNOS expression, dimerization, or phosphorylation at Ser1177 or Thr495. However, MTX/SEP 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 an involvement of eNOS uncoupling.
Conclusions: These results demonstrate, for the first time, that 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.
- © 2012 by American Heart Association, Inc.