Abstract 322: Acute Inhibition of GTP Cyclohydrolase 1 Uncouples Endothelial Nitric Oxide Synthase and Elevates Blood Pressure
Objective: GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) dictating at least partly, the balance of nitric oxide (NO) and superoxide (O2.−) produced by this enzyme. The aim of this study is to determine the effects of acute inhibition of GTPCH1 on BH4, eNOS function, and blood pressure.
Methods: The biopterin content was detected by HPLC. O2.− and NO productions were assayed by using DHE and DAF fluorescence respectively. The vessel relaxation was assayed by organ chamber. The blood pressure in wild-type (WT) or eNOS−/− mice was determined by a carotid catheter method.
Results: Exposure of bovine or mouse aortic endothelial cells to GTPCH1 inhibitors (10 mM DAHP or 1 mM NAS) for 24 hours or GTPCH1 siRNA transfection significantly reduced both BH4 and NO levels, but increased O2.− levels. This increase was abolished by 10 μM L-sepiapterin (BH4 precursor) or 1 mM L-NAME (non-selective NOS inhibitor). Incubation of isolated WT mice aortas with DAHP or NAS for 24 hours impaired acetylcholine-induced endothelium-dependent relaxation, but not endothelium-independent relaxation. Aortas from GTPCH1 siRNA-injected mice, but not their control-siRNA injected mice, also exhibited impaired endothelium-dependent relaxation. Furthermore, GT-PCH1 siRNA injection in mice reduced BH4 levels in aortas, associated with increased aortic levels of O2.−, 3-nitrotyrosine, and adhesion molecules (ICAM1 and VCAM1). In addition, an elevated mean, systolic, and diastolic blood pressure was induced by GTPCH1 siRNA injection in vivo, but not control siRNA (mean blood pressure: 114.28±4.48 vs. 136.81±2.45 mmHg) in WT mice. GTPCH1 siRNA was unable to elicit the similar effects in eNOS−/− mice, including increased oxidative stress (O2.−, 3-nitrotyrosine, ICAM1, VCAM1) and blood pressure. Finally, sepiapterin supplementation, which had no effect on high blood pressure in eNOS−/− mice, partially reversed GTPCH1 siRNA-induced elevation of systemic blood pressure in WT mice.
Conclusion: GTPCH1 via BH4 maintains normal blood pressure and endothelial function by preserving eNOS-dependent NO biosynthesis.
This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).