Acute inhibition of guanosine triphosphate cyclohydrolase 1 uncouples endothelial nitric oxide synthase and elevates blood pressure

Abstract

GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial NO synthase (eNOS) dictating, at least partly, the balance of NO and superoxide produced by this enzyme. The aim of this study was to determine the effect of acute inhibition of GTPCH1 on BH4, eNOS function, and blood pressure (BP) in vivo. Exposure of bovine or mouse aortic endothelial cells to GTPCH1 inhibitors (2,4-diamino-6-hydroxypyrimidine or N-acetyl-serotonin) or GTPCH1 small-interference RNA (siRNA) significantly reduced BH4 and NO levels but increased superoxide levels. This increase was abolished by sepiapterin (BH4 precursor) or N(G)-nitro-L-arginine methyl ester (nonselective NOS inhibitor). Incubation of isolated murine aortas with 2,4-diamino-6-hydroxypyrimidine or N-acetyl-serotonin impaired acetylcholine-induced endothelium-dependent relaxation but not endothelium-independent relaxation. Aortas from GTPCH1 siRNA-injected mice, but not their control-siRNA injected counterparts, also exhibited impaired endothelium-dependent relaxation. BH4 reduction induced by GTPCH1 siRNA injection was associated with increased aortic levels of superoxide, 3-nitrotyrosine, and adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1), as well as a significantly elevated systolic, diastolic, and mean BP in C57BL6 mice. GTPCH1 siRNA was unable to elicit these effects in eNOS(-/-) mice. Sepiapterin supplementation, which had no effect on high BP in eNOS(-/-) mice, partially reversed GTPCH1 siRNA-induced elevation of BP in wild-type mice. In conclusion, GTPCH1 via BH4 maintains normal BP and endothelial function in vivo by preserving NO synthesis by eNOS.

Figure 1

Pharmacological inhibition of GTPCH1 decreases NO release and increases eNOS-derived O₂^•− in cultured bovine aortic endothelial cells (BAECs). (A) Total biopterins and BH4 levels following a 24 h exposure to DAHP and/or NAS. N=5 *P<0.05 control vs. NAS/DHAP or both; (B) Effect of L-NAME or sepiapterin on induction of O₂^•− production by DAHP or NAS (24 h exposure). N=5, *P<0.05 NAS or DAHP vs. control; ^#P<0.05 DAHP treated vs. DAHP plus L-NAME or sepiapterin. (C) NO production following treatment with DAHP or DAHP+sepiapterin. Data are expressed as mean±SEM. N=5, *P<0.05 DAHP vs. control; ^#P<0.05 DAHP treated vs. DAHP plus sepiapterin.

Figure 2

GTPCH1-specific siRNA decreases NO release and increases eNOS-derived O₂^•− in isolated mouse aortic endothelial cells (MAECs). (A) Western blot analysis of GTPCH1 and eNOS in control and siRNA-transfected cells. The blot is representative of three blots from three independent experiments. N=3 *P<0.05 GTPCH1 siRNA vs. control or control siRNA, NS indicates P>0.05. (B) Effect of GTPCH1 siRNA on O₂^•− production. A subset of siRNA-transfected MAECs were treated with the eNOS inhibitor, L-NAME, or Tempol, a SOD mimetic. MAECs were incubated with 1 mM L-NAME or 1 mM Tempol for 24 h after the siRNA transfection. N=3 *P<0.05 GTPCH1 siRNA vs. control or control siRNA; n=5 ^#P<0.05 GTPCH1 siRNA vs. GTPCH1-siRNA plus L-NAME or plus Tempol; (C) NO release in siRNA transfected MAECs incubated with L-NAME or without L-NAME. Data are expressed as mean±SEM. N=3, *P<0.05 vs. control siRNA, NS indicates P>0.05.

Figure 3

In vivo GTPCH1 knockdown decreases both BH4 and total biopterins. (A) Western blot and RT-PCR analysis of GTPCH1 or eNOS in aorta from control siRNA- and GTPCH1 siRNA-injected mice. N=5, *P<0.05 GTPCH1 siRNA vs. control siRNA or control, NS indicates P>0.05. (B) Total biopterins and BH4 levels in aortas from siRNA-transfected mice. N=5, *P<0.05 GTPCH1 siRNA vs. control siRNA or control; (C) Endothelium–dependent relaxation in isolated aortas from control and GTPCH1 siRNA-injected mice. In a subset of experiments, aortas were incubated with sepiapterin for 1 h. Data are expressed as mean±SEM. N=5, *P<0.05 GTPCH1 siRNA vs. control siRNA or control; n=5 ^#P<0.05 GTPCH1 siRNA vs. GTPCH1 treated with sepiapterin.

Figure 4

In vivo GTPCH1 knockdown induces eNOS-dependent increases in superoxide anions, ICAM-1, VCAM-1 and 3-nitrotyrosine (3-NT). (A) Aortic O₂^•− production in WT and eNOS^−/− mice injected with GTPCH1 siRNA or control siRNA. N=5 ^*P<0.05, control siRNA vs. GTPCH1-siRNA, NS indicates P>0.05. (B) Immunocytochemical staining of 3-NT, VCAM1, and ICAM1 in wild type and eNOS^−/− mice treated with control siRNA or GTPCH1-specific siRNA. N=5 ^*P<0.05, control siRNA vs. GTPCH1-siRNA, NS indicates P>0.05.

Figure 5

In vivo GTPCH1 knockdown elevates arterial blood pressure in an eNOS-dependent manner. Mean blood pressure (BP), systolic blood pressure, and diastolic blood pressure in control or GTPCH1 siRNA-injected wild type (WT) or eNOS^−/− mice supplementation with or without sepiapterin (10 mg/kg/day for 7 days, I.P). The blood pressure was measured by a carotid catheter method. Data are expressed as mean±SEM (n=4 or 5). *P<0.05 GTPCH1 siRNA vs. control siRNA, ^#P<0.05 GTPCH1 siRNA vs. GTPCH1 siRNA treated with sepiapterin.