Tetrahydrobiopterin prevents chronic ischemia-related lower urinary tract dysfunction through the maintenance of nitric oxide bioavailability

Figures

• 

  Figure 1. Typical cystometrogram recordings in Control, CBI and CBI/BH4 group rats. Micturition interval is significantly shorter in the CBI group than in the other groups. Scale bars represent 5 min and 5 cm H2O.

• 

  Figure 2. Contractile responses to KCl, electrical field stimulation, ATP and carbachol in control, CBI and CBI/BH4 rats (n = 10 each). Mean contractile responses to KCl, 1 mM ATP, electrical field stimulation and carbachol at concentrations from 1 μM to 1 mM were significantly weaker in the CBI group than in the other groups. Values represent mean ± standard error of the mean (SEM). Asterisk indicates statistical significance at the p < 0.05 level. †p < 0.05 in CBI versus Control groups. ††p < 0.05 in CBI versus Control and CBI/BH4 groups.

• 

  Figure 3. (A) Elastica Masson staining of the same cross-sections of common iliac artery from rats in the Control (10 rats, 20 vessels), CBI (10 rats, 20 vessels) and CBI/BH4 (10 rats, 20 vessels) groups (100 × magnification; scale bars, 200 μm). Bar graphs show average wall thickness of the common iliac artery in Control, CBI and CBI/BH4 groups. Elastica Masson staining of cross-sections of common iliac artery from the CBI group demonstrates mean arterial wall thickness with neointimal formation is significantly greater in the CBI group than in the other groups. Values represent mean ± SEM. *p < 0.05. (B) Elastica Masson staining of arterioles (upper panel) and muscle layer (lower panel) in bladder from Control (A,D), CBI (B,E) and CBI/BH4 (C,F) groups (400 × magnification; scale bars, 50 μm). Bar graphs show the percentage of collagen in the muscle layer of the Control, CBI and CBI/BH4 groups. Elastica Masson staining of bladder arterioles shows obvious arterial wall thickening compared with other groups. In the CBI group, the percentage of collagen in the muscle layer is significantly increased as compared with other groups. Values represent mean ± SEM. *p < 0.05. (C)…

• 

  Figure 4. Expression of signaling targets at the protein level in the bladder. Groupings of gels/blots were cropped from different parts of the same gel. We washed the membranes by WB Stripping Solution Strong to allow two uses. With the first wash, anti-HIF-1α, anti-GCH-1, anti-SR, anti-DHFR or anti-eNOS was used. With the second wash, anti-β actin was used every time. Expression of HIF-1α, an oxidative stress marker, is significantly increased in the CBI group as compared with the other groups. Expression of eNOS is significantly higher in the CBI group and CBI/BH4 group than in the Control group. In the CBI group, expression of DHFR, which can regenerate BH4 from BH2 via the salvage pathway of BH4 synthesis, is significantly increased compared with the other groups. No significant differences in SR or GCH-1 expression are evident among the three groups. Each bar represents the mean ± SEM of 6–9 determinations, each from a different bladder. *p < 0.05. HIF-1α: hypoxia-inducible factor 1; GCH-1: guanosine triphosphate cyclohydrolase 1; SR: sepiapterin reductase; DHFR: dihydrofolate reductase; eNOS: endothelial nitric oxide synthase.