The % Tetrahydrobiopterin (BH4) and Serine 1177 Phosphorylation Is Reduced in Sickle Transgenic Mice.

Tetrahydrobiopterin (BH4) is an essential co-factor of nitric oxide synthase (NOS), the enzyme that generates nitric oxide (NO) from arginine and oxygen. NO plays an essential role in maintaining vascular tone and endothelial function, reducing levels of adhesion molecules and platelet aggregation, and reducing inflammation; all of which are critical in sickle cell disease (SCD). In addition to BH4, arginine, calmodulin, intracellular Ca⁺⁺ levels, hsp90, phosphorylation at serine 1177 and threonine 495, and monomer/dimer equilibrium have all been shown to modulate NO and superoxide production by eNOS. Arginine levels are decreased in plasma in SCD. We have demonstrated that plasma arginine is reduced in sickle transgenic mice and that arginine supplementation partially restores both red cell and vascular function. Channon et al have demonstrated that, in atherosclerosis and diabetes, enhanced superoxide production is associated with BH4 depletion; superoxide can interfere with both NO production and, by reacting with NO, with bioavailability. We hypothesized that a similar depletion of BH4 occurs in SCD and that treatment protective of tissue BH4 levels may interrupt the cycle of polymerization, vaso-occlusion and inflammation that is the origin of pathology in SCD. We report here depletion of BH4 in tissue and plasma of several lines of sickle transgenic mice and the effect of hypoxia on % BH4 and serine 1177 phosphorylation in NY1DD mice, a mild sickle transgenic mouse that becomes more severe when exposed to hypoxia. Only BH4 is active as a NOS cofactor, the oxidized forms BH2 and biopterin (B) are inactive. Results are presented as %BH4 (BH4/(BH4+BH2+B)). We find that %BH4 is the same in C57BL and NY1DD mice under room air in plasma and brain. After exposure to 4 days of hypoxia (8% O²), % BH4 is reduced from 75.7%±3.8% to 53.7%±3.8% in plasma, p<0.004 and from 82.0%±3.7% to 68.7%±3.7% in brain, p<0.006. At the same time total biopterin (BT= BH4+BH2+B) increased in plasma, but remained constant in brain. We also measured eNOS phosphorylated at serine 1177 in brain in NY1DD mice. We find that the % phosphorylation decreases from 45.0%±7.9% to 28.3%±7.3%, p<0.007 after 4 days of hypoxia, but that at the same time total eNOS and total nNOS nearly doubled. BERK mice express exclusively HbS and, under normoxia, we found decreased %BH4 in brain in BERK mice (58%±3.3% vs 76.7%±3.0% for C57, p<0.003) with constant BT. In red cells of BERK mice, the %BH4 is about the same as C57BL (81.7% vs 76.7%, respectively), but BT doubled from 124 to 257, p>0.01. In conclusion, increased total expression of eNOS and nNOS and decreased %BH4 with decreased % phosphorylation at serine 1177 of eNOS suggest that NO production in brain in NY1DD mice after hypoxia may be severely compromised. Percent BH4 is also reduced in brain of BERK mice under normoxia, which suggests that BH4 is reduced in more severe mice under room air. Plasma and red cell %BH4 and BT do not have a simple relation to tissue levels, but do respond to pathology and may be the only readily measurable variable in human studies. The multiple pathways for disregulation of NOS in SCD involve not only NO scavenging by plasma free Hb and arginine deficiency, but also reduced bioavailability of BH4.