Effect of methotrexate on pteridine-contents and relaxation response of the aorta from quinonoid-dihydropteridine reductase (QDPR) knockout mice

Abstract

Tetrahydrobiopterin (BH4) is not only an essential cofactor for aromatic L-amino acid hydroxylases but also all types of nitric oxide (NO) synthases. NO synthesized in the endothelial cells is a vasodilator, and contributes to the physiological function of blood vessels.BH4 is catalyzed to quinonoid-dihydrobiopterin (qBH2) in the coupling reaction of aromatic amino acids-hydroxylation, then reduced to BH4 by quinonoid-dihydrobiopterin reductase (QDPR). In the other pathway, qBH2 is oxidized to BH2 non-enzymatically, and reduced to BH4 by dihydrofolate reductase (DHFR). We established Qdpr gene knockout (Qdpr^-/-) mice, and administered methotrexate (MTX) to modify BH4/BH2 ratio in the tissue and analyzed vascular function.

We used four to six months old male C57BL6/Jcl background mice. Pteridine contents were quantified using HPLC-fluorescence detection after separation by a reverse phase column. Nitrate and nitrite was quantified by NO₂/NO₃ Assay Kit-FX (Dojindo). MTX (2 mg/kg) was administered intraperitoneally 2 hours before tissue-sampling. The ring segments were prepared from the thoracic aortas and vascular function was assayed using multi wire myograph system (DMT). Phenylalanine (Phe) content was determined by L-8500 amino acid analyzer (Hitach).

BH4 and BH2 contents in the aorta of wild type mice were 1.45± 0.09 pmol/mg protein and 0.31± 0.04 pmol/mg protein, respectively (BH4/BH2=4.69). Those contents in the aorta of Qdpr^-/- mice were 4.79± 0.86 pmol/mg protein and 1.61± 0.33 pmol/mg protein, respectively (BH4/BH2=2.97). There was no significant difference between two genotypes in the vascular relaxation by the cumulative addition of acetylcholine and sodium nitroprusside. BH4 content in the aorta of MTX-administered Qdpr^-/- mice was 3.00± 0.43 pmol/mg protein, and BH2 was 2.81± 0.54 pmol/mg protein (BH4/BH2=1.07). However, there was no significant difference between four groups (PBS (vehicle)-administered wild type mice, MTX-administered wild type mice, PBS-administered Qdpr^-/- mice, and MTX-administered Qdpr^-/-mice.) in the vascular relaxation and contents of NO metabolites in the plasma. Phe content in the plasma of Qdpr^-/- mice was 11.8 times higher than that of wild type mice.

BH4/BH2 ratio in the vessel was less affected by disruption of QDPR gene and inhibition of DHFR compared to those in the liver and plasma.