Effects of a Novel Xanthine Oxidoreductase Inhibitor on Increasing ATP in Mouse Vascular Smooth Muscle Cells and High Precision Analysis of Intracellular Purine Compounds by HPLC Using an Internal Standard

Abstract

Xanthine oxidoreductase (XOR) is an enzyme that catalyzes uric acid production by catabolizing hypoxanthine, an intermediate product of the synthesis and degradation of purine nucleotides, including adenosine 5'-triphosphate (ATP). Considering that hypoxanthine is utilized for purine nucleotide synthesis via the salvage pathway, XOR inhibition is expected to increase the hypoxanthine levels and enhance ATP production, leading to the treatment of various diseases. We investigated the effects of NP-1250-HU, a novel potent XOR inhibitor, on the intracellular ATP and purine levels. In the mouse vascular smooth muscle-derived cell line, NP-1250-HU decreased the uric acid levels and increased the hypoxanthine levels at concentrations of 0.3–3.0 µM. The addition of 2.0 µM NP-1250-HU to the medium significantly increased the intracellular ATP levels under hypoxic conditions and recovered cell viability upon respiratory chain inhibition. Compared to other XOR inhibitors, 2.0 µM NP-1250-HU and 10 µM febuxostat showed similar increases in the ATP levels under hypoxic conditions, but 50 µM allopurinol did not. These results suggest that NP-1250-HU contributes to increasing the ATP levels via the salvage pathway by increasing the intracellular hypoxanthine levels. This paper briefly describes a high-performance liquid chromatography method for analyzing purine compounds using caffeine as an internal standard.