Mycophenolic Acid Simultaneously Reduces Intracellular GTP and Tetrahydrobiopterin Levels in Neuro-2A Cells

Abstract

Tetrahydrobiopterin (BH₄) is involved in neural, immune and lipid functions as a natural cofactor for the aromatic amino acid hydroxylases¹, the O-alkylglycerolipid cleavage enzyme², and nitric oxide synthases^3–5. In contrast to other coenzymes, BH₄ is thought to be a regulator of these enzymes, because its intracellular concentration is within the range capable of affecting enzyme activities and is known to increase in response to the action of cytokines^6–9 and in G/S boundary in rat thymocytes¹⁰, and to decrease with the differentiation of erythroid cells^11,12. BH₄ is synthesized from GTP by sequential actions of GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase. The biosynthesis of BH₄ is mainly regulated at the step of GTP cyclohydrolase I, a ratelimiting enzyme whose activity is increased by a number of cytokines^13–17. In addition to the probable regulation of its transcription¹⁰, we proposed that GTP cyclohydrolase I is regulated according to the availability of GTP, since we have observed cooperative binding of GTP to this enzyme¹⁸. To determine whether or not the level of intracellular GTP is within a range that can affect GTP cyclohydrolase I activity, we examined the effect of changes in the GTP level on the level of BH₄ ¹⁹; IMP dehydrogenase inhibitors, which inhibit the rate-limiting and committing step in de novo synthesis of GTP, were used to reduce the level of intracellular GTP, and guanine or guanosine was used to increase it. These experiments provided evidence that the intracellular GTP concentration in rat PC-12 pheochromocytoma cells and human IMR-32 neuroblastoma cells is the minimum required to elicit the maximal activity by GTP cyclohydrolase I. This supports the theory that GTP might regulate of the reaction catalyzed by GTP cyclohydrolase I¹⁹. In this study, we attempted to confirm and characterize the GTP-BH₄ relationship using mouse Neuro-2a neuroblastoma cells.