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 synthases3–5. 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 cytokines6–9 and in G/S boundary in rat thymocytes10, and to decrease with the differentiation of erythroid cells11,12. 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 cytokines13–17. 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.