GTP cyclohydrolase I feedback regulatory protein is expressed in serotoninneurons and regulates tetrahydrobiopterin biosynthesis

Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalani ne, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit , the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L-phenylalanine specifically stimulates tetrahydrobiopterin synthesis by di splacing tetrahydrobiopterin from the GTPCH-GFRP complex. To explore the ro le of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double-label in situ hybridization. GFRP mRNA expression wa s abundant in serotonin neurons of the dorsal raphe nucleus but was undetec table in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrai n. Two species of GFRP mRNA differing by similar to 20 nucleotides in lengt h were detected in brainstem but not in other tissues, with the longer, mor e abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mR NA, although GTPCH mRNA was abundant in both. Primary neuronal cultures wer e used to examine the role of GFRP-mediated regulation of GTPCH on tetrahyd robiopterin synthesis within brainstem serotonin neurons and midbrain dopam ine neurons. L-phenylalanine increased tetrahydrobiopterin levels in seroto nin neurons to a maximum of twofold in a concentration-dependent manner, wh ereas D-phenylalanine and L-tryptophan were without effect. In contrast, te trahydrobiopterin levels within cultured dopamine neurons were not altered by L-phenylalanine. The time course of this effect was very rapid, with a m aximal response observed within 60 min. Inhibitors of tetrahydrobiopterin b iosynthesis prevented the L-phenylalanine-induced increase in tetrahydrobio pterin levels. 7,8-Dihydroneopterin, a reduced pteridine capable of inhibit ing GTPCH in a GFRP-dependent manner, decreased tetrahydrobiopterin levels in cultures of both serotonin and dopamine neurons. This inhibition was rev ersed by L-phenylalanine in serotonin but not in dopamine neurons. Our data suggest that GTPCH activity within serotonin neurons is under a tonic inhi bitory tone mediated by GFRP and that tetrahydrobiopterin levels are mainta ined by the balance of intracellular concentrations of tetrahydrobiopterin and L-phenylalanine. In contrast, although tetrahydrobiopterin biosynthesis within dopamine neurons is also feedback-regulated, L-phenylalanine plays no role, and therefore tetrahydrobiopterin may have a direct effect on GTPC H activity.