Localization of GTP cyclohydrolase in monoaminergic but not nitric oxide-producing cells

The first and rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis is GTP cyclohydrolase (GTPCH). BH4 serves as the essential cofactor for aromatic L-amino acid hydroxylases, such as tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), as well as for nitric oxide synthase (NOS). We hypothesized that to provide access to the cofactor, a close association exists between BH4-synthesizing and BH4-dependent enzymes, and we determined the relationship among GTPCH, neuronal NOS (nNOS), and TH in rat brain and adrenal gland using immunohistochemistry and in situ hybridization. Analyses of adjacent sections revealed specific localization of GTPCH in TH-containing cells of the substantia nigra, ventral tegmental area, hypothalamus, locus ceruleus, and adrenal medulla, and also in TPH-containing cells of the dorsal raphe nucleus and pineal gland. Thus, BH4 can be synthesized in all monoaminergic cells and is readily available for the enzymes requiring it. In contrast, analysis of adjacent sections showed that nNOS was not colocalized with GTPCH. Scattered nNOS-positive cells were found in the cortex, striatum, cerebellum, and olfactory bulb, all areas that receive monoaminergic innervation. The absence of GTPCH in nNOS cells suggests that nitric oxide-producing cells may either obtain biopterin from monoamine-containing processes which terminate in close proximity, or take up biopterin released into the blood. Double labelling of the same section for TH and nNOS revealed the TH nerve terminals connecting with the nNOS-positive cell bodies, suggesting the possibility that the BH4-containing nerve terminals may directly donate this cofactor to the nNOS-containing cells.