Immunocytochemical localization of GTP cyclohydrolase I in the brain, adrenal gland, and liver of mice

Abstract

GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), the cofactor of phenylalanine, tyrosine, and tryptophan hydroxylases, the enzymes that synthesize tyrosine, catecholamines (dopamine, noradrenaline, and adrenaline), and serotonin, respectively. We produced for the first time polyclonal antibody with highly sensitive immunoreactivity against an oligopeptide of rat enzyme, GEPERELPRPGA, by immunization of rabbits with the peptide conjugated to hemocyanin by glutaraldehyde. The specificity of the antibody was confirmed by Western blot analysis. Using this antibody specific for GCH, we observed strong GCH immunostaining in the liver cells, in the dopamine-, noradrenaline-, adrenaline-, or serotonin-containing cells of the brain, and in the adrenal gland of mice. Immunocytochemical studies revealed GCH to be localized in monoamine-containing perikarya in the periglomerular cells of the olfactory bulb, zona incerta, arcuate nucleus, ventral tegmental area, substantia nigra pars compacta, locus ceruleus, nucleus tractus solitarius, area postrema, and ventrolateral area of the medulla oblongata. GCH immunostaining was particularly strong in serotoninergic nuclei, such as dorsal and median raphe nuclei, nucleus raphe pallidus, and nucleus raphe magnus. By immunoelectron microscopy, GCH-labeled cytoplasm and microtubules in the processes were observed ultrastructurally, but no staining was found in the mitochondria, and Golgi apparatus. Immunostaining was observed neither in the group D neurons that contain only aromatic amino acid decarboxylase without tyrosine hydroxylase, nor in glial cells and endothelial cells. These results indicate the abundant presence of GCH in catecholaminergic and serotoninergic neurons as well as in the adrenal medulla and liver, where BH4 is synthesized as the cofactor of tyrosine, tryptophan, and phenylalanine hydroxylases.

Serotonin and Blood Pressure Regulation

“…An interesting connection between adrenal gland and brain 5-HT synthesis was made by Miller et al (1980), who demonstrated that brain tryptophan utilization was determined by the presence or absence of the adrenal gland. GTP cyclohydrolase is present in the adrenal gland, and this makes the tetrahydrobiopterin that is a necessary cofactor of the tyrosine, tryptophan, and phenylalanine hydroxylases (Nagatsu et al, 1995). Thus, there is potential for 5-HT to modify blood pressure through actions in the adrenal gland.…”

Catecholamines and Serotonin Are Differently Regulated by Tetrahydrobiopterin

“…Western Blotting-Antibodies were made against purified bovine TH (26), purified bovine AADC (23), and synthetic peptide for rat GCH (27). Monoclonal antibody against TPH was purchased from Oncogene.…”

“…The brains were dissected and sliced at 40 m, and their adrenal glands and kidneys, at 25 m, with a cryostat after having been rinsed with sucrose. Sections were pretreated with 0.5% hydrogen peroxide (H 2 O 2 ), blocked with 5% normal swine serum or 5% normal goat serum, and incubated with rabbit anti-TH serum (26), rabbit anti-AADC serum (26), rabbit anti-GCH serum (27) and mouse anti-TPH monoclonal antibody (Ab-1, 0nco-gene, Cambridge) diluted to 1:100,000, 1:20,000, 1:1000, and 1 ng/l respectively, followed by biotinylated goat anti-rabbit IgG or biotinylated anti-mouse IgG and avidin-biotin peroxidase complex (Vectastain ABC kit, Funakoshi). Then the sections were visualized by a reaction with 3,3Ј-diaminobenzidine tetrahydrochloride and 0.003% H 2 O 2 .…”

Tyrosine hydroxylase replacement in experimental Parkinson’s disease with transvascular gene therapy

“…GTP-cylcohydrolase (GTPCH) is the rate-limiting enzyme in the pathway leading to the production of tetrahydrobiopterin, and cultured rat glioma cells 26 and cancer cell lines 27 produce the GTPCH enzyme, although glial cells in brain do not normally express the GTPCH gene. 28 Both clone 877 and clone 951 produce TH enzyme activity in either cultured rat glioma cells or cultured human U87 glioma cells (Table 1). There is a 5-to 8-fold higher level of TH gene expression in human cells, which are targeted with the HIRmAb, as opposed to TH gene expression in rat glioma cells, which are targeted with the TfRmAb.…”