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

  • I. Nagatsu
  • H. Ichinose
  • M. Sakai
  • K. Titani
  • M. Suzuki
  • T. Nagatsu
Full Papers

Summary

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, GFPERELPRPGA, 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 micoscopy, 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.

Keywords

GTP cyclohydrolase I tyrosine hydroxylase tryptophan hydroxylase phenylalanine hydroxylase tetrahydrobiopterin liver adrenal medulla brain mouse immunocytochemistry 

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References

  1. Blau N, Thony B, Heizmann CW, Dhondt J-L (1993) Tetrahydrobiopterin efficiency: from phenotype to genotype. Pteridines 4: 1–10Google Scholar
  2. Dinernan JL, Dawson TM, Schell MJ, Snowman A, Snyder SH (1994) Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity. Proc Natl Acad Sci USA 91: 4214–4218Google Scholar
  3. Duch DS, Bowers SW, Woolf JH, Nichol CA (1984) Biopterin cofactor biosynthesis: GTP cyclohydrolase, neopterin and biopterin in tissues and body fluids of mammalian species. Life Sci 35: 1895–1901Google Scholar
  4. Hashimoto R, Ozaki N, Ohta T, Kasahara Y, Kaneda N, Nagatsu T (1990) Plasma tetrahydrobiopterin levels in patients with psychiatric disorders. Neuropsychobiology 23: 140–143Google Scholar
  5. Hatakeyama K, Inoue Y, Harada T, Kagamiyama H (1991) Cloning and sequencing of cDNA encoding rat GTP cyclohydrolase I. The first enzyme of the tetrahydrobiopterin biosynthetic pathway. J Biol Chem 266: 765–769Google Scholar
  6. Hirayama K, Lentz SI, Kapatos G (1993) Tetrahydrobiopterin cofactor biosynthesis: GTP cyclohydrolase I mRNA expression in rat brain and superior cervical ganglia. J Neurochem 61: 1006–1014Google Scholar
  7. Hökfelt T, Martensson R, Björklund A, Kleinau S, Goldstein M (1984) Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy. Classical transmitters in the CNS, vol 2, part 1. Elsevier, Amsterdam, pp 277–379Google Scholar
  8. Ichinose H, Ohye T, Takahashi E, Seki N, Hori T, Segawa M, Nomura Y, Endo K, Tanaka H, Tsuji S, Fujita K, Nagatsu T (1994) GTP cyclohydrolase I gene as a causative gene for hereditary progressive dystonia with marked diurnal fluctuation. Nature Genet 8: 236–242Google Scholar
  9. Jaeger CB, Ruggier DA, Albert VR, Joh TH, Reis DJ (1984) Immunocytochemical localization of aromatic-L-aminoacid decarboxylase. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy. Classical transmitters in the CNS, vol 2, part 1. Elsevier, Amsterdam, pp 387–408Google Scholar
  10. Janssens SP, Shimouchi A, Quertermous T, Bloch DB, Bloch KD (1992) Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem 267: 14519–14522Google Scholar
  11. Kaufman S (1959) Studies on the mechanism of the enzymatic conversion of phenylalanine to tyrosine. J Biol Chem 234: 2677–2682Google Scholar
  12. Kaufman S (1985) Hyperphenylalaninaemia caused by defects in biopterin metabolism. J Inner Metab Dis 8 [Suppl 1]: 20–27Google Scholar
  13. Kosaka T, Kosaka K, Hataguchi Y, Nagatsu I, Wu J-Y, Ottersen OP, Storm-Mathisen J, Hama K (1987) Catecholaminergic neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivities in various brain regions of the rat. Exp Brain Res 66: 191–210Google Scholar
  14. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685Google Scholar
  15. Lovenberg W, Jequire E, Sjoerdsma A (1967) Tryptophan hydroxylase: measurement in pineal gland, brain stem and carcinoid tumor. Science 155: 217–219Google Scholar
  16. Merrifield WB (1970) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 85: 2149–2154Google Scholar
  17. Nagatsu I, Kondo Y, Inagaki S, Karasawa N, Kato T, Nagatsu T (1977) Immunofluorescent studies on tyrosine hydroxylase: application for its axoplasmic transport. Acta Histochem Cytochem 10: 494–499Google Scholar
  18. Nagatsu I, Sakai M, Yoshida M, Nagatsu T (1988) Aromatic L-amino acid decarboxylase-immunoreactive neurons in and around the cerebrospinal fluid-contacting neurons of the central canal do not contain dopamine or serotonin in the mouse and rat spinal cord. Brain Res 475: 91–102Google Scholar
  19. Nagatsu I, Kobayashi K, Fujii T, Komori K, Sekiguchi K, Titani K, Fujita K, Nagatsu T (1990a) Antibodies raised against different oligopeptide segments of human dopamine-β-hydroxylase. Neurosci Lett 120: 141–145Google Scholar
  20. Nagatsu I, Komori K, Takeuchi T, Sakai M, Yamada K, Karasawa N (1990b) Transient tyrosine hydroxylase-immunoreactive neurons in the region of the anterior olfactory nucleus of pre- and postnatal mice do not contain dopamine. Brain Res 511: 55–62Google Scholar
  21. Nagatsu I, Yamada K, Karasawa N, Sakai M, Takeuchi T, Kaneda N, Sasaoka T, Kobayashi K, Yokoyama M, Nomura T, Katsuki M, Fujita K, Nagatsu T (1991) Expression in brain sensory neurons of the transgene in transgenic mice carrying human tyrosine hydroxylase gene. Neurosci Lett 127: 91–95Google Scholar
  22. Nagatsu I, Yamada K, Karasawa N, Kaneda N, Sasaoka T, Kobayashi K, Fujita K, Nagatsu T (1993a) Non-catecholaminergic neuronal expression of human tyrosine hydroxylase in the brain of transgenic mice with special reference to aromatic L-amino acid decarboxylase. In: Naoi M, Palvez HS (eds) Tyrosine hydroxylase. VSP Science Press, Zeist The Netherlands, pp 37–57Google Scholar
  23. Nagatsu I, Yamada K, Sakai M, Karasawa N (1993b) Immunocytochemistry and in situ hybridization of catecholamine-synthesizing enzymes and the related neurotransmitters. In: Parvez SH, Naoi M, Nagatsu T, Parvez S (eds) Methods in neurotransmitter and neuropeptide. Elsevier, Amsterdam, pp 151–183Google Scholar
  24. Nagatsu I, Ichinose H, Sakai M, Nagatsu T (1994) Evidence for localization of GTP cyclohydrolase I immunoreactivity in the mouse brain, adrenal gland and liver. Neurosci Res [Suppl 19]: S67 (Abstract)Google Scholar
  25. Nagatsu T, Levitt M, Udenfriend S (1964) Tyrosine hydroxylase: the initial step in norepinephrine biosynthesis. J Biol Chem 239: 2910–2917Google Scholar
  26. Nagatsu T, Yamaguchi T, Kato T, Sugimoto T, Matsuura S, Akino M, Nagatsu I, Iizuka R, Narabayashi H (1981) Biopterin in human brain and urine from controls and parkinsonian patients: application of a new radioimmunoassay. Clin Chim Acta 109: 305–311Google Scholar
  27. Nagatsu T, Sawada M, Yamaguchi T, Sugimoto T, Matsuura S, Akino M, Nakazawa N, Ogawa H (1984) Radioimmunoassay for neopterin in body fluids and tissues. Anal Biochem 141: 472–480Google Scholar
  28. Nagatsu T, Horikoshi T, Sawada M, Nagatsu I, Kondo T, Iizuka R, Narabayashi H (1986) Biosynthesis of tetrahydrobiopterin in Parkinsonian human brain. Adv Neurol 45: 223–226Google Scholar
  29. Nagatsu T, Matsuura S, Sugimoto T (1989) Physiological and clinical chemistry of biopterin. In: deStevens G (ed) Medical research reviews, vol 9, no 1. John Wiley & Sons, New York, pp 25–44Google Scholar
  30. Nathan C, Xie Q (1994) Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915–918Google Scholar
  31. Niederwieser A, Blau N, Wang M, Joller P, Atares M, Cardesa-Garcia J (1984) GTP cyclohydrolase I deficiency, a new enzyme defect causing hyperphenylalaninemia with neopterin, biopterin, dopamine, and serotonin deficiencies and muscular hypotonia. Eur J Pediatr 141: 208–214Google Scholar
  32. Nomura T, Ichinose H, Sumi-Ichinose C, Nomura H, Hagino Y, Fujita K, Nagatsu T (1993) Cloning and sequencing of cDNA encoding mouse GTP cyclohydrolase I. Biochem Biophys Res Commun 191: 523–527Google Scholar
  33. Nygaard TG, Marsden CD, Duvoisin RC (1988) Dopa-responsive dystonia. In: Fahn S, Marsden CD, Calne DB (eds) Advances in neurology, vol 50. Raven Press, New York, pp 377–384Google Scholar
  34. Nygaard TG, Wilhelmsen KC, Risch NJ, Brown DL, Trugman JM, Gilliam TC, Fahn S, Weeks DE (1993) Linkage mapping of dopa-responsive dystonia (DRD) to chromosome 14q. Nature Genet 5: 386–391Google Scholar
  35. Sawada M, Horikoshi T, Masada M, Akino M, Sugimoto T, Matsuura S, Nagatsu T (1986) A sensitive assay of GTP cyclohydrolase I activity in rat and human tissues using radioimmunoassay of neopterin. Anal Biochem 154: 361–366Google Scholar
  36. Sawada M, Hirata Y, Arai H, Iizuka R, Nagatsu T (1987) Tyrosine hydroxylase, tryptophan hydroxylase, biopterin, and neopterin in the brains of normal controls and patients with senile dementia of Alzheimer type. J Neurochem 48: 760–764Google Scholar
  37. Segawa M, Hosaka A, Miyagawa F, Nomura Y, Imai H (1976) Hereditary progressive dystonia with marked diurnal fluctuation. In: Eldridge R, Fahn S (eds) Advances in neurology, vol 14. Raven Press, New York, pp 215–233Google Scholar
  38. Steinbusch HWM (1981) Distribution of serotonin-immunoreactivity in the central nervous system of the rat—cell bodies and terminals. Neuroscience 6: 557–618Google Scholar
  39. Takeuchi Y, Kimura H, Sano Y (1982) Immunohistochemical demonstration of the distribution of serotonin neurons in the brainstem of the rat and cat. Cell Tiss Res 224: 247–267Google Scholar
  40. Togari A, Ichinose H, Matsumoto S, Fujita K, Nagatsu T (1992) Multiple mRNA forms of human GTP cyclohydrolase I. Biochem Biophys Res Commun 187: 359–365Google Scholar
  41. Uchida K, Tsuzaki N, Nagatsu T, Kohsaka S (1992) Tetrahydrobiopterin-dependent functional recovery in 6-hydroxydopamine-treated rats by intracerebral grafting of fibroblasts transfected with tyrosine hydroxylase cDNA. Dev Neurosci 14: 173–180Google Scholar
  42. Werner-Felmayer G, Werner ER, Fuchs D, Hausen A, Reibnegger G, Schmidt K, Weiss G, Wachter H (1993) Pteridine biosynthesis in human endothelial cells. J Biol Chem 268: 1842–1846Google Scholar
  43. Williams AC, Levine RA, Chase TN, Lovenberg W, Calne DB (1980) CFS hydroxylase cofactor levels in some neurological diseases. J Neurol Neurosurg Psychiatry 43: 735–738Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • I. Nagatsu
    • 1
  • H. Ichinose
    • 2
  • M. Sakai
    • 1
  • K. Titani
    • 2
  • M. Suzuki
    • 2
  • T. Nagatsu
    • 2
  1. 1.Department of Anatomy, School of MedicineFujita Health UniversityAichiJapan
  2. 2.Institute for Comprehensive Medical Science, School of MedicineFujita Health UniversityAichiJapan

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