Stimulation of the Brain NO/Cyclic GMP Pathway by Peripheral Administration of Tetrahydrobiopterin in the hph-1 Mouse *Laura Canevari, *²John M. Land, *John B. Clark, and *²Simon J. R. Heales Departments of *Neurochemistry and ² Clinical Biochemistry, Institute of Neurology and National Hospital for Neurology and Neurosurgery, London, England Abstract: Mutations in GTP-cyclohydrolase I (GTP-CH) have been identified as causing a range of inborn errors of metabolism, including dopa-responsive dystonia. GTP-CH catalyses the first step in the biosynthesis of tetrahydrobiopterin (BH 4 ), a cofactor necessary for the synthesis of catecholamines and serotonin. Current ther- apy based on monoamine neurotransmitter replacement may be only partially successful in correcting the neuro- logical deficits. The reason might be that BH 4 is also a cofactor for nitric oxide synthase. Using a strain of mu- tant GTP-CH-deficient (hph-1) mice, we demonstrate that in addition to impaired monoamine metabolism, BH 4 de- ficiency is also associated with diminished nitric oxide synthesis in the brain (as evaluated by measuring the levels of cyclic GMP), when compared with wild-type animals. We have found a decline in the levels of BH 4 with age in all animals, but no gender-related differences. We found a strong association between the levels of BH 4 and cyclic GMP in hph-1 mice but not in wild-type animals. We also demonstrate that acute peripheral administration of BH 4 (100 mol/kg s.c.) in hph-1 mice significantly elevated the brain BH 4 concentration and subsequently cyclic GMP levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that BH 4 administration should be considered in BH 4 deficiency states in addition to monoamine replacement therapy. Key Words: Nitric oxide synthase —Cyclic GMP—Tetrahydrobiopterin— Dopa-responsive dystonia—GTP-cyclohydrolase defi- ciency—hph-1 mouse. J. Neurochem. 73, 2563–2568 (1999). GTP-cyclohydrolase I (GTP-CH) is the enzyme re- sponsible for the first committed step in the synthesis of tetrahydrobiopterin (BH 4 ). Mutations in the gene for this enzyme are the cause of a group of neurological disor- ders: hereditary progressive dystonia, also known as dopa-responsive dystonia (DRD), when the mutation is heterozygous (autosomal dominant); and the more severe GTP-CH-deficient hyperphenylalaninemia, when ho- mozygous (autosomal recessive). Compound heterozy- gotes have also been described (Furukawa et al., 1998a). GTP-CH-deficient patients present with modest to severe reductions in CSF levels of biopterin and neopterin (Niederwieser et al., 1984; Nygaard et al., 1994). The levels of 5-hydroxyindoleacetic acid and homovanillic acid, products of serotonin and dopamine metabolism, respectively, are also reduced (Niederwieser et al., 1984; Nygaard et al., 1994). BH 4 is an essential cofactor for the aromatic amino acid monooxygenases, including trypto- phan-5-hydroxylase, tyrosine-3-hydroxylase, and phe- nylalanine hydroxylase, which are required for the syn- thesis of serotonin and dopamine, and for the metabolism of phenylalanine (Kaufman, 1978). Currently, patients are treated by administration of L-DOPA and 5-hydroxytryptophan, precursors of dopa- mine and serotonin, respectively, plus dietary phenylal- anine restriction if required. Although typically benefi- cial, these treatments are often only partially effective, and despite the correction of the neurotransmitter deficit and the peripheral hyperphenylalaninemia (McInnes et al., 1984; Tanaka et al., 1989), some symptoms persist, ranging from mental retardation to uncontrolled move- ments, rigor, and hypersalivation (Endres, 1985). BH 4 is also an essential cofactor for all known iso- forms of nitric oxide (NO) synthase (NOS) (Tayeh and Marletta, 1989), and an impairment in the metabolism of NO might be responsible for the persistence of neuro- logical symptoms in GTP-CH-deficient patients. Numer- ous important functions, largely mediated via the stimu- lation of guanylate cyclase to produce cyclic GMP (cGMP), have been identified for NO in the brain, e.g., as an intercellular messenger in memory formation and synaptic plasticity, in the modulation of ion channels and receptors such as the NMDA type and neurotransmitter release, and in activation of protein kinases and phos- phodiesterases (Garthwaite, 1991; Garthwaite and Boul- ton, 1995). NO is a very short-lived molecule, but its Received May 28, 1999; revised manuscript received August 5, 1999; accepted August 5, 1999. Address correspondence and reprint requests to Dr. L. Canevari at Department of Neurochemistry, Institute of Neurology, Queen Square, London WC1N 3BG, U.K. Abbreviations used: BH 4 , tetrahydrobiopterin; cGMP, cyclic GMP; DRD, dopa-responsive dystonia; GTP-CH, GTP-cyclohydrolase I; NO, nitric oxide; NOS, nitric oxide synthase. 2563 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry