Tetrahydrobiopterin is the essential cofactor for tyrosine and tryptophan hydroxylase, the rate-limiting enzymes in the synthesis of the monoamine neurotransmitters dopamine, norepinephrine and serotonin. Tetrahydrobiopterin is also required by the family of nitric oxide synthases for the synthesis of the gaseous neurotransmitter nitric oxide from arginine. Inherited cell-type specific deficiencies in human monoamine neurotransmission exist that are the direct result of genetic defects in tetrahydrobiopterin synthesis. whether deficiencies in neuronal nitric oxide production can result from similar genetic defects remains to be determined. This renewal application outlines a broad series of experiments performed in the intact animal and in tissue culture that are designed to further our understanding of the role of tetrahydrobiopterin in the control of monoamine and nitric oxide synthesis.
Specific Aim 1 characterizes gene expression for tetrahydrobiopterin biosynthetic enzymes within identified monoamine neurons using in situ hybridization and immunoautoradiographic techniques. It is expected that these studies will generate important new information on the distribution of these enzymes across populations of monoamine neurons.
Specific Aim 2 investigates the regulation of tetrahydrobiopterin synthesis within monoamine neurons. Changes in gene expression will be quantitated in neurons from representative monoamine cell groups following a challenge with the monoamine-depleting drug reserpine. Monoamine-containing neurons maintained in tissue culture will be used to investigate the second messenger systems involved in the long and short-term regulation of tetrahydrobiopterin synthesis.
Specific Aim 3 characterizes gene expression for tetrahydrobiopterin biosynthetic enzymes within nitric oxide neurons using in situ hybridization and immunoautoradiographic techniques and should yield important new information on the distribution of these enzymes across populations of nitric oxide neurons that contain different isotypes of nitric oxide synthase.
Specific Aim 4 studies the short and long term regulation of tetrahydrobiopterin synthesis within two different populations of nitric oxide neurons maintained in tissue culture. These studies will help to define the cellular mechanisms that regulate tetrahydrobiopterin synthesis within nitric oxide neurons and will address the potential ability of tetrahydrobiopterin to modulate neuronal nitric oxide production. An overall understanding of the control of tetrahydrobiopterin biosynthesis within specific populations of monoamine and nitric oxide neurons should help facilitate research on clinical disorders hypothesized to involve these neurotransmitters.
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