This continuation proposal focusses on the regulation of tryptophan hydroxylase (Trp H) activity in the serotonergic neurons of the rat brain. Since this enzyme is rate limiting in the synthesis of the transmitter, serotonin, (5-HT) its activity is central for determining the availability of this important substance, which is now implicated as a mediator in a wide variety of functions including mood or affect, anxiety, antinociception, sleep, temperature regulation and central control of blood pressure. Synthesis of 5-HT is enhanced in response to increased 5-HT neuronal impulse flow by a mechanism that may involve activation of Trp H. Thus enzyme prepared from stimulated cortical 5-HT projections shows an activation that is frequency dependent, and reversed by alkaline phosphatase. Inhibition of 5-HT firing on the other hand decreases enzyme activity, again by an alkaline phosphatase reversible process. Experiments are now planned 1) to further characterize the changes in kinetic behaviour of supernatant preparations of the enzyme in response to different levels of 5- HT neuronal firing in chloral hydrate anaesthetized male Sprague Dawley rats in different regions of CNS, with the natural cofactor, 6R-L-erythro-5,6,7,8-tetrahydrobiopterin; 2) to examine the role of candidate transmitter substances, microinfused stereotaxically into the dorsal raphe nucleus on the regulation of enzyme activity in ascending 5-HT projections supplied by this nucleus in awake rats; 3) to examine the effects of various transmitters or putative transmitters on tryptophan hydroxylase activity after incubation with slice preparations; 4) to establish whether in vitro changes in activity and kinetic properties of supernatant enzyme in response to these manipulations are expressed in vivo; 5) to initiate studies aimed at determining whether the activation of enzyme in response to physiological stimuli results from a direct phosphorylation of the enzyme. These latter studies will require an antibody to tryptophan hydroxylase. 5-HT metabolites and 5-HTP, generated in the Trp H assay, are isolated and quantitated by HPLC with electrochemical detection. Important insights should be gained from this work regarding cellular and molecular mechanisms for maintenance of 5-HT levels and hence 5-HT mediated function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS014090-09
Application #
3395393
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1979-12-01
Project End
1990-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
9
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Type
Overall Medical
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Corley, Karl C; Phan, Tam-Hao; Daugherty, Wilson P et al. (2002) Stress-induced activation of median raphe serotonergic neurons in rats is potentiated by the neurotensin antagonist, SR 48692. Neurosci Lett 319:1-4
Dilts, R P; Novitzki, M R; Phan, T H et al. (1996) Neurotensin inhibits the activation of midbrain serotonergic neurons produced by random inescapable sound. Brain Res 742:294-8
Dilts, R P; Boadle-Biber, M C (1995) Differential activation of the 5-hydroxytryptamine-containing neurons of the midbrain raphe of the rat in response to randomly presented inescapable sound. Neurosci Lett 199:78-80
Singh, V B; Kalimi, M; Phan, T H et al. (1994) Intracranial dehydroepiandrosterone blocks the activation of tryptophan hydroxylase in response to acute sound stress. Mol Cell Neurosci 5:176-81
Singh, V B; Corley, K C; Krieg, R J et al. (1994) Sound stress activation of tryptophan hydroxylase blocked by hypophysectomy and intracranial RU 38486. Eur J Pharmacol 256:177-84
Boadle-Biber, M C (1993) Regulation of serotonin synthesis. Prog Biophys Mol Biol 60:1-15
Boadle-Biber, M C; Singh, V B; Corley, K C et al. (1993) Evidence that corticotropin-releasing factor within the extended amygdala mediates the activation of tryptophan hydroxylase produced by sound stress in the rat. Brain Res 628:105-14
Corley, K C; Singh, V B; Phan, T H et al. (1992) Effect of gepirone on increases in tryptophan hydroxylase in response to sound stress. Eur J Pharmacol 213:417-25
Singh, V B; Hao-Phan, T; Corley, K C et al. (1992) Increase in cortical and midbrain tryptophan hydroxylase activity by intracerebroventricular administration of corticotropin releasing factor: block by adrenalectomy, by RU 38486 and by bilateral lesions to the central nucleus of the amygdala. Neurochem Int 20:81-92
Singh, V B; Onaivi, E S; Phan, T H et al. (1990) The increases in rat cortical and midbrain tryptophan hydroxylase activity in response to acute or repeated sound stress are blocked by bilateral lesions to the central nucleus of the amygdala. Brain Res 530:49-53

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