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.
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