Tryptophan hydroxylase (TrpH), the rate limiting enzyme in the synthesis of 5-hydroxytryptamine (5-HT) in CNS, converts tryptophan to 5-hydroxytryptophan (5-HTP) in a reaction that also requires molecular oxygen and reduced pterin cofactor. The project focusses on factors that may regulate the activity of this enzyme and hence 5-HT synthesis in brain in vivo with particular emphasis on the contribution that changes in its kinetic behaviour may make. 5-HT neuronal impulse flow increases both the hydroxylation of tryptophan in vivo, and the in vitro activity of cortical TrpH and an analysis of the effects of neuronal firing on the kinetic properties of the enzyme is thus a major focus of the project. These studies will be extended to other 5-HT projection areas, particularly spinal cord where evidence exists that regulatory properties of TrpH may be different. The effects on enzyme activity of blockade of 5-HT impulse flow will also be examined. Comparisons will be made of the properties of enzyme activated in vivo with those of enzyme activated in vitro under phosphorylating conditions. The possibility will also be explored that administration into the 5-HT neuronal nuclei of other substances (largely peptides) that may serve a transmitter/modulator role there can influence enzyme activity in the terminal projections of 5-HT neurons e.g. through effects on 5-HT neuronal activity. Finally regulation of enzyme activity in terminal regions versus the cell body and between different terminal fields will be compared. This study is prompted by evidence that various peptides can alter enzyme activity in slices or synaptosomes presumably as a result of direct receptor mediated interactions rather than via effects on 5-HT neuronal firing rate. Enzyme activity will be measured in vitro under subsaturating concentrations of artificial reduced pterin cofactor, 6-methyl-5,6,7,8-tetrahydropterin (6MPH4) or the natural cofactor tetrahydrobiopterin and also in vivo, by the accumulation of 5-HTP in the presence of a decarboxylase inhibitor in order to determine whether increases in activity detected in vitro are expressed in vivo. The 5-HTP formed in each case will be isolated by high performance liquid chromatography (HPLC) and quantitated by electrochemical detection (EC). This study should enhance our understanding of the regulation of 5-HT synthesis, a central monoamine transmitter, which is known to be involved in modulating or regulating a wide variety of functions including mood or affect.
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