Evidence that tyrosine hydroxylase does not have enzymatic activity in neurons of the supraoptic nucleus.

Introduction: Although numerous neurons expressing tyrosine hydroxylase (TH), the first enzyme of catecholamine synthesis, were discovered in the supraoptic nucleus (SON) of hypothalamus in animals almost forty years ago, mainly under osmotic stress, the functional significance of TH there remains unclear. Therefore, the aim of this study was to test our hypothesis that TH in these neurons has enzymatic activity capable of converting L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). Methods: In this study, 8-10 week old male C57BL/6 mice (n = 77), intact (n = 20), and intraperitoneally injected with 0.9% NaCl (n = 29) or 8.5% NaCl (n = 28) were used. To assess gene expression and synthesis of TH, as well as the hypothetical synthesis of L-DOPA in neurons of the SON, the following methods were used: immunohistochemistry for vasopressin (VP), laser microdissection of individual VP-immunopositive neurons, polymerase chain reaction (PCR), Western blotting, and high-performance liquid chromatography with electrochemical detection of dopamine and L-DOPA. Results: The genes for TH, a key-protein required for L-DOPA synthesis in dopaminergic neurons, is expressed in neurons of the SON in both intact, control and salt-loaded mice. However, some data suggest that TH in neurons of the SON does not have enzymatic activity. Indeed, in intact mice and in mice with varying degrees of osmotic stress: (i) the SON did not show expression of the gene encoding guanosine triphosphate cyclohydrolase 1, the first enzyme in synthesis of tetrahydrobiopterin, a cofactor responsible for regulating the enzymatic activity of TH, and (ii) the amount of L-DOPA remained at the same very low level, regardless of the TH content in SON neurons. We suggest that TH in neurons of the SON does not have enzymatic activity, since, as we have shown, the TH molecule in the SON, unlike TH in dopaminergic neurons of the substantia nigra, could possibly not have an N-terminus motif responsible for regulation of its enzymatic activity. Conclusion: Despite the expression of the TH gene in neurons of the SON, the protein it encodes does not have enzymatic activity, most probably due to the absence of the N-terminus, characteristic of TH in dopaminergic neurons.