The longterm objective of this research is to define the hormonal, neurohormonal, and neuromodular control of the molecular and cellular mechanisms involved in the biosynthesis and release of hypothalamic dopamine. To achieve this objective, the role of ovarian steroid hormones, prolactin, vasoactive intestinal hormone, opiate-like peptides, serotonin, nerve growth factor, and inorganic cations on dopamine secretion in hypothalamic dopaminergic cells will be studied. The effect of these agents on the level of tyrosine hydroxylase (TH) mRNA, the mass of TH, the in situ enzymic activity of TH, and phosphorylation of TH in the tuberoinfundibular dopaminergic neurons as well as the release of dopamine into hypophysial portal blood will be investigated. TH mRNA will be quantified using a single stranded (32P)-labelled RNA probe complementary to TH mRNA. The radiolabelled RNA probe, cRNA, will be synthesized using TH cDNA, pTH.4, that was subcloned into the Pst I/Eco RI sites of pSP65 vector. The circular plasmid is linearized with Hind III, and the linear DNA templates transcribed with SP6 RNA polymerase. TH mRNA will be quantified using a solution hybridization/S1 nuclease assay and the (32P)cRNA probe. The mass of TH in hypothalamic dopaminergic neurons will be quantified using an immunoblot procedure and densitometric spectrometry. Purified TH will be used as the reference standard. The in situ enzymic activity of TH in hypothalamic dopaminergic neurons will be assayed using an L-dihydroxyphenylalanine (DOPA) accumulation procedure. The activity will be expressed as moles of DOPA synthesized per hr per mole of TH. The release of dopamine will be evaluated on the basis of the rate of release of dopamine into hypophysial portal blood. The findings of this study will provide insight into the mechanisms controlling neurosecretion by dopaminergic neurons of the brain. An understanding of the molecular and cellular events underlying the secretion of dopamine is of foremost importance in the development of an understanding of the development as well as the degenerative effects of aging on dopamine secreting neurons. An inability to secrete sufficient dopamine can result in Parkinsonism, a disease that afflicts one percent of all persons older than 65 years of age.
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