The most common pituitary abnormality is hyperprolactinemia, or the overproduction and secretion of prolactin (PRL) by lactotroph cells in the anterior pituitary. In women this usually results in amenorrhea and galactorrhea. Hyperprolactinemia may be the result of macro or microadenomas of the pituitary, although in many cases the etiology is not known. PRL secretion is controlled by numerous factors found in the hypothalamus, some which act directly on the lactotroph to inhibit or release PRL, and some which exert their effect on neurons in the hypothalamus. The long-term objective is to examine the role of hypothalamic systems in regulation of PRL during several different physiological states.
Specific aims are: 1) to examine the catalytic activity of tyrosine hydroxylase (TH) the initial and rate limiting enzyme controlling synthesis of DA in TIDA neurons during pregnancy, lactation, hyper and hypoprolactinemia; 2) to measure TH gene expression during these states; 3) to determine whether acute changes in TH related to physiological state are due to alteration in the phosphorylation state of the TH molecule; 4) to determine the role of calmodulin-dependent and cAMP dependent kinases in acute TH activation in TIDA neurons, and what physiological factors regulate this; 5) to establish a long-term TIDA neuronal culture system in order to assess regulation of TH directly; 6) to evaluate the regulation of PRL gene expression in the lactotroph. Neuronal cultures are established in defined media using hypothalamic tissue of rat fetuses. Phosphorylation state of TH is determined in vitro by incorporation of inorganic phosphate into TH. DA synthesis is measured by accumulation of L-dihydroxyphenylalanine in the hypothalamus after addition of a decarboxylase inhibitor. The relative differences in mRNA concentration and enzyme quantity for tyrosine hydroxylase is evaluated by in situ hybridization and immunocytochemistry, respectively. PRL mRNA levels are determined by dot blot analysis. Elevated levels of PRL or placental lactogen-I (PL-I) in the rat is accomplished by injection of tumor cells that specifically secrete PRL (MMQ) or PL-1 (Rcho) into rats or by infusing PL-I itself. Dispersed pituitary cells are used in culture to assess the effect of PL and PRL feedback on responsiveness of lactotrophs to prolactin releasing factors. Hormone levels are determined by radioimmunoassays. Monoamines are measured by HPLC.
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