The effects of rat CRF, arginine vasopressin (VP), oxytocin (OXY), and isoproterenol (ISO) on the biosynthesis and release of pro-ACTH/endorphin-derived peptides by monolayer cultures of rat anterior pituitary cells in complete serum-free medium (CSFM) were studied. When cells were exposed to hormone for 3 h, CRF, VP, OXY, and ISO were each able to stimulate secretion of immunoactive hormone into culture medium. To determine the effects of chronic secretagogue exposure on corticotrope function, cultures were exposed to hormone for 14 days, and total hormone production was measured by immunoassay (cumulative hormone secreted plus cell hormone content). In the absence of CRF, total hormone production increased 3.6 Â± 0.2-fold (mean Â± SEM) over the period from 2âÂ€Â“14 days; chronic CRF treatment brought about a 7.9 Â± 0.7-fold increase in total hormone production over the same period (P 0.0025) or a 2.2-fold increase over control cells. Total hormone production was not affected by chronic treatment with VP (100 nM), OXY (100 nM), or ISO (100 nM); the response of the cells to chronic CRF treatment was unaltered by chronic inclusion of VP, OXY, or ISO. To examine the chronic effects of secretagogues more directly, anterior pituitary cells were grown in control CSFM or in CSFM containing CRF or VP for 7 days and then incubated in medium containing radiolabeled amino acid for 15 min. The newly synthesized pro-ACTH/ endorphin was quantified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Cells grown in CSFM containing CRF synthesized 1.9 times more labeled pro-ACTH/endorphin that cells grown in control CSFM or in CSFM containing VP. Chronic exposure of anterior pituitary cultures to 8-bromo-cAMP stimulated both synthesis and release of pro-ACTH/endorphin-derived peptides, suggesting that a secretagogue capable of producing a sustained elevation in intracellular cAMP levels will stimulate prohormone synthesis.
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