Proadrenocorticotropin/Endorphin Production and Messenger Ribonucleic Acid Levels in Primary Intermediate Pituitary Cultures: Effects of Serum, Isoproterenol, and Dibutyryl Adenosine 3′,5′-Monophosphate*

Abstract

Long term primary rat intermediate pituitary cells cultured in complete serum-free medium (CSFM) were previously shown to exhibit a low basal secretory rate and maintain a stable cellular level of immunoactive pro-ACTH/endorphin (PAE)-related peptides. The present studies used biosynthetic labeling techniques to demonstrate that the biosynthesis of PAE-derived peptides declined as a function of the time the cultures were maintained in CSFM. Compared to hormone biosynthesis in freshly isolated cells, the ability of melanotropes maintained in CSFM to synthesize hormone declined approximately 3-fold after 5 days and 7-fold after 10 days. The endoproteolytic processing of PAE was not changed. This diminution in biosynthetic rate was not observed in serum-supplemented cultures; the rates of hormone secretion and hormone biosynthesis were both substantially higher in cultures maintained in serum-containing medium, suggesting that stimulatory factors were present in serum supplements. Chronic treatment of intermediate pituitary cultures in CSFM with 100 microM (Bu)2cAMP or 100 nM isoproterenol mimicked the effects of serum on total PAE production, hormone biosynthesis, and PAE mRNA levels. The diminished biosynthetic ability of cultures maintained in CSFM for several days could be restored by subsequent chronic (B mu)2cAMP or isoproterenol treatment. Taken together with previous information, these results suggest that melanotropes maintained in CSFM exist in a functionally basal state and that stimulatory agents, in addition to inhibitory inputs such as dopamine, exert significant regulatory control over intermediate pituitary lobe function in vivo.