The acute and chronic effects of secretagogues activating cAMP-dependent pathways (CRH and cAMP) and activating cAMP-independent pathways [phenylephrine and phorbol 12-myristate 13-acetate (PMA)] on anterior pituitary function were examined in serum-free cultures. Applied acutely, PMA produced a greater stimulation of ACTH/endorphin secretion than CRH or cAMP. However, the effects of CRH and cAMP on secretion were maintained for up to 12 days, while those of PMA and phenylephrine diminished rapidly. Secretagogue effects on pro-ACTH/endorphin biosynthesis were determined by immunoprecipitation of biosynthetically labeled beta-endorphin-related peptides. Cultures exposed to CRH or cAMP and [3H]tyrosine for 12 h produced 1.7 +/- 0.2- and 1.6 +/- 0.1-fold more newly synthesized beta-endorphin-related material than control cells. Cultures exposed to phenylephrine or PMA synthesized 1.3 +/- 0.1- and 1.4 +/- 0.1-fold more peptide than control cells. Exposure of cells to CRH or cAMP for 12 days increased pro-ACTH/endorphin biosynthesis to a greater extent than the 12-h treatment (3.0 +/- 0.1- and 2.5 +/- 0.3-fold over control value, respectively). Exposure to phenylephrine or PMA for 12 days had the same effect on pro-ACTH/endorphin biosynthesis as exposure for 12 h. After acute or chronic secretagogue exposure, the cells secreted relatively more newly synthesized beta-lipotropin than beta-endorphin. Levels of pro-ACTH/endorphin mRNA in cultures treated acutely (12 h) or chronically (12 days) with CRH, cAMP, or phenylephrine changed in parallel with rates of pro-ACTH/endorphin biosynthesis. In contrast, chronic exposure to PMA stimulated biosynthesis while reducing pro-ACTH/endorphin mRNA levels. In summary, these results suggest that factors that activate cAMP-dependent pathways are more powerful stimulators of pro-ACTH/endorphin biosynthesis than factors that activate cAMP-independent pathways; the cAMP-dependent pathway may be primarily responsible for regenerating depleted hormone reserves.