We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2), insulin-like growth factor-I (IGF-I), or agents which alter intracellular cAMP levels, such as cholera toxin plus isobutylmethylxanthine (CT + IBMX) and 8-Br-cAMP, results in the up-regulation of cellular levels of the progesterone receptor, an effect believed to be mediated through the activation of estrogen receptor (ER) and phosphorylation pathways. We have therefore undertaken studies using transient transfection of these uterine cell cultures with a simple estrogen-responsive reporter gene in order to determine the ability of these agents to stimulate ER-mediated gene transcription directly. We also compared the ability of these same agents to alter the phosphorylation state of the endogenous uterine ER protein. Plasmid DNA containing two tandem estrogen responsive elements and a TATA box linked to the chloramphenicol acetyl transferase (CAT) gene was introduced into immature rat uterine cells grown in primary culture. Treatment of transfected cells with 10(-9) M E2, CT (1 micrograms/ml) + IBMX (10(-4) M), 8-Br-cAMP (10(-4) M), or IGF-I (20 ng/ml) resulted in an 8- to 10-fold induction of CAT activity. CAT activity stimulated by all agents was nearly completely suppressed by coincubation with the antiestrogen ICI 164,384 (ICI) or the protein kinase (PK) inhibitor H8. CAT activity induced by 8-Br-cAMP was more readily suppressed by ICI than that induced by E2, indicating that ER in cells exposed to 8-Br-cAMP is either unoccupied or minimally occupied by ligand. The level of ER phosphorylation in uterine cells was increased 3- to 5-fold upon exposure to E2, CT + IBMX, 8-Br-cAMP, or IGF-I. Of interest, the antiestrogen ICI also elicited a similar increase in overall ER phosphorylation. The PK inhibitors H8 and PKI suppressed the increase in overall ER phosphorylation stimulated by these agents by 50-75%. The results of our study indicate that E2, IGF-I, and agents which raise intracellular cAMP are able to stimulate ER-mediated trans-activation and ER phosphorylation. The fact that antiestrogen (ICI) evokes a similar increase in ER phosphorylation without a similar increase in transcription activation indicates that an increase in overall ER phosphorylation does not necessarily result in increased transcriptional activity.(ABSTRACT TRUNCATED AT 400 WORDS)