Glucocorticoids have been shown repeatedly to inhibit the release of prolactin (PRL) in the rat but their site and mode of action is unknown. In the present study, we used an in vitro model to examine the requirement for protein synthesis for dexamethasone to suppress the release of immunoreactive (ir)-PRL release from the rat pituitary gland. In addition we have performed a series of in vitro and in vivo experiments to investigate the potential role in this regard of lipocortin 1 (LC1), a protein shown previously not only to mediate aspects of the anti-inflammatory and anti-proliferative actions of the glucocorticoids but also to contribute to the regulatory actions of the steroids in the brain-neuroendocrine system. In vitro, the release of ir-PRL from rat anterior pituitary tissue initiated by submaximal concentrations of VIP (10 nM). TRH (10 nM) or the adenyl cyclase activator forskolin (100 microM) was reduced significantly (p < 0.01) by preincubation (2 h) of the tissue with dexamethasone (0.1 microM). By contrast, ir-PRL release evoked by a submaximal concentration of the L-Ca2+ channel opener BAY K8644 (10 microM) was unaffected by the steroid although readily antagonised (p < 0.01) by nifedipine (1-100 microM). Exposure of the pituitary tissue to dexamethasone (0.1 microM) also caused a pronounced and highly significant increase in de novo protein synthesis, as assessed by the incorporation of 14C-lysine into the tissue (p < 0.001). This response was reduced markedly by the inclusion of the RNA and protein synthesis inhibitors, actinomycin-D (0.5 micrograms/ml) or cycloheximide (1.0 micrograms/ml), in the incubation medium (p < 0.001), both of which also effectively abrogated (p < 0.01) the dexamethasone-induced inhibition of the release of ir-PRL evoked by TRH. VIP and forskolin. Lipocortin I was readily detectable by Western blotting in protein extracts of freshly excised anterior pituitary tissue: a small proportion of the protein was found to be attached to the outer surface of the cells where it was retained by a Ca(2+)-dependent mechanism. Exposure of the tissue in vitro to dexamethasone (0.1 microM) or corticosterone (0.1 microM) but not 17 beta-oestradiol (0.1 microM) caused a pronounced increase in the amount of LC1 attached to the outer surface of the cells and concomitant decrease in the LC1 content of the intracellular LC1 pool. Addition of an N-terminal LC1 fragment. LC11-188 (10 pg-10 ng/ml), to the incubation medium reduced significantly (p < 0.01) the increases in ir-PRL release induced in vitro by VIP (10 nM) and forskolin (100 microM). By contrast, at all concentrations tested. LC11-188 (10 pg-10 ng/ml) failed to influence (p < 0.05) the highly significant (p < 0.01) ir-PRL response to TRH (10 nM). Similarly, the inhibitory actions of dexamethasone (0.1 microM) on the release of ir-PRL induced by VIP (10 nM) or forskolin (100 microM) but not by TRH (10 nM) were substantially reversed (p < 0.01) by a specific monoclonal anti-LC1 antibody while an isotype-matched control antibody was without effect. In vivo, rats pretreated with either a polyclonal anti LC1 antiserum (anti-LC1 pAb, 1 ml/day s.c. for 2 days) or a corresponding volume of non-immune sheep serum (NSS) responded to stress (laparotomy under ether anaesthesia) with significant (p < 0.05) increases in the serum ir-PRL concentration. In the NSS-treated group, the ir-PRL response to stress was effectively inhibited by dexamethasone (100 micrograms/kg i.p.) which had no effect on the pre-stress serum ir-PRL concentration. By contrast, in rats pretreated with anti-LC1 pAb dexamethasone failed to block the stress-induced release of ir-PRL. The results show clearly that the inhibitory actions of dexamethasone on PRL release are dependent on de novo protein synthesis and provide novel evidence for the involvement of both LC1-dependent and LC1-independent mechanisms.