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.