AN ANTISENSE OLIGODEOXYNUCLEOTIDE TO LIPOCORTIN-1 REVERSES THE INHIBITORY ACTIONS OF DEXAMETHASONE ON THE RELEASE OF ADRENOCORTICOTROPIN FROM RAT PITUITARY TISSUE IN-VITRO

Our previous studies have demonstrated that lipocortin 1 (LC1, also ca lled annexin 1) is an important mediator of glucocorticoid action in t he neuroendocrine system, particularly with regard to the powerful inh ibitory actions of the steroids on the secretion of ACTH and its hypot halamic releasing hormones. In the present study, we have used an anti sense oligodeoxynucleotide (ODN) unique to LC1 to investigate further the role of this protein in the regulatory effects of dexamethasone on ACTH release in vitro from rat anterior pituitary cells. Pituitary ce lls dispersed with collagenase retained their functional and morpholog ical integrity in vitro and sequestered ODNs in a time-dependent manne r from the incubation medium. LC1 was readily detected in the cells by Western blot analysis or by immunoprecipitation/autoradiography after preloading with S-35-methionine/cysteine; the bulk of the protein was contained within an intracellular pool but a small amount was attache d to the outer cell surface (pericellular). Dexamethasone (100 nM, 2.5 h) initiated de novo synthesis of LC1; it also increased the amount o f LC1 in the pericellular pool detected by either method and caused a concomitant decrease in intracellular LC1. The responses to the steroi d were prevented by the inclusion in the medium of an LC1 antisense OD N (50 nhl, 3.5 h) but the corresponding sense and scrambled ODN sequen ces were inert. None of the ODN sequences tested influence the express ion of annexin 5 in the pituitary tissue. CRH-41 (100 pM-1 mM), forsko lin (1 nM-1 mM) and an L-Ca2+-channel opener BAY K8644 (100 pM-1 mu M) initiated concentration dependent increases in immunoreactive- (ir-) ACTH release from the pituitary cells that were reduced (P < 0.01) by preincubation with dexamethasone (100 nM, 2.5 h). The inhibitory effec ts of the steroid were reversed by the LC1 antisense ODN (50 nM, P < 0 .01), whereas the LC1 sense and scrambled control sequences (50 nw) we re both ineffective in this respect (P > 0.05). The results add furthe r support to the view that the acute inhibitory effects of glucocortic oids on the secretion of ACTH by the pituitary gland are dependent on the generation of lipocortin 1.