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An Antisense Oligodeoxynucleotide to Lipocortin 1 Reverses the Inhibitory Actions of Dexamethasone on the Release of Adrenocorticotropin from Rat Pituitary Tissue in Vitro¹

Department of Pharmacology (A.D.T., H.C.C., J.C.B.), Charing Cross and Westminster Medical School, London, W6 8RF, United Kingdom; Department of Human Anatomy (J.F.M.), The University of Oxford, Oxford, OX1 3QX, United Kingdom; Department of Biochemical Pharmacology R.J.F.), The William Harvey Research Institute, St. Bartholomew’s, and the Royal London School of Medicine and Dentistry at Queen Mary and Westfield College, London, EC1M 6BQ, United Kingdom

Address all correspondence and requests for reprints to: Professor Julia Buckingham, Department of Pharmacology, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF, United Kingdom. E-mail: j.buckingham{at}cxwms.ac.uk

Our previous studies have demonstrated that lipocortin 1 (LC1, also called annexin 1) is an important mediator of glucocorticoid action in the neuroendocrine system, particularly with regard to the powerful inhibitory actions of the steroids on the secretion of ACTH and its hypothalamic releasing hormones. In the present study, we have used an antisense 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 cells dispersed with collagenase retained their functional and morphological integrity in vitro and sequestered ODNs in a time-dependent manner from the incubation medium. LC1 was readily detected in the cells by Western blot analysis or by immunoprecipitation/autoradiography after preloading with ³⁵S-methionine/cysteine; the bulk of the protein was contained within an intracellular pool but a small amount was attached to the outer cell surface (pericellular). Dexamethasone (100 nM, 2.5 h) initiated de novo synthesis of LC1; it also increased the amount of LC1 in the pericellular pool detected by either method and caused a concomitant decrease in intracellular LC1. The responses to the steroid were prevented by the inclusion in the medium of an LC1 antisense ODN (50 nM, 3.5 h) but the corresponding sense and scrambled ODN sequences were inert. None of the ODN sequences tested influence the expression of annexin 5 in the pituitary tissue. CRH-41 (100 pM-1 mM), forskolin (1 nM-1 mM) and an L-Ca²⁺-channel opener BAY K8644 (100 pM-1 µM) 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 effects of the steroid were reversed by the LC1 antisense ODN (50 nM, P < 0.01), whereas the LC1 sense and scrambled control sequences (50 nM) were both ineffective in this respect (P > 0.05). The results add further support to the view that the acute inhibitory effects of glucocorticoids on the secretion of ACTH by the pituitary gland are dependent on the generation of lipocortin 1.

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