Dexamethasone inhibits the release of TSH from the rat anterior pituitary gland in vitro by mechanisms dependent on de novo protein synthesis and lipocortin 1.
Glucocorticoids have been shown repeatedly to inhibit the secretion of TSH in experimental animals and in man but their site and mode of action are unknown. In the present study, we have used an in vitro model to examine the effects of dexamethasone on the resting and pharmacologically evoked secretion of TSH by the rat anterior pituitary gland, and to show how they are influenced by inhibitors of RNA/protein synthesis. In addition, we have investigated the potential role of lipocortin 1 (LC1), a protein shown previously to contribute to glucocorticoid action in several systems, as a mediator of the glucocorticoid-induced suppression of TSH release in our in vitro preparation. The significant (P < 0.01) increases in the release of immunoreactive (ir)TSH from rat anterior pituitary tissue initiated by submaximal concentrations of TRH (10 nmol/l), vasoactive intestinal polypeptide (VIP, 10 nmol/l) or the adenyl cyclase activator, forskolin (100 mumol/l) were reduced significantly (P < 0.05) by preincubation of the tissue with dexamethasone (0.1 mumol/l). In contrast, irTSH secretion evoked by a submaximal concentration of the L-Ca2+ channel opener BAY K8644 (10 mumol/l) was unaffected by the steroid, although readily antagonised (P < 0.01) by nifedipine (1-100 mumol/l). Inclusion of actinomycin D (1.78 mumol/l) or cycloheximide (0.8 mumol/l), inhibitors of RNA and protein synthesis respectively, in the medium effectively abrogated the inhibitory effects of dexamethasone (0.1 mumol/l) on the secretory responses to TRH (10 nmol/l), VIP (10 nmol/l) and forskolin (100 mumol/l). LC1 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 to dexamethasone (0.1 mumol/l) caused a pronounced increase in the amount of cellular LC1 attached to the outer surface of the cells and a concomitant decrease in the intracellular LC1 pool. Progesterone (0.1 mumol/l) and aldosterone (0.1 mumol/l) were also weakly active in this regard, but thyroxine and tri-iodothyronine (0.1 mumol/l) were not. Addition of an N-terminal LC1 fragment, LC1(1-188) (0.05-0.53 pmol/l) to the incubation medium reduced significantly (P < 0.01) the increases in irTSH release induced by TRH (10 nmol/l), VIP (10 nmol/l) and forskolin (100 mumol/l), but failed to influence (P < 0.05) those initiated by BAY K8644 (10 mumol/l). Furthermore, the inhibitory actions of dexamethasone (0.1 mumol/l) on the release of irTSH provoked by TRH (10 nmol/l), VIP (10 nmol/l) and forskolin (100 mumol/l) were substantially reversed (P < 0.01) by a specific monoclonal anti-LC1 antibody, while an isotype-matched control antibody was without effect. The results show clearly that dexamethasone, a semi-synthetic glucocorticoid, acts at the pituitary level to inhibit the neurochemically evoked release of irTSH. They also provide novel evidence that the inhibitory actions of the steroid are dependent upon de novo RNA/protein synthesis and that they involve an LC1 dependent mechanism.
Department of Pharmacology, Charing Cross and Westminster Medical School, London, UK.
A D
AD
Taylor
R J
RJ
Flower
J C
JC
Buckingham
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D017305:Annexin A1 / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003864:Depression, Chemical; D003907:Dexamethasone / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D014176:Protein Biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013972:Thyrotropin / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D017305:Annexin A1; D002121:Calcium Channel Blockers; D005576:Colforsin; D014660:Vasoactive Intestinal Peptide; D013973:Thyrotropin-Releasing Hormone; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D003907:Dexamethasone; D013972:Thyrotropin