Abstract:
:The utility of the established ACTH secreting mouse pituitary tumor cell line AtT20 for investigating early glucocorticoid inhibition was examined. Three different strains of the cell line D1, D16v, and D16:16, respectively, were analyzed. In initial studies CRF and phorbol esters were used as secretagogues to examine the properties of hormone secretion. In a perifusion system (cells in suspension) D1 cells failed to respond to the secretagogues, whereas both D16v and D16:16 cells were responsive. However, hormone release declined upon repeated exposure to secretagogue in both D16v and D16:16 cells and similar data were obtained when cells adhering to cover slips were perifused. In static incubation D16:16 cells gave more consistent results especially with respect to inhibition by glucocorticoids and were used in all subsequent studies. Synthetic glucocorticoids acting through the type II receptor inhibited CRF-induced ACTH release within 45 min; at 120 min, stimulated release was strongly (80-90%) suppressed. In contrast, no consistent inhibition by corticosterone could be found. In the presence of glycyrrhetinic acid, an inhibitor of 11 beta-hydroxysteroid dehydrogenase, a high concentration of corticosterone (10 microM) did produce a slight inhibition of ACTH release. Dexamethasone also inhibited ACTH release induced by the calcium channel activator compound (+)202-791. The accumulation of cAMP in response to CRF was not altered by dexamethasone. The inhibitory effect of synthetic glucocorticoids on ACTH release was prevented by blockers of messenger RNA (actinomycin D, dichlorobenzimidazole ribofuranoside) or protein (puromycin) biosynthesis, indicating the induction of new proteins. Immunoblotting for lipocortin I (annexin I) and chromogranin A revealed no induction by dexamethasone of any of these proteins in D16:16 cells. Messenger RNA encoding lipocortin I was not detectable and was not induced by treatment with dexamethasone in D16:16 cells. These data show that the AtT20 D16:16 strain is a useful model for early glucocorticoid action, which is mediated by type II receptors and involves the induction of new protein(s). Notably, induction of lipocortin I messenger RNA or protein could not be detected at a time when the inhibitory effect of glucocorticoids on stimulated hormone secretion was maximal.
journal_name
Endocrinologyjournal_title
Endocrinologyauthors
Woods MD,Shipston MJ,Mullens EL,Antoni FAdoi
10.1210/endo.131.6.1332850subject
Has Abstractpub_date
1992-12-01 00:00:00pages
2873-80issue
6eissn
0013-7227issn
1945-7170journal_volume
131pub_type
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