Corticotropin-releasing factor (CRF) stimulates 45Ca2+ uptake in the mouse corticotroph cell line AtT-20.
Corticotropin-releasing factor (CRF) stimulates adrenocorticotropin (ACTH) release via the adenylate cyclase/cAMP-dependent protein kinase system. Because calcium is necessary for receptor-mediated release of ACTH, we have examined the effect of CRF on 45Ca2+ uptake in a corticotroph cell line model, AtT-20. Treatment of AtT-20 cells with CRF (10(-9)-10(-6) M) resulted in dose- and time-dependent increases in 45Ca2+ uptake, up to 2.2-fold above control values. The effect was statistically significant at 1 min and persisted for at least 10 min. Treatment with forskolin (1-30 microM), 8-Br-cAMP (0.5 mM), cholera toxin (CT, 100 ng/ml) and K+ (20 mM) also increased cell-associated 45Ca2+. The effect of K+ was completely blocked by nifedipine (100 microM), whereas the effects of CRF (10(-8) M) were only partially inhibited by this calcium channel antagonist. These data suggested a role of voltage-dependent calcium channels in 45Ca2+ uptake. Short term pretreatment (1-2 h) of AtT-20 cells with CRF (10(-8) M) significantly desensitized both CRF-stimulated cAMP accumulation and ACTH release, but did not attenuate CRF-stimulated 45Ca2+ uptake. Pretreatment with CRF (10(-8) M) for 4 h did not alter CT- or forskolin-stimulated cAMP accumulation and ACTH release. This suggests that the molecular mechanisms of desensitization are proximal to adenylate cyclase. Conversely, long term pretreatment (24 h) of AtT-20 cells with CRF (10(-8) M) induced significant desensitization of CRF-stimulated 45Ca2+ uptake. These results indicate that CRF stimulates calcium uptake in AtT-20 cells via cAMP-dependent and cAMP-independent mechanisms, and that the cellular mechanisms involved in desensitization of cAMP accumulation and ACTH release and those involved in desensitization of calcium uptake are qualitatively different.
Tojo
K
K
Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
Abou-Samra
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AB
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D002132:Calcium Radioisotopes / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003346:Corticotropin-Releasing Hormone / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D051379:Mice; D009543:Nifedipine / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002132:Calcium Radioisotopes; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000324:Adrenocorticotropic Hormone; D002772:Cholera Toxin; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D009543:Nifedipine