Expression of the L-type Ca(2+) channel in AtT-20 cells is regulated by cyclic AMP.
Activation of adenylyl cyclase by corticotropin-releasing hormone (CRH) stimulates secretion of adrenocorticotropin (ACTH) in rat anterior pituitary corticotropes and in the murine AtT-20 cell line. The stimulation of secretion is mediated by cAMP and is largely dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels. To investigate whether CRH and cAMP also increase expression of the L-type Ca(2+) channel in AtT-20 cells, an RNase protection assay was used to measure the alpha(1C) mRNA that encodes the pore-forming subunit of the L-type Ca(2+) channel. The alpha(1C) mRNA level was measured by autoradiographic densitometry and normalized to the beta-actin mRNA level in the same sample. The alpha(1C) mRNA was not changed by 24-hour treatment with CRH (10-500 nM). A 24-hour treatment with 1 mM 8Br-cAMP significantly increased the alpha(1C) mRNA by 40% over its control. The stimulatory effect was blocked by 2 microM actinomycin D and was, therefore, dependent on gene transcription. The measured half-life of the alpha(1C) mRNA, after inhibition of transcription, was 4.7 +/- 0.3 h in control and 5.2 +/- 0.6 h in the presence of 8Br-cAMP. Thus the 8Br-cAMP- induced increase in alpha(1C) mRNA could be due to an increase in alpha(1C) gene transcription or to a transcriptionally regulated increase in a protein that helps to stabilize alpha(1C) mRNA. Finally, to determine if the increased mRNA was followed by an increase in production of L-type Ca(2+) channels, the binding of [(3)H]PN200-110 to Ca(2+) channel proteins was assayed in AtT-20 membrane fragments. 8Br-cAMP increased [(3)H]PN200-110 binding sites by 32% (B(max) 36.0 +/- 1.2 fmol/mg protein in control vs. 47.4 +/- 3.2 fmol/mg protein in 8Br-cAMP-treated cells) but did not change the K(d). These studies show that both alpha(1C) mRNA and L-type Ca(2+) channel protein are increased in AtT-20 cells by cAMP.
Xie
J
J
Department of Anatomy, University of Texas Medical Branch at Galveston, Galveston, Tex., USA. jixie@utmb.edu
Nagle
G T
GT
Childs
G V
GV
Ritchie
A K
AK
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D003609:Dactinomycin / Q000494:pharmacology; D004095:Dihydropyridines / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D007700:Kinetics; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011863:Radioimmunoassay; D051381:Rats; D012260:Ribonucleases / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects
D000900:Anti-Bacterial Agents; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D012333:RNA, Messenger; D003609:Dactinomycin; D015124:8-Bromo Cyclic Adenosine Monophosphate; C038806:1,4-dihydropyridine; D000324:Adrenocorticotropic Hormone; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D012260:Ribonucleases