Expression of the L-type Ca2+ 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 Ca2+ influx through voltage-gated L-type Ca2+ channels. To investigate whether CRH and cAMP also increase expression of the L-type Ca2+ 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 Ca2+ 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 mu M 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 Ca2+ channels, the binding of [H-3]PN200-110 to Ca2+ channel proteins was assayed in AtT-20 membrane fragments. 8Br-cAMP increased [H-3]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 Ca2+ channel protein are increased in AtT-20 cells by cAMP.