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 α1C mRNA that encodes the pore-forming subunit of the L-type Ca2+ channel. The α1C mRNA level was measured by autoradiographic densitometry and normalized to the β-actin mRNA level in the same sample. The α1C 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 α1C mRNA by 40% over its control. The stimulatory effect was blocked by 2 µM actinomycin D and was, therefore, dependent on gene transcription. The measured half-life of the α1C 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 α1C mRNA could be due to an increase in α1C gene transcription or to a transcriptionally regulated increase in a protein that helps to stabilize α1C mRNA. Finally, to determine if the increased mRNA was followed by an increase in production of L-type Ca2+ channels, the binding of [3H]PN200-110 to Ca2+ channel proteins was assayed in AtT-20 membrane fragments. 8Br-cAMP increased [3H]PN200-110 binding sites by 32% (Bmax 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 Kd. These studies show that both α1C mRNA and L-type Ca2+ channel protein are increased in AtT-20 cells by cAMP.

Keywords:
Ca2+ channels, cAMP, AtT-20 cells, Molecular neuroendocrinology, Corticotropin-releasing hormone, Corticotropin
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