Expression of the L-type Ca2+ channel in AtT-20 cells is regulated by cyclic AMP

Jiangang Xie, Gregg T. Nagle, Gwen V. Childs, Aileen K. Ritchie

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

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 α(1c) mRNA that encodes the pore-forming subunit of the L-type Ca2+ channel. The α(1c) mRNA level was measured by autoradiographic densitometry and normalized to the β-actin mRNA level in the same sample. The α(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 α(1c) 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 α(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 α(1c) mRNA could be due to an increase in α(1c) gene transcription or to a transcriptionally regulated increase in a protein that helps to stabilize α(1c) 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% (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 α(1c) mRNA and L-type Ca2+ channel protein are increased in AtT-20 cells by cAMP.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalNeuroendocrinology
Volume70
Issue number1
DOIs
StatePublished - Jan 1 1999
Externally publishedYes

Keywords

  • AtT-20 cells
  • Ca channels
  • Corticotropin
  • Corticotropin-releasing hormone
  • Molecular neuroendocrinology
  • cAMP

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Endocrinology
  • Endocrine and Autonomic Systems
  • Cellular and Molecular Neuroscience

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