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 Citations (Scopus)

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

Fingerprint

Cyclic AMP
Messenger RNA
Corticotropin-Releasing Hormone
Proteins
Adrenocorticotropic Hormone
Cytoprotection
Densitometry
Ribonucleases
Adenylyl Cyclases
Genes
Half-Life
Actins
Binding Sites
Cell Line
Membranes

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

Cite this

Expression of the L-type Ca2+ channel in AtT-20 cells is regulated by cyclic AMP. / Xie, Jiangang; Nagle, Gregg T.; Childs, Gwen V.; Ritchie, Aileen K.

In: Neuroendocrinology, Vol. 70, No. 1, 01.01.1999, p. 1-9.

Research output: Contribution to journalArticle

Xie, Jiangang ; Nagle, Gregg T. ; Childs, Gwen V. ; Ritchie, Aileen K. / Expression of the L-type Ca2+ channel in AtT-20 cells is regulated by cyclic AMP. In: Neuroendocrinology. 1999 ; Vol. 70, No. 1. pp. 1-9.
@article{d4f12ee4b59a46998a024ebed3735230,
title = "Expression of the L-type Ca2+ channel in AtT-20 cells is regulated by cyclic AMP",
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.",
keywords = "AtT-20 cells, Ca channels, Corticotropin, Corticotropin-releasing hormone, Molecular neuroendocrinology, cAMP",
author = "Jiangang Xie and Nagle, {Gregg T.} and Childs, {Gwen V.} and Ritchie, {Aileen K.}",
year = "1999",
month = "1",
day = "1",
doi = "10.1159/000054454",
language = "English (US)",
volume = "70",
pages = "1--9",
journal = "Neuroendocrinology",
issn = "0028-3835",
publisher = "S. Karger AG",
number = "1",

}

TY - JOUR

T1 - Expression of the L-type Ca2+ channel in AtT-20 cells is regulated by cyclic AMP

AU - Xie, Jiangang

AU - Nagle, Gregg T.

AU - Childs, Gwen V.

AU - Ritchie, Aileen K.

PY - 1999/1/1

Y1 - 1999/1/1

N2 - 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.

AB - 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.

KW - AtT-20 cells

KW - Ca channels

KW - Corticotropin

KW - Corticotropin-releasing hormone

KW - Molecular neuroendocrinology

KW - cAMP

UR - http://www.scopus.com/inward/record.url?scp=0032768099&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032768099&partnerID=8YFLogxK

U2 - 10.1159/000054454

DO - 10.1159/000054454

M3 - Article

C2 - 10420088

AN - SCOPUS:0032768099

VL - 70

SP - 1

EP - 9

JO - Neuroendocrinology

JF - Neuroendocrinology

SN - 0028-3835

IS - 1

ER -