Regulation of human β1-adrenergic receptors and their mRNA in neuroepithelioma SK-N-MC cells

Effects of agonist, forskolin, and protein kinase A

Suleiman Bahouth, Kevin M. Sowinski, John J. Lima

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

We determined the effect of long-term exposure to β-agonists on β1-adrenergic receptors (β1-AR) in human neuroepithelioma SK-N-MC cells because earlier studies have indicated that β1-AR in this cell line are resistant to agonist-induced down-regulation. Exposing SK-N-MC cells to isoproterenol for 24 hr reduced the density of β1-AR by 72%, whereas forskolin, an activator of all the isoforms of adenylyl cyclase, failed to affect the density of β1-AR. Measurement of β1-AR mRNA levels by the ribonuclease protection assay revealed that isoproterenol-induced down-regulation of β1-AR was associated with a sharp decline in β1-AR mRNA, while forskolin also failed to affect this parameter. The differences between the effects of isoproterenol and forskolin on β1-AR were unrelated to cyclic AMP levels, since both agents increased cyclic AMP equally. Next, we determined the role of cyclic AMP-dependent protein kinase A (PKA) in this phenomenon. Inhibition of PKA by its specific inhibitor, H-89 {N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl}, markedly reduced the magnitude of the isoproterenol-mediated down-regulation of the β1-AR and its mRNA. Transient expression of the catalytic subunit of PKA in SK-N-MC cells down-regulated β1-AR independently of isoproterenol. Therefore, PKA is central to the effect of β-agonists in down-regulating β1-AR, and its spacial compartmentalization and access to the receptor appear to be essential components of its action.

Original languageEnglish (US)
Pages (from-to)1211-1220
Number of pages10
JournalBiochemical Pharmacology
Volume62
Issue number9
DOIs
StatePublished - Nov 1 2001

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Colforsin
Cyclic AMP-Dependent Protein Kinases
Adrenergic Receptors
Messenger RNA
Isoproterenol
Down-Regulation
Cyclic AMP
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits
Ribonucleases
Adenylyl Cyclases
Assays
Protein Isoforms
Cells
Cell Line

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Pharmacology

Cite this

Regulation of human β1-adrenergic receptors and their mRNA in neuroepithelioma SK-N-MC cells : Effects of agonist, forskolin, and protein kinase A. / Bahouth, Suleiman; Sowinski, Kevin M.; Lima, John J.

In: Biochemical Pharmacology, Vol. 62, No. 9, 01.11.2001, p. 1211-1220.

Research output: Contribution to journalArticle

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abstract = "We determined the effect of long-term exposure to β-agonists on β1-adrenergic receptors (β1-AR) in human neuroepithelioma SK-N-MC cells because earlier studies have indicated that β1-AR in this cell line are resistant to agonist-induced down-regulation. Exposing SK-N-MC cells to isoproterenol for 24 hr reduced the density of β1-AR by 72{\%}, whereas forskolin, an activator of all the isoforms of adenylyl cyclase, failed to affect the density of β1-AR. Measurement of β1-AR mRNA levels by the ribonuclease protection assay revealed that isoproterenol-induced down-regulation of β1-AR was associated with a sharp decline in β1-AR mRNA, while forskolin also failed to affect this parameter. The differences between the effects of isoproterenol and forskolin on β1-AR were unrelated to cyclic AMP levels, since both agents increased cyclic AMP equally. Next, we determined the role of cyclic AMP-dependent protein kinase A (PKA) in this phenomenon. Inhibition of PKA by its specific inhibitor, H-89 {N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl}, markedly reduced the magnitude of the isoproterenol-mediated down-regulation of the β1-AR and its mRNA. Transient expression of the catalytic subunit of PKA in SK-N-MC cells down-regulated β1-AR independently of isoproterenol. Therefore, PKA is central to the effect of β-agonists in down-regulating β1-AR, and its spacial compartmentalization and access to the receptor appear to be essential components of its action.",
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