Regulation of human beta(1)-adrenergic receptors and their mRNA in neuroepithelioma SK-N-MC cells: effects of agonist, forskolin, and protein kinase A.
We determined the effect of long-term exposure to beta-agonists on beta(1)-adrenergic receptors (beta(1)-AR) in human neuroepithelioma SK-N-MC cells because earlier studies have indicated that beta(1)-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 beta(1)-AR by 72%, whereas forskolin, an activator of all the isoforms of adenylyl cyclase, failed to affect the density of beta(1)-AR. Measurement of beta(1)-AR mRNA levels by the ribonuclease protection assay revealed that isoproterenol-induced down-regulation of beta(1)-AR was associated with a sharp decline in beta(1)-AR mRNA, while forskolin also failed to affect this parameter. The differences between the effects of isoproterenol and forskolin on beta(1)-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 beta(1)-AR and its mRNA. Transient expression of the catalytic subunit of PKA in SK-N-MC cells down-regulated beta(1)-AR independently of isoproterenol. Therefore, PKA is central to the effect of beta-agonists in down-regulating beta(1)-AR, and its spatial compartmentalization and access to the receptor appear to be essential components of its action.
Bahouth
S W
SW
Department of Pharmacology and Pharmaceutical Sciences, Colleges of Medicine and Pharmacy, The University of Tennessee Health Sciences Center, Memphis, TN 38163, USA. sbahouth@utmem.edu
Sowinski
K M
KM
Lima
J J
JJ
D000322:Adrenergic Agonists / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides; D014407:Tumor Cells, Cultured
D000322:Adrenergic Agonists; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D012333:RNA, Messenger; D018342:Receptors, Adrenergic, beta-1; D013449:Sulfonamides; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide