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 cell s 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 t o isoproterenol for 24 hr reduced the density of beta (1)-AR by 72%, wherea s forskolin, an activator of all the isoforms of adenylyl cyclase, failed t o affect the density of beta (1)-AR. Measurement of beta (1)-AR mRNA levels by the ribonuclease protection assay revealed that isoproterenol-induced d own-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 diffe rences between the effects of isoproterenol and forskolin on beta (1)-AR we re unrelated,to cyclic AMP levels, since both agents increased cyclic AMP e qually. 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-regulatio n of the beta (1)-AR and its mRNA. Transient expression of the catalytic su bunit of PKA in SK-N-MC cells down-regulated beta (1)-AR independently of i soproterenol. Therefore, PKA is central to the effect of beta -agonists in down-regulating beta (1)-AR, and its spacial compartmentalization and acces s to the receptor appear to be essential components of its action. (C) 2001 Elsevier Science Inc. All rights reserved.