Characterization and Regulation of β1‐Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Abstract

Intact human neuroepithelioma SK-N-MC cells bound the β-adrenergic antagonist (–)-[³H]-CGP 12177 with a K_D of 0.13 nM and a B_max of 17,500 sites/cell. When the cells were exposed to β-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol norepinephrine > epinephrine, which is indicative of a β₁-subtype receptor. Membranes prepared from the cells bound (–)-3-[¹²⁵I]iodocyanopindolol with a K_D of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the β₁-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the β₂-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only β1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the β₁- and β₂-adrenergic receptors revealed the presence of a very strong β₁-adrenergic receptor mRNA signal, while under the same conditions no β₂-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of β₁-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.