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Agonist and Cyclic AMP‐Mediated Regulation of β1‐Adrenergic Receptor mRNA and Gene Transcription in Rat C6 Glioma Cells

Abstract: Exposure of rat C6 glioma cells to either agonists or agents that increase cyclic AMP levels leads to down‐regulation of β1‐adrenergic receptors (β1AR) as measured by loss of radioligand binding sites. The present study examines the influence of isoproterenol and forskolin treatment on levels of β1AR mRNA, mRNA stability, and gene transcription rate. Isoproterenol treatment of C6 cells altered β1AR mRNA levels in a biphasic manner; i.e., short‐term exposure (30–60 min) increased by 50%, whereas longer exposure (2–6 h) decreased by 50% the levels of β1AR mRNA. The extent of both the up‐ and down‐regulation was dependent on agonist concentration. Similar regulation of β1AR mRNA was observed in forskolin‐treated cells. Pretreatment of the cells with Pseudomonas exotoxin A, a potent inhibitor of protein synthesis, completely blocked isoproterenol‐ and forskolin‐mediated down‐regulation of β1AR mRNA, and thereby potentiated the increase in receptor mRNA up to fourfold over the 6‐h time course. The mechanisms underlying β1AR mRNA down‐regulation were examined. The half‐life of β1AR mRNA was slightly increased (from 61 to 77 min) after a 2‐h exposure of the cells to either isoproterenol or forskolin. Nuclear run‐on analysis demonstrated that the rate of β1AR gene transcription was increased after isoproterenol incubation for 60 min, but then decreased after 90–240 min, consistent with the time course for up‐ and down‐regulation of β1AR mRNA. Isoproterenol treatment (120 min) also decreased the level of β1AR nascent transcripts, purified by affinity chromatography of RNA isolated from 4‐thiouridine‐pulsed cells. The results demonstrate that β1AR mRNA has a relatively short half‐life and that agonist regulation of β1AR mRNA is mediated by activation of the cyclic AMP system. Moreover, the results indicate that agonist regulation of β1AR mRNA occurs at the level of β1AR gene transcription, not mRNA stability. Finally, the observed requirement for protein synthesis indicates that β1AR mRNA down‐regulation may be mediated by the induction of a repressor of the β1AR gene.