β adrenergic receptor repopulation of C6 glioma cells after irreversible blockade and down regulation

Abstract

C₆ glioma cells possess β adrenergic receptors coupled with adenylate cyclase which can be irreversibly blocked by bromoacetylaminomethylpindolol (Br‐AAM‐pindolol), a β adrenergic antagonist. With 1 μM Br‐AAM‐pindolol, more than 80% of β adrenergic receptors, labeled by (³H)‐dihydroalprenolol ((³H)‐DHA), were blocked. After this blockade, new β adrenergic receptors were synthesized only during cell division. However, at cell confluency when the cell number was constant, turnover of β adrenergic receptors was barely detectable. Cycloheximide (1 μg/ml) inhibited cell growth as well as reappearance of β adrenergic receptors. A 90% loss of β adrenergic receptors in C₆ glioma cells was obtained after down‐regulation for 15 h with 10 μM isoproterenol, a β adrenergic agonist. After removal of the agonist, recovery of β‐adrenergic‐sensitive adenylate cyclase was complete within 2 to 3 days, whereas β adrenergic receptors reached 90% of control value within 6 days. The half‐life of the receptor recovery was 2 to 3 days. Pretreatment of C₆ glioma cells by Br‐AAM‐pindolol and subsequent cell exposure to isoproterenol indicated that down regulation and recovery of unblocked β adrenergic receptors did occur; however isoproterenol did not accelerate the biosynthesis of β adrenergic receptors. The recovery of both biological response and β adrenergic receptor occupancy was restored both in the presence or absence of cycloheximide (1 μg/ml), a concentration which blocked 90% of protein synthesis. Our results suggest that reappearance of β adrenergic receptors in C₆ glioma cells, following isoproterenol‐induced down regulation, was not due to synthesis of new receptors but to recycling of the β adrenergic receptors.