Use of a density shift method to assess beta-adrenergic receptor synthesis during recovery from catecholamine-induced down-regulation in human astrocytoma cells.
Exposure of postconfluent 1321N1 human astrocytoma cells to 1.0 microM isoproterenol for 12-24 hr results in a 90% loss of beta-adrenergic receptors. Upon removal of agonist, recovery of beta-receptors to control levels occurs within 72 hr. The recovery of receptors is completely blocked by cycloheximide [R. C. Doss, J. P. Perkins, and T. K. Harden, J. Biol. Chem. 256:12281-12286 (1981)]. In contrast cycloheximide does not block recovery of beta-receptors after down-regulation in preconfluent cultures. To determine unambiguously if beta-receptor synthesis accounts for the recovery of receptors after down-regulation, post confluent cultures were incubated with isoproterenol and then transferred to agonist-free medium containing either normal or "heavy" (2H, 13C, 15N) amino acids. The rate and extent of beta-receptor recovery were similar in both normal and heavy amino acid-containing medium. When beta-receptors that had recovered in the heavy amino acid-containing medium were labeled with 125I-cyanopindolol, solubilized in Lubrol PX, and subjected to centrifugation on a 5-15% sucrose density gradient, they exhibited an increased mass compared to beta-receptors that recovered in the presence of normal amino acids. These results confirm that the density shift method is a useful approach for the study of beta-receptor synthesis and that new receptor synthesis occurs during recovery of beta-receptors from catecholamine-induced down-regulation in postconfluent cultures.
D001205:Ascorbic Acid / Q000494:pharmacology; D001254:Astrocytoma / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D020120:Iodocyanopindolol; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D010869:Pindolol / Q000031:analogs & derivatives / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D011943:Receptors, Adrenergic, beta; D020120:Iodocyanopindolol; D010869:Pindolol; D007545:Isoproterenol; D001205:Ascorbic Acid