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Use of a density shift method to assess beta-adrenergic receptor synthesis during recovery from catecholamine-induced down-regulation in human astrocytoma cells

GL Waldo, RC Doss, JP Perkins and TK Harden

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.

Volume 26, Issue 3, pp. 424-429, 11/01/1984
Copyright © 1984 by American Society for Pharmacology and Experimental Therapeutics




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Copyright © 1984 by the American Society for Pharmacology and Experimental Therapeutics