Molecular Pharmacology, Vol 10, 563-581, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

The Reaction of [³H]Norepinephrine with Particulate Fractions of Cells Responsive to Catecholamines

¹ Department of Pharmacology, School of Medicine, University of Virginia, Charlottesville, Virginia 229O3

Some previous reports of [³H]norepinephrine binding to beta adrenergic receptors were reexamined in cultured glial cells (C6), other clonal cell lines, and canine cardiac microsomes. There was no correlation between [³H]norepinephrine binding to particulate preparations from five clones and the ability of catecholamines to stimulate cyclic 3',5'-AMP synthesis in intact cells. While the [³H]norepinephrine binding with particulate fractions required hours for completion, cyclic AMP accumulation rates in intact cells were maximal within seconds even at low hormone concentrations. The [^3H]norepinephrine binding reaction could only be blocked by those adrenergic drugs with a catechol moiety. There was no apparent pharmacological specificity. Furthermore, o- and p-dihydroxybenzene derivatives (1-10 µM), but not meta derivatives (e.g., catechol and hydroquinone, but not resorcinol), blocked the reaction of [³H]norepinephrine with the particulate fractions. Since effective inhibitors thus appeared to be reducing agents, ascorbic acid and sodium metabisulfite were tested. They were found to be potent inhibitors of the reaction in micromolar concentrations. When tested in intact cells, many compounds able to block binding (dopa, 5-hydroxydopa, pyrogallol, catechol, hydroquinone, ascorbate, metabisulfite) neither stimulated (at 0.1 mM) nor blocked (at 10⁵:1 excess) catecholamine-stimulated cyclic AMP synthesis. Inhibition by EDTA of the [³H]norepinephrine binding reaction was reversed by micromolar Cu⁺⁺, Fe⁺⁺, and Mn⁺⁺ but not by Ca⁺⁺, Mg⁺⁺, and Ba⁺⁺. Depletion of oxygen in reaction mixtures with N₂ inhibited binding. The reaction of [³H]norepinephrine with cell particulate fractions could not be reversed after prolongee exposure to 1 N HCl, 5% trichloroacetic acid or 1 mM norepinephrine. After incubation little of the ³H in the medium was identifiable as [³H]norepinephrine. We hypothesize that observed [³H]norepinephrine binding to cell fractions involves oxidation and subsequent covalent reaction with macromolecules and, as such, does not represent binding to the beta adrenergic receptor. Conditions are discussed under which true binding of catecholamines to beta adrenergic receptors might be measured.

Note:
ACKNOWLEDGMENTS The authors are indebted to Drs. T. C. Westfall, M. J. Peach, and G. Brooker for advice and criticism. We are also happy to acknowledge the expert technical assistance of Mses. Hannah Anderson and Kathleen Gallagher.