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 to 10 μM), but not meta derivatives (e.g., pyrocatechol 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, pyrocatechol, hydroguinone, ascorbate, metabisulfite) neither stimulated (at 0.1 mM) nor blocked (at 105:1 excess) catecholamine stimulated cyclic AMP synthesis. Inhibition by edetic acid 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 prolonged 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. It is suggested 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 bindings of catecholamines to beta adrenergic receptors might be measured.
|Original language||English (US)|
|Number of pages||19|
|State||Published - Jan 1 1974|
ASJC Scopus subject areas
- Molecular Medicine