Ascorbic acid prevents nonreceptor "specific" binding of [3H]-5-hydroxytryptamine to bovine cerebral cortex membranes
[3H]-5-Hydroxytryptamine ([3H]-5-HT) decomposes rapidly when exposed to air in solution at physiological pH if antioxidants are not present. The decomposition products appear to bind to two saturable sites on brain membranes (apparent Kd values = 1-2 and 100-1000 nM). This binding mimics "speci...
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Published in | The Journal of pharmacology and experimental therapeutics Vol. 240; no. 3; pp. 701 - 711 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
Bethesda, MD
American Society for Pharmacology and Experimental Therapeutics
01.03.1987
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Subjects | |
Online Access | Get full text |
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Summary: | [3H]-5-Hydroxytryptamine ([3H]-5-HT) decomposes rapidly when exposed to air in solution at physiological pH if antioxidants
are not present. The decomposition products appear to bind to two saturable sites on brain membranes (apparent Kd values =
1-2 and 100-1000 nM). This binding mimics "specific" ligand/receptor binding in that it is inhibited by 10 microM unlabeled
5-HT. This inhibition is not competitive, but rather is due to the prevention of [3H]-5-HT breakdown by excess unlabeled 5-HT.
Unlike genuine ligand/receptor binding, the binding of [3H]-5-HT breakdown products is essentially irreversible and does not
display a tissue distribution consistent with binding to authentic 5-HT receptors. [3H]-5-HT decomposition can be eliminated
by the inclusion of 0.05 to 5 mM ascorbic acid. At these concentrations ascorbic acid is not deleterious to reversible [3H]-5-HT
binding. When [3H] 5-HT exposure to air occurs in the presence of brain membranes, the apparent antioxidant activity of brain
membranes themselves affords protection against [3H]-5-HT degradation equal to ascorbic acid. This protection is effective
below final [3H]-5-HT concentrations of 10 nM. Above 10 nM [3H]-5-HT, addition of ascorbic acid or other antioxidants is necessary
to avoid the occurrence of additional low affinity (apparent Kd = 15-2000 nM) binding sites that are specific but nonetheless
irreversible. When care is taken to limit [3H]-5-HT oxidation, the only reversible and saturable specific binding sites observed
are of the 5-HT1 high affinity (Kd = 1-2 nM) type. Radioligand oxidation artifacts may be involved in previous reports of
low affinity (Kd = 15-250 nM) [3H]-5-HT binding sites in brain membrane preparations. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0022-3565 1521-0103 |