Dual effects of ascorbate on serotonin and spiperone binding in rat cortical membranes

Ascorbate-induced lipid peroxidation, as measured by malonyldialdehyde (MDA) production, caused irreversible decreases in Bmax of both [3H]5-HT and [3H]spiperone binding. CaCl2 (4 mM) inhibited ascorbate-induced MDA formation at ascorbate concentrations greater than 0.57 mM, but not at less than or equal to 0.57 mM. Under the standard assay conditions (5.7 mM ascorbate and 4 mM CaCl2), CaCl2 inhibited the MDA production caused by ascorbate by 88%, and the loss in [3H]5-HT binding by 57%. Ascorbate still decreased [3H]5-HT binding when lipid peroxidation was completely inhibited by EDTA. This additional effect of ascorbate was reversible after washing the membranes. Other reducing agents (dithiothreitol, glutathione, and metabisulfite) also decreased the binding of [3H]serotonin. In contrast, [3H]spiperone binding was not affected by ascorbate in the absence of lipid peroxidation or by other reducing agents. These experiments demonstrate that ascorbate has a dual and differential effect on serotonin binding sites. First, ascorbate-induced lipid peroxidation irreversibly inactivates both [3H]5-HT and [3H]spiperone binding. Second, independent of lipid peroxidation, there is a direct, reversible effect of ascorbate on [3H]serotonin but not on [3H]spiperone binding, which is probably due to the difference in the biochemical nature of the two serotonin binding sites.