Dual Effects of Ascorbate on Serotonin and Spiperone Binding in Rat Cortical Membranes

Abstract: Ascorbate-induced lipid peroxidation, as measured by malonyldialdehyde (MDA) production, caused irreversible decreases in B_max of both ³H]5-HT and ³H]spiperone binding. Cacl₂ (4mM) inhibited ascorbateinduced MDA formation at ascorbate concentrations >0.57 mM, but not at ≤ 0.57 mM. Under the standard assay conditions (5.7 mM ascorbate and 4mM CaCl₂), Cacl₂ inhibited the MDA production casued by ascorbate by 88%, and the loss in ³H]5-HT binding by 57%. Ascorbate still decreased 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 peroxiation 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.