Identification of Ascorbate as an Endogenous Substance That Irreversibly Inhibits Binding of Dihydropyridine Calcium Channel Blockers

Endogenous material present in heat-denatured extracts of rat brain that inhibited the binding of [3H]-isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-metho xyca rbonyl-2,6-dimethyl-3-pyridinecarboxylate ([3H]-PN200-110) to calcium channels in brain membranes was purified. Spectrophotometric analysis of material purified by strong anion-exchange and reverse-phase chromatography showed an absorption maximum at 266 nm at pH 7.0 that shifted to 245 nm at pH 2.0. This pH-dependent spectral shift was indistinguishable from that of ascorbic acid. Samples of the purified extract contained ascorbic acid; however, the inhibition of binding by purified material was always greater than the inhibition seen with equivalent concentrations of ascorbate, implying the presence of additional inhibitory factors. Attempts to detect and identify such inhibitory substances by chromatography showed that inhibition activity was coincident with the presence of ascorbate, and the inhibitory activity of purified material was abolished after treatment with ascorbic acid oxidase. Iron enhanced the inhibition produced by ascorbate, and chemical analysis of purified preparations revealed the presence of iron. Studies comparing the potency of the purified material with that of a mixture of ascorbate plus iron showed that the content of ascorbate and iron in the purified brain extract is sufficient to explain the observed inhibition of binding of [3H]PN200-110.