Evidence for an ascorbate shuttle for the transfer of reducing equivalents across chromaffin granule membranes

Adrenal chromaffin granules must shuttle reducing equivalents from the cytosol inward to reduce ascorbic acid oxidized during norepinephrine biosynthesis by intragranular dopamine-beta-hydroxylase. A transmembrane electron shuttle between the external (cytosolic) and intragranular ascorbate pools was demonstrated in vitro in intact bovine chromaffin granules undergoing tyramine- or dopamine-stimulated dopamine-beta-hydroxylase turnover. Incubation of intact chromaffin granules with tyramine results in a time-dependent decrease in reduced intragranular ascorbate and production of octopamine. The rate of ascorbate oxidation is a function of the extragranular concentrations of tyramine over the range 50 microM to 2 mM and is 95% inhibited by addition of the dopamine-beta-hydroxylase inhibitor disulfiram. The stoichiometry of octopamine synthesized/ascorbate oxidized closely approximates unity. The presence of extragranular dopamine also induces oxidation of intragranular ascorbate which is inhibited by blocking dopamine transport with reserpine. On the other hand, incubation with octopamine, which is also transported by the granules, causes no net decrease in reduced intragranular ascorbate. The presence of 400 microM extragranular ascorbate abolishes the observed tyramine-induced intragranular ascorbate oxidation. The addition of ascorbate extragranularly 30 min after addition of tyramine reverses the oxidation of intragranular ascorbate. The measurement of [14C]ascorbate distribution ratios in granule pellets and supernatants indicates that there is no transmembrane transport of ascorbate. Extravesicular NADH had no significant effect on matrix ascorbate levels during beta-hydroxylation. These data provide new in vitro evidence that chromaffin granules shuttle reducing equivalents inwardly from an extra- to an intravesicular ascorbate pool and that cytosolic ascorbate is the source of the intragranular reducing equivalents required during norepinephrine biosynthesis.