Ascorbic acid and Mg-ATP co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules

Ascorbic acid and Mg-ATP were found to regulate norepinephrine biosynthesis in intact secretory vesicles synergistically and specifically, using the model system of isolated bovine chromaffin granules. Dopamine uptake into chromaffin granules was shown to be unrelated to the presence of Mg-ATP and ascorbic acid at external dopamine concentrations of 7.5 and 10 mM. Under these conditions of dopamine uptake, norepinephrine biosynthesis was enhanced 5-6-fold by Mg-ATP and ascorbic acid compared to control experiments with dopamine only. Furthermore, norepinephrine formation was enhanced approximately 3-fold by ascorbic acid and Mg-ATP together compared to norepinephrine formation in granules incubated with either substance alone. The action of Mg-ATP and ascorbic acid together was synergistic and independent of dopamine content of chromaffin granules as well as of dopamine uptake. The apparent Km of norepinephrine formation for external ascorbic acid was 376 microM and for external Mg-ATP was 132 microM, consistent with the larger amounts of cytosolic ascorbic acid and ATP that are available to chromaffin granules. Other physiologic reducing agents were not able to increase norepinephrine biosynthesis in the presence or absence of Mg-ATP. In addition, maximum enhancement of norepinephrine biosynthesis occurred only with the nucleotide ATP and the cation magnesium. The mechanism of the effect of ascorbic acid and Mg-ATP on norepinephrine biosynthesis was investigated and appeared to be independent of a positive membrane potential. The effect was also not mediated by direct action of ADP, ATP, or magnesium on the activity of soluble or particulate dopamine beta-monooxygenase. These data indicate that Mg-ATP and ascorbic acid specifically and synergistically co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules, independent of substrate uptake. Although the mechanism is not known, the data are consistent with the possibility that the chromaffin granule ATPase mediates these effects.