Ascorbic acid regulation of norepinephrine biosynthesis in isolated chromaffin granules from bovine adrenal medulla

The effect of ascorbic acid on the conversion of dopamine to norepinephrine was investigated in isolated chromaffin granules from bovine adrenal medulla. Ascorbic acid was shown to double the rate of [³H]norepinephrine formation from [³H]dopamine, despite no demonstrable accumulation of ascorbic acid into chromaffin granules. The enhancement of norepinephrine biosynthesis by ascorbic acid was dependent on the external concentrations of dopamine and ascorbate. The apparent K(m) of the dopamine β-hydroxylation system for external dopamine was approximately 20 μM in the presence or absence of ascorbic acid. However, the apparent maximum velocity of norepinephrine formation was nearly doubled in the presence of ascorbic acid. By contrast, the apparent K(m) and V(max) of dopamine uptake into chromaffin granules were not affected by ascorbic acid. Norepinephrine formation was increased by ascorbic acid when the concentration of ascorbate was 200 μM or higher; a concentration of 2 mM appeared to induce the maximal effect under the experimental conditions used here. The effect of ascorbic acid on conversion of dopamine to norepinephrine required Mg-ATP-dependent dopamine uptake into chromaffin granules. In contrast to ascorbic acid, other reducing agents such as NADH, glutathione, and homocysteine wer unable to enhance norepinephrine biosynthesis. These data suggest that ascorbic acid provides reducing equivalents for hydroxylation of dopamine despite the lack of ascorbate accumulation into chromaffin granules. These findings imply the functional existence of an electron carrier system in the chromaffin granule which transfer electrons from external ascorbic acid for subsequent intragranular norepinephrine biosynthesis.