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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 1, 2003 as doi:10.1096/fj.02-1167fje. |
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,2
* Clinical Neurocardiology Section, National Institute of Neurological Disorders and Stroke;
Genetic Diseases Research Branch, National Human Genome Research Institute;
Molecular and Clinical Nutrition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA; and
Departments of
Endocrinology and
** Anatomy, Heinrich-Heine-University, Düsseldorf, Germany
2Correspondence: Department of Endocrinology, University of Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany. E-mail: stefan.bornstein{at}uni-duesseldorf.de
SPECIFIC AIMS
Vitamin C is a powerful antioxidant and an essential cofactor for enzymes involved in catecholamine biosynthesis. Mice deficient in sodium-dependent vitamin C transporters (SVCT2) die within a few minutes of birth, with respiratory failure and brain hemorrhage revealing a hitherto unrecognized requirement for ascorbic acid in the perinatal period. Among the various tissues of the body, the adrenals contain some of the highest levels of ascorbic acid and are particularly sensitive to deficiency of ascorbic acid. We therefore hypothesized that severe decreases in brain and sympathoadrenal levels of catecholamines might account for the lethality of this deficiency. To address this hypothesis we compared cardiac, brain, and adrenal tissue levels of catecholamines and adrenal chromaffin cell structure in transgenic mice lacking SVCT2 with those of their wild-type littermates.
PRINCIPAL FINDINGS
1. Increased ratios of tissue dopamine to norepinephrine in heart and adrenals of SVCT2 null mice are consistent with mildly impaired dopamine ß-hydroxylase activity
In catecholamine synthesis, ascorbic acid functions as an electron donor for the enzyme dopamine ß-hydroxylase, which converts dopamine to norepinephrine. We investigated the ratio of dopamine to norepinephrine in the autonomic nervous system of these animals. Impaired activity of dopamine ß-hydroxylase in sympathetic nerves of SVCT2 null mice was manifest by a 100% increase in cardiac tissue levels of dopamine, a small nonsignificant decrease (14%) in tissue norepinephrine, and a 250% increase in the ratio of tissue dopamine to norepinephrine. There was also a smaller 82% increase in the ratio of tissue dopamine to norepinephrine in the adrenals of SVCT2 null mice compared with wild-type mice. However, rather than reflecting a build-up of dopamine precursor, this increased ratio reflected a 50% decrease in adrenal levels of norepinephrine.
There was no difference in the ratio of dopamine to norepinephrine in brain tissue. These data confirm previous in vitro studies suggesting a role of ascorbic acid as a cofactor for dopamine ß-hydroxylase. The changes in tissue catecholamines, however, indicated only minor changes in the activity of dopamine ß-hydroxylase, establishing that the enzyme can function in vivo in the presence of markedly reduced ascorbic acid levels.
2. Severe impairment of adrenal chromaffin cell function in SVCT2 null mice
The adrenals show the largest falls in ascorbic acid levels in mice with deficiency of SVCT2. We therefore hypothesized that the adrenals are particularly sensitive to deficiency of ascorbic acid and that this would be reflected by altered tissue levels of catecholamines.
Epinephrine accounted for 62%, norepinephrine 36%, and dopamine 2% of the total catecholamine content in the adrenal glands of wild-type mice. In SVCT2 null mice total adrenal contents of epinephrine were reduced by 81%, while contents of norepinephrine were reduced by 50% and those of dopamine were unchanged (Fig. 1
A, B). Thus, in SVCT2-deficient mice, norepinephrine, and not epinephrine, was the predominant catecholamine present in adrenal glands, accounting for 57% of the total catecholamine content.
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Adrenomedullary cells in wild-type animals had the characteristic ultrastructural features of neuroendocrine cells with an ample presence of membrane bound, dense cored secretory vesicles ranging from 60 to 400 nm in greatest dimension, and a rough endoplasmic reticulum. In adrenals from wild-type animals, the cytoplasm was filled with both types of secretory granules (Fig. 1C
). In contrast, in chromaffin cells of SVCT2 null mice, the number of secretory granules was markedly reduced (Fig. 1D-F
). The remaining vesicles were primarily elongated electron dense norepinephrine-containing vesicles. Secretory granules were frequently found lining up along the cell membrane or entrapped in RER (Fig. 1F
). Chromaffin cells in the process of programmed cell death were frequently found in the adrenal medulla of SVCT2 null mice. Characteristic signs of apoptosis included cell condensation and nuclear fragmentation and lysis (Fig. 1D, E
). Apoptotic bodies engulfing chromaffin vesicles could be seen in cells undergoing programmed cell death (Fig. 1E
).
3. Altered adrenocortical hormone production and morphology contribute to the impairment of adrenal chromaffin cell function
Since adrenal catecholamine biosynthesis is dependent on intact adrenocortical function, we measured plasma ACTH and corticosterone levels and analyzed adrenocortical structure. Serum corticosterone concentrations were mildly reduced by 31% in SVCT2 null mice compared with wild-type animals and appeared to follow a reduction in ACTH levels in SVCT2 null mice.
Adrenocortical cells of wild-type animals exhibited ample mitochondria, smooth endoplasmic reticulum, and liposomes characteristic of steroid-producing cells. The mitochondria demonstrated internal vesicular membranes. There was a reduction in mitochondria and smooth endoplasmic reticulum, and increased abundance of liposomes in the SVCT2 null mice compared with wild-type mice. The internal membranes of the mitochondria presented in a tubulo-lamellar pattern characteristic of adrenocortical cells in a hypofunctional state.
CONCLUSIONS AND SIGNIFICANCE
Our study shows that deficiency of the plasma membrane ascorbic acid transporter SVCT2 and the associated marked decreases in tissue levels of ascorbic acid result in derangements of catecholamine systems that are largely restricted to the adrenal medulla. This and the morphological abnormalities indicate an important role for ascorbic acid in adrenal chromaffin cell function.
Since ascorbic acid is a necessary cofactor for dopamine ß-hydroxylase, we expected that the neurochemical phenotype in SVCT2 null mice might be similar to that in mice lacking dopamine ß-hydroxylase, where tissue levels of the product of the enzyme norepinephrine are markedly decreased and those of the precursor dopamine markedly increased. We also hypothesized that the lethality of both deficiency states might be secondary to severely impaired norepinephrine synthesis. The neurochemical phenotype in SVCT2 null mice did exhibit some of the features of that in dopamine ß-hydroxylase deficiency, but was markedly less severe, suggesting that impaired catecholamine synthesis is unlikely to account for the lethality of the deficiency.
Despite marked reductions in tissue ascorbic acid levels, levels of norepinephrine in heart and brain of SVCT2 null mice were not significantly lower than in their wild-type littermates.
The consistently increased ratios of tissue dopamine to norepinephrine in heart and adrenals of SVCT2 null mice are consistent with some impairment of dopamine ß-hydroxylase activity, a finding that supports observations that vitamin C is required for full function of the enzyme in vitro. The mild to moderate nature of the changes in tissue norepinephrine and dopamine indicates, however, that dopamine ß-hydroxylase can function in the presence of much lower levels of ascorbic acid than those normally found in vivo.
The relatively moderate 50% decrease in adrenal norepinephrine contrasted with a surprisingly larger 81% decrease in adrenal epinephrine in SVCT2 null mice. This and the lack of increase in dopamine normally observed in dopamine ß-hydroxylase deficiency states suggest that the decreases in adrenal catecholamine contents in SVCT2 null mice are not simply due to impaired dopamine ß-hydroxylase activity, but may also result from a more general effect on catecholamine synthesis. Alternatively, ascorbic acid may have other functions that could affect the stability of vesicular stores of catecholamines and their turnover. This latter possibility is consistent with the results of our morphological studies, which showed decreased catecholamine containing secretory granules in adrenal chromaffin cells from SVCT2 null mice.
An important role of glucocorticoids in the regulation of epinephrine biosynthesis is well established. We therefore analyzed adrenocortical function in SVCT2-deficient mice and observed moderate reductions in plasma ACTH and corticosterone levels. These alterations suggested central and peripheral defects in the hypothalamic-pituitary-adrenocortical-axis that might contribute to the substantial decrease in adrenal epinephrine content in SVCT null mice.
Adrenal chromaffin cells in SVCT2 null mice exhibited frequent signs of apoptosis by electron microscopy, a gold standard technique for the analysis of programmed cell death. Characteristic signs of apoptosis included cell condensation, nuclear fragmentation, lysis, and apoptotic bodies engulfing cell organelles such as secretory granules. In addition, chromaffin cells demonstrated an accumulation of glycogen, which has been associated with cells in a hypofunctional state. Therefore, loss of antioxidant capacity induced by depletion of ascorbic acid in the SVCT null mice may lead to an increased rate of apoptosis, further impairing adrenomedullary function.
In summary, our findings establish a crucial role for ascorbic acid for the adrenal stress system particularly during early life.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1167fje; doi: 10.1096/fj.02-1167fje 
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