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(The FASEB Journal. 1999;13:1185-1194.)
© 1999 FASEB

Adrenomedullary function is severely impaired in 21-hydroxylase-deficient mice

S. R. BORNSTEIN*,1, T. TAJIMA*,2, G. EISENHOFER{dagger}, A. HAIDAN{ddagger} and G. AGUILERA*

* Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development and
{dagger} National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-1862, USA; and
{ddagger} Department of Internal Medicine, University of Leipzig, Germany

1Correspondence: Developmental Endocrinology Branch, NICHD, NIH, Bldg. 10, Rm. 10n262, 10 Center Drive MSC 1862, Bethesda, Maryland 20892-1862, USA. E-mail: Bornstes{at}mail.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Deficiency of 21-hydroxylase (21-OH), one of the most common genetic defects in humans, causes low glucocorticoid and mineralocorticoid production by the adrenal cortex, but the effect of this disorder on the adrenomedullary system is unknown. Therefore, we analyzed the development, structure, and function of the adrenal medulla in 21-OH-deficient mice, an animal model resembling human congenital adrenal hyperplasia. Chromaffin cells of 21-OH-deficient mice exhibited ultrastructural features of neuronal transdifferentiation with reduced granules, increased rough endoplasmic reticulum and small neurite outgrowth. Migration of chromaffin cells in the adrenal to form a central medulla was impaired. Expression of phenylethanolamine-N-methyltransferase (PNMT) was reduced to 27 ± 9% (P<0.05), as determined by quantitative TaqMan polymerase chain reaction, and there was a significant reduction of cells staining positive for PNMT in the adrenal medulla of the 21-OH-deficient mice. Adrenal contents of epinephrine were decreased to 30 ± 2% (P<0.01) whereas norepinephrine and dopamine levels were reduced to 57 ± 4% (P<0.01) and 50 ± 9% (P<0.05), respectively. 21-OH-deficient mice demonstrate severe adrenomedullary dysfunction, with alterations in chromaffin cell migration, development, structure, and catecholamine synthesis. This hitherto unrecognized mechanism may contribute to the frequent clinical, mental, and therapeutic problems encountered in humans with this genetic disease.—Bornstein, S. R., Tajima, T., Eisenhofer, G., Haidan, A., Aguilera, G. Adrenomedullary function is severely impaired in 21-hydroxylase-deficient mice.


Key Words: 21-OH deficiency • adrenal medulla • catecholamine synthesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CONGENITAL ADRENAL HYPERPLASIA (CAH)3 due to 21-hydroxylase (21-OH) deficiency is among the most common genetic disorders in humans (1) . The defective or absent enzyme results in glucocorticoid and mineralocorticoid deficiency. Classical 21-OH deficiency is a life-threatening disorder, causing severe salt wasting and eventually death if not diagnosed and treated promptly (1 , 2) . The decreased glucocorticoid production results in activation of the hypothalamic-pituitary-adrenal (HPA) axis with hypersecretion of pituitary corticotrophin (ACTH), hyperplasia of the adrenal cortex, and overproduction of precursor steroids, which are shunted into the androgen biosynthetic pathway (3) . This causes symptoms and signs of androgen excess including hirsutism, acne, disproportionate advancement of bone age, menstrual problems, diminished bone mass, infertility, and learning disabilities (1 , 4 5 6 7) .

Despite advances in our understanding of the molecular events causing congenital adrenal hyperplasia, patients with this disorder continue to have problems reflecting inadequacy of the current therapeutic approach (2 , 8 9 10 11) . The complex pathophysiology of 21-OH deficiency is far from being understood, and replacement therapy with glucocorticoids and mineralocorticoids often fails to normalize dysfunction of the HPA axis, growth, development, and well-being, suggesting alterations in other systems (4 , 5 , 9 , 12 , 13) .

The adrenocortical and adrenomedullary systems are intimately linked both anatomically and functionally in the adrenal gland (14 , 15) . From in vitro studies, it is well established that glucocorticoids are required for the survival and maintenance of chromaffin cells and their ability to produce epinephrine (16 , 17) . The effect of impaired glucocorticoid secretion on sympathoadrenomedullary function has never been studied in 21-OH deficiency or other types of CAH. In this study, the 21-OH-deficient mice, an animal model of CAH, were used to analyze this problem. The mice that have a deletion of the CYP21 gene and impaired 21-OH activity are a model for the human classic 21-OH deficiency that occurs in ~1:14,000 live births due to gene deletion or gene conversion (1 , 13) . As in human disease, the lack of glucocorticoids results in adrenocortical hyperplasia and accumulation of precursors (18) . The majority of affected mice die within a week if not treated with glucocorticoids and mineralocorticoids (19) . Immunohistochemistry and electron microscopy were used to analyze both adrenocortical and adrenomedullary structures in this animal model. Adrenal catecholamine levels were determined by liquid chromatography. The expression of mRNA for phenylethanolamine-N-methyl transferase (PNMT), the enzyme responsible for epinephrine production was quantitatively assessed by TaqMan polymerase chain reaction (PCR).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and experimental protocols
Heterozygous 21-deficient mice (H-2 aw18 haplotype), kindly provided by Dr. Toshihiro Shiroishi, Institute of Genetics, Shizuoka-ken, Japan, and wild-type C5BL10J mice purchased from Jackson Labs (Bar Harbor, Maine) were maintained according to NIH guidelines with a 12 h light-dark cycle and free access to food and water. The presence of a vaginal plug on the morning after mating was set as day 0.5 of gestation. As described previously, the gestation period in heterozygous mice was 1–2 days longer than that expected in wild-type mice (17 , 18) . All dams were treated with 5 µg of dexamethasone/day subcutaneous (s.c.) from gestational day 20 until delivery in order to prevent death of homozygous pups at birth. Pups were treated with corticosterone (5 µg/day) and fludrocortisone (0.025 µg/day) s.c. until day 14, followed by corticosterone in the drinking water until day 21. Animals were analyzed at 1 wk and 1 month of age. Each animal was killed within 3 min of being removed from its home cage and the procedures were performed sequentially in a separate room. All animal protocols were approved by the Animal Users Care Committee, NICHD, NIH.

Determination of genotype and 21-OH deficiency
Genomic DNA was extracted from livers or tails using standard procedures described previously (17) . A 950 bp cDNA fragment encoding exons 3 to 9 of the mouse CYP21 cDNA was prepared by PCR using 500 ng of total adrenal RNA (prepared with TRIzol reagent, Life Technologies, Gaithersburg, Md.) and the GeneAmpRNA PCR kit (Perkin Elmer, Foster City, Calif.). The upstream primer was 5'-GAAAGATGGACTTGGACCTGTCCT-3' and the downstream primer was 5'-AGGGTAGTCATAGCCGGAGAT-3'. PCR was performed using 500 ng of mouse adrenal RNA as a template under the following conditions: 30 cycles, 1 min at 94°C; 1 min at 58°C; and 3 min extension at 72°C. The blunt-ended PCR product was cloned with the use of a TA cloning kit (InVitrogen, Carlsbad, Calif.) and used to prepare random primer radiolabeled probes for Southern blot analysis of Bgl-II digests of the genomic DNA (17) . As previously shown, wild-type mice showed two bands corresponding to the active CYP21-B and the functionally inactive CYP21-A genes. Homozygous mice allowed a single smaller molecular band containing CYP21-A, whereas heterozygotes showed the three bands.

Electron microscopy
Adrenal glands were removed, dissected, and fixed for 3 h in 2% formaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. Tissue slices were postfixed for 90 min (2% OsO4 in 0.1 M cacodylate buffer pH 7.3), dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined at 80 kV in a Philips EM 301.

Immunohistochemistry
For specific staining of chromaffin cells, paraffin sections of adrenals were immunostained using a mouse anti-tyrosine hydroxylase antibody (Boehringer, Mannheim, Germany). Immunohistochemistry for PNMT was performed using a monoclonal rabbit anti-mouse antibody (courtesy of C. Grothe, Freiburg, Germany). Sections were deparaffinized and pretreated in the microwave in 10 mM citrate buffer pH 6.0 for 3 x 5 min (PNMT) or in Tris-buffered saline, pH 7.6 (TBS) containing 0.5% Triton X-100 for 2 min (tyrosine hydroxylase). Endogenous peroxidase activity was quenched by incubation in 0.5% hydrogen peroxide in TBS containing 10% methanol for 20 min. Immunostaining was performed by the avidin-biotin technique using the UniTect immunohistochemistry detection system (Dianova, Hamburg, Germany). After preincubation with 2% normal horse serum for 30 min, sections were incubated with the anti-tyrosine hydroxylase antibody diluted 1:10 in TBS containing 2% normal horse serum at 4°C overnight. For immunostaining of PNMT, nonspecific binding was blocked by 3% normal goat serum for 45 min. Subsequently, sections were incubated with the PNMT-antibody at 4°C overnight. The antibody was diluted 1:250 in Dako Antibody Diluent (Dako, Hamburg, Germany) containing 1% goat serum and 0.3% Triton X-100. After rinsing in TBS, sections were incubated with biotinylated link antibodies for 30 min and avidin-biotin-peroxidase complex for 30 min. Visualization of the immune complex was achieved by incubating the sections in AEC (3-amino-9-ethyl-carbazole) chromogen System (Dianova) for 15 min and counterstaining with hematoxylin.

TaqMan PCR
For quantitation of PNMT mRNA expression, we applied the novel technique of Real Time Quantitative PCR (TaqMan PCR) (20) using the 7700 Sequence Detector (Perkin Elmer Applied Biosystems). The amount of PNMT mRNA was measured using adrenocortical RNA and the following primers and TaqMan-probe designed from the mouse PNMT gene sequence (GenBank L12687) by Primer Express (PE): forward 5'-GTC GGG ACG GGT TCT CAT T-3'; reverse 5'-CCA AGA AGT CTG TCA TGG TGA TG-3'; TaqMan-probe 5' (FAM)-CTC CGG CCC CAC CAT ATA TCA GCT G-(TAMRA) 3'. 18S RNA levels were detected with the TaqMan ribosomal RNA control reagents (PE).

Total RNA was isolated from the adrenals of wild-type and 21-OH-deficient mice using the RNAeasy kit from QIAGEN (Valencia, Calif.). Traces of DNA were removed by digestion with RNase free DNase A (Boehringer, Indianapolis, Ind.) A one-step reverse transcription-PCR (RT-PCR) was performed according to the protocol supplied with the TaqMan Gold RT-PCR kit (PE). Reactions contained 1 x TaqMan buffer A, 5.5 mM MgCl2, 0.3 mM each dATP, dCTP, and dGTP, 0.6 mM dUTP, 0.4 U/µl RNase inhibitor, 0.025 U/µl AmpliTaq Gold DNA polymerase, and 0.25 U/µl MultiScribe reverse transcriptase. Primers and probes were added at the following concentrations: 900 nM for the PNMT forward and reverse primer, 200 nM for the PNMT TaqMan probe, and 50 nM for 18 S primers and probe. After reverse transcription at 49°C for 30 min, AmpliTaq Gold was activated at 95°C for 10 min. Thermal cycling proceeded with 40 cycles of 95°C for 15 s and 60°C for 1 min. Input RNA amounts were calculated with relative standard curves for both PNMT and 18S. The amount of PNMT mRNA was corrected by division by the amount of 18S RNA in each sample.

Catecholamine measurements
Tissues were homogenized in 5 volumes of ice-cold 0.4 M perchloric acid containing 0.5 nM EDTA. Homogenates were centrifuged at 3000 rpm at 4°C, and the supernatants were stored in aliquots until assayed. Catecholamines in supernatants were determined by liquid chromatography with electrochemical detection after a batch alumina extraction, as described previously in detail (21) .

Plasma steroid levels
Blood was collected from newborn mice by decapitation at 9:00 AM using nonheparinized capillary tubes. Plasma corticosterone levels were determined quantitatively by high-performance liquid chromatography (HPLC) as described previously (22) . Plasma progesterone concentrations were determined using commercial kit reagents from Diagnostic Systems Laboratories, Inc. (Webster, Tex.). Controls were used in the low and high section of the standard curve. The inter- and intra-assay coefficients of variation were both < 5%.

Statistical analysis
Data are presented as mean ± SE and were analyzed by Student's t test or the Mann-Whitney-U test, depending on the distribution pattern of the data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adrenal morphology
Mice deficient in 21-OH showed marked changes in adrenal structure compared with wild-type mice (Fig. 1A, B ). The adrenal cortex of mutant mice was markedly enlarged with hyperplasia of adrenocortical cells. There was no normal zona glomerulosa and fasciculata-like cells extended to the capsule. The migration of chromaffin cells in the center of the gland was incomplete, with single cells and islets of chromaffin cells remaining within the adrenal cortex as demonstrated by the presence of tyrosine hydroxylase or PNMT (not shown) stained cells (Fig. 1 C). In addition, chromaffin cells interspersed within the cortical tissue formed thin cellular extensions. These alterations were present in both 1-wk-old and 1-month-old mice.



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  		Figure 1. Morphological changes of adrenals of adult 21-OH-deficient mice compared with wild-type animals. A) Wild-type adrenals demonstrate a normal zonation with a capsule (CAP), outer zona glomerulosa (ZG), zona fasciculata (ZF); zona reticularis (ZR), and an inner zona medullaris (ZM) (x200). B) The adrenal cortex of the 21-OH-deficient mice is hyperplastic with enlarged adrenocortical cells (small arrows). There is no regular zonation, with fasciculata-like cells reaching to the outer capsule (large arrows). Islets and single chromaffin cells identified by immunostaining for tyrosine hydroxylase are located within the cortex. C) Cortical cells and chromaffin tissue are intermingled and form direct cellular contact (large arrows) (x200). Compared to wild-type animals in 21-OH-deficient mice, chromaffin cells show an irregular staining pattern for tyrosine hydroxylase. Some cells form tyrosine hydroxylase positive cellular extensions representing outgrowth of small neurites (small arrows).

Electron microscopy
At the ultrastructural level, adrenocortical cells of wild-type mice demonstrated normal smooth endoplasmic reticulum, liposomes, and the characteristic mitochondrial structure, elongated with tubulolamellar cristae in glomerulosa cells and round with tubulovesicular cristae in fasciculata/reticularis cells. Adrenocortical cells of 21-OH-deficient mice showed enlarged mitochondria with scarce internal membranes, frequently demonstrating myelin-type figures and lipidic inclusions.

Chromaffin cells of wild-type animals had the characteristic ultrastructural feature of neuroendocrine cells with an ample presence of membrane-bound, secretory granules so-called dense-core vesicles ~60 to 400 nm in greatest dimension. (Fig. 2 A, C) Two principal types of granule-containing cells were found in the normal adrenal medulla: 1) epinephrine containing large, round or elongated medium-density granules with a particulate substructure; and 2) small norepinephrine containing electron-dense granules within a large lucent vacuole. (Fig. 2C ) In the 21-OH-deficient mice, there was a conspicuous depletion of secretory vesicles. (Fig. 2B ) The remaining granules were predominantly electron-dense norepinephrine granules within a large lucent vacuole (Fig. 2D ). Chromaffin cells were frequently in contact with adrenocortical cells and formed small neurite-like outgrowths. Cells contained large areas of rough endoplasmic reticulum (RER). Frequently, the RER was dilated and vesiculated.



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  		Figure 2. Electronmicrograph of chromaffin cells of 1-wk-old 21-OH-deficient mice as compared with wild-type animals stained with uranyl acetate and lead citrate. A) On the ultrastructural level, the cytoplasm of chromaffin cells of controls is filled with densely grouped catecholamine storing secretory vesicles 50 to 450 nm in size. NUC, nucleus; bar = 0.5 µM. B) In the 21-OH-deficient mice, there is a conspicuous reduction in number of secretory granules. Remaining granules are frequently found lining up at the cell membrane (arrowhead). Cells contain a large amount of rough endoplasmic reticulum (RER) with dilated cisternae. The RER is frequently found in the perinuclear area. A shrunken nucleus with condensed chromatin as an early sign of programmed cell death can be seen (arrows). Chromaffin cells are in direct contact with adrenocortical cells. Adrenocortical cells demonstrate liposomes (LIP) and large round mitochondria with sparse tubulovesicular internal membranes (MIT) (bar = 0.5 µM). C) In wild-type animals, the majority of cells contain secretory vesicles with round or elongated medium-density granules with a granular substructure representing epinephrine-storing-vesicles (arrows). D) In 21-OH-deficient mice, the remaining granules are predominantly electron-dense, norepinephrine-containing vesicles lying in large lucent vacuoles (small arrows). The empty vacuoles endowed with ribosomes are not secretory granules but vesiculated RER (large arrows). MIT, mitochondria; bar = 0.1 µM.

PNMT expression
PNMT mRNA expression in homozygous 21-OH-deficient mice was significantly reduced to 27 ± 9% (P<0.05) compared with wild-type mice, as shown by TaqMan PCR. (Fig. 3 ) Concomitantly, the number of chromaffin cells staining for PNMT was markedly decreased in the 21-OH-deficient mice. (Fig. 4 A, B). There was no remarkable change in the number of chromaffin cells staining for tyrosine hydroxylase in 21-OH-deficient mice compared with wild-type mice.



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  		Figure 3. Quantitative PNMT mRNA in the adrenals of wild-type animals and of 21-OH-deficient mice as determined by TaqMan PCR. Amount of PNMT RNA is expressed as percent of reduction of wild-type levels (n=4, or P<0.05).



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  		Figure 4. Immunoreactive PNMT in the adrenal medulla of 1-month-old wild-type (A) and homozygous 21-OH-deficient mice (B). The number of cells staining positive for PNMT is markedly reduced in the 21-OH-deficient mice.

Adrenal catecholamine levels
Consistent with the altered chromaffin cell structure, adrenal catecholamine levels were significantly reduced in 21-OH-deficient mice. Dopamine levels in the 21-OH-deficient mice were significantly reduced to 50 ± 9% (P<0.05) and norepinephrine levels to 57 ± 4% (P<0.01) compared with the levels in wild-type animals. (Fig 5 A, B). Epinephrine levels in homozygous 21-OH-deficient mice were 1258 ± 92 ng/adrenal compared with 4227 ± 529 ng/adrenal in controls (P<0.01), representing a reduction to 30 ± 2% of the levels in the controls. (Fig. 5C ).



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  		Figure 5. Adrenal catecholamine levels in wild-type and homozygous 21-OH-deficient mice. Dopamine (A), norepinephrine (B), and epinephrine (C) levels were determined by liquid chromatography. Data are expressed as mean ± SE (bars); catecholamine production: ng/adrenal or µM/adrenal; n = 4 in each group (*P<0.05, **P<0.01).

Plasma steroid levels
Serum corticosterone levels as determined by HPLC were significantly reduced in 21-OH-deficient mice compared with wild-type mice (6.58±1.1 ng/ml vs. 44.4±6.2 ng/ml). Plasma progesterone levels were markedly elevated in 21-OH-deficient mice compared with wild-type mice (0.9±0.1 ng/ml vs. 269.2±32.4 ng/ml).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The findings of our study indicate that glucocorticoid insufficiency in 21-OH-deficient mice causes major structural changes in the adrenomedullary system and impairment of adrenal catecholamine production.

There was a fourfold reduction in adrenal epinephrine levels and a twofold decrease in norepinephrine levels. This finding was consistent with a significant reduction in PNMT mRNA and PNMT protein as well as epinephrine-secreting vesicles in the adrenal medullae of 21-OH-deficient mice. In late embryonic, neonatal, and adult chromaffin cells, PNMT expression is regulated by glucocorticoids (16 , 17) . In vitro, glucocorticoids have been shown to induce the enzyme PNMT, which is necessary for the production of epinephrine in medullary cells (23 24 25) . Epinephrine deficiency has been reported in hypocorticotropic hypopituitary children (26) . Therefore, the absence of normal adrenocortical corticosterone secretion is likely to be responsible for an impaired production of adrenal catecholamines in 21-OH-deficient mice. Clearly, other corticosterone precursors that are overproduced in these animals cannot compensate for the loss of corticosterone. Also, exogenous replacement of synthetic corticosteroids in the 7-day-old mice did not restore chromaffin cell function. This is in line with reduced epinephrine levels in patients with adrenocortical insufficiency due to Addison's disease that are adequately replaced with hydrocortisone (27) . The decreased levels of dopamine, norepinephrine, and epinephrine indicate that reduction of epinephrine was not due simply to reduced PNMT, but probably also involved decreased tyrosine hydroxylation. This agrees with previous reports, demonstrating that glucocorticoids are also involved in the induction of tyrosine hydroxylase (28 29 30) . Nevertheless, the predominant decrease in epinephrine compared with norepinephrine and dopamine are consistent with the major effect on PNMT. This suggests that circulating glucocorticoid levels are not sufficient to maintain normal adrenomedullary function, and the local influence of very high glucocorticoid levels directly from the adrenal cortex is necessary for a normal chromaffin cell function. Thus, local intra-adrenal levels of glucocorticoids after replacement therapy are not sufficient to normalize the function of the adrenal medulla.

In addition to the severely impaired adrenal catecholamine production, the adrenals of the 21-OH-deficient mice demonstrated major structural changes. As sympathetic principal neurons, adrenomedullary chromaffin cells originate from neural crest precursor cells and migrate into the adrenal `anlagen' where they later differentiate into chromaffin cells in the adrenal medulla under the influence of adrenocortical steroids (17 , 31) . A third cell type, small intensely fluorescent (SIF) cells, with intermediate characteristics between neurons and chromaffin cells have been described in culture studies (32 33 34) . While converting to neurons, adrenal chromaffin cells transiently assume an intermediate phenotype resembling SIF cells, underscoring the remarkable plasticity of chromaffin cells and the importance of environmental factors in neural crest development. It is interesting that the phenotype of the chromaffin cells in the 21-OH-deficient mice resembled the SIF cells, with formation of small neurites, reduction of catecholamine-storing vesicles, and ample rough endoplasmic reticulum. Furthermore, in contrast to the central location of normal adrenal medullae in wild-type mice, chromaffin cells in the adrenals from 21-OH-deficient mice were intermingled with the hyperplastic cortical tissue, suggesting a defect in the migration process.

Does this finding in 21-OH-deficient mice relate to the situation in human with CAH? In contrast to the 21-OH-deficient mice, which have very low corticosterone levels, the adrenals of CAH patients can synthesize a baseline amount of cortisol due to increased ACTH secretion and adrenal hyperplasia. However, once the CAH patients receive adequate replacement therapy with exogenous glucocorticoids, the suppression of plasma ACTH levels and of adrenal hyperplasia results in extremely low endogenous intra-adrenal cortisol production (<1 µg/dl) (35) . In a preliminary study, we found significantly decreased 24 h urinary epinephrine concentrations in patients with classical 21-OH deficiency upon adequate steroid replacement therapy (S. R. Bornstein, G. Eisenhofer, and D. Merke, unpublished observation). This strongly suggests that the adrenomedullary impairment described in 21-OH-deficient mice also occurs in humans with this common genetic disorder.

The defect in adrenomedullary function may have several implications. Clearly, the two endocrine systems within the adrenal form a functional unit, and an alteration in one system will certainly affect the other (15) . It has recently been demonstrated that chromaffin cells are in close cellular contact with adrenocortical cells (14 , 36 37 38) , immune cells (39 , 40) , nerve cells (41) , and endothelial cells (42) in normal adrenals. These paracrine intra-adrenal interactions within different components of the adrenal are important for basal hormone production and play a role in the development, cell proliferation, circadian rhythm and zonation of the adrenal gland (15 , 43 44 45 46 47 48 49 50 51 52 53 54 55 56) . Therefore, it is possible that adrenomedullary insufficiency in CAH also influences adrenocortical differentiation and that impaired adrenomedullary function due to glucocorticoid deficiency may contribute to the lack of zonation found in these animals.

Second, an ACTH-independent, neuroadrenocortical regulation mediated through the sympathoadrenal system has been demonstrated to be important in acute stress, fine-tuning of the adrenals, early development, and chronic adaptation to stress in inflammation, sepsis, and mental disorders (15 , 43 , 44 , 57 58 59 60) . Therefore, a defect in the neuroadrenocortical regulation will contribute to an impaired adaptation to stress in patients with 21-OH deficiency. This may be particularly important during early infancy, when the adrenal neocortex has to adapt to extrauterine life after disruption of the fetoplacental unit by adjusting glucocorticoid levels (61) .

Third, adrenomedullary dysfunction will contribute to the hypoglycemia that is a common problem in CAH patients, and a defective adrenal medulla can also aggravate the blood pressure problems in patients with congenital adrenal hyperplasia (62 , 63) .

Finally, similar to the adrenal medulla, glucocorticoid deficiency may also affect epinephrine synthesis in the brain (16 , 64 , 65) . Epinephrine has been reported to play an important role in tonic regulation of arousal, reward, and sensitivity to environmental stimuli and subjective well-being (66 , 67) . It is a critical factor in mental task performance and attention-deficit hyperactivity disorder (68 69 70) . Therefore, it is possible that a defect in epinephrine production is responsible for the high frequency of language/learning disabilities in children with congenital adrenal hyperplasia (5 , 71) .

Conventional treatment with glucocorticoid and mineralocorticoid replacement cannot restore the alteration in the HPA axis and the sympathoadrenomedullary system. Note that recent studies in our laboratory have shown that adenovirus-mediated transfer of the human CYP21 gene to 21-OH-deficient mice successfully restore corticosterone formation in the adrenal. Concomitant with the increase in glucocorticoid synthesis, we could demonstrate that adrenal zonation, including formation of a normal medulla and PNMT expression, could be partially normalized (unpublished results.)

In conclusion, our data demonstrate that glucocorticoid and mineralocorticoid deficiency as a result of the 21-OH defect not only affects the HPA axis and the renin-angiotensin-aldosterone system, but also severely compromises the adrenomedullary system. This is important for a better definition of symptoms and therapeutic strategies in 21-OH deficiency and should be considered in the clinical management of this common genetic disorder.


   ACKNOWLEDGMENTS
 
Supported in part by grants from Deutsche Forschungsgemeinschaft [EH 161/2–4] and by a Heisenberg grant, both to Dr. S.R.B. We would like to thank D. Merke, U. Lopatin, and M. Connors for helpful comments.


   FOOTNOTES
 
2 Department of Pediatrics, School of Medicine, Hokkaido University, Sapporo Kita-ku N15 W7, Japan

3 Abbreviations: ACTH, corticotrophin; CAH, congenital adrenal hyperplasia; HPA, hypothalamic-pituitary-adrenal; HPLC, high-performance liquid chromatography; 21-OH, 21-hydroxylase; PCR, polymerase chain reaction; PNMT, phenylethanolamine-N-methyltransferase; RER, rough endoplasmic reticulum; RT, reverse transcription; SIF, small intensely fluorescent; TBS, Tris-buffered saline.

Received for publication November 4, 1998. Revision received December 12, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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