¹Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

²Clinical Neurocardiology Section and Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA

³The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA, USA

⁴Institute of Neuroanatomy, Heinrich-Heine University, Düsseldorf, Germany

⁵Department of Anatomy, Heinrich-Heine University, Düsseldorf, Germany

⁶Department of Endocrinology, Heinrich-Heine University, Düsseldorf, Germany

Correspondence to: M Yoshida-Hiroi, Clinical Neurocardiology Section and Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bldg 10, Room 6N252, 10 Center Drive, Bethesda, Maryland 20892-1620, USA. E-mail: hiroim@ninds.nih.gov

Corticotropin-releasing hormone (CRH) is both a main regulator of the hypothalamic-pituitary-adrenocortical axis and the autonomic nervous system. CRH receptor type 1 (CRHR1)-deficient mice demonstrate alterations in behavior, impaired stress responses with adrenocortical insufficiency and aberrant neuroendocrine development, but the adrenal medulla has not been analyzed in these animals. Therefore we studied the production of adrenal catecholamines, expression of the enzyme responsible for catecholamine biosynthesis neuropeptides and the ultrastructure of chromaffin cells in CRHR1 null mice. In addition we examined whether treatment of CRHR1 null mice with adrenocorticotropic hormone (ACTH) could restore function of the adrenal medulla. CRHR1 null mice received saline or ACTH, and wild-type or heterozygous mice injected with saline served as controls. Adrenal epinephrine levels in saline-treated CRHR1 null mice were 44% those of controls (P<0.001), and the phenylethanolamine N-methyltransferase (PNMT) mRNA levels in CRHR1 null mice were only 25% of controls (P <0.001). ACTH treatment increased epinephrine and PNMT mRNA level in CRHR1 null mice but failed to restore them to normal levels. Proenkephalin mRNA in both saline- and ACTH-treated CRHR1 null mice were higher than in control animals (215.8% P <0.05, 268.9% P <0.01) whereas expression of neuropeptide Y and chromogranin B did not differ. On the ultrastructural level, chromaffin cells in saline-treated CRHR1 null mice exhibited a marked depletion in epinephrine-storing secretory granules that was not completely normalized by ACTH-treatment. In conclusion, CRHR1 is required for a normal chromaffin cell structure and function and deletion of this gene is associated with a significant impairment of epinephrine biosynthesis.

Molecular Psychiatry (2002) 7, 967-974. doi:10.1038/sj.mp.4001143

CRH receptor type 1; chromaffin cell; catecholamine; ACTH; adrenal; mouse

Introduction

Corticotropin-releasing hormone (CRH) is a major coordinator of the hypothalamic-pituitary-adrenocortical (HPA) axis.¹ CRH released from the paraventricular nucleus (PVN) of the hypothalamus activates CRH receptors on anterior pituitary corticotropes, resulting in secretion of adrenocorticotropic hormone (ACTH) which in turn activates the synthesis and release of glucocorticoids from the adrenal cortex.^1,2 CRH acts through two G-protein-coupled receptors, CRHR1 and CRHR2, with CRHR1 being the major receptor mediating the activation of the HPA axis in rodents. Genetic manipulation of each element of the CRH-system is associated with various degrees of impairment of adrenocortical structure and function.^{3,4,5,6,7,8,9,10,11}

CRH-deficient mice lack the normal diurnal glucocorticoid rhythm and present with adrenocortical atrophy and severely impaired glucocorticoid release to the stress of restraint, ether and fasting.⁴ Similarly, CRHR1 null mice demonstrate adrenal atrophy and a reduced stress-induced secretion of corticosterone.^6,7 CRHR2 null mice present with normal corticosterone secretion to acute stress but show early termination of ACTH release and an altered recovery phase of the HPA reponse with elevated corticosterone levels.⁸

There is a functional interdependence between the two endocrine cell systems in the adrenal gland.¹² While catecholamines and neuropeptides produced in the adrenal medulla regulate adrenocortical function, adrenocortical steroids influence the structure and function of the chromaffin cells in the adrenal medulla.^2,13,14 Glucocorticoids induce the enzyme responsible for catecholamine biosynthesis and glucocorticoid receptor-deficient mice have no adrenal epinephrine production.¹⁵ Likewise, patients with adrenocortical insufficiency due to Addison's disease or 21-OH deficiency have low plasma epinephrine levels and adrenomedullary dysplasia.^16,17,18 Recent studies demonstrated low epinephrine levels and a decrease in adrenal PNMT expression in CRH-deficient mice^4,6,7 and adrenomedullary atrophy has been reported in CRHR1 null mice.⁶

CRHR1 receptors are expressed on chromaffin cells¹⁹ and a direct action of CRH on chromaffin cell function has been described.^20,21 CRHR1 null mice are an interesting model to analyze the role of the CRH system for the functioning of the adrenal medulla. Therefore we studied the production of adrenal hormones, expression of adrenal enzymes, StAR, PNMT, neuropeptides and the ultrastructure of adrenal cells in CRHR1-deficient mice. In addition we examined whether treatment of CRHR1 null mice with ACTH could restore the adrenal structure and function.

Materials and methods

Mice

The mice in which exons 5-8 of the CRHR1 were replaced with a PCK-neomycin-resistant cassette (CRHR1 null)⁷ and their wild-type littermates used in these experiments were bred from a colony at the Salk Institute for Biological Research. The CRHR1 genotypes were identified by PCR analysis using DNA isolated from tail samples.⁷ All animals were bred under pathogen-free conditions, fed on a normal diet and maintained in the transgenic animal facility of the Salk Institute. Maintenance and experiments were conducted according to the Institutional Animal Care and Use committee at the Salk Institute for Biological Research, La Jolla, CA, USA.

Mice were weaned at 21 days of age. Starting at weaning, seven CRHR1 null mice received normal saline (50 l) and six null mice were treated with 0.4 U of ACTH (ACTHar gel, Rhone-Poulenc Rorer, Collegeville, PA, USA). Mice were injected subcutaneously at 4 pm, 2 h before lights turned off at 6 pm during a 12:12 h light cycle. Ten wild-type or heterozygous mice injected with normal saline served as controls. Fourteen days later, mice were restrained for up to 4 min while blood was obtained through the retroorbital sinus. Serum was separated from blood samples and assayed for corticosterone (ICN Diagnostics, Costa Mesa, CA, USA). Thymus glands were dissected and weighed. After killing, adrenal glands for PCR were carefully removed with surrounding fat with microforceps and cleaned of extraneous material. The residual fat and tissue is not a confound for the subsequent measurements because data are expressed per gland. The left adrenals from each pair were immediately snap frozen and stored at -80°C until homogenized. The right adrenals were fixed for microscopy.

Assay of tissue catecholamines

Adrenal concentrations of catecholamines (norepine- phrine, epinephrine, dopamine, and dihydroxy- phenylalanine) were quantified by high-performance liquid chromatography (HPLC) with electrochemical detection.²² Adrenals were homogenized in 500 l of cold 0.4 M perchloric acid containing 0.5 mM EDTA. Homogenized samples were centrifuged (3000 rpm for 30 min at 4°C) and supernatants collected and stored at -80°C until assayed. Concentrations of catecholamines in supernatants were determined after extraction using an alumina adsorption method described previously.²²

Total RNA isolation and TaqMan PCR

Total RNA was isolated from the adrenal glands of CRHR1 null and wild-type mice using the microRNA isolation kit (Stratagene, La Jolla, CA, USA). RT-PCR experiments were carried out according to the THERMOSCRIPT RT-PCR system kit (GIBCO, Gaithersburg, MD, USA) after treatment with deoxyribonuclease I (GenHunter Corp, Nashville, TN, USA). Total RNA (1 g) of adrenal glands of each group were reverse transcribed to complementary DNA (cDNA) by a reaction containing 2 mM deoxynucleotide mix, 100 mM DTT, 40 units RNase inhibitor, 50 ng random primer and 15 units thermoscript reverse transcriptase. The reaction was run at 25°C for 10 min and 50°C for 50 min, heated at 85°C for 5 min, and then cooled to 4°C.

To quantify the expression of phenylethanolamine N-methyltransferase (PNMT), steroidogenic acute regulatory protein (StAR), neuropeptide Y (NPY), chromogranin B and proenkephalin, we applied the TaqMan PCR using the 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster, CA, USA) as described previously.²³ Reactions contained 1´ TaqMan Universal PCR Master Mix, 900 nM of forward and reverse primers for PNMT, StAR, NPY, chromogranin B or proenkephalin, and 200 nM of TaqMan probe (Table 1). Thermal cycling proceeded with 40 cycles of 95°C for 15 s and 60°C for 1 min. Input RNA amounts were calculated with multiplex comparative method for both the mRNAs of interest (PNMT, StAR, NPY, chromogranin B or proenkephalin) and 18S.

Histology and electron microscopy

The adrenals were embedded in tissue tek. Five mm sections were mounted on poly-L-Lysin (Sigma, Munich, Germany) coated slides. After air drying, sections were fixed in acetone for 15 min. The sections were stained with hematoxylin-eosin and screened for infiltration.

For ultrastructural investigations, samples of tissue were fixed with 2% formaldehyde and 2% glutaraldehyde. Thereafter, the samples were incubated in 0.1 M phosphate buffer at pH 7.3 for 3 h. Samples 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 mounted on 200-mesh uncoated nickel grids. Sections were stained with uranyl acetate and lead Na citrate for 24 h and examined under a HITACHI electron microscope H-600 (Hitachi, Japan) and photographed.

Statistical analysis

Data analysis was performed by one-way ANOVA and Fisher's Protected Least Significant Difference for catecholamine. Expressions of mRNA were analyzed using Kruskal-Wallis test. Results are expressed as the mean ± SEM. Statistical significance was defined as P <0.05.

Results

Hormonal analysis

Serum corticosterone concentrations, 2 h after lights on, in saline-treated CRHR1 null mice were significantly lower than those of control mice (6.1 ± 3.6 ng ml^-1 vs 46.4 ± 13.3 ng ml^-1, P <0.01). Corticosterone concentrations in ACTH-treated CRHR1 null mice were equally low (8.3 ± 4.5 ng ml^-1), likely due to the 16 h interval between injection and sampling. Nonetheless, ACTH treatment appeared to provide a physiological adrenal stimulation during the dark phase of the light cycle. As expected in corticosteroid-deficient animals, thymus weights in saline-treated CRHR1-null mice were significantly larger than those in controls (0.079 ± 0.006 vs 0.05 ± 0.014 g, P <0.01). ACTH-treatment of the CRHR1-null mice restored this glucocorticoid-sensitive tissue (0.058 ± 0.0042 g).

Adrenal catecholamine levels

Adrenal epinephrine levels were 56% lower in saline-treated CRHR1 null mice (1318.5 ± 172.5 ng adrenal gland^-1; AD, P <0.001) than controls (3025.4 ± 234.2 ng AD^-1) (Figure 1). Adrenal epinephrine levels in ACTH-treated CRHR1 null mice were increased (2374.6 ± 209.1 ng AD^-1) (P <0.05) to levels greater than in saline-treated CRHR1 null mice, but were not fully restored to the level in control mice. Adrenal norepinephrine levels among the three groups were not different (1129.5 ± 112.5, 1166.9 ± 150.2, 1415.2 ± 66.0 ng AD^-1 in saline-treated CRHR1 null mice, ACTH-treated CRHR1 null mice, and controls, respectively).

Expression of PNMT, StAR, NPY, chromogranin B and proenkephalin mRNA in adrenal glands

Expression of PNMT mRNA in adrenals of saline-treated CRHR1 null mice was markedly reduced compared to controls (22.5 ± 3.1% reduction of control, P <0.01) (Figure 2). PNMT expression levels in ACTH-treated CRHR1 null mice were 42.7 ± 9.1% less than those in controls (P <0.05). Expression of StAR mRNA in CRHR1 null mice was distinctly decreased less than controls (28.0 ± 6.4% reduction of control, P <0.001). StAR expression levels in ACTH-treated CRHR1 null mice was blunted more than saline-treated CRHR1 null mice but clearly less than that control (42.1 ± 9.0% reduction of control, P <0.01). NPY and chromogranin B mRNA expression did not differ among the three groups. The level of proenkephalin in both saline and ACTH-treated CRHR1 null mice was increased compared to controls (215.8 ± 31.9%, P <0.05; 268.9 ± 22.3%, P <0.01).

Ultrastructural analysis of the adrenal glands

Adrenocortical cells of control mice demonstrated irregularly shaped nuclei and the characteristic mitochondrial structures with tubulovesicular cristae and ample smooth endoplasmic reticulum (SER). Adrenocortical cells contained few liposomes and exhibited some filopodia on the cell surface. (Figure 3a). Saline-treated CRHR1 null mice displayed an increase in liposomes compared with controls (Figure 3b). The amount of SER and the number of mitochondria were reduced, consistent with decreased corticosterone production in these animals. Also, the internal mitochondrial membranes were reduced and exhibited a more tubular arrangement characteristic of adrenocortical cells in a hypofunctional state. Following ACTH injections, adrenocortical structure and ultrastructure were restored (Figure 3c). Cortical cells demonstrated an increase in filopodia, SER and mitochondria. The mitochondrial membranes displayed a dense vesicular pattern, reflecting an adequate trophic effect of ACTH on the adrenal cortex.

Adrenomedullary cells in controls had the characteristic ultrastructural features of neuroendocrine cells with an ample presence of membrane bound, secretory ranging from 60 to 400 nm in greatest dimension, and rough endoplasmic reticulum (RER) (Figure 4a). The two principal types of chromaffin vesicles were epinephrine and norepinephrine. Epinephrine-containing vesicles were large, round or elongated medium-density. Norepinephrine-containing vesicles were small and electron-dense. In controls the cytoplasm was filled with both types of secretory granules. In contrast, in chromaffin cells in saline-treated CRHR1 null mice, the number of secretory granules was clearly reduced. The remaining granules were smaller and primarily electron dense (Figure 4b). Adrenomedullary cells in ACTH-treated CRHR1 null mice displayed an increase in medium-density secretory vesicles and RER but normal chromaffin structure was not restored (Figure 4c).

Discussion

CRH is a crucial regulator of the neuroendocrine, autonomic and behavioral response to stress.^1,24,25,26 In the present study we demonstrate a significant impairment of chromaffin cell function and structure in CRHR1-deficient mice. In accordance with a reduced epinephrine production there was a decline in PNMT expression and depletion in epinephrine storing secretory granules in chromaffin cells. ACTH injections at physiologically relevant doses in the CRHR1 null mice only partially restored chromaffin cell structure and function to that observed in controls.

CRHR1 deletion and ACTH replacement had expected effects on the ultrastructural parameters of the adrenal cortex. CRHR1 null animals demonstrated a reduced number of internal mitochondrial membranes and SER with an increase in liposomes. There is a well-established correlation between the production sites for steroidogenesis in adrenocortical cells, as indicated by the number and density of inner mitochondrial membranes and SER, and the biochemical activity of these cells.^27,28 Likewise, the increase in liposomes, the storage organelles for cholesterol, reflect reduced steroid biosynthesis and a hypofunctional adrenocortical state.²⁹ Consistent with these assumptions, ACTH injections at a dose sufficient to restore thymus weight induced a strong increase in the number of mitochondria and increased density of their internal membranes while the number of liposomes were markedly reduced. This was associated with a significant increase in adrenal epinephrine production. However, ACTH failed to fully restore corticosterone level, StAR expression, chromaffin cell structure and PNMT expression in these animals. The mechanism behind this disparity of ACTH replacement effects reflects the complex regulation of chromaffin cell functions. Adequate intraadrenal concentrations of catecholamines are required for full chromaffin cell activity. For example, steroidogenic factor 1 (SF-1) heterozygous mice have impaired-induced glucocorticoid production coupled with a significant defect in adrenomedullary development and catecholamine production.³⁰

The use of non-peptide CRHR1 antagonist, antalarmin reduces plasma Epi and NE responses to stress in primates.³¹ In addition, antalarmin blocks the central CRHR1-mediated hypertension in rats.³² Epi levels were decreased in CRHR1 null mice. NE levels did not change compared to controls in this study. The cause of the difference in NE levels between the two studies is unclear. However, CRH null mice have a low Epi level associated with decreased expression of PNMT in the adrenal medulla.³³ Thus, the cause may reflect a species difference. Glucocorticoids exert marked and complex influences on PNMT gene transcription. For example, stimulation of the PNMT gene by the neural crest factor activating protein-2 (AP-2) requires a direct interaction with ligand-activated type II glucocorticoid receptors.³⁴ In addition, the PNMT gene contains overlapping consensus elements for the promoter selective transcription factor (Sp 1) and the immediate early gene transcription factor Egr-1 that are capable of differentially activating PNMT gene expression.³⁵ Sp 1, Egr-1 and the glucocorticoid receptor and AP-2 function cooperatively to stimulate PNMT promoter activity through poorly delineated mechanisms.³⁶

Glucocorticoid control of PNMT gene transcription and protein synthesis do not fully account for changes in PNMT expression in the present study. The capacity of ACTH injections to increase epinephrine biosynthesis while PNMT expression remains markedly decreased suggests that corticosteroids can posttranscriptionally regulate PNMT protein expression.³⁷ Altered PNMT expression in CRHR1 null mice may reflect direct effects of gene deletion in adrenal chromaffin cells. Chromaffin cells express CRHR1 receptors and there is an intraadrenal CRH/ACTH system under feedback control of glucocorticoids.^12,19 Exogenous CRH preserves chromaffin cell morphology in vitro and stimulates catecholamine production in dispersed rat chromaffin and human pheochromocytoma cells.²¹ Hypoxia-induced adrenal chromaffin cell changes are partially reversed with exogenous CRH. Thus deficits in chromaffin cell in saline and ACTH-treated CRHR1 null mice may reflect inadequate stimulation of the adrenal CRHR1.

To further analyze the functional state of the chromaffin cells in CRHR1-null mice, we performed a quantitative mRNA analysis of proenkephalin, NPY and chromogranin B. Proenkephalin, NPY and chromogranins were chosen because they are major components stored with catecholamines in chromaffin cells.^38,39,40,41 While met-enkephalin constitutes the most abundant neuropeptide produced by chromaffin cells and correlates with catecholamines, NPY is regulated in a differential manner, following neural activation.^42,43,44 In ACTH and saline-treated CRHR1 null mice, there was an increase in pro-enkephalin RNA levels. Studies that examined the level of regulation of proenkephalin synthesis by catecholamine-depleting agents in vitro have produced mixed results, with some groups reporting decreases in proenkephalin mRNA, and others an increase after incubation with reserpine and/or tetrabenzamine.^45,46,47,48 Therefore, this increase in neuropeptide production may be the consequence of an increased pre-ganglionic stimulation of the chromaffin cells in CRHR1 null mice. Interestingly, these results are consistent with tyrosine hydroxylase-deficient mice that have blunted catecholamine secretion and low corticosterone production.⁴⁹ The expression of chromogranin B, a major vesicle compound in the adrenal medulla of mice, and NPY were not significantly altered in the CRHR1 null mice. Chromogranin B levels have been shown to be unaffected by hypophysectomy and consequently low corticosterone levels and were not significantly affected by increased neuronal activity generated with insulin treatment.⁴⁴ This concurs with our findings in the CRHR1 null mice and confirms the notion that different classes of secretory proteins presented in chromaffin vesicles are regulated in a differential manner in vivo.

The finding of impaired adrenal catecholamine and neuropeptide production in CRHR1 null mice should be considered when interpreting the behavioral abnormalities in these animals. We have recently reported transgenic animals overexpressing PNMT and high epinephrine levels in the brain.⁵⁰ These animals had metabolic alterations and were highly aggressive, sometimes escalating in the killing of caged males.⁵¹ On the other hand, epinephrine-deficient animals created by deletion of the enzyme responsible for the conversion of norepinephrine to epinephrine (PNMT) did not exhibit major changes of viability, metabolism, or behavior.⁵²

In conclusion, CRHR1 is required for normal chromaffin cell development and function and deletion of this gene is associated with a significant impairment of epinephrine biosynthesis. Novel preclinical and clinical strategies employing non-peptidic antagonists for CRHR1 should keep in mind the functional interdependence of the endocrine stress response in vivo.

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Figure 1 Concentrations of adrenal catecholamines in CRHR1 null mice and control mice. Mean ± SEM, asterisks: statistically significant differences between concentrations of CRHR1 null mice and controls,* P <0.05, **P <0.01, ***P <0.001; EPI, epinephrine; NE, norepinephrine.

Figure 2 Expression of PNMT, StAR, NPY, chromogranin B and proenkephalin mRNA in adrenal grands. The expression of PNMT, StAR, NPY, chromogranin B and proenkephalin mRNA were determined by quantitative TaqMan PCR in the adrenal grands of CRHR1 null mice and controls. Results are standardized to expression of controls mRNA (100%). Mean ± SEM, asterisks: statistically significant differences between expressions of CRHR1 null mice and controls, *P <0.05, **P <0.01, ***P <0.001; Chro B, chromogranin B; proE, proenkephalin.

Figure 3 Adrenocortical ultrastructure of CRHR1 null mice (+/- ACTH) and controls. In wild-type animals adrenocortical cells exhibit numerous round vesicular mitochondria (Mit) and filopodia (arrows) but few liposomes (Lip) (a). In contrast in CRHR1 null mice the number of lipid droplets are increased while the amount of mitochondria and smooth endoplasmic reticulum (Ser) is reduced (b). ACTH treatment restores adrenocortical cell morphology with a marked increase in mitochondria and the cell surface presents numerous filopodia. Liposomes are reduced (c). Nuc = nucleus, bar = 1 m, cells stained with uranyl acetate in all three panels.

Figure 4 Adrenomedullary ultrastructure of CRHR1-null mice (+/- ACTH) and controls. In wild-type animals the cytoplasm of chromaffin cells is filled with chromaffin vesicles (CV) of both granular/epinephrine and electron-dense/ norepinephrine storing secretory granules 50-450 nm in diameter. Cells contain rough endoplasmic reticulum (Rer) and cristae-like mitochondria (Mit) (a). In the CRHR1 null mice there is a reduction in chromaffin vesicles with the remaining granules presenting as electron dense (b). ACTH treatment fails to restore normal chromaffin ultrastructure in the CRHR1 null mice (c). In all three panels the bar represents 1 m.

Table 1 Sequences of primers and TaqMan probes