Molecular Psychiatry (2002) 7, 967–974 2002 Nature Publishing Group All rights reserved 1359-4184/02 $25.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Chromaffin cell function and structure is impaired in corticotropin-releasing hormone receptor type 1-null mice M Yoshida-Hiroi1,2, MJ Bradbury3, G Eisenhofer2, N Hiroi1,5, WW Vale3, GE Novotny4, HG Hartwig5, WA Scherbaum6 and SR Bornstein6 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, Du¨sseldorf, Germany; 5Department of Anatomy, Heinrich-Heine University, Du¨sseldorf, Germany; 6Department of Endocrinology, Heinrich-Heine University, Du¨sseldorf, Germany 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 adreno- cortical insufficiency and aberrant neuroendocrine development, but the adrenal medulla has not been analyzed in these animals. Therefore we studied the production of adrenal cat- echolamines, expression of the enzyme responsible for catecholamine biosynthesis neuro- peptides and the ultrastructure of chromaffin cells in CRHR1 null mice. In addition we exam- ined 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 Ͻ0.001), and the phenylethan- olamine N-methyltransferase (PNMT) mRNA levels in CRHR1 null mice were only 25% of con- trols (P Ͻ0.001). 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 Ͻ0.05, 268.9% P Ͻ0.01) 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 epine- phrine biosynthesis. Molecular Psychiatry (2002) 7, 967–974. doi:10.1038/sj.mp.4001143 Keywords: CRH receptor type 1; chromaffin cell; catecholamine; ACTH; adrenal; mouse Introduction through two G-protein-coupled receptors, CRHR1 and CRHR2, with CRHR1 being the major receptor Corticotropin-releasing hormone (CRH) is a major coor- mediating the activation of the HPA axis in rodents. dinator of the hypothalamic-pituitary-adrenocortical Genetic manipulation of each element of the CRH-sys- (HPA) axis.1 CRH released from the paraventricular tem is associated with various degrees of impairment nucleus (PVN) of the hypothalamus activates CRH of adrenocortical structure and function.3–11 receptors on anterior pituitary corticotropes, resulting CRH-deficient mice lack the normal diurnal gluco- in secretion of adrenocorticotropic hormone (ACTH) corticoid rhythm and present with adrenocortical atro- which in turn activates the synthesis and release of glu- phy and severely impaired glucocorticoid release to the cocorticoids from the adrenal cortex.1,2 CRH acts stress of restraint, ether and fasting.4 Similarly, CRHR1 null mice demonstrate adrenal atrophy and a reduced Correspondence: M Yoshida-Hiroi, Clinical Neurocardiology Sec- stress-induced secretion of corticosterone.6,7 CRHR2 tion and Surgical Neurology Branch, National Institute of Neuro- null mice present with normal corticosterone secretion logical Disorders and Stroke, National Institutes of Health, Bldg to acute stress but show early termination of ACTH 10, Room 6N252, 10 Center Drive, Bethesda, Maryland 20892- 1620, USA. E-mail: hiroimȰninds.nih.gov release and an altered recovery phase of the HPA 8 Received 22 October 2001; revised 18 February 2002; accepted reponse with elevated corticosterone levels. 21 February 2002 There is a functional interdependence between the Chromaffin cell function in CRHR1-null mice M Yoshida-Hiroi et al 968 two endocrine cell systems in the adrenal gland.12 measurements because data are expressed per gland. While catecholamines and neuropeptides produced in The left adrenals from each pair were immediately the adrenal medulla regulate adrenocortical function, snap frozen and stored at −80°C until homogenized. adrenocortical steroids influence the structure and The right adrenals were fixed for microscopy. function of the chromaffin cells in the adrenal med- ulla.2,13,14 Glucocorticoids induce the enzyme respon- Assay of tissue catecholamines sible for catecholamine biosynthesis and glucocort- Adrenal concentrations of catecholamines (norepine- icoid receptor-deficient mice have no adrenal phrine, epinephrine, dopamine, and dihydroxy- epinephrine production.15 Likewise, patients with phenylalanine) were quantified by high-performance adrenocortical insufficiency due to Addison’s disease liquid chromatography (HPLC) with electrochemical or 21-OH deficiency have low plasma epinephrine lev- detection.22 Adrenals were homogenized in 500 ␮lof els and adrenomedullary dysplasia.16–18 Recent studies cold 0.4 M perchloric acid containing 0.5 mM EDTA. demonstrated low epinephrine levels and a decrease in Homogenized samples were centrifuged (3000 rpm for adrenal PNMT expression in CRH-deficient mice4,6,7 30 min at 4°C) and supernatants collected and stored and adrenomedullary atrophy has been reported in at −80°C until assayed. Concentrations of catecholam- CRHR1 null mice.6 ines in supernatants were determined after extraction CRHR1 receptors are expressed on chromaffin cells19 using an alumina adsorption method described pre- and a direct action of CRH on chromaffin cell function viously.22 has been described.20,21 CRHR1 null mice are an inter- esting model to analyze the role of the CRH system for Total RNA isolation and TaqMan PCR the functioning of the adrenal medulla. Therefore we Total RNA was isolated from the adrenal glands of studied the production of adrenal hormones, CRHR1 null and wild-type mice using the microRNA expression of adrenal enzymes, StAR, PNMT, neuro- isolation kit (Stratagene, La Jolla, CA, USA). RT-PCR peptides and the ultrastructure of adrenal cells in experiments were carried out according to the THER- CRHR1-deficient mice. In addition we examined MOSCRIPT RT-PCR system kit (GIBCO, Gaithersburg, whether treatment of CRHR1 null mice with ACTH MD, USA) after treatment with deoxyribonuclease I could restore the adrenal structure and function. (GenHunter Corp, Nashville, TN, USA). Total RNA (1 ␮g) of adrenal glands of each group were reverse transcribed to complementary DNA (cDNA) by a reac- Materials and methods tion containing 2 mM deoxynucleotide mix, 100 mM Mice DTT, 40 units RNase inhibitor, 50 ng random primer The mice in which exons 5–8 of the CRHR1 were and 15 units thermoscript reverse transcriptase. The replaced with a PCK-neomycin-resistant cassette reaction was run at 25°C for 10 min and 50°C for (CRHR1 null)7 and their wild-type littermates used in 50 min, heated at 85°C for 5 min, and then cooled to these experiments were bred from a colony at the Salk 4°C. Institute for Biological Research. The CRHR1 geno- To quantify the expression of phenylethanolamine types were identified by PCR analysis using DNA iso- N-methyltransferase (PNMT), steroidogenic acute regu- lated from tail samples.7 All animals were bred under latory protein (StAR), neuropeptide Y (NPY), chromo- pathogen-free conditions, fed on a normal diet and granin B and proenkephalin, we applied the TaqMan maintained in the transgenic animal facility of the Salk PCR using the 7700 Sequence Detector (Perkin-Elmer Institute. Maintenance and experiments were conduc- Applied Biosystems, Foster, CA, USA) as described ted according to the Institutional Animal Care and Use previously.23 Reactions contained 1× TaqMan Univer- committee at the Salk Institute for Biological Research, sal PCR Master Mix, 900 nM of forward and reverse La Jolla, CA, USA. primers for PNMT, StAR, NPY, chromogranin B or pro- Mice were weaned at 21 days of age. Starting at enkephalin, and 200 nM of TaqMan probe (Table 1). weaning, seven CRHR1 null mice received normal Thermal cycling proceeded with 40 cycles of 95°C for saline (50 ␮l) and six null mice were treated with 0.4 U 15 s and 60°C for 1 min. Input RNA amounts were cal- of ACTH (ACTHar gel, Rhone-Poulenc Rorer, Col- culated with multiplex comparative method for both legeville, PA, USA). Mice were injected subcutane- the mRNAs of interest (PNMT, StAR, NPY, chromo- ously at 4 pm, 2 h before lights turned off at 6 pm dur- granin B or proenkephalin) and 18S. ing a 12:12 h light cycle. Ten wild-type or heterozygous mice injected with normal saline served as controls. Histology and electron microscopy Fourteen days later, mice were restrained for up to The