Expression of 11Β-Hydroxylase and Aldosterone Synthase Genes in The
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321 Expression of 11-hydroxylase and aldosterone synthase genes in the rat brain S M MacKenzie1, C J Clark1, R Fraser1, C E Gómez-Sánchez2, J M C Connell1 and E Davies1 1MRC Blood Pressure Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow G11 6NT, UK 2Department of Internal Medicine, University of Missouri–Columbia and the Harry S Truman Memorial Veterans Hospital, Missouri 65201–5297, USA (Requests for offprints should be addressed toSMMacKenzie, MRC Blood Pressure Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow G11 6NT, UK; Email: [email protected]) ABSTRACT The terminal stages of cortisol and aldosterone immunolocalised in paraffin-embedded rat adrenal production in the human adrenal gland are catalysed and brain sections using mouse monoclonal anti- by the enzymes 11-hydroxylase and aldosterone bodies. Negative controls utilised a mouse mono- synthase, which are encoded by the CYP11B1 and clonal antibody raised against a non-mammalian CYP11B2 genes respectively. Recent studies have epitope. In the brain, 11-hydroxylase and aldoster- suggested that aldosterone and cortisol are also one synthase were detected in the cerebellum, made in other tissues such as the brain, heart and especially the Purkinje cells, as well as the vascular system and may play a role in cardiovascu- hippocampus. The specificities of the 11- lar homeostasis. The aim of this study was to hydroxylase and aldosterone synthase antibodies confirm the presence of these enzymes and localise were confirmed by positive immunostaining of the them precisely in the rat brain. relevant regions of the adrenal cortex. Reverse transcription-polymerase chain reaction This is the first direct evidence that steroid (RT-PCR)/Southern blotting confirmed transcrip- hydroxylases involved in the final stages of tion of CYP11B1 and CYP11B2 in whole brain corticosteroid biosynthesis are present in specific and hypothalamus minces from Wistar–Kyoto rats. regions of the central nervous system. 11-Hydroxylase and aldosterone synthase were Journal of Molecular Endocrinology (2000) 24, 321–328 INTRODUCTION 11-Hydroxylase is localised in the zona fasciculata and is regulated by adrenocorticotrophin (ACTH). Synthesis and secretion of mineralocorticoids and Aldosterone synthase is unique to the zona glucocorticoids from cholesterol occurs in the glomerulosa and is regulated by the renin– adrenal cortex, with the initial step, the conversion angiotensin system (RAS) (Oertle & Müller 1993). of cholesterol to pregnenolone, performed by the The genes encoding 11-hydroxylase (CYP11B1) CYP11A1 gene product, the side-chain cleavage and aldosterone synthase (CYP11B2) have a high enzyme. The major glucocorticoid in man is percentage of sequence identity (Imai et al. 1990, cortisol; in the rat, it is corticosterone. Aldosterone Nomura et al. 1993, Zhang & Miller 1996). is the major mineralocorticoid in both species. It is Both aldosterone and cortisol (or corticosterone) synthesised in the zona glomerulosa of the adrenal have important effects on electrolyte balance and cortex while cortisol or corticosterone are made blood pressure by their actions on the kidney and mainly in the zona fasciculata (White et al. 1992, the vasculature (Fraser et al. 1989). In addition Vinson et al. 1995). The final steps of cortisol and to these systemic effects, however, it is clear that aldosterone biosynthesis are catalysed by 11- they exert significant central effects. Three types hydroxylase and aldosterone synthase respectively. of evidence exist. First, direct intracerebral Journal of Molecular Endocrinology (2000) 24, 321–328 Online version via http://www.endocrinology.org 0952–5041/00/024–321 2000 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/30/2021 04:06:01PM via free access 322 and others · Brain 11-hydroxylase and aldosterone synthase 1. Oligonucleotide primer sequences used for PCR amplification of cDNA Length of product Primer Sequence (5–3) (bp) CYP11B1/2/3 Upstream AAC TCC GTG GCC TGA GAC G 342 Downstream GCT GTG TGG TGG ACT TGA AC Adrenodoxin Upstream GAC TCT CTG CTA GAT GTT GTG ATT 289 Downstream ATT CTT GCT CAT GTC AAC AGA CTG TCG CYP11A1 Upstream CAA CAT CAC AGA GAT GCT GGC AGG 583 Downstream CTC AGG CAT CAG GAT GAG GTT GAA administration of doses too small to affect systemic opposed to gene transcription, and more precise concentrations elicit effects which may differ from information on their location within the brain have their classical systemic effects. For example, small been obtained by immunohistochemistry. doses of aldosterone raise blood pressure in the rat without the accompanying changes in extracellular sodium or potassium concentrations (Gómez- MATERIALS AND METHODS Sánchez 1997) characteristic of systemic admin- istration. Secondly, the brain is abundantly supplied RNA isolation with corticosteroid receptors. The glucocorticoid receptor is widely distributed but the mineralo- Tissues from adult female Wistar–Kyoto rats fed corticoid receptor is particularly concentrated in the on normal diets were snap frozen in liquid nitrogen hippocampus and hypothalamus (De Kloet et al. for subsequent RNA extraction. Total RNA was 1998). Thirdly, there is increasing evidence to extracted from these tissues according to the suggest that corticosteroids can be synthesised in standard RNAzol B reagent protocol (Biogenesis the brain. Both CYP11B1 and CYP11B2 appear Ltd, Poole, Dorset, UK). The quality and con- to be transcribed in brain tissue (Strömstedt & centration of the resulting RNA was confirmed by Waterman 1995). There is also some evidence that spectrophotometry and electrophoresis on ethidium brain tissue in vitro converts precursors to end bromide-stained agarose gels. products (Gómez-Sánchez et al. 1997) although de novo synthesis from cholesterol has yet to be RT-PCR demonstrated. Rates of synthesis are likely to be small in comparison to those of the adrenal cortex. Oligonucleotide primers (Oswel, Southampton, However, since the brain is highly sensitive to Hants, UK) homologous to parts of the CYP11B1/ corticosteroids (see above), low level, local synthesis B2, adrenodoxin and CYP11A1 genes were pro- may be physiologically important. duced (Table 1). RT-PCR amplification was We investigated the transcription of CYP11B1, performed using the GeneAmp RNA PCR kit CYP11B2 and CYP11A1 in several rat tissues. (Perkin Elmer, Warrington, UK) and oligo-d(T) Evidence of CYP11A1 expression in extra-adrenal primers. cDNA was transcribed according to the tissues would support the de novo formation of standard kit protocol. Amplification of CYP11A1 corticosteroids. In addition, we also tested for the and adrenodoxin were performed under identical transcription of adrenodoxin, a cofactor required conditions, following the method of Strömstedt & by mitochondrial cytochrome P450 enzymes such Waterman (1995). There was a final extension step as aldosterone synthase, 11-hydroxylase and the of 7 min at 74 C, after Nomura et al. (1993). side-chain cleavage enzyme. Controls had reverse transcriptase enzyme omitted, Evidence of corticosteroid synthesis in the rat or water substituted for RNA. brain has so far been obtained by reverse transcription-PCR (RT-PCR) or by conversion of steroid precursors to end products in tissue Southern blotting homogenates. To assess its potential role, it is RT-PCR products were run on 2% agarose gels and necessary to define more precisely the site(s) of Southern blotted to Hybond-N+ membrane synthesis. With this in mind, evidence of aldoster- (Amersham Life Science, Amersham, Bucks, UK). one synthase and 11-hydroxylase production, as CYP11B1 and CYP11B2 transcript products were Journal of Molecular Endocrinology (2000) 24, 321–328 http://www.endocrinology.org Downloaded from Bioscientifica.com at 09/30/2021 04:06:01PM via free access Brain 11-hydroxylase and aldosterone synthase · and others 323 distinguished by hybridising with radiolabelled performed using the DAKO Catalysed Signal oligonucleotide probes. The CYP11B1-specific Amplification (CSA) System Peroxidase Kit oligonucleotide was 5-TAA ACA TTC AGT CCA (DAKO Ltd, Cambridge, Cambs, UK). Primary ATA-3; the CYP11B2-specific oligonucleotide was antibody was incubated overnight at 4 C. Im- 5-TGG ATG TCC AGC AAA GTC-3 (position munostaining was developed with 3,3- 556–574 bp, exon III). CYP11A1 (the gene for diaminobenzidine tetrahydrochloride (DAKO Ltd). P450scc) transcripts were identified using the Sections were then counterstained with filtered oligonucleotide 5-GGT GGA GTC TCA GTG modified Harris haematoxylin solution (Sigma- TCT CCT TGA TGC TGG CTT TGA G-3.An Aldrich Co. Ltd, Poole, Dorset, UK) for 5 min, adrenodoxin-specific radiolabelled oligonucleotide dehydrated, and mounted in DPX (BDH Labora- was not required as any resulting bands were clearly tory Supplies, Lutterworth, Leics, UK). Control visible on ethidium bromide-stained gels under UV sections were incubated with mouse IgG1 negative light. control culture supernatant (DAKO Ltd). In addition, specificity of the monoclonal antibodies was confirmed by incubating them with an excess of Monoclonal antibody preparation their respective antigenic polypeptides (Alta Bio- Synthetic peptides corresponding to hydrophilic science, Birmingham, UK), thus quenching any areas of aldosterone synthase and 11-hydroxylase antibody binding. exhibiting minimal homology were prepared by Research Genetics Inc. (Huntsville, AL, USA). The aldosterone synthase monoclonal antibody was RESULTS