Steroid Hydroxylases in the Rat Brain: Evidence of Gene Expression and Enzyme Activity

Aldosterone and corticosterone are the main mineralocorticoid and glucocorticoid products of the rat adrenal cortex. They share a common biosynthetic pathway until the fmal stage where the substrate deoxycorticosterone (DOC) is converted to either aldosterone or corticosterone by the actions of the enzymes aldosterone synthase (CYP11B2) or 11beta-hydroxylase (CYP11B1) respectively. These enzymes are the products of highly homologous genes whose expression, along with that of other components of the corticosteroidogenic pathway, was long thought to be confined to the adrenal cortex. In recent years, however, due to the advent of more sensitive molecular biological techniques, evidence has accumulated to suggest that this is not the case and that certain extra-adrenal tissues may be capable of autonomous aldosterone and corticosterone production. In chapter 3, RT-PCR was used to detect transcripts from the CYP11B1 and CYP11B2 genes in a number of different tissue types. The transcription of genes encoding other components of corticosteroidogenesis, such as the side-chain cleavage enzyme and adrenodoxin, was also examined. Of the tissues examined, only brain tissue was found to contain transcripts from all these genes. Chapter 4 describes attempts using an SDS-polyacrylamide gel electrophoresis and immunoblotting technique to show that these transcripts are translated within brain tissue to result in the enzymes themselves. This utilised two monoclonal antibodies raised against non-homologous regions of the rat aldosterone synthase and 11beta-hydroxylase enzymes. However, no evidence of translation in extra-adrenal tissue was obtained using this technique, despite the identification of a positive band corresponding to 11beta-hydroxylase in adrenal tissue fractions. In chapter 5, the same antibodies were used to detect aldosterone synthase and 11beta-hydroxylase within extra-adrenal tissues by immunostaining methods. Using adrenal tissue sections, the two antibodies were found to be highly specific for their respective antigens and apparently free of the cross-reactivity which might be expected between such highly homologous enzymes. Positive staining was also produced in brain tissue, where the enzymes were found to colocalise within the hippocampus and the cerebellum. Although previously published work had established that steroidogenesis occurs in the glial cells of the central nervous system, strong positive staining within the Purkinje cells of the cerebellum in this study presented strong evidence for neuronal expression. Rigorous control experiments confirmed the specificity of these results. Attempts to produce specific staining within heart tissue were unsuccessful. Primary cultures of rat fetal hippocampal neurons permitted the study of CYP11B1 and CYP11B2 expression within a homogenous neuronal cell type. This work is described in chapter 6. CYP11B1 and CYP11B2 transcription and translation within these neuronal cells was demonstrated by the techniques used previously. In addition, the substrate DOC was incubated with the cells for 24-hour periods. Upon extraction and partial purification, the aldosterone and corticosterone content of the cell medium was measured by radioimmunoassay and shown to be significantly higher than that of control medium incubated in the absence of DOC. In summary, this thesis provides compelling evidence of the production of aldosterone and corticosterone within the rat central nervous system. It also includes detailed information concerning the distribution of the aldosterone synthase and 11beta- hydroxylase enzymes that produce these steroids. Finally, it demonstrates that neuronal cells cultured from the fetal rat hippocampus are capable of converting DOC into their respective products. These findings could have profound implications for the diverse physiological processes that are regulated by corticosterone and aldosterone