Zone-specific localization of cytochrome P45011B1 in human adrenal tissue by PCR-derived riboprobes

Abstract

Cytochrome P45011B1 (11 beta-hydroxylase) was detected in the human adrenal cortex and in human adenomas by in situ hybridization methods. Specific riboprobes were generated by in vitro transcription of 11 beta-hydroxylase--specific synthetic oligonucleotides with attached T7 and SP6 polymerase promotors. [35S]- and digoxigenin-labeled riboprobes were hybridized to sections of an aldosterone-producing adenoma (APA), the non-tumour portion of the corresponding adrenal gland, and two adenomas not related to hyperaldosteronism using standard protocols and varying washing conditions. After exposure of the radiolabeled sections to X-ray film, the signals were quantified and compared by statistical tests. Following autoradiography or immunohistochemical detection of the digoxigenin cytochrome P45011B1 mRNA was clearly localized in the zona fasciculata/reticularis of non-tumour portion of an human adrenal with an APA. Zona glomerulosa, medulla and connective tissue were free of label. As revealed by the semi-quantitative analysis, 11 beta-hydroxylase mRNA signals in the APA were significantly lower than those in the attached non-tumour portion and the other two adenomas. The results confirm known observations on the occurrence of cytochrome P45011B1 in the adrenal cortex of other species, but show, contrary to several immunohistochemical studies, that the enzyme is obviously not expressed in the zona glomerulosa.