Tadashi Ogishima

Molecular cloning of a cDNA encoding aldosterone synthase cytochrome P-450 in rat adrenal cortex

Using an oligonucleotide probe designed on the basis of the N‐terminal amino acid sequence of purified rat aldosterone synthase cytochrome P‐450 [(1989) J. Biol. Chem. 264 10935] we have isolated from rat adrenal cDNA library a 2687 base pair cDNA that encodes a protein of 500 amino acid residues. The deduced amino acid sequence contained the regions well conserved among all cytochrome P‐450s sequenced to date, and also a portion (residues 25–44) which was identical to the N‐terminal peptide sequence of rat aldosterone synthase cytochrome P‐450. These results indicate that the cDNA encodes a precursor form of rat aldosterone synthase cytochrome P‐450.

Localization of p450aldo and p45011β in normal and regenerating rat adrenal cortex

A novel layer of cells that do not contain both P450aldo and P45011 beta has been discovered between the zonae glomerulosa and fasciculata of the rat adrenal cortex. Since P450aldo and P45011 beta are the enzymes responsible for the formation of aldosterone and corticosterone, respectively, the cells in that zone are presumably inert in synthesizing both aldosterone and corticosterone, in other words, the layer is composed of cells that have no zone-specific endocrine function as an adrenocortical component. Cytologically, the layer consists of tightly packed cells, which contain a lesser amount of lipid droplet than the cells in the other zones, and appears as a white ring or a white zone in the double immunostaining with anti P450aldo and anti P45011 beta. Upon angiotensin II-stimulation evoked by Na-deficiency, the number of the zona glomerulosa cells expressing P450aldo increases for the initial 2 or 3 days and then the P450aldo-containing zona glomerulosa cells begin to proliferate. Thus angiotensin II serves as a proliferator of the zona glomerulosa cells of the rat adrenal cortex. During the period, the thickness of the white zone decreases for initial 3 days and becomes constant after 5 or 6 days, being about 5% of the total cell number of the adrenal cortex. When localization of replicating cells was examined in the adrenal cortex, they were found to be concentrated in and around the white zone. Then the pulse-chase experiments with BrdU showed that the labeled cells migrated out of the white zone and into the zonae fasciculata and reticularis. The localization of the replicating cells in the regenerating adrenal cortex was also around the region between the zonae glomerulosa and fasciculata. On the basis of these findings, we suggest that the newly discovered cell layer (the white zone) is the stem cell zone of the rat adrenal cortex.

Structural differences in 5′-flanking regions of rat cytochrome P-450aldo and P-45011β genes

Two rat genomic clones, one for cytochrome P-450aldo and the other for P45011β, were isolated and characterized. The two genes, encoding structurally homologous proteins, were closely similar in their intron-exon organizations. Their 5′-flanking regions, however, contained only a few homologous regions. A putative cyclic AMP responsive element, TGACGTGA, was found in the P-450aldo gene, but this sequence was altered at two positions in the P-45011β gene. S1 nuclease protection assay revealed a single transcription initiation site for the P450aldo gene, while multiple sites were found for the P-45011β gene. These results suggest that transcriptional regulation of the rat P-450aldo and P-45011β genes is due to differences in the sequences of their 5′-flanking regions.