Expression of 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the human fetal adrenal.

OBJECTIVE: To understand better the steroidogenic capacity of the human fetal adrenal (HFA), we evaluated the expression of 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the fetal zone and neocortex of the HFA using a specific RNase protection assay. METHODS: Adrenal glands were obtained at the time of elective termination of pregnancy. Whole adrenals (n = 7) were frozen in liquid nitrogen, and subsequently total RNA extraction was performed by tissue homogenization followed by guanidinium/chloroform purification. In addition, RNA was obtained from separated fetal zone (n = 4) and neocortex (n = 4) tissues obtained by dissection. RNase protection assays were then performed using radiolabeled complementary RNA probes generated by T7 RNA polymerase directed against transcripts for CYP11B1, CYP11B2, and actin, the latter of which was used as a control for RNA integrity. Transcripts also were examined using a reverse transcription polymerase chain reaction (RT-PCR) protocol specific for CYP11B1 or CYP11B2. RESULTS: The RNase protection assay was designed to distinguish specific bands that corresponded to CYP11B1 (232 bp), CYP11B2 (262 bp), and actin (221 bp). RNA isolated from whole HFA was observed to have high levels of CYP11B1 transcript, whereas CYP11B2 was not detected. Dissected neocortex and fetal zones were found to contain transcript for CYP11B1 using both the RNase protection assay and RT-PCR analysis. In contrast, using the RNase protection assay, CYP11B2 mRNA was not observed in the RNA from the fetal zone, but after prolonged exposure there was a band corresponding in size to CYP11B2 observed in RNA from the neocortex. Using the more sensitive RT-PCR method, transcript for CYP11B2 was found in both neocortex and fetal zone. CONCLUSION: The HFA expresses low levels of CYP11B2 in accordance with its low production of mineralocorticoid. The expression of CYP11B1 in the fetal zone is intriguing because this enzyme is not necessary for the production of C19 steroids. Definition of the molecular mechanisms controlling expression of the CYP11B genes will be necessary to determine why the HFA differentially expresses these isoenzymes.

Fish testicular 11beta-hydroxylase: cDNA cloning and mRNA expression during spermatogenesis.

We isolated and characterized a cDNA encoding testicular 11beta-hydroxylase, cytochrome P450(11beta) from the Japanese eel (Anguilla japonica) testis. The cDNA contains an open reading frame that encodes a protein of 511 amino acids. The predicted amino acid sequence shares 38-48% homology with those of adrenal P450(11beta) from mammals and frog. Transient expression in COS 1 cells confirmed that the protein encoded by this cDNA had P450(11beta) activity. Northern blotting revealed a single 1.8 kb long transcript of P450(11beta). This transcript was not found in immature eel testes prior to an injection with human chorionic gonadotropin (hCG), but it was present in eel testes after hCG injection.

Cloning and expression of cytochrome P450(11 beta) of porcine adrenal cortex.

Four cDNA clones were isolated from a porcine adrenal gland library by using a bovine cytochrome P450(11 beta) cDNA fragment as a probe. Nucleotide sequences of the four clones overlapped with each other. The deduced amino acid sequences indicated that these clones were derived from a porcine P450(11 beta) cDNA. Consecutive alignment of these clones covered almost 70% of a coding region of the cDNA, but its 5'-terminus was missing. The adrenal mRNA was reverse-transcribed, and polymerase chain reaction was used to obtain a cDNA fragment including the 5'-terminus. A cDNA constructed from this fragment and the isolated four fragments covered the entire apparent open reading frame of the enzyme, which was thus concluded to comprise 503 amino acids including a putative extension peptide of 24 amino acids at the NH2-terminus. The amino acid sequence was 82% identical to that of bovine P450(11 beta)-3. The cDNA was transfected into COS-7 cells, and steroidogenic activity of the cells was measured. The cells not only converted 11-deoxycorticosterone to corticosterone and 18-hydroxycorticosterone, but also produced aldosterone. Thus we conclude that the primary sequence of porcine P450(11 beta) which plays a role in the biosynthesis of glucocorticoids as well as mineralocorticoids was determined.

The role of ascorbic acid in the function of the adrenal cortex: studies in adrenocortical cells in culture.

To investigate the role of ascorbic acid in the function of the adrenal cortex, we studied the effects of ascorbate on the regulation of 11 beta-hydroxylase in culture. When primary bovine adrenocortical cells were cultured in a serum-free defined medium in the absence of ACTH, 11 beta-hydroxylase activity declined with a half-time of about 40 h. When 50 microM cortisol, which acts as a pseudosubstrate for 11 beta-hydroxylase, was added to such cultures, 11 beta-hydroxylase activity declined with a half-time of about 6 h. Ascorbate (5 mM) markedly reduced the rate of loss of 11 beta-hydroxylase activity in the presence of cortisol. Previous studies showed that phenolic and sulfoxide antioxidants, which also prevent loss of 11 beta-hydroxylase activity, inhibited the enzyme at concentrations somewhat higher than those required for protective activity. However, ascorbate at concentrations from 10 microM to 5 mM did not inhibit 11 beta-hydroxylase. The same range of ascorbate concentrations added to cells during a 24-h preincubation with cortisol showed increasing prevention of loss of 11 beta-hydroxylase activity. Ascorbate and a lowered concentration of oxygen were synergistic in their protective action. At 2% oxygen, 5 mM ascorbate almost completely prevented loss of 11 beta-hydroxylase activity in the presence of 50 microM cortisol. 11 beta-Hydroxylase activity was reinduced over a period of 5 days in third passage cultures by addition of 1 microM ACTH in defined lipoprotein-free medium. Addition of ascorbate enhanced the reinduction about 2-fold. The action of ascorbate in prevention of pseudosubstrate-mediated loss of activity and in enhancing reinduction of 11 beta-hydroxylase is specific; neither alpha-tocopherol nor selenium prevented loss of 11 beta-hydroxylase in the presence of cortisol or enhanced reinduction of 11 beta-hydroxylase in the presence of ACTH. As an additional test of specificity, it was shown that reinduction of 17-hydroxylase activity was completely unaffected by ascorbate, selenium, or alpha-tocopherol, and addition of cortisol to cultures with high 17-hydroxylase did not result in any loss of enzyme activity. Thus, a major function of ascorbate in the adrenal cortex is as a protective compound for cytochrome.