18-OH DOC Research Articles

P450c11B3 mRNA, Transcribed from a Third P450c11 Gene, Is Expressed in a Tissue-specific, Developmentally, and Hormonally Regulated Fashion in the Rodent Adrenal and Encodes a Protein with Both 11-Hydroxylase and 18-Hydroxylase Activities

The rat genome contains four P450c11 genes. One of these (CYP11B1) encodes P450c11 beta, which is the steroid 11 beta-hydroxylase found solely in the adrenal zona fasciculata/reticularis, and is responsible for the conversion of 11-deoxycorticosterone to corticosterone. A second P450c11 gene (CYP11B2) encodes P450c11AS, which is the aldosterone synthase found solely in the adrenal zona glomerulosa. P450c11AS has three activities, 11 beta-hydroxylase, 18-hydroxylase, and 18-oxidase, and is responsible for the conversion of 11-deoxycorticosterone to aldosterone. Recently, two more rat P450c11 genes, P450c11B3 and P450c11B4, were cloned. P450c11B4 appears to be a pseudogene, as two exons are replaced by unrelated DNA. P450c11B3 closely resembles P450c11 beta in mRNA and encoded amino acid sequences, predicting a protein of 498 amino acids. However, the expression of this mRNA and protein have not been demonstrated to date. We now demonstrate that this P450c11B3 mRNA is expressed in the adrenal gland several days after birth and is not expressed during fetal development or in the adult rat adrenal. Like P450c11 beta mRNA, P450c11B3 mRNA is expressed in the zona fasciculata/reticularis and not in the zona glomerulosa. However, the regulation of P450c11B3 mRNA expression is different from that of P450c11 beta mRNA, in that its abundance is decreased by ACTH in a sex-dependent fashion. Transfection of eukaryotic cells with a vector expressing P450c11B3 shows that this form of P450c11 can convert 11-deoxycorticosterone (DOC) to corticosterone and thus has the same enzymatic activity as P450c11 beta. In addition, P450c11B3 can convert DOC to 18-OH DOC and corticosterone to 18-OH corticosterone and thus has 18-hydroxylase activity similar to P450c11AS, but it lacks detectable 18-oxidase activity. Thus, P450c11B3 catalyzes 11 beta- and 18-hydroxylation and thus has a spectrum of activities midway between P450c11 beta and P450c11AS.

The regulation of plasma 18-hydroxy 11-deoxycorticosterone in man.

18-hydroxy 11-deoxycorticosterone (18-OH DOC), a weak mineralocorticoid, was estimated by a radioimmunoassay procedure after purification in 49 patients with hypertension and 38 normal control subjects. The sensitivity of the method was 2-4 pg; there was no detectable blank, and the precision was 9-10%. In normal subjects the absolute plasma levels were similar to those of aldosterone. ACTH administration produced a 23-fold increase, and sodium restriction resulted in a 4-fold increase (5.4+/-0.7-20.5+/-3.0 ng/dl). On the other hand, the plasma levels of 18-OH DOC declined by nearly 50% with upright posture or angiotensin II infusion. During both of these procedures, plasma aldosterone levels significantly increased. Patients with normal and low renin hypertension had similar changes in plasma 18-OH DOC levels with sodium restriction. However, the mean high sodium level in the normal renin essential hypertension group (11.6+/-1.6 ng/dl) was significantly greater (P is less than 0.001) than in the control group (5.4+/-0.7 ng/dl). In addition, at least 22% and perhaps as high as 37% of the hypertensive subjects had levels greater than the upper limits of normal on a high sodium intake. Differences between the groups were less impressive in the sodium-restricted studies. There were no significant differences in age, duration of hypertension, sodium balance, serum sodium, potassium, or blood urea nitrogen in those patients who had elevated levels of plasma 18-OH DOC. Patients with primary aldosteronism had levels within the normal range on both dietary intake. However, in contrast to the other groups there were no significant changes in the plasma levels with sodium restriction. Thus, a significant number of patients with essential hypertension presumably have an alteration in 18-OH DOC secretion.

Mineralocorticoid secretion in essential hypertension with normal and low plasma renin activity.

In 19 control subjects, 33 patients with essential hypertension and normal plasma renin activity (PRA) and 11 patients with low PRA, secretory rates of 18-hydroxy-11-deoxy-corticosterone (18-OH DOC), 11-deoxycorticosterone (DOC) and corticosterone were measured. Patients with low PRA were significantly older and had higher arterial pressure and slightly lower plasma potassium levels than patients with normal PRA. Mean 18-OH DOC secretion rate was higher in patients with normal PRA (603 +/- 112 SEM mug/24 hr) than in control subjects (219 +/- 19) and considerably higher (P less than 0.001) in patients with low PRA (1800 +/- 472). DOC and corticosterone secretion rates were within normal limits in most hypertensive patients. Plasma aldosterone was significantly higher in the hypertensive population than in control subjects whereas no significant difference was observed between the low- and normal-renin groups. A significant (P less than 0.01) mutual positive correlation was found between the secretion rates of 18-OH DOC, DOC and corticosterone in patients with low plasma renin activity. In contrast, there was no correlation between the secretion rates of the three mineralocorticoids in control subjects and patients with normal plasma renin activity. These data suggest a biosynthetic variation of the mineralocorticoid pathways in essential hypertension.

IS THERE A STEROID HORMONE CAPABLE OF PRODUCING HYPERTENSION IN MAN?

The hypothesis that a steroid can produce hypertension in man was tested in an 18 yr. old male with dexamethasone-suppressible hypertension. Reinvestigation after 6 yrs. of little or no treatment demonstrated that the hypertension and mild hyperaldosteronism were promptly decreased by a small dose of dexamethasone. During dexamathasone treatment, when aldosterone secretion was suppressed to less than normal and he was normotensive, steroids were given by constant infusion in an attempt to reproduce the hypertension of the dexamethasone-free state. Neither 5 days of aldosterone (A) nor 18-hydroxy desoxycorticosterone (18-OH DOC) at 1 mg/day nor DOC at 30 mg/day caused hypertension. However, Na+ retention and K+ loss was observed during A and DOC infusion. Hypertension was produced within 5 days during infusion of ACTH or oral metyrapone. The hypertensive effect of the latter was eliminated by addition of aminogluthethimide treatment. These studies suggest that an ACTH dependent steroid other than A, 18-OH DOC, or DOC may be the cause of the hypertension in this patient.

Mineralocorticoid receptors in salt-susceptible and salt-resistant rats.

The abnormal secretion of the adrenalsteroid 18-OH DOC is currently suspected of beingimplicated in some cases of human essential hypertension,and in genetic hypertension in saltsensitiverats. In such rats, the secretion rates ofaldosterone and desoxycorticosterone are similar tothose in the salt-resistant strain. From the studiesreported in this paper the additional differencesbetween the two strains necessary to allow developmentof hypertension are not differences inmineralocorticoid effector mechanisms. Both sensitiveand resistant rats have similar concentrations(∼ 3 × 10−14 moles/mg protein) of renal cytoplasmicmineralocorticoid receptors, which in both specieshave a similar affinity for aldosterone (Kdiss 37C ∼ 10−9M). By competition experiments, it hasbeen shown in both strains that 18-OH DOC has anaffinity for the mineralocorticoid receptor ∼ 1.5%that of aldosterone. Finally, no differences can bedemonstrated between the strains in terms of thetransfer of receptor-steroid complex from cytoplasmto n...

IS THERE A STEROID HORMONE CAPABLE OF PRODUCING HYPERTENSION IN MAN?

The hypothesis that a steroid can produce hypertension in man was tested in an 18 yr. old male with dexamethasone-suppressible hypertension. Reinvestigation after 6 yrs. of little or no treatment demonstrated that the hypertension and mild hyperaldosteronism were promptly decreased by a small dose of dexamethasone. During dexamethasone treatment, when aldosterone secretion was suppressed to less than normal and he was normotensive, steroids were given by constant infusion in an attempt to reproduce the hypertension of the dexamethasone-free state. Neither 5 days of aldosterone (A) nor 18-hydroxy desoxycorticosterone (18-OH DOC) at 1 mg/day nor DOC at 30 mg/day caused hypertension. However, Na+ retention and K+ loss was observed during A and DOC infusion. Hypertension was produced within 5 days during infusion of ACTH or oral metyrapone. The hypertensive effect of the latter was eliminated by addition of aminoglutethimide treatment. These studies suggest that an ACTH dependent steroid other than A, 18-OH DOC, or DOC may be the cause of the hypertension in this patient. The aminoglutethimide data suggest that it is a steroid, and the metyrapone studies suggest that steroid is an 11 desoxysteroid. Urine and blood collected under ACTH stimulation and metyrapone treatment is a rich source from which we may characterize this hormone.

The biosynthesis and secretion of aldosterone by the rat adrenal zona glomerulosa, and the significance of the compartmental arrangement of steroids.

ABSTRACT The formation of aldosterone was studied in rat adrenal capsules (zona glomerulosa) using a technique of incubation with dialysis. A number of features of aldosterone biosynthesis was observed which appeared to be inconsistent with the concept that corticosterone was a key intermediate in the pathway for aldosterone formation. These were as follows: In incubations with [3H]pregnenolone and [14C]acetate as precursors the [3H]specific activity of aldosterone was only one-fifth of the value for corticosterone, whereas the [14C]specific activities for the same products were similar. Pretreatment with cortisol, to suppress ACTH secretion, reduced the subsequent in vitro secretion of corticosterone but not of aldosterone. Pretreatment with Frusemide, a diuretic, stimulated secretion of aldosterone but not of corticosterone. Neither effect was correlated with any change in the transformation of [3H]pregnenolone into either compound. Aldosterone formed from endogenous precursors or from [14C]acetate tended to be bound to a non-dialysable component in the zona glomerulosa. The binding was reduced with the addition of ACTH to the incubation flask. This contrasts with our previously reported observations on corticosterone (which is preferentially formed from 'free' precursors), but it is similar to 18-hydroxydeoxycorticosterone (18-OH DOC) which appears to be formed preferentially from 'bound' precursors. From these experiments it appears that an alternative pathway for aldosterone biosynthesis may exist which tilises bound precursors (including possibly 18-OH DOC) instead of free precursors such as corticosterone.

A dissociated effect of Amino-Glutethimide on the mineralocorticoid secretion in man

Amino-Glutethimide (AG) decreases the secretion rates of aldosterone in control subjects. DOC secretory rate changes are less consistent (with an increase in 3 out of 5) and 18-OH DOC increases evidently in all 5 subjects where measured. Urinary sodium excretion increases after AG. With dexamethasone added to AG to suppress the compensatory increase in ACTH, the DOC and 18-OH DOC secretion rates decrease to the initial levels and further sodium diuresis occurs. Similar changes in urinary sodium excretion occurred in two hypertensive patients, but the blood pressure decreasing effect of AG was not consistent. These results indicate that zonal differences in the response of the adrenal cortex to AG probably exist. The zona glomerulosa, producing aldosterone, is more susceptible to the inhibitory action of AG, while other mineralocorticoids from the zona fasciculata (18-OH DOC and, in part, also DOC) increase as a consequence of the compensatory increase in ACTH. The combination of AG and dexamethasone may prove as a useful tool in suppressing the excessive mineralocorticoid secretion.

In vitro effects of ACTH, angiotensins, serotonin and potassium on steroid output and conversion of corticosterone to aldosterone by isolated adrenal cells.

Adrenal cells have been prepared, using crude collagenase, from the separated capsular and decapsulated portions of adrenals from intact rats. Microscopic examination and consideration of steroid outputs suggest that preparations from decapsulated glands consist of zona fasciculata-reticularis cells, while those from capsular glands consist mainly of zona glomerulosa cells with some zona fasciculata contamination. The yield of cells from the 2 preparations was of the order of 2 ×105 cells/adrenal for decapsulated glands and 1 ×lO5/adrenal for capsular glands. The endogenous output of 18-OH B, aldosterone, 18-OH DOC and corticosterone from the incubations of capsular and decapsulated gland cells has been examined. Three of the 4 steroids are produced by both types of cells; aldosterone production is virtually confined to capsular gland cells. The effect of ACTH, angiotensins, potassium and serotonin added in vitro on the output of corticosterone by decapsulated gland cells and that of corticosterone and al...

Testosterone inhibition of estradiol-induced stimulation of adrenal 11-beta- and 18-hydroxylation.

SummaryAdrenal homogenates from intact and castrated male and female rats treated with 1 μg 17β-estradiol iv were tested for ability to convert exogenous DOC to B and 18-OH DOC. Estradiol, which causes a significant increase in hydroxylating capacity in intact female rats, produced no such increase in intact males. Orchiectomized males, however, responded to estradiol with increased ability to hydroxylate. The estradiol response was lost after administration of testosterone to both orchiectomized males and intact females.

Studies on the Enzymatic Systems of Rat Adrenal in Early Postnatal Period : II. The Effect of Nadph on Progesteron-4-C14Hydroxylation and Pentose Cycle Activity

AbstractThe effect of NADPH producing sys em (G6P and NADP) on the hydroxylating capacity of rat adrenals was investigated during the early postnatal period. It was observed that the addition of the NADPH producing system to the adrenals of 16 days old rats increases the conversion of progesterone-4-14C to corticosterone twofold, and to 18-OH DOC as much as threefold. Such an effect was not observed either in the G6P itself nor in the NADP. The presence of active HMC was found in the adrenals of young rats : its activity around the 16th day became moderately increased.