Late Steps Of Aldosterone Biosynthesis Research Articles

Late steps of aldosterone biosynthesis: sheep are not rats.

1. The last three steps of aldosterone biosynthesis have been demonstrated to be catalysed by a single enzyme, referred to as CYP11B (or P450(11) beta) in cow, pig, sheep and bullfrog and as CYP11B2 (or P450aldo) in rat, human, mouse and hamster. 2. The related enzyme CYP11B1 (also referred to as P450(11) beta) in rat, human, mouse and hamster does not have aldosterone synthesis activity, but no such enzyme has been reported in the cow, pig or sheep to date. 3. Exclusive aldosterone secretion in the zona glomerulosa (ZG) of the adrenal cortex in species such as rat, human, mouse and hamster could be ascribed to the restricted distribution of CYP11B2 to the same region in the adrenal cortex. 4. In other species, such as cow, pig and sheep, the CYP11B enzyme is expressed throughout the adrenal cortex and, thus, the exclusive aldosterone biosynthesis in the ZG could not be explained simply by the distribution of the enzyme. 5. We have shown in the sheep that potassium loading and acute sodium depletion stimulate the CYP11B transcript levels, which are not further increased by chronic sodium depletion. 6. The predominant CYP11B in the sheep adrenal cortex catalyses the synthesis of aldosterone from deoxycorticosterone (DOC) in vitro, is expressed throughout the adrenal cortex and the corresponding transcript levels are increased by K+ loading or sodium depletion. In short, as far as the last step of aldosterone biosynthesis is concerned, sheep are different from rats. In the rat, the CYP11B2 transcript or protein is elevated by K+ loading or sodium depletion, but not the CYP11B1 transcript or protein. 7. We propose that during severe sodium deficiency there is a switch in the aldosterone pathway to one preferentially involving 18-OH-DOC and not corticosterone.

Regulation of Aldosterone Biosynthesis: A Continual Challenge

Aldosterone secretion by the zona glomerulosa of the adrenal cortex is directly influenced by a large, growing number of different physiologic agents. Their action includes: 1) acute stimulation or inhibition of early biosynthetic steps; 2) long-term activation or suppression of late biosynthetic steps; and 3) induction of growth or atrophy of zona glomerulosa cells. In rats, adaptation of late steps of aldosterone biosynthesis to sodium and potassium intake is mediated by the induction or repression of a second form of cytochrome P-45011 beta, which differs from the main form of the enzyme by a lower molecular weight and a greater range of catalytic properties.

Divergent effects of dietary chloride restriction on aldosterone biosynthesis and the renin-angiotensin system in rats

The effects of dietary chloride restriction - alone or combined with sodium and potassium restriction - on aldosterone biosynthesis and the renin-angiotensin system were studied in rats. Treatment with a chloride-deficient diet led to a temporary decrease in the capsular adrenal conversions of corticosterone to 18-hydroxycorticosterone and aldosterone (manifest after 2 weeks but not longer apparent after 3 weeks), which was accompanied by a progressive rise in plasma renin activity and a moderate fall in plasma potassium. Combined restriction of sodium, potassium and chloride elicited a decreased activity of the enzyme(s) involved in late steps in aldosterone biosynthesis, an elevation of plasma renin activity to excessively high levels and a substantial hypokalaemia. Chloride repletion of these rats by the addition of NH4Cl or CaCl2 to their drinking fluid stimulated aldosterone biosynthesis while lowering plasma renin activity and raising plasma potassium. According to these observations, dietary chloride deficiency is another example of an experimental situation in which a high activity of the renin-angiotensin system contrasts with an unchanged or suppressed aldosterone biosynthesis. Most likely, this divergence is at least partly due to hypokalaemia which is induced during long-term chloride deficiency by a yet unknown mechanism.

Effects of angiotensin, prostaglandin e 2 and indomethacin on the early and late steps of aldosterone biosynthesis in isolated adrenal cells

The involvement of prostaglandins in the regulation of aldosterone biosynthesis was investigated in isolated adrenal glomerulosa cells. Cells were treated with cyanoketone to inhibit the 3β-hydroxy-steroid dehydrogenase and isolate the early step of aldosterone synthesis and the late step. Angiotensin II and PGE 2 stimulated the synthesis of aldosterone in a concentration-related manner. The stimulation by both compounds was inhibited by indomethacin, a prostaglandin synthesis inhibitor. Indomethacin also inhibited arachidonic acid-stimulation of 6-keto PGF 1α synthesis, whereas cyanoketone was without effect. Both angiotensin II and PGE 2 stimulated the synthesis of pregnenolone (the early step) in a concentration-related manner. At higher concentrations, angiotensin II also stimulated the conversion of [ 3H]corticosterone to [ 3H]aldosterone (the late step). PGE 2 did not alter the late step significantly. Indomethacin had no effect on either biosynthetic step when added alone. However, it inhibited the angiotensin- and PGE 2-stimulated pregnenolone synthesis by 41 and 59%, respectively ( P < 0.05). Indomethacin did not alter angiotensin stimulation of the conversion of corticosterone to aldosterone. These findings indicate that PGE 2 increases the synthesis of aldosterone by stimulating the conversion of cholesterol to pregnenolone. Indomethacin inhibits angiotensin II- and PGE 2-induced steroidogenesis at the same early biosynthetic step. These findings suggest that indomethacin may act by a mechanism other than a reduction in the concentration of prostaglandins.

Sites of action of beta-melanocyte stimulating hormone in aldosterone biosynthesis in the rat.

The sites of action of beta-melanocyte stimulating hormone (beta-MSH) on aldosterone biosynthesis were studied using collagenase-dispersed adrenal glomerulosa cells from rats maintained on either normal or sodium-deficient diets for 2 weeks. Isolated cells were treated with a cyanoketone derivative (WIN 19,578) to isolate the early and late steps in aldosterone biosynthesis. WIN 19,578 (1 microM) completely blocked aldosterone production stimulated by sodium depletion, AII, ACTH, and beta-MSH. beta-MSH (1 microM) significantly stimulated pregnenolone production (early step) and the conversion of corticosterone to aldosterone (late step) in aldosterone biosynthesis. The effect of beta-MSH was similar to AII and ACTH. Sodium depletion enhanced the effect of beta-MSH only on the late step in aldosterone biosynthesis. In conclusion, beta-MSH stimulates both the early and late steps of aldosterone biosynthesis. These results suggest that beta-MSH or peptides containing beta-MSH may play a role in the regulation of aldosterone production.

Role of the renin-angiotensin system in the regulation of late steps in aldosterone biosynthesis by sodium intake of potassium-deficient rats.

The role of the renin-angiotensin system in the adaptation of late steps in aldosterone biosynthesis to sodium intake was studied in potassium-deficient rats. Capsular portions of adrenal glands were incubated with [3H]corticosterone and conversion to aldosterone and 18-hydroxycorticosterone was measured by double isotope dilution and multiple paper chromatography. Sodium loading of sodium- and potassium-depleted rats resulted in a rapid and extensive fall in PRA but only in a delayed and gradual suppression of aldosterone biosynthesis. Treatment with the converting enzyme inhibitor, captopril, did not affect aldosterone biosynthesis in rats with established sodium and potassium deficiency, but blocked the stimulation of the conversion of corticosterone to aldosterone and 18-hydroxycorticosterone by sodium restriction of potassium-depleted rats. Infusion of a high dose of angiotensin II into potassium-deficient rats stimulated aldosterone biosynthesis depending upon the concurrent sodium intake. Accordingly, the renin-angiotensin system plays an important but limited role in the control of late steps of aldosterone biosynthesis by sodium intake. Angiotensin II seems to be essential for the induction but not for the maintenance of a high activity of the enzyme(s) involved in the conversion of corticosterone to aldosterone during combined sodium and potassium restriction. The sensitivity of the zona glomerulosa to the long term stimulatory action of angiotensin II varies with the sodium intake and appears to be regulated by the plasma potassium concentration and unknown other mediators.

A potassium-induced mitochondrial protein related to aldosterone biosynthesis.

Late steps of aldosterone biosynthesis, i.e., the conversions of corticosterone to 18-hydroxycorticosterone and aldosterone, are catalyzed by a mitochondrial cytochrome P-450. Resumption of potassium intake by potassium-depleted rats resulted within 2 days in a marked stimulation of these conversions, as reflected by increased production of aldosterone and 18-hydroxycorticosterone and decreased outputs of deoxycorticosterone, corticosterone, and 18-hydroxy-11-deoxycorticosterone by incubated capsular portions of the adrenal glands. The stimulation of aldosterone biosynthesis was accompanied by the appearance of a protein with a molecular weight of about 49,000 in the mitochondria of the zona glomerulosa but not of the inner zones of the adrenal cortex. Over 48 h of potassium repletion, the amount of this protein increased in parallel with the activity of the final steps of aldosterone biosynthesis. According to its molecular weight, its zone specificity, and the time course of its appearance, this protein might represent the steroid 18-methyl oxidase (cytochrome P-450CMO for corticosterone methyl oxidase) that catalyzes the conversion of corticosterone to 18-hydroxycorticosterone and aldosterone.

Stimulation of aldosterone biosynthesis by experimental edema: role of the kidneys.

The role of the kidneys in the stimulation of aldosterone biosynthesis by sodium sequestration was investigated in potassium-depleted rats. After 2 wk on a potassium-deficient diet, rats were treated by subcutaneous injections of polyethylene glycol or formalin and were then kept on sucrose and water for 24 h. Either type of experimental edema markedly enhanced the conversions of tritiated deoxycorticosterone and corticosterone to aldosterone and 18-hydroxycorticosterone by incubated capsular portions of the adrenal glands and partially normalized the deranged pattern of endogenous corticosteroid output in response to serotonin. Bilateral nephrectomy completely blocked these effects of edema on the zona glomerulosa. When uremia was induced by bladder resection, with the kidneys left intact, edema still significantly stimulated aldosterone biosynthesis. Irrespective of increases in the plasma creatinine, the plasma potassium remained at uniformly low levels in all experimental groups of animals. According to these observations, experimental edema stimulates late steps of aldosterone biosynthesis in potassium-depleted rats by mediation of the kidneys, most likely through the renin-angiotensin system.

Loci of Action of Regulators of Aldosterone Biosynthesis in Isolated Glomerulosa Cells

The effects of angiotensin II (All), ACTH, and potassium on early and late steps of aldosterone biosynthesis were studied in collagenase-dispersed cells from rat adrenal capsules and dog zona glomerulosa. Isolated cells were incubated with a cyanoketone derivative (WIN 19,578) to isolate the nearly and late portions of the biosynthetic pathway. In this way, the formation of pregnenolone and later steps of biosynthetic conversion in the absence of endogenous precursors could be measured independently during stimulation by the three regulators. During incubation for 3 h with 10 nM All, 10 nM ACTH, or 15 mM potassium, marked stimulation of pregnenolone production was observed in glomerulosa cells of both species. In rat glomerulosa cells, each of the three regulators also stimulated conversion of exogenous corticosterone to aldosterone. In dog glomerulosa cells, All and potassium, but not ACTH, stimulated the conversion of corticosterone to aldosterone. In rat glomerulosa cells, aldosterone production from e...

The effects of potassium on early and late steps in aldosterone biosynthesis in cells of the zona glomerulosa.

It has been clearly established that potassium can modulate aldosterone biosynthesis. However, the site of action of potassium on the aldosterone biosynthetic pathway has not been definitively established. This study was designed to examine in vitro the effects of potassium on the isolated early phase and the isolated late phase of aldosterone biosynthesis in adrenal cell suspensions. When the potassium concentration was increased in glomerulosa cell suspensions from 0 to 6 mEq/liter, a significant increase in aldosterone production occurred (P < 0.01). However, when the potassium concentration was increased from 6 to 12 mEq/liter, changes observed in aldosterone production were small and inconsistent. The early phase of aldosterone biosynthesis was then isolated, using trilostane to inhibit the conversion of pregnenolone to progesterone and, therefore, to aldosterone. Increasing the potassium concentration of trilostane- treated glomerulosa cell suspensions either from 0 to 6 mEq/liter or from 6 to 12 mE...

Angiotensin stimulates both early and late steps in aldosterone biosynthesis in isolated bovine glomerulosa cells

The present study was designed to examine the effect of angiotensin on both the early and the late phases of aldosterone biosynthesis. In order to isolate the early phase (the formation of pregnelone). it was necessary to identify an agent that would inhibit the 3β-hydroxysteroid dehydrogenase, 4–5 eneisomerase enzyme system which facilitates the conversion of pregnenolone to progesterone. Such an agent was found in trilostane. Treatment of zona glomerulosa cells with trilostane resulted in accumulation of pregnenolone, and the addition of angiotensin increased the accumulation of pregnenolone still further. This observation demonstrates that angiotensin stimulates aldosterone biosynthesis at a point prior to pregnenolone formation. The late phase of aldosterone biosynthesis was isolated by using aminoglutethimide to inhibit the formation of pregnenolone by cells of the zona glomerulosa. Aldosterone was formed when aldosterone precursors occurring in the biosynthetic pathway from pregnenolone onward were added. When angiotensin was added along with deoxycorticosterone, the conversion of deoxycorticosterone to aldosterone was enhanced ( P < 0.05). This observation demonstrates that angiotensin stimulates aldosterone formation at a point distal to deoxycorticosterone formation. Thus, angiotensin stimulates aldosterone biosynthesis at at least 2 independent points, one early and one late in the biosynthetic pathway.

Regulation of aldosterone secretion by the renin-angiotensin system during sodium restriction in rats.

The role of angiotensin II as mediator of the aldosterone response to short periods of sodium restriction was studied in rats by administration of a converting enzyme inhibitor to block formation of the octapeptide throughout the duration of decreased sodium intake. In control animals, short-term sodium restriction caused increased levels of adrenal receptors for angiotensin II, with enhancement of early and late steps in aldosterone biosynthesis and elevation of plasma aldosterone concentration. Each of these changes induced by sodium deficiency was abolished during blockade of angiotensin II formation by continuous infusion of the converting enzyme inhibitor, SQ 14,225. The absolute dependence of adrenal glomerulosa cell responses on angiotensin II formation indicates that the renin-angiotensin system is the primary regulator of aldosterone secretion during physiological fluctuations in sodium intake.

Effects of treatment with mineralocorticoids on late steps of aldosterone biosynthesis in the rat

Treatment of rats with either 9 α-fluorocortisol or deoxycorticosterone trimethyl acetate for two weeks resulted in a marked decrease in the conversion of tritiated corticosterone and deoxycorticosterone to 18-hydroxy corticosterone and aldosterone and, to a lesser extent, also in the conversion of added deoxycorticosterone and 11-deoxycortisol to 11 β-hydroxylated derivatives by incubated capsular adrenal tissue (‘zona glomerulosa’). It did not affect the rate of steroid 18- and 11 β-hydroxylations by the decapsulated portions (‘zona fasciculata-reticularis’) of the glands. When incubated with serotonin, capsular adrenals of mineralocorticoid-treated rats produced less aldosterone but more deoxycorticosterone from endogenous precursors than capsular adrenals of control animals. Such effects of mineralocorticoids were not observed in animals kept on a sodium-deficient diet. However, in rats kept on a potassium-deficient diet, the administration of either mineralocorticoid induced an additional decrease in the capsular adrenal conversion of tritiated corticosterone to 18-hydroxycorticosterone and aldosterone. According to these observations, mineralocorticoids decrease the activity of two enzymes involved in the late stages of aldosterone biosynthesis, i.e. zona glomerulosa 18-hydroxylase and 11 β-hydroxylase, by a yet unknown mechanism, which depends on an unrestricted sodium intake. Similar effects had been previously induced in rats by sodium loading or potassium restriction.

Renin suppression by DOC and NaCl in the rat.

Renin suppression by DOC and NaCl in the rat.