Conversion Of Corticosterone Research Articles

Effects of Dehydroepiandrosterone Sulfate on the Conversion of Corticosterone into 11-Dehydrocorticosterone in Stress: A Regulatory Scheme

In male rats, repeated but not single exposures to stress increased the conversion of corticosterone (CS) to 11-dehydrocorticosterone (11-DHCS), particularly on the background of administration of dehydroepiandrosterone sulfate (DHEAS). Naltrexone given 20 min before DHEAS at a dose of 0.1 mg/kg, at which it selectively blocks mu opioid receptors, prevented this effect of DHEAS, which is evidence that it is mediated by mu opioid receptors. This action of DHEAS involved endogenous ACTH and was thus mediated by central regulatory mechanisms. Our results, along with published data, lead to the first proposed scheme for the physiological regulation of the interconversion of CS and 11-DHCS in conditions of repeated stress with the involvement of DHEAS and mu opioid receptors.

Inhibitory effects of digoxin and ouabain on aldosterone synthesis in human adrenocortical NCI‐H295 cells

The present study was to investigate the effects and action mechanisms of digoxin and ouabain on steroidogenesis in human adrenocortical NCI-H295 cells. Administration of digoxin or ouabain for 24 h decreased the basal and angiotensin II (Ang II)-stimulated release of aldosterone by NCI-H295 cells. The conversions of corticosterone (substrate of cytochrome P450 aldosterone synthase, P450c11AS) to aldosterone or deoxycortisol (substrate of cytochrome P450 11beta-hydroxylase, P450c11beta) to cortisol were reduced by digoxin or ouabain. The basal and 22-hydroxy-cholesterol (a membrane-permeable cholesterol, substrate of cytochrome P450 side-chain cleavage enzyme, P450scc)-stimulated pregnenolone release in mitochondria was inhibited by digoxin or ouabain. Digoxin or ouabain suppressed the basal and Ang II-stimulated protein expression of steroidogenic acute regulatory (StAR) protein and P450scc. Incubation of digoxin or ouabain for 24 h reduced P450c11AS mRNA expression in NCI-H295 cells. Digoxin or ouabain (10(-6) M, 24 h)-treated cells showed a lower resting intracellular Ca2+ concentration ([Ca2+]i) and an attenuated response of [Ca2+]i to Ang II. Since no significant cytotoxicity was observed at 10(-6) M digoxin or ouabain, the digoxin- or ouabain-induced decrease of aldosterone or cortisol release was not associated with cytotoxicity. These results demonstrate that digoxin or ouabain inhibits the aldosterone or cortisol release via reduction of P450c11AS or P450c11beta and P450scc activities, inhibition of StAR and P450scc protein expression, suppression of P450c11AS mRNA expression, and attenuation of Ca2+ mobilization in NCI-H295 cells.

11β‐HYDROXYSTEROID DEHYDROGENASE TYPE I ENZYME IN THE HEARTS OF NORMOTENSIVE AND SPONTANEOUSLY HYPERTENSIVE RATS

1. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) converts glucocorticoids to their inactive 11-keto metabolites. The ubiquitous expression of the NADP-dependent isoform (11 beta HSD1) suggest an important role in modulating glucocorticoid action, but little is known about 11 beta HSD1 gene expression and enzymatic activity in the rat heart. 2. In the present study rat cardiac 11 beta HSD1 activity and ontogeny of gene expression have been characterized. The addition of NADP, but not NAD, to heart homogenates resulted in significant increases in the metabolism of both corticosterone and cortisol, with the former substrate displaying far greater metabolism. Both 11 beta HSD1 gene expression and enzyme activity increased in parallel from low levels at 1 week of age to maximal levels at 8 weeks, with no further change by 16 weeks of age. 3. We also compared the activity of 11 beta HSD1 in the hearts of male and female spontaneously hypertensive rats (SHR) with normotensive Wistar-Kyoto (WKY) controls. Enzyme activity in the pooled atria of female SHR was significantly higher than in male SHR atria (7.6 +/- 0.6% conversion of corticosterone vs 4.5 +/- 0.5%; P < 0.05). The left ventricles of female WKY rats contained significantly less 11 beta HSD activity than either male WKY rats or female SHR (8.6 +/- 0.8% conversion vs 17 +/- 1.4 and 13.6 +/- 0.5%, respectively; P < 0.05). In the right ventricle, female WKY rats also had significantly less enzyme activity than either female SHR or male WKY rats (4.9 +/- 0.7 vs 10.0 +/- 1.7 and 10.2 +/- 1.4%; P < 0.05). 4. These results clearly show that the rat heart contains significant amounts of the 11 beta HSD1 enzyme and that this activity is sexually dimorphic. Furthermore, significant differences were observed between a normotensive and hypertensive strain of rat. The relevance of these observations to the aetiology and maintenance of hypertension remains to be explored.

O2 dependence of pregnenolone and aldosterone synthesis in mitochondria from bovine zona glomerulosa cells

Hypoxia in vivo results in a decrease in aldosterone not accounted for by extra-adrenal controllers. We have demonstrated that aldosteronogenesis but not cortisol synthesis in the whole cell is O2 sensitive. In the intact glomerulosa cell, this sensitivity is located in the late pathway step catalyzed by conversion of corticosterone to aldosterone (P-450aldo), whereas the early pathway catalyzed by conversion of cholesterol to pregnenolone (P-450scc) is not inhibited until PO2 is very low. Because P-450aldo and P-450scc are mitochondrial enzymes that depend on the same NADPH-specific electron transport proteins, we hypothesized that O2 sensitivity would be independent of energy production and expressed in isolated mitochondria. We measured the conversion of exogenous 25(OH)-cholesterol to pregnenolone and of exogenous corticosterone to aldosterone in the presence of cyanoketone in mitochondria isolated from bovine zona glomerulosa cells and exposed to an experimental gas (1-100% O2) vs. a room air control. Pregnenolone production was not affected until PO2 was < 35 Torr and decreased to almost nil when PO2 was < 30 Torr. In contrast, aldosterone production increased under hyperoxia and decreased under moderate decreases in O2. The conversion of corticosterone to aldosterone was maintained at approximately 50% of control, even when PO2 was < 20 Torr. The sensitivity of the aldosterone pathway to changes in O2 within the physiological range appears to reside in the mitochondrial late pathway (i.e., P-450aldo) and is not significantly influenced by cytosolic regulators of steroidogenesis or by limitation of reducing equivalents.

Inhibition of aldosterone synthesis in rat adrenal cells by nicotine and related constituents of tobacco smoke.

Previous work has shown that nicotine and related constituents of tobacco smoke inhibit selected P450 enzymes in the glucocorticoid and sex steroid synthetic pathways. Because aldosterone synthesis is also cytochrome P450 dependent, we hypothesized a similar inhibitory action on aldosterone production. In this study we examined the effects of nicotine, anabasine (a related alkaloid), and cotinine (the major metabolite of nicotine) on in vitro aldosterone synthesis. Freshly isolated rat adrenal cells were assayed for corticosterone and aldosterone production in the basal state and after stimulation with ACTH or angiotensin-II (ANG-II). The addition of nicotine, anabasine, and cotinine in concentrations up to 100 microM did not inhibit stimulated corticosterone production. However, there was a potent dose-dependent inhibitory action of all three tobacco compounds on aldosterone production. The relative inhibitory potency was: cotinine > anabasine > nicotine. When employed at a concentration of 100 microM, the three compounds inhibited ACTH-stimulated aldosterone synthesis by 75%, 44%, and 21%, respectively. ANG-II-stimulated aldosterone synthesis was inhibited by 92%, 78%, and 62%, respectively. The plasma cotinine concentration range attained in tobacco smokers is between 1-10 microM. When tested with [3H]corticosterone and [3H]progesterone as exogenous substrates, 1-10 microM cotinine caused a significant dose-dependent inhibition of ACTH- and ANG-II-stimulated aldosterone synthesis. Cotinine substantially blocked the conversion of corticosterone to 18-hydroxycorticosterone, implicating the 18-hydroxylase or corticosterone 18-methyloxidase-I (CMO-I) step as the major site of inhibition. In summary, our results indicate that tobacco compounds cause direct and specific inhibition of aldosterone synthesis, primarily at the CMO-I step. This enzymatic blockade would be expected to result in activation of the renin-angiotensin system in vivo. We postulate that chronic stimulation of the renin-angiotensin system by this mechanism might contribute to the cardiovascular damage that occurs with long term tobacco use.

Rat liver 11 beta-hydroxysteroid dehydrogenase complementary deoxyribonucleic acid encodes oxoreductase activity in a mineralocorticoid-responsive toad bladder cell line

The mineralocorticoid receptor displays equal affinity for aldosterone and corticosterone. It has been proposed that aldosterone selectivity in vivo is achieved by the conversion of corticosterone into its inactive metabolite 11-dehydrocorticosterone by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). To test this hypothesis, we transfected rat liver 11 beta HSD cDNA into TBM cells, a sodium-transporting cell line. These cells respond equally well to aldosterone and corticosterone, indicating that endogenous 11 beta HSD is expressed at low levels in TBM cells. Although exogenous rat liver 11 beta HSD was expressed at high levels in transfected cells, mineralocorticoid selectivity was not observed. By contrast, the biologically inactive 11-dehydrocorticosterone was readily converted into corticosterone, a potent agonist for sodium transport. Our results indicate that rat liver 11 beta HSD behaves predominantly as a reductase in TBM cells. Another 11 beta HSD isoform is likely to be responsible for the dehydrogenase reaction in aldosterone-responsive cells.

Rat liver 11 beta-hydroxysteroid dehydrogenase complementary deoxyribonucleic acid encodes oxoreductase activity in a mineralocorticoid-responsive toad bladder cell line.

The mineralocorticoid receptor displays equal affinity for aldosterone and corticosterone. It has been proposed that aldosterone selectivity in vivo is achieved by the conversion of corticosterone into its inactive metabolite 11-dehydrocorticosterone by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). To test this hypothesis, we transfected rat liver 11 beta HSD cDNA into TBM cells, a sodium-transporting cell line. These cells respond equally well to aldosterone and corticosterone, indicating that endogenous 11 beta HSD is expressed at low levels in TBM cells. Although exogenous rat liver 11 beta HSD was expressed at high levels in transfected cells, mineralocorticoid selectivity was not observed. By contrast, the biologically inactive 11-dehydrocorticosterone was readily converted into corticosterone, a potent agonist for sodium transport. Our results indicate that rat liver 11 beta HSD behaves predominantly as a reductase in TBM cells. Another 11 beta HSD isoform is likely to be responsible for the dehydrogenase reaction in aldosterone-responsive cells.

The conversion of corticosterone to aldosterone is the site of the oxygen sensitivity of the bovine adrenal zona glomerulosa.

The dissociation of renin and aldosterone observed during hypoxia in vivo has been attributed to a direct inhibition of low oxygen on adrenal zona glomerulosa function. We have demonstrated that the adrenal zona glomerulosa production of aldosterone in vitro is directly proportional to a wide range of oxygen concentrations in the physiological range but that cortisol production from coincubated fasciculata cells is not oxygen sensitive. The present study examined the hypothesis that the sensitivity to O2 is limited to the aldosteronogenic late pathway. In order to localize the site of oxygen sensitivity, we measured endogenous pregnenolone production (early pathway) and the conversion of exogenous corticosterone to aldosterone (ALDO) (i.e. 18-hydroxylase activity) in adrenal cells treated with cyanoketone (3-beta-hydroxy-steroid dehydrogenase inhibitor). Acutely dispersed bovine adrenal glomerulosa cells (four experiments in pentuplicate) were incubated under low (5%) vs. normal (21%) O2 in the presence of cyanoketone (CK; 1 microM) and/or the following: corticosterone (500 ng/ml), angiotensin II (ANG II; 10 nM), or dibutyryl cAMP (1 mM). Conversion of exogenous corticosterone to ALDO in the presence of CK was inhibited by 41 +/- 1% under low O2. This was similar to the inhibitory effect of low O2 on ANG II-stimulated aldosterone production from endogenous precursors in the absence of CK (52 +/- 11% inhibition). Basal, ANG II-, and cAMP-stimulated endogenous pregnenolone production was not significantly reduced by low O2. In another experiment, glomerulosa cells were incubated under 5, 13, or 50% vs. 21% O2 in the presence of CK (1 microM) and different concentrations of corticosterone (10-1000 ng/ml). ALDO production was significantly inhibited by low O2 when corticosterone was greater than or equal to 500 ng/ml and ALDO was significantly augmented by high O2 when added corticosterone was 1000 ng/ml. We conclude that the conversion of corticosterone to ALDO (i.e. 18-hydroxylase) appears to be the primary site of oxygen sensitivity since 1) pregnenolone production was unaffected and 2) the magnitude of the inhibition of the conversion of corticosterone to ALDO by low O2 in the presence of CK was similar to the inhibition of ALDO production from endogenous precursors in the absence of CK. These studies demonstrate that oxygen sensitivity of the steroidogenic pathway is a unique, constitutive property of 18-hydroxylase, the enzyme which catalyzes the conversion of corticosterone to ALDO. We propose that the sensitivity of 18-hydroxylase to oxygen accounts for the dissociation of renin and aldosterone during hypoxia in vivo.

11 beta-Hydroxysteroid dehydrogenase mRNA expression in rat kidney

11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) protects nonspecific renal mineralocorticoid receptors from exposure to circulating glucocorticoid in vivo by catalyzing the conversion of corticosterone to inactive 11-dehydrocorticosterone. Although 11 beta-OHSD bioactivity and aldosterone binding sites are found in distal tubular cells, mineralocorticoid receptor and 11 beta-OHSD immunoreactivities are not colocalized. However, there are several kidney isoforms of 11 beta-OHSD, not all of which may be immunoreactive, whereas only a single mRNA species has been described. Using in situ hybridization we found 11 beta-OHSD mRNA is highly expressed in all renal tubular epithelia in the rat. It is therefore likely that 11 beta-OHSD is colocalized with mineralocorticoid receptors in distal tubular cells.

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11β-/18-hydroxylase from porcine adrenal

Highly purified cytochrome P-450 11β-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [ 3H] aldosterone from [ 3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11β-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11β-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H + and 3 moles of oxygen per mole of aldosterone produced: C 21 H 30 O 3 + 3 NADPH + 3 H + + 3 O 2 → C 21 H 28 O 5 + 3 NADP + + 4 H 2 O

Synthesis of aldosterone by a reconstituted system of cytochrome P-45011 beta from bovine adrenocortical mitochondria.

When corticosterone was incubated with cytochrome P-45011 beta purified from bovine adrenocortical mitochondria in the presence of adrenodoxin, NADPH-adrenodoxin reductase and an NADPH generating system, aldosterone as well as 18-hydroxycorticosterone were formed with turnover numbers of 0.23 and 1.1 nmol/min/nmol P-450, respectively. Phospholipids extracted from adrenocortical mitochondria remarkably enhanced the activity of aldosterone formation by the cytochrome P-45011 beta-reconstituted system. The apparent Km and turnover number were estimated to be 6.9 microM and 2.0 nmol/min/nmol P-450 for aldosterone formation in the presence of the lipidic extract. When 18-hydroxycorticosterone was tested as a substrate, cytochrome P-45011 beta showed catalytic activity for aldosterone synthesis with an apparent Km and turnover number of 325 microM and 5.3 nmol/min/nmol P-450, respectively. Carbon monoxide and metyrapone inhibited the production of aldosterone from corticosterone and that from 18-hydroxycorticosterone. These results suggest that conversion of corticosterone and of 18-hydroxycorticosterone to aldosterone occurs through P-45011 beta-catalyzed reaction.

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.

A Role for β-Melanotropin in the Regulation of Aldosterone Secretion during Sodium Deficiency in the Rat

1. The effect of sodium deficiency on adrenal sensitivity to β-melanotropin (β-melanocyte stimulating hormone; β-MSH) and the effect of β-MSH on a late step of aldosterone biosynthesis were studied using collagenase-dispersed rat adrenal cells. 2. Sodium depletion enhanced the sensitivity of the adrenal glomerulosa cells to β-MSH, resulting in a shift of the dose-response curve to the left, so that doses of β-MSH within the physiological range caused significant stimulation of aldosterone production. 3. The aldosterone level obtained by maximum doses of β-MSH was similar to that obtained after angiotensin (‘angiotensin II’; ANGII) during sodium depletion. 4. Sodium depletion did not change the corticosterone response to β-MSH by decapsular cells. 5. Similarly to ANGII, β-MSH significantly stimulated the conversion of exogenous corticosterone into aldosterone in the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase, WIN 19,578. 6. These data suggest that β-MSH or peptides containing β-MSH may play a role in the regulation of aldosterone secretion during sodium depletion in the rat and that β-MSH increases aldosterone production partly by stimulating the late step in aldosterone biosynthesis, the conversion of corticosterone into aldosterone.

Regulatory actions of angiotensin II on receptors and steroidogenic enzymes in adrenal glomerulosa cells.

The trophic effects of angiotensin II (AII) on adrenal glomerulosa function were studied by prolonged infusion of the Ile5-octapeptide in intact and 14-day hypophysectomized rats. The administration of a nonnatriuretic dose of AII (50 ng/ min) for 6 days elevated blood levels of AII 3-fold and increased the plasma aldosterone concentration from 9.6 ± 1.2 to 29.7 ± 5.7 ng/dl in intact rats and from 7.5 ± 1.5 to 31.6 ± 7.4 ng/dl in hypophysectomized rats. AII receptors measured in adrenal glomerulosa fractions from hypophysectomized rats were increased 2-fold, from 1408 ± 138 to 2270 ± 324 fmol/mg. Significant increases in enzymes of the aldosterone biosynthetic pathway, more marked in hypophysectomized animals, were observed after AII infusion. Mitochondrial cholesterol content and pregnenolone formation were increased by 30% in intact rats and by 100% and 240%, respectively, in hypophysectomized rats. The conversion of corticosterone to aldosterone was also increased, by 100% in intact rats and by 180% in...

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...

Adrenocortical function in rats with inherited hypothalamic diabetes insipidus (Brattleboro strain)

Abstract Steroidogenic function was studied in cells isolated from the zonae glomerulosa and fasciculata/reticularis of normal and sodium deficient Brattleboro rats. Compared with those of non-DI rats, z. glomerulosa cells of rats with diabetes insipidus (DI) produced more corticosterone and were less sensitive to in vitro stimulation by angiotensin II, potassium and ACTH while their aldosterone secretory response was more sensitive to dietary sodium changes. These differences of DI and non-DI z. glomerulosa function have been analyzed with regard to quantitative and qualitative changes in enzymes responsible for cholesterol cleavage and for the conversion of corticosterone to aldosterone. Z. fasciculata/reticularis cells from DI rats were less responsive to ACTH than those of non-DI cells.

Cytochrome P-450 and 18-oxygenase system from beef adrenocortical mitochondria. — Spectral and kinetic studies

Summary The transformation of corticosterone to aldosterone and the binding of corticosterone to cytochrome P-450 were studied in beef adrenal mitochondria. The corticosterone concentration giving a half maximal rate of aldosterone synthesis was found to be 1.510 −5 M in beef adrenal mitochondrial preparations. 7.010 −5 M was the concentration at which half maximal binding of corticosterone to cytochrome P-450 was obtained. pH 7.0 was found optimal for both the binding of corticosterone to cytochrome P-450 and its transformation to aldosterone. 2M urea diminished the binding of corticosterone to cytochrome P-450 by 78% and the conversion of corticosterone to aldosterone by 72%. It seem that in vitro factors favouring the binding of corticosterone to cytochrome P-450 also favour the transformation of corticosterone to aldosterone.

Effects of postpuberal orchiectomy on adrenal reductase activity and corticosterone secretion in the rat.

SummaryGonadectomy of adult male rats resulted in hypertrophy of the adrenal glands. Total adrenal steroid (blue tetrazoli-um positive) production in vitro was similarly augmented. Corticosterone secretion both in vitro and in vivo was unaffected. The ratio of corticosterone to total steroid, therefore, gradually declined following removal of the testes. This effect is explained by a concomitant increase in adrenal 5a-reductase activity after orchiectomy, resulting in greater conversion of corticosterone to 5a-reduced metabolites.The authors are grateful to Mrs. Nancy Swygert and Mrs. Andrea Newsome for expert technical assistance.

The effect of hypophysectomy on the conversion of corticosterone to aldosterone in the sodium-depleted rat.

Sodium depletion stimulates aldosterone secretion and the last steps in biosynthesis, the conversion of corticosterone to aldosterone. In the sodium-depleted rat, hypophysectomy markedly lowers aldosterone production within two days but does not lower the conversion of corticosterone to aldosterone. Presumably, hypophysectomy reduces the rate of earlier steps in biosynthesis. {Endocrinology 90: 827, 1972) T7ARLY histological studies demonstrated that ^ the zona glomerulosa maintains a normal or nearly normal appearance following hypophysectomy, which suggested that this zone of the adrenal gland is free of pituitary control. Moreover, sodium depletion widened the zona glomerulosa in the hypophysectomized animal as it did in the normal animal (1,2). Subsequent studies have shown, however, that the functional integrity of the zona glomerulosa was strikingly dependent upon intact pituitary function (3). It now appears that ACTH, growth hormone, and possibly other factors are important in maintaining normal aldosterone responses to sodium depletion (2,4,5). Previous studies from this laboratory have shown that in the sodium-depleted animal, aldosterone secretion in vivo, or aldosterone production in vitro fell markedly within two days following hypophysectomy (4). The conversion of corticosterone to aldosterone is a slow and possibly rate-limiting step in aldosterone biosynthesis which is stimulated by sodium deficiency (6). The purpose of this study was to determine whether the final step in aldosterone biosynthesis was normal or impaired in the sodium-depleted, hypophysectomized animal. Methods Male Sprague-Dawley rats, weighing 160-210 g were placed on a diet containing only a trace quantity of sodium and 132 mEq/kg potassium. After 6-7 days on this low sodium diet the rats were hypophysectomized transaurally, using the Hoffman-Reiter Hypophysectomy apparatus. Controls included those animals in which the hypophysectomy procedure failed to yield pituitary tissue, and some in which the ear drum was perforated only. The rats were then returned to their cages, and continued on the sodium-free diet for an added two days. Tetracycline (25 mg/100 ml) was added to the distilled drinking water one day before hypophysectomy and continued until sacrifice to improve overall survival. In other experiments it has been determined that this dose of tetracycline has no effect upon the rate of aldosterone production by either control or low sodium rats. On the day of sacrifice, those animals who had evidence of hypophysectomy at surgery, and an empty sella at autopsy were classed as hypophysectomized. Those sham operated rats, plus those failures at surgery which had an intact pituitary at autopsy were classed as intact controls. All equivocal animals were excluded. Corticosterone production was also reduced in the hypophysectomized animals. The rats were sacrificed by decapitation and the Received July 21, 1971. 1 Camm Research Institute, Wayne, New Jersey. 2 H. Newman & Company, Skokie, Illinois. 3 Boyd, J. E., and R. B. Page, unpublished observation. 828 NOTES AND COMMENTS Endo • 1972 Vol 90 • No 3 EFFECT OF HYPOPHYSECTOMY UPON ALDOSTERONE SECRETION, ALDOSTERONE PRODUCTION AND THE CONVERSION OF CORTICOSTERONE TO ALDOSTERONE

Studies on the biosynthesis of aldosterone using the sheep adrenal transplant: effect of sodium depletion on the conversion of corticosterone to aldosterone.

SUMMARY The effect of sodium depletion on the conversion of corticosterone to aldosterone has been examined in vivo using the adrenal transplants of two sheep. [3H]Corticosterone was infused continuously directly into the adrenal gland via the carotid artery over a period of 30 min. and the total adrenal effluent was collected via the jugular vein in six consecutive 5-min. samples. The conversion of [3H]corticosterone to [3H]aldosterone and the endogenous output of aldosterone was measured in each sample using a double isotope derivative method and the specific activity of the aldosterone calculated. Radioactive conversion of B → aldosterone reached equilibrium within 10 min. of the start of infusion and remained constant over a period of 10–25 min. Aldosterone secretion was also constant during the first 25 min. of infusion. In the same sheep the mean percentage conversion increased as aldosterone secretion rose over a range of 2–12 μg./hr. With more severe sodium depletion, i.e. with aldosterone secretion rates of 12–16 μg./hr., conversion decreased to that found in the sodium replete state. The specific activity of the aldosterone was constant throughout the mildly deplete range (2–12 μg./hr.) but fell with severe sodium depletion. In the sodium replete range (0–2 μg./hr.) before the introduction of a parotid fistula, the specific activity was the same as in the mildly deplete state. After the introduction of a parotid fistula the specific activity increased as the secretion decreased from 2 to 0 μg. The validity of the approach and interpretation of the results in terms of the biosynthetic pathways involved are discussed.