Involvement of the corticoadrenal hormones in the pheromonal restoration of reproductive activity in aging rats

The involvement of the adrenal progesterone and corticosterone in the early gonadotropin secretion associated with the pheromonal restoration of ovarian cyclic activity (PRCA) in aging female rats is studied. PRCA is induced by male urinary pheromones and is preceded by an alpha-adrenergic-mediated release of the hypothalamic decapeptide luteinizing hormone-releasing hormone and plasma increases of estradiol, progesterone and the gonadotropins luteinizing hormone and follicle stimulating hormone. Aging reproductive Wistar female rats were used to study the effects of bilateral adrenalectomy and of a subcutaneous injection of the antisteroid RU486 on plasma levels of corticosterone, progesterone and gonadotropins in rats stimulated with nasal spraying of male urine (MU) or saline. The results demonstrate that progesterone and corticosterone released by MU are from adrenal origin, and that these adrenal secretory products are critical for MU-induced increase of gonadotropins. This suggests that olfactory stimulation of ACTH release stimulates adrenal release of progesterone and corticosterone, and both trigger the events that initiate the activation of the hypothalamus-pituitary-ovarian axis that leads to PRCA.     

Adrenal steroidogenesis in the guinea pig: effects of androgens.

In humans, the onset of adrenache has been found to occur with the appearance of the zona reticularis, the inner zone of the adrenal cortex. Since an increase in the volume of adrenal cortex during maturation in the guinea pig has been associated with the growth of the zona reticularis, we were interested in investigating the changes in adrenal steroidogenesis during maturation in this species. In addition, the effect of androgens on adrenal steroidogenesis was studied. We demonstrated that between 1 and 10 weeks of age, a period of maximal growth of the adrenals in the guinea pig, there is a decrease in the concentrations of adrenal pregnenolone, cortisol, dehydroepiandrosterone, testosterone, androstenedione, and 11 beta-hydroxyandrostenedione, suggesting lower steroid production by the guinea pig adrenals. In plasma, we observed that the concentration of 11 beta-hydroxyandrostenedione (the sole C19 steroid present after castration) remained unchanged during maturation, while cortisol and corticosterone were lower between 1 and 4 weeks of age. Although castration as well as the administration of the antiandrogen flutamide had no effect on adrenal steroidogenesis, dihydrotestosterone caused an inhibition of cortisol and corticosterone levels in the adrenals while the concentrations of progestins (namely, pregnenolone, 17-hydroxypregnenolone, progesterone, and 17-hydroxyprogesterone) tended to increase in the adrenals, thus suggesting that dihydrotestosterone induces a blockade in the steroidogenic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androstenedione-mediated inhibition of 11 beta-hydroxylation in monolayer cell cultures of fetal calf adrenals

The possibility that the formation of androstenedione by fetal calf adrenal cells in culture is linked to their decreased ability to form cortisol and corticosterone was investigated. Fetal calf adrenal cells metabolise radioactive adrostenedione to two major products which coelute on thin layer chromatography with 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone. When the cells are incubated with 11-deoxycortisol or 11-deoxycorticosterone in the presence of androstenedione there is a dose dependant inhibition of cortisol and corticosterone formation. Further studies with progesterone showed an accumulation of 11-deoxycortisol and 11-deoxycorticosterone in cells incubated simultaneously with androstenedione. The results demonstrate that exogenous androstenedione can have dramatic effects on steroidogenesis in the fetal calf adrenal and suggest that the accumulation of androstenedione in the medium of cultured andrenocortical cells is responsible, at least in part, for the decreased formation of cortisol and corticosterone.     

Enhanced secretion of oxytocin from bovine granulosa cells treated with adrenal steroids

Bovine granulosa cells were exposed in vitro to various adrenal steroids (cortisol, cortisone, corticosterone, aldosterone; 1 mumol/l), in the presence and absence of stimulation by ascorbic acid (0.5 mmol/l), to determine the possible effects of these hormones on ovarian oxytocin and progesterone secretion. Only cortisol produced a consistent stimulation of the cells; the response was dose-related over the range 0.01 to 1.0 mumol/l and was greatly enhanced in the presence of ascorbate. The secretion of oxytocin was stimulated to a greater extent and with more consistency than was that of progesterone. Although the secretion of oxytocin could be stimulated by cortisol on the day of treatment, the cells also showed a delayed and persistent response to exposure earlier in the culture. It is concluded that cortisol may directly stimulate the secretion of ovarian oxytocin in the cow and that granulosa cells may respond in such a way as to smooth out the effects of short-term fluctuations in cortisol concentration.     

Effect of prenatal stress on the pituitary-adrenal axis in blue foxes

Handling is a source of stress for farm bred blue foxes. The influence of handling during the late gestation period on the pituitary--adrenal axis was studied in 10-day old male and female blue foxes. Cortisol and progesterone were measured by radioimmunoassay in the plasma, adrenal homogenates, and in vitro incubates from animals of both sexes. Adrenals were incubated in vitro in the absence or presence of ACTH. In addition, the adrenal weight and plasma concentration of ACTH were assessed. In cubs of both sexes, the adrenal weight was decreased after prenatal stress. The plasma concentration of progesterone and the adrenal cortisol in vitro production were elevated in the prenatally stressed female cubs, as compared to the control, along with the adrenal progesterone in vitro production in prenatally stressed male cubs. The adrenal cortisol and progesterone content and plasma ACTH and cortisol concentrations were not affected by prenatal stress. In conclusion, the results of this study suggest that the prenatal stress induced by handling pregnant vixens can affect the pituitary--adrenal axis in neonatal offspring, this effect being more pronounced in female cubs.     

Ovine fetal adrenal maturation at term and during fetal ACTH administration: evidence that the modulating effect of cortisol may involve cAMP

Exogenous ACTH1-24 promotes adrenal maturation in fetal sheep, and this effect appears to be modulated in part by cortisol (Challis et al. 1985). We have examined whether similar changes in adrenal metabolism of progesterone occur with ACTH-induced labour as at spontaneous term and whether the site of cortisol modulation is on adrenal steroidogenesis or at the level of cAMP generation. Chronically catheterized fetal sheep were infused in utero for 100 h between days 127 and 131 of pregnancy with P-ACTH, P-ACTH + metopirone, P-ACTH + metopirone + cortisol, or saline. After 100 h the metabolism of [3H]progesterone was measured in adrenal homogenates. Similar incubations were performed with adrenal tissue from fetal sheep at day 130 of pregnancy and at spontaneous labour. In the treatment groups of sheep, cAMP output by dispersed adrenal cells in response to ACTH added in vitro was also determined. Similar qualitative patterns of [3H]progesterone metabolism were found in adrenal homogenates after in vivo ACTH or at term. At both times there was an increase in cortisol and in total 17 alpha-hydroxycorticosteroid accumulation and also evidence for increased activity of 11 beta-hydroxylase enzyme. The formation of total 17 alpha-hydroxycorticosteroids was not affected significantly by concurrent treatment in vivo with metopirone +/- cortisol. The accumulation of cAMP in vitro was increased after in vivo ACTH, attenuated after ACTH + metopirone, but statistical significance over controls was restored after ACTH + metopirone + cortisol treatment. We conclude that ACTH-induced labour and spontaneous parturition in sheep is associated with qualitatively similar changes in progesterone metabolism by the fetal adrenal gland.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of aldosterone and corticosterone by the adrenals of Brattleboro rats in response to ACTH administration

The secretions of aldosterone and corticosterone in response to administration of 0,5 mUI of (1,24) ACTH (synacthène-Ciba) were measured in the adrenal venous blood of 15 Brattleboro female rats genetically lacking vasopressin and in 15 Long-Evans female rats, pretreated with dexamethasone. The secretions of aldosterone and corticosterone increased according to a similar profile in the two groups of animals: maximum values were 20-30 min. after ACTH injection; however the steroidogenic secretion of the adrenal cortex was always about 50% less in the Brattleboro female rats than in Long-Evans female rats. This result suggests mainly that vasopressin may be involved in the mechanisms which control the in vivo production of aldosterone by the adrenal glomerulosa cells.     

Sex differences in adrenal function in the lizard Cnemidophorus sexlineatus: II. Responses to acute stress in the laboratory.

Circulating concentrations of plasma corticosterone and gonadal steroids were measured in intact and gonadectomized male and female lizards (Cnemidophorus sexlineatus) following acute stress (handling) in the laboratory. There was a significant increase in plasma corticosterone after stress. Whereas intact females exhibited greater concentrations of corticosterone relative to intact males, ovariectomized females exhibited lower concentrations of corticosterone relative to castrated males. In addition to sex differences in corticosterone responses to gonadectomy, progesterone was elevated by stress in both intact and ovariectomized females but not in males. Corticosterone adjusted for castration and handling in males was negatively correlated with the plasma androgen level. The adrenal responsiveness of males to acute stress may be attenuated by androgens presumably secreted by the testis. Not only does adrenal function influence reproduction, but adrenal responses differ between males and females, and appear to be influenced by the gonadal axis. The sex differences in adrenal responses to stress likely reflect different reproductive strategies and nutritional requirements of males and females during the breeding season.     

Effect of Prenatal Stress on the Pituitary–Adrenal Axis in Blue Foxes

Handling is a source of stress for farm bred blue foxes. The influence of handling during the late gestation period on the pituitary–adrenal axis was studied in 10-day old male and female blue foxes. Cortisol and progesterone were measured by radioimmunoassay in the plasma, adrenal homogenates, and in vitro incubates from animals of both sexes. Adrenals were incubated in vitro in the absence or presence of ACTH. In addition, the adrenal weight and plasma concentration of ACTH were assessed. In cubs of both sexes, the adrenal weight was decreased after prenatal stress treatment. The plasma concentration of progesterone and the adrenal cortisol in vitro production were elevated in the prenatally stressed female cubs, as compared to the control, along with the adrenal progesterone in vitro production in prenatally stressed male cubs. The adrenal cortisol and progesterone content and plasma ACTH and cortisol concentrations were not affected by prenatal stress. In conclusion, the results of this study suggest that the prenatal stress induced by handling pregnant vixens can affect the pituitary–adrenal axis in neonatal offspring, this effect being more pronounced in female cubs.     

The effects of progestins,mineralocorticoids, glucocorticoids,and steroid solubility on the induction of sexual receptivity in rats

In the first experiment, progesterone and its 5α-reduced metabolite, 5α-dihydroprogesterone, dissolved in two different vehicles were compared for their effectiveness in facilitating lordosis behavior in ovariectomized estrogen-primed rats. When dissolved in oil vehicle, 5α-dihydroprogesterone was less effective than progesterone. However, when dissolved in Tween 80 solution, the two progestins were equally effective. In the second experiment, adrenal corticoids dissolved in Tween 80 solution were tested for their relative ability to facilitate sexual receptivity. Progesterone, desoxycorticosterone, and desoxycorticosterone acetate were equally effective in facilitating sexual receptivity. Aldosterone, corticosterone, and corticosterone acetate were no more effective than the vehicle in facilitating sexual receptivity.     

Enzymatic sulfation of steroids III. The sulfation of corticosterone by the glucocorticoid sulfotranferases of rat liver cytosol

A radioisotopic assay for the cytoplasmic corticosterone sulfotransferase activity of rat liver was developed. The steroid inhibits the enzyme reaction. For reliable results, a complex assay method, using three different corticosterone concentrations, each studied with several different amounts of enzyme, was necessary. This ‘mosaic’ assay compensates for observed biological, gonadal and seasonal enzyme fluctuations. Cytosols from female rats contain 6–9-times the enzyme activity found in males. The sulfation product with both sexes is corticosterone-21-sulfate.The effects of castration and of androgen administration on hepatic cortisol and corticosterone sulfation were compared in female rats. Ovariectomy resulted in 20–32% and 25–35% decreases of hepatic corticosterone and cortisol sulfotransferase activity, respectively. Androgen administration caused 37–55% and 40–60% decreases of sulfation of the twoo steroids. The data suggest the equivalence of hepatic cortisol and corticosterone sulfotransferases.Fractionation of cytosols from female rats, on DEAE-Sephadex A-50 columns, resolved three peaks of corticosterone sulfotransferase activity which eluted concurrently with the hepatic cortisol sulfotransferase I, II and III. They appear to be the same enzymes. Cytosol from males contained cortisosterone sulfotransferase activity due to mostly to sulfotransferase III. Sulfotransferases I and II appear to have higher turnover numbers for hepatic cortisol than for corticosterone. The reverse is true for sulfotransferase III.     

Enzymatic sulfation of steroids. II. The sulfation of corticosterone by the glucocorticoid sulfotransferases of rat liver cytosol

A radioisotopic assay for the cytoplasmic corticosterone sulfotransferase activity of rat liver was developed. The steroid inhibits the enzyme reaction. For reliable results, a complex assay method, using three different corticosterone concentrations, each studied with several different amounts of enzyme, was necessary. This "mosaic" assay compensates for observed biological, gonadal and seasonal enzyme fluctuations. Cytosols from female rats contain 6--9-times the enzyme activity found in males. The sulfation product with both sexes is corticosterone-21-sulfate. The effects of castration and of androgen administration on hepatic cortisol and corticosterone sulfation were compared in female rats. Ovariectomy resulted in 20--32% and 25--35% decreases of hepatic corticosterone and cortisol sulfotransferase activity, respectively. Androgen administration caused 37--55% and 40--60% decreases of sulfation of the two steroids. The data suggest the equivalence of hepatic cortisol and corticosterone sulfotransferases. Fractionation of cytosols from female rats, on DEAE-Sephadex A-50 columns, resolved three peaks of corticosterone sulfotransferase activity which eluted concurrently with the hepatic cortisol sulfotransferases I, II and III. They appear to be the same enzymes. Cytosol from males contained cortisosterone sulfotransferase activity due mostly to sulfotransferase III. Sulfotransferases I and II appear to have higher turnover numbers for hepatic cortisol than for corticosterone. The reverse is true for sulfotransferase III.     

Melatonin and adrenal cortex steroid production: in vivo and in vitro studies.

The present study was designed to clarify the interaction between the pineal melatonin and adrenal cortex steroid production. Experiments with male rats under chronic stress conditions (sleep deprivation) revealed that melatonin circadian pattern was fully destroyed and daytime plasma concentration were significantly elevated. Constant illumination (2500 lux) during the nighttime was not able to suppress melatonin production in the stressed animals. Plasma concentration of corticosterone were increased in the stressed rats as well. The modulatory effect of melatonin on corticosterone and progesterone production by rat adrenals was studied in a superfusion system. During melatonin challenge progesterone secretion was two-three fold elevated with no effect on corticosterone content in the plasma samples. Pineal cytoplasmic glucocorticoid and progesterone receptors were investigated as well. A specific binding was not observed in that case. Presented data support the existence of direct communication between the pineal and adrenal glands.     

Validation of the mechanisms proposed for the stimulatory and inhibitory effects of progesterone on gonadotropin secretion in the estrogen-primed rat: a possible role for adrenal steroids.

The stimulatory and inhibitory effects of progesterone on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion were found to be dependent on the length of estrogen exposure in ovariectomized estrogen-primed rats. Progesterone suppressed LH and FSH secretion when administered 16 hours after a single injection of estradiol to ovariectomized rats. If the estradiol treatment was extended over 40 hours by two injections of estradiol 24 hours apart, progesterone administration led to a highly significant elevation of both serum LH and FSH levels 6 hours later. In addition to the direct stimulatory effect on LH and FSH release, progesterone, when injected 1 hour before, was able to antagonize the suppressive effect of a third injection of estradiol on LH and FSH release. In the immature ovariectomized estrogen-primed rat, 10 IU of ACTH brought about a release of progesterone and corticosterone 15 minutes later and LH and FSH 6 hours later. Progesterone, but not corticosterone, appeared to be responsible for the effect of ACTH on gonadotropin release. The synthetic corticosteroid triamcinolone acetonide brought about LH and FSH release in the afternoon, while cortisol, similar to corticosterone, was unable to do so. Nevertheless, triamcinolone acetonide and cortisol brought about increased secretion of FSH the following morning.     

Ultrastructural evidence for endogenous testosterone immunoreactivity in the pituitary gland of the rat

Summary Several attempts have been made to localize steroids by means of immunocytological techniques. However, these methods were found inadequate for detecting steroids bound to their receptors. To localize endogenous testosterone (T) in its target cells at the ultrastructural level, an immunocytological technique was performed on ultrathin sections obtained by cryo-ultramicrotomy. T was detected in the pituitary glands obtained from intact male or female rats and castrated rats, but not in castrated + adrenalectomized rats. Animals were also injected either with testosterone, with other steroids (estradiol, progesterone, corticosterone) or with an androgen antagonist (cyproterone acetate). In addition, some ultrathin sections were preincubated either with phosphate buffers of various pH, corticosterone, cyproterone acetate solution, or with T solution. The content of T in the pituitary before and after fixation was measured by radioimmunoassay; it decreased after fixation. T immunoreactivity was localized in the gonadotropic cells only, both in the male and female rats. At the subcellular level, the immunoreactivity was detected in the cytoplasmic matrix and in the nucleus. Immunoreactive T disappeared 1) in rats after castration+adrenalectomy; by means of radioimmunoassay no T was measured in these pituitary glands; 2) in rats injected with 25 (g/rat of cyproterone acetate; 3) after preincubation of pituitary sections on a drop of cyproterone acetate (1 × 10^-6 M). The immunocytological reaction was not modified when the rats were injected with estradiol, progesterone or corticosterone (1 mg/rat), or after preincubation of the sections with corticosterone (1 × 10^-3 M), or a buffer solution at pH 7.6. Lower or higher pH values led to a strong decrease in the immunoreactivity. After injection of T (15 g/rat) the immunocytological reaction was more abundant in the nucleus and less in the cytoplasm. The immunoreactivity was again observed when the sections were preincubated with cyproterone acetate solution and then with T solution. These data suggest that T can be detected by means of immunocytochemistry. It is probably bound to a specific binding site.This work was presented in part at the VI^th International Congress on Hormonal Steroids, Jerusalem, 1982     

The neonatal glucocorticoid treatment-produced long-term changes of the pituitary-adrenal function and brain corticosteroid receptors in rats.

Two distinct periods of sensitivity to elevated glucocorticoid hormone levels during postnatal development of the pituitary-adrenal axis were studied. Wistar rats were injected subcutaneously (s.c.) with cortisol (1 mg/kg) on postnatal days 1-5 or 14-18. The steroid treatment during the first postnatal week resulted in a decrease of the morning basal and stress-induced plasma corticosterone levels in 30 day-old male rats, as well as in rats that were injected with cortisol on the third postnatal week. Stress-induced corticosterone levels in 90-day old cortisol-treated rats were determined in blood samples drawn from the tail vein before the restraint stress, immediately after the 20-min long stress, then 60 and 180 min afterwards. Only the rats treated with cortisol during the third week showed a prolonged stress-induced corticosterone secretion, with the highest corticosterone level in 180 min after the restraint stress. The early neonatal cortisol treatment had no effect on (3)H-corticosterone binding in all studied brain areas of the 90-day old rats. The rats treated with cortisol at the 14-17th postnatal days showed a significantly lower (3)H-corticosterone binding in the frontal cortex, hippocampus, and hypothalamus. These findings suggest that the third week of life in rats is more sensitive to elevated levels of corticosterone than the first one. The high level of glucocorticoids at this period has long-term effects on the efficiency of the negative feedback mechanisms provided by hypothalamus-pituitary-adrenal axis.     

Trend analysis of serum progesterone, deoxycorticosterone, deoxycorticosterone sulfate, cortisol, corticosterone, 18-hydroxydeoxycorticosterone and estradiol in early neonates

To elucidate the origin and regulatory mechanism of deoxycorticosterone (DOC) and deoxycorticosterone sulfate during fetal life, the levels of serum DOC, DOC sulfate, progesterone, cortisol, corticosterone and 18-hydroxydeoxycorticosterone (18OH-DOC) were determined in the fraction separated on high performance liquid chromatogram (HPLC) by radioimmunoassay (RIA) using the serum from normal newborn. Elimination curves both of serum DOC and DOC sulfate showed two phases: rapidly decreasing and slowly decreasing ones. Both serum DOC and DOC sulfate correlated with progesterone (r = 0.340, p less than 0.01; r = 0.737, p less than 0.01, respectively). They also correlated with cortisol (DOC, r = 0.467, p less than 0.01; DOC sulfate, r = 0.549, p less than 0.01, respectively). Serum DOC reached normal adult levels by 16 hrs after birth. However serum DOC sulfate concentration was maintained high throughout the entire early neonatal period. On the contrary, the changes in serum cortisol, corticosterone and 18OH-DOC showed a peak surge in the initial phase after delivery. Both serum corticosterone and 18OH-DOC correlated with cortisol (r = 0.518, p less than 0.01; r = 0.410, p less than 0.01, respectively). These findings suggest that, in the fetus, serum DOC and DOC sulfate are mainly produced at extraadrenal sites isolated from normal mineralocorticoids synthesis and after birth they begin to be formed at adrenal glands.     

Sex differences in adrenocortical structure and function. III. The effects of postpubertal gonadectomy and gonadal hormone replacement on adrenal cholesterol sidechain cleavage activity and on steroids biosynthesis by rat adrenal homogenates.

In rats postpubertal orchiectomy results in an increase in the adrenal weight, testosterone replacement restores the adrenal weight to the normal level. Neither ovariectomy (8 weeks of duration) nor estradiol replacement has an effect on adrenal weight in female rats. Pregnenolone synthesis as well as corticosterone and blue tetrazolium-positive steroids secretion is significantly higher in homogenates of adrenals from female rats than from males. Orchiectomy results in a marked increase in pregnenolone biosynthesis, testosterone replacement restores the value to the normal levels. Neither ovariectomy nor estradiol replacement has an effect on pregnenolone synthesis in v i t r o. In both sexes gonadectomy causes a marked decrease in corticosterone output by adrenal homogenates, concomitantly the increase in the adrenal 5alpha-reductase activity is observed. The ratio of secreted corticosterone to pregnenolone is significantly lower in gonadectomized rats of both sexes than in control animals. Estradiol or testosterone replacement inhibits the adrenal 5alpha-reductase activity and restores the corticosterone output as well as corticosterone/pregnenolone ratio to the normal values. The above described findings show that the sex differences in steroids secretion by the rat adrenal are partially conditioned by a cholesterol sidechain cleavage activity. Testosterone inhibits this activity while estradiol under applied experimental conditions has no effect on the cholesterol sidechain cleavage activity.     

Influence of ACTH secreting tumor (MtTF4) on fetal rat adrenal gland steroidogenesis in vitro in prolonged pregnancy

Pregnant female rats with ACTH secreting tumor (MtTF4) have prolonged pregnancy and cannot deliver. The fetuses of tumor bearing females have in prolonged pregnancy on days 24 and 25 of pregnancy greater body weight and smaller adrenal weight as compared to intact fetuses of the 22nd day of pregnancy. The fetal adrenal glands converted to vitro 4-14C progesterone to radioactive 11-deoxycorticosterone (DOC), corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), 18-hydroxy-corticosterone (18-OH-B) and aldosterone. Fetal adrenal glands in prolonged pregnancy synthetized in vitro less amount of radioactive DOC, B and 18-OH-DOC. A negative relationship exists between the maternal corticosterone which passes the placenta to fetuses and corticosteroidogenesis of fetal adrenal glands. These results indicate the possibility that fetal rat adrenal glands with their corticosteroids participate in pregnancy and influence normal delivery.     

ACTH sensitivity of isolated human pathological adrenocortical cells: variability of responses in aldosterone, corticosterone, deoxycorticosterone and cortisol production

In vitro aldosterone, deoxycorticosterone, corticosterone and cortisol production of human adrenocortical cells derived from adenomas (Conn's syndrome, Cushing's syndrome), from hyperplastic adrenals (Cushing's syndrome) and from adrenals surrounding aldosteronoma are described. Cells from adenomas causing either Cushing's syndrome or Conn's syndrome harboured the highest basal and ACTH-stimulated corticosteroid production. Adrenocortical cells derived from micronodular hyperplasia causing Cushing's syndrome and cells from cortisol producing adenoma displayed predominantly cortisol and corticosterone secretion both under basal conditions and following stimulation with ACTH. Aldosteronoma cells showed highly variable aldosterone, deoxycorticosterone, corticosterone and cortisol response to ACTH. However, in aldosteronoma cell suspensions, the basal and ACTH-stimulated ratios of aldosterone to cortisol were increased when compared to ratios of steroids produced by cells from other adrenal tissues. Chronic treatment with spironolactone of patients with Conn's syndrome before surgery was associated with a decreased ratio of aldosterone to corticosterone, revealing that 18-hydroxylase in aldosteronoma cells may be inhibited during long-term therapy. Non-tumorous cells isolated from adrenals surrounding aldosteronoma displayed less aldosterone prior to and after stimulation with ACTH than aldosteronoma cells.