Mechanism of dissociation of cortisol and adrenal androgen secretion after removal of adrenocortical adenoma in patients with Cushing's syndrome

We investigated the mechanism of dissociation of cortisol and dehydroepiandrosterone sulfate (DHEA-S) secretion by the adrenal glands after the removal of an adrenal gland containing an adrenocortical adenoma in a patient with Cushing's syndrome. After removal of the adrenocortical adenoma, the serum cortisol rapidly decreased from 24.6 +/- 6.4 micrograms/dl (mean +/- SD, n = 6) to 0.7 +/- 0.5 micrograms/dl. Serum DHEA-S levels were 15 +/- 14 micrograms/dl and 6 +/- 9 micrograms/dl before and after surgery, respectively, and significantly lower than the control values. Serum cortisol levels reverted to normal levels 1.5 to 3 years after the surgery. On the other hand, DHEA-S levels reverted to normal 5 to 7 years after the serum cortisol levels had normalized. Monolayer cultures of normal human adrenal cells obtained at adrenalectomy in patients with advanced breast cancer and atrophic adrenal cells adjacent to the adrenocortical adenoma in patients with Cushing's syndrome were used to study the mechanism of the dissociation of cortisol and DHEA-S secretion. ACTH caused significant increases in the productions of pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17-OH-P5), 17-hydroxyprogesterone (17-OH-P4), DHEA, DHEA-S, androstenedione (delta 4-A), and cortisol. The amounts of 17-OH-P5 and 17-OH-P4 produced by ACTH in atrophic adrenal cells were significantly greater than those in normal adrenal cells. The amounts of DHEA, DHEA-S and delta 4-A produced by ACTH in atrophic adrenal cells were significantly smaller than those of normal adrenal cells. The conversion rate of 17-OH-[3H]P5 to 17-OH-[3H]P4 and 11-deoxy-[3H] cortisol was higher in atrophic adrenal cells than in normal adrenal cells, but the conversion rate to [3H]DHEA, [3H]DHEA-S and [3H]delta 4-A was significantly lower in atrophic adrenal cells than in normal adrenal cells. These results suggest that the dissociation of cortisol from DHEA-S after the removal of adrenocortical adenoma is a probably due to diminished C17,20-lyase activity in the remaining atrophic adrenal gland.     

Effect of ACTH and prolactin on dehydroepiandrosterone, its sulfate ester and cortisol production by normal and tumorous human adrenocortical cells

The effect of ACTH and prolactin on the synthesis of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) was studied in cell suspensions of "normal" and tumorous (adenoma) human adrenal cortex. A stimulation of DHEA and no response of DHEAS production by ACTH in "normal" adrenocortical cell suspension was observed. However ACTH stimulated both DHEA and DHEAS synthesis in tumorous adrenocortical cells. Prolactin did not influence either the basal or the ACTH stimulated DHEA and DHEAS production of adrenocortical cells irrespective of their origin. Our results are compatible with the concept that the biosynthesis of DHEA is under ACTH control, while other factor(s) regulate(s) the sulfate pathway of DHEA secretion under normal conditions. In tumorous adrenocortical cells DHEA may be regulated--at least partly--by ACTH. Prolactin seems to have no direct effect on DHEA and DHEAS synthesis. It is postulated that the relationship between serum prolactin and DHEAS (or DHEA) levels observed by several authors might be an extraadrenal effect of prolactin on adrenal androgens.     

Chronic treatment with corticotropin increases the capacity of rabbit adrenocortical cells to convert pregnenolone into androgens

The present study was conducted to evaluate whether the previously demonstrated enhancement in adrenocortical androgen secretion in rabbits chronically treated with ACTH results, in addition to an increased pregnenolone production, from a more efficient conversion of this precursor of steroidogenesis into androgens. To this end, the adrenocortical cells from 14 control and 14 ACTH-treated rabbits (ACTH 1-24,200 micrograms s.c. daily for 12 days) were incubated either in the presence of different concentration of ACTH or with pregnenolone added in amounts from 0.5 to 250 micrograms. The total steroidogenic potency (maximal response to ACTH) was significantly enhanced for cells from ACTH-treated animals, as was the ACTH-induced production of dehydroepiandrosterone (DHEA), DHEA-sulfate, androstenedione and testosterone. In addition the production of these androgens from given amounts of exogenous pregnenolone was also significantly increased. The maximal capacity of adrenocortical cells to convert pregnenolone into androgens averaged (for ACTH-treated vs control group) 130 +/- 34 vs 43 +/- 10 pmol for DHEA, 138 +/- 43 vs 46 +/- 14 pmol for DHEA-sulfate, 99 +/- 31 vs 10 +/- 2 pmol for androstenedione and 8.0 +/- 2.6 vs 2.4 +/- 0.3 pmol for testosterone (P less than 0.001 for all androgens). The addition of ACTH to adrenocortical cells incubated with pregnenolone did not modify the maximal capacity of conversion of pregnenolone into androgens, which was in both experimental groups similar to that documented in the absence of ACTH. Thus, while an acute stimulatory effect of ACTH on adrenocortical steroidogenesis is devoid of any influence on the activity of the post-pregnenolone pathway of androgen synthesis, the chronic exposure of adrenocortical cells to ACTH lead to increased activity of steroidogenic pathway involved in the conversion of pregnenolone into androgens.     

An oxidized derivative of linoleic acid stimulates dehydroepiandrosterone production by human adrenal cells.

We previously reported that an oxidized derivative of linoleic acid stimulated steroidogenesis in rat adrenal cells. This derivative was also detected in human plasma, and was positively correlated with visceral adiposity and plasma DHEA-S. The present study sought to characterize the effects of this derivative, 12,13-epoxy-9-keto-(10- trans)-octadecenoic acid (EKODE), on steroid production by normal human adrenocortical cells obtained during clinically-indicated adrenalectomy. Cell suspensions were incubated in the presence of varying concentrations of EKODE and ACTH. EKODE (16 microM) significantly increased DHEA production by 28% under basal conditions and by 25% in the presence of a low concentration of ACTH (0.2 ng/ml). The effect on DHEA was absent at a higher ACTH concentration (2.0 ng/ml). EKODE decreased cortisol production by 16% (low ACTH) and 25% (high ACTH), but was without effect on cortisol under basal conditions. The results suggest that EKODE affects adrenal DHEA production in the human, possibly by modulating steroidogenic enzyme activity. We postulate that excess visceral fat delivers fatty acids to the liver, where oxidized derivatives are formed that modulate adrenal steroidogenesis. This may be an important phenomenon in the genesis of changes in adrenal function associated with syndromes of obesity, especially those that include androgen excess.     

Alpha-human natriuretic polypeptide (alpha-hANP) specific binding sites in bovine adrenal gland

The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. alpha-hANP did not inhibit basal aldosterone secretion. alpha-hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of ACTH (10(-8) M)-stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of [125I]alpha-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of alpha-hANP.     

Effects of glucagon and glucagon-like peptide-1 on glucocorticoid secretion of dispersed rat adrenocortical cells.

The effects of glucagon and glucagon-like peptide-1 (GLP-1) on the secretory activity of rat adrenocortical cells have been investigated in vitro. Neither hormones affected basal or agonist-stimulated aldosterone secretion of dispersed rat zona glomerulosa cells or basal corticosterone production of zona fasciculata-reticularis (inner) cells. In contrast, glucagon and GLP-1 partially (40%) inhibited ACTH (10(-9) M)-enhanced corticosterone secretion of inner cells, maximal effective concentration being 10(-7) M. The effect of 10(-7) M glucagon or GPL-1 was suppressed by 10(-6) M Des-His1-[Glu9]-glucagon amide (glucagon-A) and exendin-4(3-39) (GPL-1-A), which are selective antagonists of glucagon and GLP-1 receptors, respectively. Glucagon and GLP-1 (10(-7) M) decreased by about 45-50% cyclic-AMP production by dispersed inner adrenocortical cells in response to ACTH (10(-9) M), but not to the adenylate cyclase activator forskolin (10(-5) M). Again this effect was blocked by 10(-6) M glucagon-A or GLP-1-A. The exposure of dispersed inner cells to 10(-7) M glucagon plus GLP-1 completely suppressed corticosterone response to ACTH (10(-9) M). However, they only partially inhibited (by about 65-70%) both corticosterone response to forskolin (10(-5) M) or dibutyryl-cyclic-AMP (10(-5) M) and ACTH (10(-9) M)-enhanced cyclic-AMP production. Quantitative HPLC showed that 10(-7) M glucagon or GLP-1 did not affect ACTH-stimulated pregnenolone production, evoked a slight rise in progesterone and 11-deoxycorticosterone release, and markedly reduced (by about 55%) corticosterone secretion of dispersed inner adrenocortical cells. In light of these findings the following conclusion are drawn: (i) glucagon and GLP-1, via the activation of specific receptors, inhibit glucocorticoid response of rat adrenal cortex to ACTH; and (ii) the mechanism underlying the effect of glucagon and GLP-1 is probably two-fold, and involves both the inhibition of the ACTH-induced activation of adenylate cyclase and the impairment of the late steps of glucocorticoid synthesis.     

Dehydroepiandrosterone and dehydroepiandrosterone sulfate production in the human adrenal during development and aging.

Dehydroepiandrosterone (DHEA) is produced in prodigious quantities by the human adrenal, principally as the 3-sulfoconjugate DHEA sulfate (DS) during intrauterine life. The fetal zone and neocortex cells of the fetal adrenal express large amounts of DHEA sulfotransferase and minimal amounts, at least until very near the end of gestation, of 3beta-hydroxysteroid dehydrogenase. This pattern of enzyme expression favors substantial secretion of DHEA/DS with minimal cortisol produced; the DHEA/DS serves as the major precursor for placental estrogen formation in human pregnancy. Aside from adrenocorticotropin, other physiologic regulators of growth and steroidogenesis in the fetal adrenal have been postulated to exist, but have yet to be identified. Whereas intrauterine stressors may activate adrenal cortisol secretion, the fetal adrenal responds to many pregnancy conditions by suppressing DHEA/DS formation. After birth, the human adrenal undergoes reorganization whereby the large, inner fetal zone regresses, and DHEA/DS production is diminished. Just prior to gonadal maturation, the human adrenal undergoes morphologic and functional changes (adrenarche) that give rise to a prominent zona reticularis that is characterized by the presence of DHEA sulfotransferase, the absence of 3beta-hydroxysteroid dehydrogenase, and an enhancement of DHEA/DS production. The adrenal of the adult responds to stress in many instances like that of the fetus: increased cortisol secretion and diminished DHEA/DS secretion. The mechanisms for this divergence in the adrenocortical pathway is unknown. With aging, there is suppression of DHEA/DS secretion, possibly as the consequence of an involution of the zona reticularis, but corticosteroid production continues unabated.     

Naloxone potentiates ACTH and angiotension II but not potassium stimulated aldosterone secretion, in vitro

The effects of naloxone on basal and ACTH, Angiotensin II (AII) and [K+] o stimulated aldosterone secretion from superfused rat adrenocortical tissue were investigated. A high dose (10(-6) M) of naloxone inhibited while a smaller dose (10(-10) M) potentiated and doses of 10(-8) or 10(-12) M naloxone were without an effect on ACTH stimulated aldosterone secretion. A potentiation of AII stimulated aldosterone secretion was observed beginning 2 hrs after 10(-6) or 10(-10) M naloxone was administered while no effect was observed with 10(-4) M naloxone. No effects of 10(-6), 10(-8), 10(-12) M naloxone were detected on aldosterone secretion stimulated by transiently elevating extracellular potassium. Naloxone from 10(-4) to 10(-12) M did not appear to significantly influence basal steroidogenic activity under these conditions. These findings demonstrate that the "opioid antagonist" naloxone has prominent actions on adrenocortical tissue. Both the specificity and lack of specificity of the action of this agent to influence the activity of the 3 secretagogues suggest that naloxone and possibly a naturally occurring endogenous ligand interacts with one or more membrane receptor distinct from the ACTH receptor. A naturally occurring ligand for this receptor could play a prominent role in the physiological regulation of adrenal steroid secretion.     

Studies on lipoprotein and adrenal steroidogenesis: II. Utilization of low density lipoprotein- and high density lipoprotein-cholesterol for steroid production in functioning human adrenocortical adenoma cells in culture

We examined the utilization of human low density lipoprotein (LDL)- and high density lipoprotein (HDL)-cholesterol for steroid production in primary monolayer culture cells from adenomas of primary aldosteronism and Cushing's syndrome and an adrenal of nodular hyperplasia of Cushing's syndrome. We compared the data obtained with findings in the case of cultured normal human adrenocortical cells. In the presence of 10(-7) M adrenocorticotropin (ACTH), the addition of either LDL or HDL to the culture medium at a cholesterol concentration of 100 micrograms/ml led to a significant increase in the daily secretion rates of cortisol, dehydroepiandrosterone sulfate (DHEA-S) and aldosterone in the adenoma and nodular hyperplasia cells, as in the normal cells. Although LDL greatly increased the secretion of steroid hormones, no significant difference in steroid secretion following the treatments with LDL and HDL were observed in these cultured cells. The contribution of endogenous cholesterol to steroid production was also high, thereby indicating that the neoplastic transformation did not have untoward effects. Cells from adenomas of primary aldosteronism secreted not only aldosterone, but also cortisol and DHEA-S. The daily secretion rates of these steroids were markedly increased when ACTH was added to the medium. With prolonged exposure to ACTH, however, the rate of aldosterone secretion showed a gradual decrease with the incubation time. This decrease might be due to the impaired conversion of corticosterone to 18-hydroxycorticosterone. In case of adenomas in patients with Cushing's syndrome, the secretion of steroid hormones varied in quantity and quality, depending on the type of plasma cortisol response to the rapid ACTH test in vivo, thereby suggesting that the adrenocortical adenoma of Cushing's syndrome might be divided into two subtypes. These results indicate that human functioning adrenocortical adenoma cells utilize plasma lipoproteins as a source of cholesterol for steroidogenesis during the prolonged stimulation of steroid secretion.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

Adrenal androgens in children with short stature

Recent data suggest that adolescent individuals with growth hormone (GH) deficiency have subnormal levels of adrenal androgens (AA). In order to determine the developmental pattern of AA in GH deficiency and to assess whether AA levels can help identify children with GH deficiency, we measured plasma concentrations of dehydroepiandrosterone (DHEA), DHEA sulfate (DHEA-S), delta 4-androstenedione (delta 4A), and cortisol in the basal state and during prolonged adrenocorticotropin (ACTH) infusion (8 h) in a group of 34 individuals, 26 males and 8 females, with short stature. Their chronological ages (CA) ranged from 1.75 to 17.5 years (median 10.35 years). The subjects were grouped into two categories according to the results of pituitary testing: group 1 = short, non-GH-deficient (n = 16), and group 2 = GH-deficient, ACTH-sufficient (n = 18). Patients in groups 1 and 2 had similar bone ages (BA: 7.2 +/- 0.7 vs. 7.5 +/- 1.0 years) and Z scores for height (-3.0 +/- 0.2 vs. -3.2 +/- 0.3 units) and height velocity (-2.5 +/- 0.4 vs. -2.6 +/- 0.2 units). For both groups there were significant increases from basal to peak levels for DHEA, DHEA-S, delta 4A and cortisol following prolonged ACTH infusion. Although both basal and peak levels of DHEA-S overlapped in groups 1 and 2 for all CA and BA, levels in group 2 tended to be lower, especially for BA greater than 10 years.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gastric inhibitory polypeptide stimulates glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase-dependent signaling pathway.

Gastric inhibitory polypeptide (GIP) is a 42-amino acid peptide, belonging to the VIP-secretin-glucagon superfamily, some members of this group are able to regulate adrenocortical function. GIP-receptor mRNA has been detected in the rat adrenal cortex, but investigations on the effect of GIP on steroid-hormone secretion in this species are lacking. Hence, we have investigated the distribution of GIP binding sites in the rat adrenal gland and the effect of their activation in vivo and in vitro. Autoradiography evidenced abundant [125I]GIP binding sites exclusively in the inner adrenocortical layers, and the computer-assisted densitometric analysis of autoradiograms demonstrated that binding was displaced by cold GIP, but not by either ACTH or the selective ACTH-receptor antagonist corticotropin-inhibiting peptide (CIP). The intraperitoneal (IP) injection of GIP dose-dependently raised corticosterone, but not aldosterone plasma concentration: the maximal effective dose (10 nmol/rat) elicited a twofold increase. GIP did not affect aldosterone and cyclic-AMP release by dispersed zona glomerulosa cells. In contrast, GIP enhanced basal corticosterone secretion and cyclic-AMP release by dispersed inner adrenocortical cells in a concentration-dependent manner, and the maximal effective concentration (10(-7) M) evoked 1.5- and 2.4-fold rises in corticosterone and cyclic-AMP production, respectively. GIP (10(-7) M) did not display any additive or potentiating effect on corticosterone and cyclic-AMP responses to submaximal or maximal effective concentrations of ACTH. The corticosterone secretagogue action of 10(-7) M GIP was abolished by the protein kinase A (PKA) inhibitor H-89 (10(-5)M), and unaffected by CIP (10(-6)M). Collectively, these findings indicate that GIP exerts a moderate but statistically significant stimulatory effect on basal glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.     

Lack of inhibitory effect of alpha-human atrial natriuretic polypeptide on cortisol secretion in cultured adrenocortical adenoma cells from the patients with Cushing's syndrome

The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on cortisol secretion by adrenocortical adenoma cells from patients with Cushing's syndrome (CS cells) in primary monolayer cultures, compared to cultured normal adrenal cells, were studied. alpha-hANP significantly inhibited cortisol secretion by human normal adrenal cells in culture, but had no direct effect on cortisol secretion from CS cells, in the presence or absence of 10(-8) M ACTH. alpha-hANP enhanced the accumulation of intracellular cyclic GMP in normal adrenal cells in culture, but not in CS cells. Visualization of [125I] iodo-alpha-hANP-specific binding sites by an in vitro receptor autoradiographic technique showed that these sites were lacking in adrenocortical adenoma tissues. These results suggest that the loss of alpha-hANP inhibitory effect on cortisol secretion in CS cells may be due to the absence of alpha-hANP receptor sites.     

Ovine prolactin potentiates the action of adrenocorticotropic hormone on the secretion of dehydroepiandrosterone sulfate and dehydroepiandrosterone from cultured bovine adrenal cells

To determine the direct effect of prolactin on adrenal androgen secretion, the daily secretions of dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone (DHEA), androstenedione and cortisol were determined in monolayer culture of bovine adrenal cells in the presence or absence of adrenocorticotropic hormone (ACTH) and/or prolactin. In the absence of ACTH ovine prolactin alone had no effect on steroid secretion during seven-day culture. Ovine prolactin, when administered in combination with ACTH, significantly potentiated the stimulatory effect of ACTH on DHEA-S and DHEA but not androstenedione secretion on the seventh day in culture. On the first day in culture prolactin showed no synergistic effect with ACTH on DHEA and DHEA-S secretion, although ACTH significantly increased DHEA and cortisol secretion. DHEA-S secretion increased as a function of prolactin concentration in the presence of ACTH. These results indicated that long-term treatment by ovine prolactin with ACTH caused the increase in adrenal androgen secretion from bovine adrenal cells. The site of action of prolactin was suggested to be the partial inhibition of adrenal 3 beta-hydroxysteroid dehydrogenase by the result of increases in DHEA-S and DHEA but not androstenedione secretion.     

The effect of ACTH therapy on serum dehydroepiandrosterone, androstenedione, testosterone and 5 alpha-dihydrotestosterone in infants

Serum concentrations of dehydroepiandrosterone (DHEA), androstenedione, testosterone, 5 alpha-dihydrotestosterone and cortisol were measured in 10 infants (age 5-22 months) before, during and after 6-weeks of ACTH therapy for infantile spasms. During therapy, their mean DHEA concentrations increased 2.3-fold, androstenedione 12.3-fold, testosterone 2.7-fold, 5 alpha-dihydrotesterone 2.5-fold and cortisol 2.9-fold compared to pre-therapy values. Serum dehydroepiandrosterone sulphate (DHEA-S) concentrations were also increased during ACTH therapy above the normal prepubertal range. Three days after the cessation of ACTH treatment, all androgens had returned to the pre-therapy level. We conclude: At least in pharmacologic doses ACTH alone stimulates adrenal androgen secretion in infants, excluding the necessity of a separate adrenal androgen stimulating hormone.     

Porcine brain natriuretic peptide receptor in bovine adrenal cortex

The action of porcine brain natriuretic peptide (pBNP) on the steroidogenesis was investigated in cultured bovine adrenocortical cells. Porcine BNP induced a significant dose-dependent inhibition of both ACTH- and A II-stimulated aldosterone secretion. 10(-8) M and 10(-7) M pBNP also significantly inhibited ACTH-stimulated cortisol and dehydroepiandrosterone (DHEA) secretions. Binding studies of [125I]-pBNP to bovine adrenocortical membrane fractions showed that adrenal cortex had high-affinity and low-capacity pBNP binding sites, with a dissociation constant (Kd) of 1.70 x 10(-10) M and a maximal binding capacity (Bmax) of 19.9 fmol/mg protein. Finally, the 135 Kd radioactive band was specially visualized in the affinity labeling of bovine adrenal cortex with disuccinimidyl suberate (DSS). These results suggest that pBNP may have receptor-mediated suppressive actions on bovine adrenal steroidogenesis, similar to that in atrial natriuretic peptide (ANP).     

Relationship between endogenous cyclic AMP production and steroid hormone secretion in chick adrenal cells

Dispersed chick adrenal cells were incubated with either ACTH, cholera toxin or forskolin. All three agents stimulated cyclic AMP accumulation and secretion of corticosterone and aldosterone by the dispersed cells. The dose-response to ACTH was similar for cyclic AMP and corticosterone but aldosterone secretion appeared to be more sensitive to ACTH stimulation. Concentrations higher than 10(-8) M of ACTH caused suppression of corticosterone output but not of cyclic AMP accumulation or aldosterone secretion. A significant cyclic AMP accumulation occurred within 30 min of exposure to ACTH whereas significant increases in steroid secretion were observed only after 30 min. An early increase (within 30 min) in cyclic AMP accumulation with both cholera toxin and forskolin was not accompanied by any significant stimulation of steroid secretion, which occurred only after 120 min. The results with the avian adrenal cells are consistent with the thesis that steroid production in the adrenocortical cells is stimulated by cyclic AMP-dependent pathways, whereas steroid release may be modulated by others.