Role of cyclic AMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E1 and dibutyryl cyclic AMP in normal adrenal cells and adrenal tumor cells from humans.

The role of the cyclic AMP-protein kinase system in mediating the steroidogenic effect of ACTH, prostaglandin E1 and dibutyryl cyclic AMP, induced similar stimulations of protein kinase activity, cyclic AMP was studied using human adrenal cells isolated from normal and adrenocortical secreting tumors. At high concentrations of ACTH, complete activation of protein kinase of normal adrenal cells was observed within 3 min, at the time when cyclic AMP production was slightly increased and there was still no stimulation of steroidogenesis. At supramaximal concentrations, ACTH, PGE1 and dibutyryl cyclic AMP and cortisol productions in adrenal cells isolated from normal and from one adrenocortical tumor. In one tumor in which the adenylate cyclase activity was insensitive to ACTH, the hormone was unable to stimulate protein kinase or steroidogenesis, but the cells responded to both PGE1 and dibutyryl cyclic AMP. In another tumor in which the adenylate cyclase was insensitive to PGE1, this compound also did not increase protein kinase activity or steroidogenesis, but both parameters were stimulated by ACTH and dibutyryl cyclic AMP. After incubation of normal adrenal cells with increasing concentrations of ACTH (0.01-100 nM) marked differences were found between cyclic AMP formation and cortisol production. However at the lowest concentrations of ACTH exerting an effect on steroid production a close linked correlation was found between protein kinase activation and cortisol production, but half-maximal and maximal cortisol production occurs at lower concentration of ACTH than was necessary to induce the same stimulation of protein kinase. Similar findings were found after incubating the adrenal cells with dibutyryl cyclic AMP (0.01-10 mM). The results implicate an important role of the cyclic AMP-protein kinase system during activation of adrenal cell steroidogenesis by low concentrations of steroidogenic compounds.     

The effects of potassium, 5-hydroxytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3′:5′-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells

Dispersed rat adrenal cells prepared from both the capsule and the decapsulated gland were used to investigate the effects on cyclic AMP accumulation of known stimuli of steroidogenesis [ACTH (adrenocorticotrophin), angiotensin II, K(+) ions and 5-hydroxytryptamine]. Since glomerulosa-cell preparations from capsular strippings are normally contaminated with a proportion of fasciculata cells, cells purified by fractionation on a bovine serum albumin gradient were also used. The results showed that: (1) ACTH and angiotensin II stimulated cyclic AMP accumulation in both fractionated and unfractionated zona fasciculata cells; (2) 5-hydroxytryptamine and an increased extracellular K(+) concentration (from 3.6 to 8.4mm) had no effect on cyclic AMP concentrations in fasciculata cell preparations; (3) the addition of ACTH, angiotensin II, 5-hydroxytryptamine or K(+) to the incubation medium resulted in increased cyclic AMP concentrations in unpurified zona glomerulosa cell preparations; (4) fractionation and hence the virtual elimination of fasciculata contamination, did not affect the response to 5-hydroxytryptamine and increased K(+) concentration. However, the responses to ACTH and angiotensin II were markedly lowered but not abolished. These results strongly suggest a link between cyclic AMP production and steroidogenesis in the zone of the adrenal gland that specifically secretes aldosterone. All four agents used stimulated both steroid output and cyclic AMP accumulation. However, at certain doses of 5-hydroxytryptamine, K(+) and angiotensin II the significant increases in corticosterone output were not accompanied by measurable increases in cyclic AMP accumulation.     

Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity

The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.     

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.     

Effects of ACTH (corticotropin) analogues on steroidogenesis and cyclic AMP in rat adrenocortical cells. Evidence for two different steroidogenically responsive receptors.

Comparative studies on the mechanism of action of ACTH1-39 and ACTH5-24 [corticotropin-(1-39)- and corticotropin-(5-24)-peptides] on isolated rat adrenal cells were performed. The relationship between stimulated steroidogenesis and cyclic AMP was very different, suggesting that cyclic AMP does not play the same role in mediating the action of the two agonists. Data from three separate experiments showed that the competitive antagonist ACTH6-24 [corticotropin-(6-24)-peptide] had mean inhibitor constants of 13.4 +/- 3.1 nM against ACTH1-39 and 3.4 +/- 1.0 nM against ACTH5-24, indicating that the steroidogenic actions of these two agonists are mediated by two different receptor types. We conclude that there are two possible mechanisms for corticotropin action, only one of which involves the necessary production of cyclic AMP.     

A Dual Pathway for Acth Steroidogenic Action in Purified Adrenocortical Cells

Abstract

Isolated adrenal fasciculata cells were purified by centrifugation through a 0-50% hyperbolic gradient of Percoll^R. The dose-dependence and kinetics of both intracellular cyclic AMP accumulation and steroido-genesis in response to ACTH_1-39 and ACTH_5-24 (corticotropin-(1-39) and corticotropin-(5-24)-peptides) were determined using purified cells. The rate of intracellular cyclic AMP formation was maximal during the first five minutes after hormone addition and remained constant or fell thereafter. Therefore intracellular cyclic AMP accumulation, assessed after 5 min., was compared with steroid output after 20 min. Maximal steroidogenesis was elicited by ACTH^5-24 without discerning a significant stimulation of intracellular cyclic AMP accumulation. ACTH_6-24 (corticotropin-(6-24)-peptide) could completely inhibit the intracellular cyclic AMP accumulation elicited by ACTH_1-39 or by ACTH_5-24 at concentrations that only partially inhibited steroidogenesis.

It is possible that there are two pathways for the steroidogenic action of ACTH, one of which is obligatorily mediated by intracellular cyclic AMP, and another which involves a different mediator.     

On the mechanism of action of cholera toxin on isolated rat adrenocortical cells Comparison with the effects of adrenocorticotropin on steroidogenesis and cyclic AMP output

The effects of cholera toxin on isolated rat adrenocortical cells have been investigated. Both steroid and cyclic AMP output from adrenal cells were increased by the toxin in a dose dependent fashion. The concentration of toxin for half maximal stimulation for both of these responses was about 40 ng/ml. Maximal steroidogenesis and cyclic AMP output was obtained with similar concentrations of the toxin. A correlation was observed between the low amounts of cyclic AMP produced in response to all doses of cholera toxin and to physiologically significant concentrations of adrenocorticotropin (ACTH) (< 0.1 munit/ml; i.e. submaximal for steroidogenesis in this system). This was in direct contrast to the much higher levels of cyclic AMP generated by concentrations of ACTH greater than 1 munits/ml. Time course studies demonstrated a time-lag between toxin addition and steroid response of at least 40 min. Binding of cholera toxin to adrenal cells was rapid and was 90% complete within 15 min at both 37 and 0°C. These data indicate that most of the delay in response to cholera toxin is due to processes subsequent to the initial binding interaction. Following the initial delay the subsequent maximal rate of steroidogenesis brought about by cholera toxin was very similar to that obtained with a concentration of ACTH that was maximal for steroidogenesis. Significant increases in cyclic AMP levels were detected about 20 min before increased steroidogenesis was apparent. Possible explanations for this result are considered. The results presented indicate great potential use for cholera toxin in the study of adrenal steroidogenic control mechanisms, particularly at the level of receptor mechanisms and the role of cyclic AMP.     

Radioactive probes for adrenocorticotropic hormone receptors

Our attempts to develop adrenocorticotropic hormone (ACTH) analogues that can be employed for ACTH receptor identification and isolation began with the synthesis of ACTH fragments containing N epsilon-(dethiobiotinyl)lysine (dethiobiocytin) amide in position 25 to be used for affinity chromatographic purification of hormone-receptor complexes on Sepharose-immobilized avidin resins. Because labeling ACTH or ACTH fragments by conventional iodination techniques destroys biological activity due to oxidation of Met4 and incorporation of iodine into Tyr2, we have prepared [Phe2,Nle4]ACTH1-24, [Phe2,Nle4,biocytin25]ACTH1-25 amide, and [Phe2,Nle4,dethiobiocytin25]ACTH1-25 amide by conventional synthetic techniques. The HPLC profiles and amino acid analyses of the final products indicate that the materials are of a high degree of purity. The amount of tertiary butylation of the Trp residue in the peptides was assessed by NMR and was found to be less than 0.5%. All three peptides are equipotent with the standard ACTH1-24 as concerns their ability to stimulate steroidogenesis and cAMP formation in bovine adrenal cortical cells. Iodination of [Phe2,Nle4]ACTH1-24, with iodogen as the oxidizing agent, has been accomplished without any detectable loss of biological activity. The mono- and diiodo derivatives of [Phe2,Nle4]ACTH1-24 have been prepared, separated by HPLC, and assayed for biological activity. Both peptides have the full capacity to stimulate steroidogenesis and cAMP production in bovine adrenal cortical cells.     

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.     

Atrial natriuretic factor regulation of cyclic GMP levels and steroidogenesis in isolated fasciculata cells of rat adrenal cortex

Isolated fasciculata cells of rat adrenal cortex, when incubated with atrial natriuretic factor (ANF), stimulated the levels of cyclic GMP and corticosterone production in a concentration-dependent manner without a rise in the levels of cyclic AMP. The ANF-dependent elevation of cyclic GMP was rapid, with a detectable increment in 30 s. ANF also stimulated the particulate guanylate cyclase. These results not only indicate the coupling of cyclic GMP and corticosterone production with ANF signal, but also demonstrate that, like the ACTH signal, cyclic AMP is not the mediator of ANF-induced adrenocortical steroidogenesis.     

Lanthanum: inhibition of ACTH-stimulated cyclic AMP and corticosterone synthesis in isolated rat adrenocortical cells

Lanthanum (La+++) is a well-known Ca++ antagonist in a number of biological systems. It was used in the present study to examine the role of Ca++ in the regulation of adenyl cyclase of the adrenal cortex by ACTH. In micromolar concentrations, .La+++ inhibited both cyclic AMP and corticosterone response of isolated adrenal cortex cells to ACTH. However, a number of intracellular processes were not affected by La+++. These include the stimulation of steroidogenesis by dibutyryl cyclic AMP, conversion of several steroid precursors into corticosterone, and stimulation of the latter by glucose. Thus, inhibition of steroidogenesis by La+++ appears to be solely due to an inhibition of ACTH-stimulated cyclic AMP formation. Electron microscope examination showed that La+++ was localized on plasma membrane of the cells and did not appear to penetrate beyond this region. Since La+++ is believed to replace Ca++ at superficial binding sites on the cell membrane, it is proposed that Ca++ at these sites plays an important role in the regulation of adenyl cyclase by ACTH. Similarities in the role of Ca++ in "excitation-contraction" coupling and in the ACTH-adenyl cyclase system raise the possibility that a contractile protein may be involved in the regulation of adenyl cyclase by those hormones which are known to require Ca++ in the process.     

The effect of calcium concentration on ACTH stimulation of steroidogenesis in mouse adrenal tumor cells

Calcium is required for ACTH stimulated steroidogenesis in adrenal tumor cells in tissue culture. In the absence of calcium, the dose of ACTH required to induce half maximum steroidogenesis was increased 30 fold. In contrast to intact adrenal glands or isolated adrenal cells, high doses of ACTH (50 mU/ml) maximally stimulated steroidogenesis in the absence of calcium. Growth for up to six days in medium with low calcium did not affect basal or ACTH induced steroidogenesis. The addition of calcium to cells incubated with ACTH produced a maximum steroidogenic response in 15 minutes. In contrast to intact adrenal glands, calcium is not required for adenosine-3′,5′-cyclic monophosphate (cyclic AMP) stimulated steroidogenesis in adrenal tumor cells. These experiments support the concept that calcium is important at the level of ACTH-membrane receptor site interaction or activation of adenyl cyclase in adrenal tumor cells.     

Effect of dexamethasone on steroidogenesis and on adrenocortical cyclic AMP and cyclic GMP responses to ACTH

The inhibitory action of dexamethasone on the adrenal steroidogenic response to ACTH was confirmed by im administration of graded doses (5, 10 and 30 ng) of synthetic beta 1-24 ACTH to young adult male rats which had received dexamethasone (0.1 mg/100 g bw) 4 hr prior to sacrifice. Following this, kinetic studies were performed by measuring plasma corticosterone, adrenocortical cyclic AMP and cyclic GMP before and 4, 12 and 30 min after administration of either 10 or 30 ng of ACTH. These doses were selected because their effects could be either completely or partially inhibited by dexamethasone. In rats without dexamethasone all the doses of ACTH which were checked induced an increase in both corticosterone and cyclic AMP and a decrease in cyclic GMP. With the smallest dose of ACTH the earlier administration of dexamethasone resulted in complete suppression of both the steroidogenic response and the cyclic AMP response. With the largest dose of ACTH both responses were diminished. In dexamethasone-treated rats the decrease in cyclic GMP was significantly less pronounced 4 min after ACTH than it was in non-treated rats. These results support the view that cyclic AMP and cyclic GMP might both be concerned with the mechanism of acute adrenal steroidogenesis.     

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa of the adrenal cortex.

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa (the capsular fraction) from the rat adrenal cortex have been studied. Although high concentrations of extracellular calcium potentiated the stimulatory action of ACTH on cyclic AMP and aldosterone output, tetracaine or verapamil inhibited aldosterone output but not cyclic AMP production during ACTH-stimulation. Lanthanum reduced both aldosterone and cyclic AMP accumulation induced by ACTH. These results suggest that an extracellular calcium would be essential in stimulating the capsular steroidogenesis without involvement of the cyclic AMP system.     

Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis.

The steroidogenic response to ACTH prostaglandin E2 (PGE2) was studied in cat adrenocortical cells dispersed with trypsin. The dose-response curves of both agents were qualitatively and quantitatively similar. Exposure to PGE2 or ACTH in the presence of labeled steroid precursor (acetate) resulted in the accumulation of comparable levels of steroid intermediates and end-product. Submaximal or maximal concentrations of ACTH and PGE-2 given simultaneously elicited a response which was no greater than that obtained with either stimulant alone. Although calcium was required for optimal PGE-2 stimulation of steroid production, this requirement with ACTH as the stimulant, but greater than with butyryl cyclic AMP. PGE-2-induced increase in the adrenal cyclic AMP was not statistically significant and was small in relation to that found with equipotent steroidogenic ACTH concentrations. The possible relationship between prostaglandins, cyclic AMP, and calcium in the action of ACTH is discussed.     

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.     

Steroidogenic effect of exogenous phospholipase C on bovine adrenal fasciculata cells

Phospholipase C (Bacillus cereus) added to the incubation medium stimulated the steroidogenic activity of bovine adrenal zona fasciculata cell suspensions to a level similar to that induced by optimal concentration of ACTH. This effect was not related to an increase of cyclic AMP; it was calcium-dependent and was also induced by an other bacterial phospholipase C (from Clostridium perfringens) whereas phospholipases A2 and D were ineffective. Phospholipid metabolism was examined in these cells after radiolabeling with [14C]-glycerol or [32P]orthophosphate. Phospholipase C induced a very fast (5 seconds) increase in cellular [14C]-1,2-diacylglycerol followed by [32P] labeling of phosphatidic acid and phosphatidylinositol. These events preceded the stimulation of steroidogenesis which was detectable after 2 minutes of incubation. These observations suggest that activation of an endogenous phospholipase C activity may be considered as an early event in the response of bovine adrenocortical cells to steroidogenic effectors such as angiotensin II and acetylcholine.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells.

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2-3 fold increase at 15-30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E2 also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise in cyclic AMP. These results suggest that the relative amounts of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

Involvement of 5-lipoxygenase metabolites in ACTH-stimulated corticosteroidogenesis in rat adrenal glands

Various lipoxygenase (LO) products of arachidonic acid (AA) have been found to have potent biological activities and modulate physiological processes in various cells including endocrine cells. However, no studies concerning LO products in adrenocortical cells have been reported. The present study was performed to investigate LO products in rat adrenocortical cells and its role in ACTH-stimulated adrenal steroidogenesis. LO metabolites produced in ACTH-stimulated rat adrenocortical cells prelabeled with [3H]AA was analyzed by reverse phase and straight phase HPLC and two 5-LO products, 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) were identified. ACTH-induced 5-HETE and LTB4 production in adrenal cells was dose dependently inhibited by AA861, a specific inhibitor of 5-LO. AA861 reduced ACTH-stimulated corticosteroid production without any change in cyclic AMP formation, while indomethacin did not affect both corticosteroid and cyclic AMP production. Reduced steroidogenesis by AA861 was reversed by the addition of 5-hydroperoxyeicosatetraenoic acid (5-HPETE). Also exogenously added 5-HPETE dose dependently augmented ACTH-stimulated corticosteroid production without any concomitant change in cyclic AMP production. However, 5-HETE and LTB4 had no such effect. These results indicate that 5-LO pathway is present in rat adrenocortical cells and its metabolites, most likely 5-HPETE, may play an important role in adrenal steroidogenesis.