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.     

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.     

Exogenous steroids alter steroidogenesis in cultured Y-1 adrenal tumor cells by actions preceding cyclic AMP

Using cultured Y-1 mouse adrenal tumor cells which produce 20 alpha-hydroxy-4-pregnen-3-one (20-DHP), it was found that 0.01 mM corticosterone and deoxycorticosterone increased basal and inhibited ACTH-induced 20-DHP production during consecutive 30 and 120 min incubations. Steroid effects were concentration-dependent and reversible. Six other steroids tested did not stimulate 20-DHP production and varied in ability to inhibit ACTH-stimulated steroidogenesis. Experiments demonstrated that 20-DHP production following treatment with cholera toxin, N,0'-dibutyryl cyclic AMP (dbcAMP), or pregnenolone was not inhibited by exogenous steroids. Corticosterone (0.01 mM) increased basal and inhibited ACTH-induced intracellular cyclic AMP (cAMP) production. Cytochalasin D, a microfilament perturbing agent, inhibited steroid-stimulated 20-DHP production, suggesting that ACTH and steroid stimulation mechanisms were similar. These findings taken together suggest that exogenous steroids can alter steroidogenesis by modifying plasma membrane adenylate cyclase activity.     

Differential response to adrenocorticotropic hormone analogs of bovine adrenal plasma membranes and cells.

The ability of three analogs of ACTH1-24 ([Gln5, Phe9] ACTH1-24, [Gln5, Ala9[Acth1-24, and [Gln5, Lys8, Phe9[ ACTH1-24) embodying tryptophan substitutions to activate the adenylate cyclase system of a bovine adrenal plasma membrane preparation was compared to the effect of the analogs on adenosine 3':5'-monophosphate (cyclic AMP) accumulation and steroidogenesis in viable bovine adrenocortical cells. The results were not comparable. Whereas the analogs antagonized the ACTH1-24-activated membrane cyclase they stimulated cyclic AMP accumulation as well as steroid production of the cells. None of the analogs inhibited steroidogenesis of ACTH1-24-stimulated cells, but two of them, at very high dose levels, inhibited cyclic AMP production. The ability of the analogs to stimulate steroidogenesis of the adrenal cells half-maximally decreased in the order tryptophan greater than phenylalanine greater than alanine, indicating that the aromaticity of the indole ring of tryptophan is necessary for maximal interaction between hormone and receptor. Both the absolute and relative steroidogenic potencies were the same for several analogs when assayed with rat adrenal cells. Although only a small fraction of the cell's potential to produce cyclic AMP was necessary to induce maximum steroid production, the relative activities of a series of analogs were the same for steroidogenesis as for cyclic AMP accumulation. Furthermore, the concentration of cyclic AMP necessary for full steroidogenesis was practically identical for a series of peptides that differed widely in potency. These findings support the postulate that cyclic AMP accumulation and steroidogenesis in adrenocortical cells are coupled processes. The differential behavior of bovine adrenal plasma membranes and bovine adrenocortical cells toward ACTH analogs indicates that structure-function studies using cyclase assays may not reflect events that take place in the intact adrenal or in cell preparations derived therefrom.     

Measurement of the dynamics of stimulation and inhibition of steroidogenesis in isolated rat adrenal cells by using column perfusion

Isolated adrenal cells were perfused in a small column by using Bio-Gel polyacrylamide beads as an inert supporting matrix, and the time-course of the response to various stimuli was observed by measuring fluorogenic 11-hydroxycorticosteroids in the effluent. A small but significant response was observed 1 min after stimulation with physiological concentrations of ACTH (adrenocorticotrophin), but the response did not start to build up rapidly for 3-4min and eventually reached a plateau after 9-10min. A similar pattern of events was observed for the decay of the steroid output on removal of ACTH. ACTH analogues, including one with a long duration of action in vivo, were found to produce responses with similar kinetics. However, cyclic AMP caused a more rapid increase in steroidogenesis and its effects were more short-lived after withdrawal. If, as present evidence suggests, cyclic AMP is produced rapidly after ACTH stimulation the delayed build-up of the steroidogenic response to ACTH would indicate that cyclic AMP may not be the intracellular mediator. When inhibitors were applied during ACTH stimulation, aminoglutethimide, which blocks mitochondrial conversion of cholesterol into pregnenolone (3beta-hydroxypregn-5-en-20-one), caused a rapid fall in steroid output (1 min), whereas cycloheximide took longer to achieve its full effect. Nevertheless, the response had fallen by 50% in 2 min, indicating a much shorter half-life than that previously reported for the labile protein implicated in steroidogenesis. In addition the rapid response to cyclic AMP makes it unlikely that steroid production is induced as a result of initiation of protein synthesis. This suggests that the labile protein plays an obligatory but permissive role in the development of the response. Column perfusion has proved to be a simple technique which can readily yield accurate data on responses of cells to stimulants and inhibitors.     

Forskolin potentiates adrenocorticotropin-induced cyclic AMP production and steroidogenesis in isolated rat adrenal cells

Forskolin, a unique diterpene which directly activates the adenylate cyclase, stimulated production of both cyclic AMP and corticosterone in isolated rat adrenal cells, in vitro. This agent also potentiated the action of adrenocorticotropin and/or cholera toxin on cyclic AMP production and steroidogenesis at lower concentrations. It augmented both an early (cyclic AMP production) and a late (steroidogenesis) action of the hormone in the adrenal gland.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Inhibitory effect of ibuprofen on the cholera toxin-induced cyclic AMP response in Chinese hamster ovary cells

Abstract Ibuprofen, an inhibitor of prostaglandin synthesis in eukaryotic cells, was shown to inhibit the accumulation of 3',5'-cyclic adenosine monophosphate (cyclic AMP) in Chinese hamster ovary (CHO) cells exposed to cholera toxin. The inhibition was dose dependent, with a dose of 100 μg/ml reducing the cholera toxin response by approximately 50%, and maximal inhibition was observed when the drug was applied to the cells simulataneously with or 1 h before the toxin. Although ibuprofen also inhibited adenylate cyclase stimulation by forskolin, suggesting a nonspecific effect, the drug had no effect on cholera toxin-induced cyclic AMP accumulation when added to the culture medium 15 min or more after the toxin.     

Adrenocortical responses to adrenocorticotrophin in the rat

The time course of plasma adrenocorticotrophin (ACTH), adrenal cyclic AMP, adrenal corticosterone, and plasma corticosterone was measured in male Sprague-Dawley rats whose endogenous release of ACTH had been blocked (1) following rapid injections of 100 and 300 ng ACTH/100 g body weight, i.v., (2) during prolonged infusions at rates of 1, 2, and 4 ng ACTH/min per 100 g body weight, and (3) after termination of 30-min infusions at rates extending from 0.06 to 8 ng ACTH/min per 100 g body weight. Following injections, the time course of the variables is similar to the one simulated from our models of adrenal cortical secretion, including the simulation of an intermediate variable of our models of the adrenal cortex cell which was presumed to correspond to cyclic AMP. However, during prolonged infusions there is an unexpected overshoot of adrenal cyclic AMP content whereas adrenal and plasma corticosterone concentrations rise to a steady-state value without overshoot. The total amount of cyclic AMP gradually increases following the three increasing infusion rates of ACTH whereas similar levels of plasma corticosterone concentrations are reached at steady state; therefore the saturation of the adrenal cortical secretion is due to a step ulterior to cyclic AMP formation in the steroidogenesis. After 30-min infusions, plasma corticosterone concentration reaches its maximal value following a rate of ACTH input which evokes only a 4-fold increase in adrenal cyclic AMP content; however, there is a 250-fold increase of adrenal cyclic AMP with respect to control value following the higher rates of infusion of ACTH.     

Action of cholera toxin on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of ⁴⁵Ca or cellular cyclic AMP. Binding of ¹²⁵I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of bindind of ¹²⁵I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in ⁴⁵Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretion or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

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.     

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.     

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.     

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.     

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.     

Comparison of the Ca ++ requirement for the steroidogenic effect of ACTH acid dibutyryl cyclic AMP in rat adrenal cell suspension

Calcium requirement for ACTH and Dibutyryl cyclic AMP (DBCAMP) stimulation of steroidogenesis was compared in rat adrenal cell suspensions. In the absence of added calcium ACTH at low concentrations (< 1 mU/ml) was ineffective; however, the calcium requirement decreased when higher concentrations of ACTH were used. This was not the case with DBCAMP. At all levels of the nucleotide tested, the Ca⁺⁺ requirement was about the same. When the cells were preincubated with EGTA, the Ca⁺⁺ requirement became more pronounced for ACTH than for DBCAMP. The results indicate that the events before the formation of cyclic AMP show a greater dependence on Ca⁺⁺ than the events following its formation.     

Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis

The steroidogenic response to ACTH and 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 PGE₂ given simultaneously elicited a response which was no greater than that obtained with either stimulant alone. Although calcium was required for optimal PGE₂ stimulation of steroid production, this requirement for calcium was less than the requirement with ACTH as the stimulant, but greater than with butyryl cyclic AMP. PGE₂-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.     

Induction of refractoriness to isoproterenol by prior treatment of C6-2B rat astrocytoma cells with cholera toxin.

Rat C6-2B astrocytoma cells responded to cholera toxin treatment with an 8-fold increase in intracellular cyclic AMP concentrations. Cyclic AMP levels began to rise 60--90 minutes after addition of the toxin and reached maximal concentrations in 3 hours. Cells exposed to cholera toxin and the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), displayed an increase in cyclic AMP of 15-fold. The peak isoproterenol response was reduced 80--90% in cells previously treated with cholera toxin. Cholera toxin-induced refractoriness was time dependent and was not altered by concurrent treatment with propranolol. Prolonged exposure of the cells to isoproterenol reduced the cyclic AMP response to cholera toxin by 80%. MIX augmented both cholera toxin-induced refractoriness and isoproterenol-induced refractoriness. Cycloheximide inhibited the full development of refractoriness to both cholera toxin and isoproterenol. These results indicate that C6-2B cell refractoriness to cholera toxin is mediated by cyclic AMP and requires new protein synthesis. Refractoriness in C6-2B cells does not appear to be agonist-specific and probably involves a common locus of action on adenylate cyclase beyond that of the membrane receptors for cholera toxin and isoproterenol.     

An investigation of the involvement of adenosine 3'':5''-cyclic monophosphate in steroidogenesis by using isolated adrenal cell column perfusion.

The involvement of cyclic AMP in corticosteroidogenesis was investigated by using isolated adrenal cell column perfusion. Steroids were produced in response to 0.5, 1.0 and 5.0 mg of cyclic AMP/ml. Analysis of the shape of the response curves indicated an inverse relationship between rate of onset of steroid production and dose. A further increase in steroid production during the washout period after the 5 mg/ml dose was considered to indicate an intracellular inhibitory effect of cyclic AMP. Release of cyclic AMP into the perfusate only occurred in response to supramaximal steroidogenic doses of ACTH (adrenocorticotrophin). A connexion between dose and response was demonstrated over a narrow concentration range. Variation in the time-lag before cyclic AMP production and in the duration of the response was marked; further, no reproducible ratio of steroid output to cyclic AMP output was shown at any level of stimulation. These results are discussed together with those of other recent investigations. It is considered that these findings do not support an obligatory role for cyclic AMP as mediator of ACTH action in the adrenal.     

The effects of corticotrophin (ACTH1-24), cyclic AMP and TPA (12-O-tetradecanoyl phorbol-13-acetate) on DNA replication and proliferation of primary rabbit adrenocortical cells in a synthetic medium

ACTH1-24 stimulated the parenchymal cells in cultures of rat adrenal cortex in serum-free synthetic HiWoBa 2000 medium to replicate DNA, enter mitosis and divide. But ACTH's principal mediator, cyclic AMP, was not a complete mitogen: the adenylate cyclase-stimulating cholera toxin and dibutyryl cyclic AMP stimulated parenchymal cells to replicate DNA but not to enter mitosis. Thus, there must have been an additional mediator of the response to ACTH1-24 that enabled the parenchymal cells to enter mitosis. This additional mediator might have been protein kinase C because a protein kinase C activator and cyclic AMP elevator, TPA, stimulated the adrenocortical parenchymal cells to replicate DNA, enter mitosis and divide.