Effect of calcium on steroidogenesis in isolated human adrenal cells (author's transl)]

Isolated adrenal cells were obtained surgically from patients with primary aldosteronism, breast cancer, or Cushing's syndrome. They were prepared by the modification of Sayers method, and incubated at 37 degrees C for 2 hours under 95% O-2-5% CO-2, in the medium of calcium-free Krebs-Ringer bicarbonate buffer containing 0.2% glucose and 0.5% bovine serum albumin, to which various doses of calcium, ACTH, dibutyryl cyclic AMP or cycloheximide were added. Steroid production was measured by the method of Silber et al. In isolated normal adrenocortical cells, 11-OHCS was produced by calcium alone in the absence of ACTH or dibutyryl cyclic AMP, while it was not produced by ACTH alone without calcium. 11-OHCS production by ACTH was decreased in the high concentration of calcium (10.16 mM, 12.70 mM). Cycloheximide partially blocked an increase in 11-OHCS synthesis induced by calcium. These data suggest that adenyl cyclase of human adrenocortical cells may be stimulated by calcium alone, supporting the notion that calcium is a second messenger. The ratio of 11-OHCS production by calcium alone to that by dibutyryl cyclic AMP was higher in adenoma cells than in normal cells. This may account for the character of autonomic steroid production in adrenocortical adenoma cells.     

Effects of ACTH and its O-nitrophenyl sulphenyl derivative on adrenocortical function in vivo.

Both ACTH and NPS-ACTH in which the single tryptophan residue of the hormone is modified were able to stimulate adrenal corticosterone concentration to the same extent in hypophysectomized rats, although a higher dose of NPS-ACTH was required. ACTH stimulated adrenal cyclic AMP levels 120-fold in hypophysectomized rats whereas NPS-ACTH caused a marginal increase. In the case of ACTH, low doses of the hormone capable of producing maximal stimulation of corticosterone synthesis did not produce any detectable change in cyclic AMP concentration. The rates of secretion of corticosterone induced by ACTH and NPS-ACTH in vivo were the same. NPS-ACTH was found to be 1.2% as potent as ACTH. The role of cyclic AMP in adrenal repair was investigated by administering equipotent doses of ACTH or NPS-ACTH to hypophysectomized rats. In adult rats both failed to produce a significant increase in adrenal weight. Adrenal function (measured by responsiveness to exogenous ACTH in vitro) was restored by NPS-ACTH but not to the same degree as ACTH. In hypophysectomized weanling rats, ACTH produced a small but significant increase in adrenal weight but NPS-ACTH did not. These results suggest that an increase in adrenal cyclic AMP may not be obligatory for the stimulation of steroidogenesis by ACTH and that some of the trophic actions of the hormone may be mediated by cyclic AMP.     

Studies on the mechanism of corticotrophin-mediated desensitization of corticosterone secretion by rat adrenocortical cells

Long-term exposure of the adrenocortical cells in vivo or in vitro to high concentrations of ACTH results in a diminution of the responsiveness of these cells to a subsequent stimulation of corticosterone release by ACTH. Conflicting studies have been published on the mechanism of this 'desensitization' phenomenon. Dispersed adrenocortical cells prepared from the hypertrophic/hyperplastic adrenal glands of rats bearing the ACTH/PRL-secreting rat pituitary tumour 7315a showed an increased basal release of corticosterone, but had lost their ability to respond further to ACTH. However, corticosterone release in response to dibutyryl cyclic AMP (dbcAMP), cholera toxin and forskolin remained intact. Pretreatment of normal rats for 3, 9 and 21 days with 50 micrograms/rat/day of a long-acting ACTH depot preparation induced a dose-dependent increase in basal corticosterone release by the adrenocortical cells prepared from these animals and a dose-dependent decrease in the sensitivity to ACTH. However, the responsiveness of the adrenocortical cells prepared from the adrenal glands of control and ACTH pretreatment rats to dbcAMP, cholera toxin and forskolin was similar. In addition, pretreatment with ACTH in vivo did not affect the sensitivity of the adrenocortical cells in vitro to calmodulin inhibition by haloperidol and 11 beta-hydroxylase inhibition by etomidate. It is concluded that long-term exposure of the adrenal gland to high concentrations of ACTH in vitro results in an excessive activation of corticosterone release by the adrenocortical cells in vitro, which is accompanied by a loss of sensitivity to ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of corticotropin receptors.

A photoaffinity label for corticotropin (ACTH) receptors was prepared by selective chemical modification of the single tryptophan residue in the hormone by reaction with 2-nitro-5-azidophenylsulfenyl chloride. The photoreactive derivative, [(2-nitro-5-azidophenylsulfenyl)-Trp9]ACTH (2,5-NAPS-ACTH), stimulated corticosterone synthesis to 60% of the maximal rate induced by ACTH in isolated rat adrenocortical cells. 2.5-NAPS-ACTH caused only a marginal stimulation of cyclic AMP production compared to the unmodified hormone. Stimulation of corticosterone production and cyclic AMP accumulation induced by ACTH were both inhibited in a competitive manner by 2,5-NAPS-ACTH. Photolysis of adrenocortical cells in the presence of 2,5-NAPS-ACTH resulted in a 40% inactivation of ACTH receptors mediating steroidogenesis, as shown by the decrease in response to subsequent stimulation with ACTH. No loss of function was observed when photolysis was conducted in the presence of the photoresistant analog [(2,4-dinitrophenylsulfenyl)-Trp9]ACTH. Covalent attachment of the hormone to the receptors was also demonstrated by photolyzing adrenocortical cells in the presence of tritiated 2,5-NAPS-ACTH of high specific radioactivity (90 Ci/mmol) and analyzing the cell proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A protein with an approximate molecular weight of 100,000 was specifically labeled by this procedure. The unique labeling of an adrenocortical cell protein and the concomitant loss of ACTH responsiveness suggest that physiologically relevant receptors are photolabeled by this method.     

Inhibition of Replication in Functional Mouse Adrenal Tumor Cells by Adrenocorticotropic Hormone Mediated by Adenosine 3′:5′-Cyclic Monophosphate

Adrenocorticotropic hormone (ACTH) inhibited replication in functional adrenal tumor cells with a concomitant stimulation of steroidogenesis and a characteristic change of morphology from a flattened to a spherical type. [(3)H]Thymidine incorporation into DNA was inhibited by about 50% 6 hours after ACTH treatment. Both cyclic AMP and dibutyryl cyclic AMP inhibited [(3)H]thymidine incorporation and caused the characteristic morphological change noted with ACTH. The extent of stimulation of steroidogenesis and the amount of inhibition of [(3)H]thymidine incorporation in response to various doses of ACTH were closely related and both were in parallel with the concentration of cyclic AMP in the cells. Cyclic GMP and cyclic IMP did not inhibit [(3)H]thymidine incorporation significantly, and did not change the morphology of the cells. AMP inhibited [(3)H]thymidine incorporation into DNA and caused the characteristic morphological change. However, AMP did not increase the cyclic AMP content of the cells. CMP, GMP, and UMP showed a significant inhibition of [(3)H]thymidine incorporation into DNA, but the extent of the inhibition was much less than that with AMP. These nucleotides did not change the morphology of the cells.     

On the role of protein synthesis in the response of adrenal tumor cells to ACTH.

Y-1 adrenal tumor cells were incubated with aminoglutethimide with and without ACTH. Greater production of pregnenolone from endogenous cholesterol was observed (after washing to remove aminoglutethimide) in mitochondria from cells incubated with aminoglutethimide and ACTH than in those from cells incubated with aminoglutethimide alone. This response was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. ACTH increased the incorporation of [3H]tyrosine into protein associated with mitochondria but not into total cell protein or protein of postmitochondrial supernatant. This response did not require aminoglutethemide block and was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. Dibutyryl cyclic AMP produced both of these responses (increased production of pregnenolone and synthesis of protein associated with mitochondria). The concentration of cycloheximide required to cause 50% inhibition of the responses to ACTH and dibutyryl cyclic AMP was approximately the same for steroidogenesis by whole cells, for production of pregnenolone by isolated mitochondria, for incorporation of [3H]tyrosine into Y-1 cell protein and for the increase in synthesis of protein associated with mitochondria produced by ACTH (0.08--0.2 microgram/ml). Disc gel electrophoresis revealed that the increased incorporation of [3H]tyrosine involved two proteins corresponding to molecular weight of approximately 27,000 and 13,000 respectively. These observations suggest that ACTH promotes synthesis of protein(s) by cytoplasmic ribosomes on stable messenger RNA, that the protein(s) becomes associated with mitochondria and that the protein(s) includes one or more which are associated with the increase in production of pregnenolone produced in mitochondria by the addition of ACTH to adrenal cells.     

Further studies on the mechanisms controlling prostaglandin biosynthesis in the cat adrenal cortex: the role of calcium and cyclic AMP.

In light of previous studies which have implicated prostaglandin (PG) formation as a link in ACTH-induced steroid production by isolated cat adrenocortical cells, experiments were carried out to provide additional information regarding the role of PGs in adrenal steroidogenesis and their interactions with calcium and cyclic AMP. Perfusion of cat adrenal glands with Locke's solution plus beta(1-24)-ACTH resulted in an immediate increase in PGF2alpha release, which rapidly declined to basal levels after the stimulus was withdrawn. In contrast, maximal rates of steroid release were manifest some 30 min after removal of ACTH. ACTH and its onitrophenyl sulfenyl derivative (NPS-ACTH) increased PG (PGF2alpha and PGE2) and steroid release by trypsin-dispersed cat cortical cells, but NPS-ACTH, unlike ACTH, did not augment cortical cyclic AMP levels. In this same preparation, indomethacin completely blocked ACTH and NPS-ACTH facilitated PGF2alpha and PGE2 release but failed to suppress steroid release markedly. Calcium-deprivation blocked PG and steroid release evoked by these two polypeptides, and depressed PG release elicited by monobutyryl cyclic AMP (bcAMP) without affecting steroid release. These experiments offer additional evidence to support the concept that PGs play a role in the mode of action of ACTH; however, they do not appear to be obligatory intermediates in the steroidogenic process. The importance of calcium in regulating PG formation is discussed with special regard for the idea that this cation has a direct action on the enzyme systems which control PG synthesis.     

Response of adrenal cells to parathyroid hormone stimulation

Dispersed adrenal cells of avian and bovine origin were incubated with human (h) 1-34 parathyroid hormone (PTH(1-34)), bovine (b) PTH(1-84), bPTH(3-34), avian (a) PTH, and with ACTH. Kidney tubular cells, the established target cells for PTH, were used for comparison. Cyclic AMP (cAMP) accumulation and steroid hormone (aldosterone and corticosterone) secretion were measured in response to the hormones. In the bovine adrenocortical cells PTH of both bovine and avian origin stimulated cAMP production, but the aPTH action was more pronounced. The maximal cAMP response of avian adrenocortical cells to aPTH was 15-fold greater than that of ACTH, but a 20-times higher concentration of aPTH was required to reach half-maximal response. Avian PTH stimulated steroid hormone secretion in the chick adrenocortical cells, but the induced secretion was similar to that induced by ACTH, despite the difference in cAMP accumulation. Human PTH(1-34) and bPTH(1-84), which stimulated cAMP production in kidney cells, and the conventional antagonist bPTH(3-34) inhibited aPTH stimulation of cAMP accumulation in avian adrenocortical cells, but did not interfere with ACTH action. Furthermore, cAMP stimulation by aPTH and ACTH in avian adrenal cells was additive. The results establish the adrenal as a target organ for PTH, and suggest that the PTH acts through specific receptors, distinct from those for ACTH.     

Stimulation of chick adrenal steroidogenesis by avian parathyroid hormone

Dispersed chick adrenocortical cells were incubated with avian parathyroid hormone (aPTH) or ACTH. Accumulation of cyclic AMP (cAMP), activity of cAMP-dependent protein kinase and the secretion of corticosterone and aldosterone, in response to these hormones, were measured. Accumulation of cAMP and activity of cAMP-dependent protein kinase were stimulated by both aPTH and ACTH as well as by cholera toxin. Cyclic AMP production followed a similar time-course when stimulated by either peptide hormone. Stimulation of steroid hormone secretion was detectable after 20 min of incubation with ACTH, but only after 40 min with aPTH. The maximal steroid hormone secretion by adrenocortical cells was similar when induced by either peptide hormone. The aPTH concentrations needed for half-maximal response of corticosterone and aldosterone secretion were higher than those for ACTH (2.5- and 2-fold respectively), but still within the physiological range. The 11 beta-hydroxylase inhibitor metyrapone inhibited the secretion of both corticosterone and aldosterone when induced by either aPTH or ACTH. The results suggest that aPTH is almost as potent as ACTH in stimulating the secretion of corticosterone and aldosterone from chick adrenocortical cells and utilizes a cAMP-dependent pathway similar to that of ACTH.     

Regulation of the fetal human adrenal cortex: effects of adrenocorticotropin on growth and function of monolayer cultures of fetal and definitive zone cells

Monolayer cultures have been prepared from both definitive and fetal zones of the human fetal adrenal cortex. Cultures from each zone consist predominately of adrenocortical cells, as determined by a specific morphological retraction response to ACTH, and by ACTH-induced inhibition of DNA synthesis and cell proliferation. Cell growth was stimulated by fibroblast growth factor. ACTH stimulated steroidogenesis in cells from each zone with an ED50 of 0.4--1.0 nM and at a maximal concentration of 5 nM. Short term stimulation of less than 24 h with ACTH produced a pattern of steroid secretion that was characteristic of the zone of origin. Definitive zone cultures produced both cortisol and dehydroepiandrosterone plus its sulfate (DHA/S), with cortisol production exceeding DHA/S production. Fetal zone cultures produced more DHA/S than cortisol. 3 beta-Hydroxysteroid dehydrogenase, delta 4,5-isomerase enzyme activity was 3-fold less in fetal than in definitive zone cultures. Long term stimulation of 1--4 days with ACTH, 8-bromo-cAMP, or cholera toxin increased steroidogenic capacity in cultures from both zones. The pattern of steroid production by definitive zone cells remained constant, but cortisol production was preferentially increased in fetal zone cells. Forty-eight-hour treatment of fetal zone cells with ACTH increased 3 beta-hydroxysteroid dehydrogenase, delta 4,5-isomerase activity 5-fold. alpha-, beta-, gamma 1-, gamma 2-, and gamma 3-MSH were not effective steroidogenic agents for either zone. These studies indicate that steroidogenic agents induce in fetal zone cells steroid production characteristic of definitive and adult adrenocortical cells.     

Characterization of adult bovine adrenocortical cells throughout their life span in tissue culture.

The characteristics of adult bovine adrenocortical cells were studied throughout their life span of 55-65 generations in monolayer culture. Over this period, the cells maintained the capacity to synthesize steroids when tested with repeated maximal doses of ACTH, prostaglandin E1, monobutyryl cAMP, or cholera toxin. Prostaglandin E1 stimulated cAMP production and steroidogenesis, and inhibited DNA synthesis, as measured by incorporation of [3H]thymidine, with dose-response characteristics that did not vary over the first 50 generations in culture. In contrast, the maximal rate of cAMP production stimulated by ACTH declined exponentially at a rate of 7% per generation. In primary and secondary cultures, ACTH stimulated steroidogenesis maximally and inhibited [3H]thymidine incorporation into DNA completely at a half-maximal effective concentration (ED50) of 0.08 nM which was two orders of magnitude less than the ED50 of 8 nM for stimulation of cAMP production. As the ACTH-stimulated maximal rate of cAMP production fell with increasing generation number, the ED50 for ACTH stimulation of steroidogenesis and inhibition of DNA synthesis increased. From about the 20th generation onward, the ability of ACTH to inhibit DNA synthesis maximally declined so that by the 40th generation, cells were completely resistant to the growth-inhibitory effects of ACTH. High-dose ACTH continued, however, to stimulate steroid production maximally over the 50 generations studied. In late passage cells, the ED50 for ACTH stimulation of steroidogenesis was 8 nM, identical to that for cAMP production. Although ACTH-stimulated cAMP production was related to both stimulation of steroidogenesis and inhibition of DNA synthesis, higher cAMP levels appeared required for inhibition of DNA synthesis than for stimulation of steroidogenesis. Mitogenic responses to fibroblast growth factor and to angiotensin II were retained throughout long term growth in culture. The progressive loss of ACTH-responsiveness was specific and a function of aging of bovine adrenocortical cells in culture.     

Studies on the dynamics of adrenocortical responses to ACTH in the rat

The transient dynamics of plasma ACTH, adrenal cyclic AMP, adrenal corticosterone and plasma corticosterone were evaluated in male Sprague-Dawley rats, whose endogenous release of ACTH had been blocked by dexamethasone: (1) 40 min after single injections of ACTH ranging from 2 to 300 ng ACTH/100 g B.W., i.V.; (2) at time intervals after single injections of 9, 37 and 300 ng ACTH/100 g B.W.; (3) during and after prolonged infusion of 4 ng ACTH/min/100 g B.W. Plasma corticosterone concentration was still at a nearly maximal level 40 min after the injection of ACTH at a dose level for which the adrenal cyclic AMP content had fallen back to a value that was scarcely above the control one; a narrow window, defined by a 2-fold increase in the dose of ACTH, represents the transition between a minimal and a maximal adrenal cyclic AMP content. The adrenal cyclic AMP transient response after injection of graded doses of ACTH increased rapidly to a peak whose amplitude was dose-dependent; the duration of the cyclic nucleotide response, however, appeared to be independent of the ACTH dose level. The adrenal corticosterone content rose rapidly, and the eventual fall was delayed by increasing doses of ACTH. The time course of the early plasma corticosterone concentrations exhibited a similar rate of increase after any dose of ACTH; in any case, a steady state whose duration was dose-dependent was eventually reached and the ensuing fall therefrom occurred at a time when the adrenal cyclic AMP had fallen to very low levels. The adrenal cyclic AMP content showed an overshoot at a time when ACTH and corticosterone had reached a constant steady state, during a prolonged infusion of ACTH; adrenal cyclic AMP stabilized during the later phase of the infusion. After removal of the infusion, plasma ACTH levels fell relatively slowly as compared with adrenal cyclic AMP, whereas corticosterone remained at a maximal level for at least 120 min.Our results, derived from experiments in vivo, support the recent proposal by Bristow et al. (1980), derived from studies in vitro, that ACTH can act via either of two types of receptor: binding to one receptor elicits steroidogenesis via cyclic AMP production whereas binding to the other receptor elicits steroidogenesis through some other mechanism.     

Steroidogenesis in isolated adrenal cells of rat. -Effects of cyanoketone on pregnenolone and corticosterone production- (author's transl)]

In order to study the effect of cyanoketone on steroidogenesis of rat adrenal, the assay technique for corticosteroids released into the incubated media of the rat adrenal cells treated with collagenase was basically investigated. Corticosterone was measured by fluorometric method and pregnenolone by radioimmunoassay. Reliability of radioimmunoassay was satisfactory. About 400,000 cells were obtained from one adrenal gland of male or female rats and sex-dependent difference in pregnenolone and corticosterone production in response to ACTH was not found. Net corticosterone production by isolated adrenal cells was related to the log of the concentration of ACTH by a sigmoid curve over the range 1 to 1000 muU/ml. The half-maximum response was observed at an ACTH concentration of 10 muU/ml, and maximum corticosterone production responding to ACTH (100-1000 muU/ml) was about 5 mug/adrenal/120 min. When cell suspensions were incubated with 1000 muU/ml of ACTH, the conversion from pregnenolone to corticosterone was inhibited 50% by cyanoketone at a concentration of 2 times 10(-8) M. The conversion was completely inhibited at a concentration of more than 10(-7) M. Cyanoketone up to a concentration of 10(-5) M seemed to have no inhibitory effect on cholesterol side-chain cleavage. In the absence of ACTH significant amount of pregnenolone was formed (about 60 ng/adrenal) by isolated adrenal cells obtained from normal adult female rats during incubation with 10(-7) M of cyanoketone for 60 min. To eliminate the possibility of the effect of endogenous ACTH which might be present in incubation medium, cell suspensions were obtained from hypophysectomized female rats. Incubations were carried out in the same condition as mentioned above and significant amount of pregnenolone was formed by cell suspension, which was about 35 ng/adrenal.     

Protein kinase C in adrenal cells: possible role in regulation of steroid synthesis

Y-1 adrenal tumor cells and rat fasciculata cells were shown to possess an enzyme with the properties of protein kinase C. Activity was stimulated by Ca2+ and phospholipid (specifically phosphatidylserine). Enzyme activity was stimulated by addition of phorbol ester to a cell homogenate (ED50 10 nM) and inhibited by trifluoperazine (ID50 10 microM). ACTH and cyclic AMP added to Y-1 cells increased the activity of protein kinase C. Dose-response curves with ACTH showed that the hormone was effective in stimulating protein kinase C at lower concentrations than those required to increase steroid synthesis. When phorbol ester was added to Y-1 cells, total kinase C activity was diminished. Neither phorbol ester nor ACTH causes redistribution of protein kinase C between membranes and cytosol. Phorbol ester also stimulates steroid production by Y-1 cells. Protein kinase C phosphorylates 5 proteins in Y-1 cells (67, 61, 32, 16 and less than 14.4 kDa). Puromycin and cycloheximide increase the activity of protein kinase C in adrenal cells. It is concluded that protein kinase C may play an ancillary role in regulation of adrenal steroid synthesis but does not mediate the classical steroidogenic response that results from activation of adenylate cyclase by ACTH.     

Studies on the responsiveness of human adrenocortical tumors to ACTH the clinical and experimental observations (author's transl)]

Six cases of Cushing's syndrome with adrenocortical tumors which showed a variety of responsiveness to ACTH were investigated in relation to their clinical pictures and laboratory findings. Abnormal responses to ACTH in tumors was analyzed by in vitro experiments with surgically obtained tumor tissues, and the ACTH responsive mechanism of the tumors was discussed. An 8 hour intravenous ACTH infusion test showed that three of these patients were ACTH responsive, and the other three unresponsive. Histological observation of the tumors revealed that ACTH responsive tumors were adenomas and that ACTH unresponsive tumors were "black adenomas" in two and a carcinoma in one. To investigate possible factors which might account for these differences in ACTH responsiveness, tumor specimens of each one of the responsive and unresponsive adenomas, and a carcinoma were subjected to in vitro studies. When incubated with ACTH or cyclic AMP, tissue sections of a responsive adenoma enhanced cortisol secretion, while that of a black adenoma failed to show any change. Steroidogenesis by carcinoma sections were significantly suppressed in the presence of ACTH or cyclic AMP. Cycloheximide abolished a stimulatory effect of ACTH and cyclic AMP on steroidogenesis in a responsive adenoma without affecting its basal secretion of cortisol. Steroidogenesis by unresponsive tumors (an adenoma and a carcinoma) were decreased by cycloheximide. Since the conversion of cholesterol to pregnenolone, the rate limiting step in steroidogenesis, takes place in adrenal mitochondria, the effect of cyclic AMP on pregnenolone formation from 14C-cholesterol by mitochondrial fractions of these tumors was examined. Cyclic AMP stimulated pregnenolone formation by mitochondrial fraction of an ACTH responsive adenoma, while with that of an unresponsive adenoma pregnenolone formation was not affected. Pregnenolone formation by cancer mitochondria was significantly suppressed by cyclic AMP. These results suggest that the unresponsiveness to ACTH of these tumors might be explained by the ineffectiveness of cyclic AMP to stimulate pregnenolone formation by tumor mitochondria, and that the steroidogenic pathway in unresponsive tumors are in an enhanced state even without cyclic AMP. It should be mentioned that all unresponsive adenomas gave a characteristic appearance of a "black adenoma". Histologically, tumors were composed of compact cells with abundant lipofuscin granules. The possible relationship between the ACTH responsiveness of adrenocortical tumors and some clinical pictures caused by them was also noticed. ACTH unresponsive adenomas resulted in shorter duration, severer conditions of the disease and higher 17-ketosteroid excretion than responsive adenomas. The growth of unresponsive tumors seemed faster than that of responsive ones.     

Differences between the growth regulatory pathways in primary rat adrenal cells and mouse tumor cell line

In this study, DNA synthesis, phosphorylation of ERK1/2 and CREB proteins, as well as induction of c-Fos protein, were examined in rat adrenocortical, glomerulosa and fasciculata/reticularis cells, as well as in the Y1 cell line. We found that FGF2 was mitogenic only in glomerulosa cells and although ACTH did not activate ERK1/2, it did activate CREB protein, indicating efficient transduction of signals initiated in the ACTH receptors of rat adrenocortical cells. The FGF2 activated ERK1/2 in rat adrenal cells by a mechanism that might be modulated by upstream PKA pathway phosphorylation of MEK and despite the nonmitogenic effect of ACTH on rat adrenal cells it effectively induces c-Fos protein. The results presented herein describe distinct differences between the ACTH and FGF2 signal transduction mechanisms seen in adrenocortical cells and those observed in the Y1 cell line, indicating that, in vitro, ACTH blockage of the mitogenic effect occurs in normal adrenal cells after induction of c-Fos protein.     

Effects of corticosterone on adrenocorticotrophin-induced mitochondrial differentiation with special reference to 11 beta- and 18-hydroxylation.

The effects of corticosterone in concentrations found in adrenal venous plasma on ACTH-induced changes in cultured cortical cells derived from foetal rat adrenals were studied. Corticosterone at a concentration of 5-7 X 10(-5) mol/l completely inhibited mitochondrial differentiation to fasciculte-like morphology. The same cultures revealed significant inhibition of 11beta- and 18-hydroxylation compared with cultures treated with ACTH only. This was shown by the reduced formation of corticosterone and 18-OH-deoxycorticosterone (48%, P less than 0-001) and simultaneous enhancement of deoxycorticosterone formation (33%, P less than 0-05) from added [4-14C]progesterone. Similar inhibition was observed when dibutyryl cyclic AMP replaced ACTH as an inducer of differentiation. Lower concentrations of corticosterone (1-2 X 10(-5) and 2-4 X 10(-5) mol/l) inhibited ACTH-stimulated formation of corticosterone and 18-OH-deoxycorticosterone from endogenous precursors. The results demonstrate that corticosterone regulates the stage of differentiation in cultured adrenocortical cells. The possible role of corticosterone in the regulation of growth and steroidogenic capacity of the adrenal cortex is discussed.     

Cyclic AMP-mediated cytoskeletal effects in adrenal cells are modified by serum, insulin, insulin-like growth factor-I, and an antibody against urokinase plasminogen activator

In adrenocortical cells in culture, increased intracellular cyclic AMP resulting from exposure to agents such as ACTH and cholera toxin causes a change in cell morphology termed 'retraction' or 'rounding'. The breakdown of actin-containing stress fibers in rounding suggested a role for microfilaments in steroidogenesis. Previously, we showed that cultured bovine adrenal cells under standard conditions (medium with 10% fetal bovine serum) do not round in response to intracellular cyclic AMP. Here, we show that these cells do round in defined, serum-free medium. Rounding was maximal within 1 h of addition of 1 nM cholera toxin and after 10 h most cells remained rounded. Cycloheximide at 100 micrograms/ml did not inhibit the response to cholera toxin. The rounding response was abolished when 10% fetal bovine serum, horse serum, or ether-extracted fetal bovine serum was included in the medium. The inhibitory effect of serum was not mimicked by growth factors with the exception that insulin and insulin-like growth factor-I (IGF-I), while not preventing rounding, accelerated the return of cells to a flattened morphology. A monoclonal antibody against urokinase plasminogen activator completely prevented rounding whereas a monoclonal antibody against tissue plasminogen activator had only a slight effect. Fluorescence visualization of F-actin with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin showed that rounding in cultured bovine adrenocortical cells resembles that defined earlier for human and rat adrenocortical cells and includes depolymerization of actin microfilaments. These cytoskeletal changes in adrenal cells are unlikely to play a role in steroidogenesis; however, they may be involved in tissue remodeling occurring as part of the indirect mitogenic effects of ACTH.