Mitochondrial localization of a phosphoprotein that rapidly accumulates in adrenal cortex cells exposed to adrenocorticotropic hormone or to cAMP

We have reported previously that a phosphoprotein, ib, is present in adrenal cortex, corpus luteum, and Leydig cells stimulated with either tissue-specific peptide hormone or with cAMP. The accumulation of protein ib in each of these cell types has been found to parallel the stimulation of steroid synthesis with respect to both time course and stimulant dose response. Thus, protein ib is a potential mediator in the acute stimulation of steroidogenesis by peptide hormone or cyclic AMP. A second protein, pb, the unphosphorylated form of ib, is synthesized constitutively in unstimulated but not stimulated cells and is not converted post-translationally to ib upon stimulation. Using two-dimensional gel electrophoresis of subcellular fractions isolated from rat adrenal cortex cells labeled with [35S] methionine, we have determined the intracellular localization of proteins p and i. We demonstrate that proteins ib and pb are localized predominantly in the mitochondria and are tightly associated with that organelle. We also find that inhibition of mitochondrial protein synthesis by chloramphenicol affects neither the accumulation of these proteins nor the stimulation of steroidogenesis. Thus, protein pb and its phosphorylated counterpart, ib, are synthesized in the cytosol and transported to the mitochondria, the site of the rate-limiting step in steroid hormone biosynthesis.     

ACTH induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol biosynthesis, and steroidogenesis in primary cultures of bovine adrenocortical cells

The current studies demonstrate that corticosteroidogenesis can be maintained by primary cultures of bovine adrenocortical cells under lipoprotein-depleted conditions. The cholesterol necessary as substrate for steroid synthesis was found to arise from de novo synthesis within these cells. Adrenocorticotropin (ACTH) increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity 5-fold within 12 h after addition to the medium. The increase in activity apparently represented accumulation of enzyme as determined by protein blotting and immunodetection. The predominant immunodetectable species of HMG-CoA reductase from bovine adrenal cells was 97,000 daltons; no higher molecular mass species was detectable. The ACTH induction of HMG-CoA reductase activity could be prevented after inhibition of cholesterol conversion to pregnenolone with clotrimazole. These results are suggestive that ACTH increases adrenocortical cholesterol biosynthesis and HMG-CoA reductase activity after conversion of a cellular pool of cholesterol and/or oxysterol into steroid. The increased rate of cholesterol biosynthesis is then capable of maintaining ACTH-promoted steroid production. This is the first study, in vitro, to demonstrate an ACTH-promoted accumulation of HMG-CoA reductase of adrenocortical cells.     

Estimation of pregnenolone synthesis in rat adrenal homogenates: some cofactor requirements: effects of stress, hypophysectomy, cortisone and ACTH.

Pregnenolone synthesis was estimated in whole adrenal homogenates incubated in the presence of cyanoketone (2alpha-cyano-4,4,17alpha-trimethyl-androst-5-en-17beta-ol-3-one). The yield of pregnenolone depended on the type of incubation medium employed. Both Ca++ and bovine serum albumin (BSA) markedly stimulated the rate of pregnenolone synthesis as did NADPH or NADPH generating system. Aminoglutethimide added in vitro inhibited cholesterol sidechain cleavage activity. Ether stress in vivo stimulated pregnenolone synthesis in vitro, and hypophysectomy of 24 hours duration resulted in a decrease. Cortisone administration for 8 days reduced the formation of pregnenolone by rat adrenal homogenates, an effect prevented by concomitant treatment with ACTH. Similarly, hypophysectomy of 8 days duration resulted in a marked diminution of pregnenolone synthesis and ACTH replacement reversed this effect. Changes in pregnenolone synthesis were paralleled by changes in corticosterone and total steroid production.     

Adrenal adenylate cyclase and steroidogenic activities of 63 day old ovine fetuses: in vitro effects of ACTH1-24 and forskolin

This study examines the activity of the adenylate cyclase system and that of some enzymes of the steroidogenic pathway of adrenal cells from 62-63 day old ovine fetuses. Synthetic corticotropin (ACTH1-24), cholera toxin and forskolin stimulated both cAMP and corticoid productions by freshly isolated adrenal cells. The cAMP response to ACTH1-24 was lower than that to forskolin. However, forskolin-induced steroidogenesis was significantly lower than the ACTH1-24-induced steroid output. Freshly isolated cells metabolized quickly [14C]-labeled pregnenolone mainly through the 17-deoxy pathway. The amounts of cortisol and of corticosterone formed, in the presence of exogenous pregnenolone, were roughly 15-fold higher than under maximal stimulation by ACTH1-24. When the cells were cultured for 6 days in the absence or presence of ACTH1-24 (10(-8) M) or forskolin (10(-5) M), a small development of the cAMP response to these factors was observed in the course of the experiment. However, the mechanism of this development appeared different, according to the conditions of culture. The amounts of corticosterone secreted on day 6 by ACTH1-24- or forskolin-treated cells were 2- to 4-fold higher than on day 1, whereas cortisol outputs were much lower on day 6 than on day 1. The response to ACTH1-24 of cells maintained in ACTH-free media decreased dramatically during the culture in terms of both cortisol and of corticosterone. On day 6 of the experiment, the metabolism of [14C]pregnenolone was lower than on day 1 under all 3 conditions of culture. Only the 3 beta-hydroxysteroid dehydrogenase/isomerase activity could be maintained by continuous treatment with forskolin. However, both ACTH1-24 and forskolin enhanced the production of pregnenolone from an endogenous substrate. In conclusion, these results present evidence that: 1) the adenylate cyclase system is not a bottleneck in the steroidogenic response to ACTH1-24 of freshly isolated adrenal cells from 62-63 day old ovine fetuses; 2) the main rate-limiting step for steroidogenesis by these cells is the availability of pregnenolone; 3) neither ACTH1-24 nor forskolin is able to maintain the activity of most enzymes involved in the metabolization of pregnenolone by cultured cells while increasing pregnenolone availability; 4) some inhibiting factors are involved in the loss of adrenal cells responsiveness to ACTH between days 50 and 100 of gestation, and they probably act mainly on the adenylate cyclase system.     

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH_1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH_1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH_1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH_1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced ³H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.     

Acute stimulation of steroidogenesis in corpus luteum and adrenal cortex by peptide hormones. Rapid induction of a similar protein in both tissues

Two-dimensional electrophoresis was used to detect a protein (ic) synthesized in rat corpus luteum cells in response to acute stimulation by human chorionic gonadotropin or dibutyryl cyclic AMP. This induced protein ic is isoelectric at pH 6.5 (isoelectric focusing) and has an apparent molecular weight of 28,000 (sodium dodecyl sulfate electrophoresis). The human chorionic gonadotropin or dibutyryl cyclic AMP dose response and time course of synthesis of the protein parallel those of progesterone synthesis in stimulated luteal cells. Additionally, cycloheximide, which inhibits the increase in progesterone formation caused by human chorionic gonadotropin or cAMP, also inhibits the synthesis of ic. Proteolytic polypeptide mapping suggests that ic has a very similar primary structure to another protein (pc), which has the same molecular weight as ic, differs from ic in pI, and is synthesized only in unstimulated cells. These polypeptide maps also demonstrate the close similarity of pc and ic to two proteins p and i, synthesized in control and in adrenocorticotropic hormone-stimulated rat adrenal cortex cells, respectively (Krueger, R. J. and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167). In both adrenal cortex and corpus luteum, binding of a tissue-specific polypeptide hormone acts via cAMP to cause increased steroidogenesis and induction of the synthesis of protein i (ic), with the same time course and hormone dose dependence. Also in both tissues, inhibition of protein synthesis at the level of translation (e.g. by cycloheximide addition) causes inhibition of i (ic) synthesis and of stimulated steroid production. This close correlation between the two different tissues in conditions which cause induction of the synthesis of these proteins suggests that the proteins may be common intermediaries in the control by polypeptide hormones of steroidogenesis in endocrine tissues.     

The influence of cytochalasin B on the response of adrenal tumor cells to ACTH and cyclic AMP.

Cytochalasin B inhibits increase in steroid synthesis by mouse adrenal tumor cells (Y-1), produced either by ACTH or cyclic AMP. Basal levels of steroid synthesis are not decreased and the inhibitor acts by decreasing the response of the side-chain cleavage step (cholesterol → pregnenolone) to ACTH. Inhibition is reversible and is seen in medium without glucose. These observations suggest that microfilaments may play a role in the response of adrenal cells to ACTH.     

Hormonal regulation of the adrenocortical cell

Monolayer cultures of bovine and human adrenocortical cells have been used to study regulation of growth and function. Homogeneous bovine adrenocortical cells exhibit a finite life span of ～60 generations in culture. Full maintenance of differentiated function (steroid hormone synthesis) requires an inducer such as ACTH and antioxidizing conditions. Full induction of differentiated function occurs only when cellular hypertrophy is stimulated by growth factors such as fibroblast growth factor and serum. ACTH and other agents that increase cellular cAMP inhibit replication but do not block growth factor-induced cellular hypertrophy. ACTH and growth factors together result in a hypertrophied, hyperfunctional cell. Replication ensues only when desensitization to the growth inhibitory effects of ACTH occurs. Cultures of the definitive and fetal zones of the human fetal adrenal cortex synthesize the steroids characteristic of the two zones in vivo. ACTH stimulates production of dehydroepiandrosterone (DHA), the major steroid product of the fetal zone, and of cortisol, the characteristic steroid product of the definitive zone. Prolonged ACTH treatment of fetal zone cultures results in a preferential increase in cortisol production so that the pattern of steroid synthesis becomes that of the definitive zone. The preferential increase in cortisol production by fetal zone cultures results from induction of 3β-hydroxysteroid dehydrogenase, Δ^4,5 isomerase activity, which is limiting in fetal zone cells. ACTH thus causes a phenotypic change in fetal zone cells to that of definitive zone cells. In both bovine and human adrenocortical cells, the principal effect of ACTH is to induce full expression of differentiated function. This occurs only under conditions where growth substances and nutrients permit full amplication.     

Evidence for 6-keto-PGF1 alpha in adrenal cortex of the rat and effects of 6-keto-PGF1 alpha and PGI2 on adrenal cAMP levels and steroidogenesis.

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF1 alpha, the hydrolysis metabolite of PGI2, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF1 alpha was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF2 alpha or PGE2. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI2 metabolite. Studies in vitro, using isolated cortical cells exposed to 6-keto-PGF1 alpha (10(-6)-10(-4)M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI2 (10(-6)-10(-4)M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering cortico-sterone production, ACTH (5-200 microU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI2 produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused in situ with PGI2 showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF1 alpha is present in the rat adrenal cortex, its precursor, PGI2, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Ca2+ signal stimulates the expression of steroidogenic acute regulatory protein and steroidogenesis in bovine adrenal fasciculata-reticularis cells

Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.     

The Involvement of Nek2 and Notch in the Proliferation of Rat Adrenal Cortex Triggered by POMC-Derived Peptides

The adrenal gland is a dynamic organ that undergoes constant cell turnover. This allows for rapid organ remodeling in response to the physiological demands of the HPA axis, which is controlled by proopiomelanocortin (POMC)-derived peptides, such as adrenocorticotropic hormone (ACTH) and N-Terminal peptides (N-POMC). In the rat adrenal cortex, POMC-derived peptides trigger a mitogenic effect, and this process increases cyclins D and E, while inhibiting p27Kip1. The goal of the present study was to further explore the mitogenic effect of ACTH and synthetic N-POMC_1–28 peptides by investigating the differences in the expression of key genes involved in the cell cycle of the rat adrenal cortex, following inhibition of the HPA axis. Moreover, we evaluated the differences between the inner and outer fractions of the adrenal cortex (ZF-fraction and ZG-fraction) in terms of their response patterns to different stimuli. In the current study, the inhibition of the HPA axis repressed the expression of Ccnb2, Camk2a, and Nek2 genes throughout the adrenal cortex, while treatments with POMC-derived peptides stimulated Nek2, gene and protein expression, and Notch2 gene expression. Furthermore, Notch1 protein expression was restricted to the subcapsular region of the cortex, an area of the adrenal cortex that is well-known for proliferation. We also showed that different regions of the adrenal cortex respond to HPA-axis inhibition and to induction with POMC-derived peptides at different times. These results suggest that cells in the ZG and ZF fractions could be at different phases of the cell cycle. Our results contribute to the understanding of the mechanisms involved in cell cycle regulation in adrenocortical cells triggered by N-POMC peptides and ACTH, and highlight the involvement of genes such as Nek2 and Notch.     

Sites of action of beta-melanocyte stimulating hormone in aldosterone biosynthesis in the rat

The sites of action of beta-melanocyte stimulating hormone (beta-MSH) on aldosterone biosynthesis were studied using collagenase-dispersed adrenal glomerulosa cells from rats maintained on either normal or sodium-deficient diets for 2 weeks. Isolated cells were treated with a cyanoketone derivative (WIN 19,578) to isolate the early and late steps in aldosterone biosynthesis. WIN 19,578 (1 microM) completely blocked aldosterone production stimulated by sodium depletion, AII, ACTH, and beta-MSH. beta-MSH (1 microM) significantly stimulated pregnenolone production (early step) and the conversion of corticosterone to aldosterone (late step) in aldosterone biosynthesis. The effect of beta-MSH was similar to AII and ACTH. Sodium depletion enhanced the effect of beta-MSH only on the late step in aldosterone biosynthesis. In conclusion, beta-MSH stimulates both the early and late steps of aldosterone biosynthesis. These results suggest that beta-MSH or peptides containing beta-MSH may play a role in the regulation of aldosterone production.     

Cyclic AMP-independent effects of ACTH on glomerulosa cells of the rat adrenal cortex

The aim of the present paper is to point out the complexity of ACTH action in glomerulosa cells of the adrenal cortex. We demonstrate that the increase in cAMP production induced by ACTH is the result of a balance between activation of adenylyl cyclase and direct modulation of a PDE2 phosphodiestease activity, an effect mediated by inhibition of cGMP content. Moreover, Ca²⁺ is essential for cAMP production and aldosterone secretion, but its exact primary action is not clearly determined. We recently described that ACTH activated a chloride channel, via the Ras protein, which can be involved in steroidogenesis. ACTH also increases tyrosine phosphorylation of several proteins. These data, together with those of phospholipase C activation, indicate that ACTH action in the adrenal is complex, and most certainly not limited to cAMP production, in particular for the low concentrations of the hormone.

Some years ago, cAMP was considered to be the unique second messenger of ACTH action; now it becomes more and more evident that ACTH triggers complex signaling pathways using several second messengers in a closely interacting way. The most predominant point is that these signals are observed for low concentrations of ACTH.     

Effects of protease inhibitors on adenylate cyclase activation and aldosterone production in rat adrenal zona glomerulosa cells

It has been shown that serine proteases are involved in aldosterone and 18-hydroxycorticosterone production by the rat adrenal zona glomerulosa in response to a variety of stimulants. From evidence presented for various tissues, including the rat adrenal cortex, the observation that adenylate cyclase can be activated by proteolytic enzymes and inhibited by protease inhibitors has led to the suggestion that serine proteases may also be involved in the hormonal stimulation of adenylate cyclase. In studies designed to test this hypothesis using protease inhibitors, only high concentrations (greater than 10(-4) M) of TAME (p-tosyl-L-arginine methyl ester) inhibited ACTH stimulated steroid and cAMP production in rat adrenal glomerulosa cells. TPCK (tosyl-L-phenylalanine chloromethylketone) and TLCK (tosyl-L-lysine chloromethylketone) were found to have a similar effect at very high concentrations (10(-2) M) but had no effect at the serine protease inhibitory concentration of 5 X 10(-6) M. Other protease inhibitors tested had no effect on ACTH-stimulated cAMP but the inhibitory effect of high concentrations of protease inhibitors on ACTH-stimulated adenylate cyclase was duplicated by the polyanion dextran sulphate. The results suggest that the inhibitors act through non-specific membrane effects and that proteases are not involved in the activation of zona glomerulosa adenylate cyclase by ACTH. In view of these findings it is concluded that a more rigorous approach should be applied to the use of protease inhibitors in whole cell systems, and that the concept of hormonal activation of adenylate cyclase via proteolytic events, which is based on studies with such inhibitors, should be reconsidered.     

Angiotensin stimulation of adrenal fasciculata cells

In this paper we provide evidence to show that the pathways by which adrenocorticotropic hormone (ACTH) and angiotensin II (AII) stimulate steroidogenesis in bovine fasciculata cells are only partially independent. Both hormones have the same intrinsic activity but a 500-fold higher dose of AII is required to achieve 50% stimulation of steroidogenesis. Whereas ACTH acts by way of cAMP, AII appears to operate through protein kinase C. The phorbol ester, 12-O-tetradecanoylphorbol-13 acetate (TPA), and the calcium ionophore, A23187, each stimulate steroidogenesis and, when added together, act synergistically. To test the relationship between the ACTH and AII pathways, we added the two hormones simultaneously and measured steroid production. When the hormones were present at submaximal concentrations, their effects were additive. At maximal doses, steroid production was 40% above that elicited by either hormone alone. In contrast to the action of AII in the glomerulosa cell where it inhibits ACTH-stimulated cAMP formation, AII causes no inhibition in the fasciculata. Cycloheximide inhibits steroidogenesis stimulated by AII or a mixture of TPA and A23187. Scatchard analysis of the binding of 125I-AII to particulates from adrenal cortical fasciculata indicates the presence of a single class of binding sites (Kd = 0.6 X 10(-8) M). Binding is not inhibited by ACTH. Biotin-containing AII analogs that bind specifically to the particulates have been evaluated as potential tools for avidin-biotin affinity chromatography of the receptor. One of these, [N epsilon-6-(biotinylamido)hexyllys1, Val5] AII, is a promising candidate for receptor isolation.