Impact of architectural disruption on adrenocortical steroidogenesis in vitro

The adrenal cortex is an architecturally complex tissue, with cellular zonation thought to determine steroidogenesis. The impact that disruption of this tissue's architecture has on steroidogenesis in vitro, particularly adrenal androgen (AA) production, is unclear. We hypothesized that the extent of architectural disruption during tissue preparation would impact the study results. To test this hypothesis, we compared adrenocortical steroidogenesis in freshly prepared tissue slices, minces, and cell suspensions. Normal human adrenals (n = 5, three males and two females, age range 17-43 yr) were obtained at the time of organ donation. The three adrenal tissue preparations were incubated in serum-free medium with 10 microM pregnenolone substrate +/- 1 microM ACTH. The production of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, and cortisol in the media were measured by radioimmunoassay. Initial time course intubations using adrenals from a single donor generally demonstrated that minces and suspensions had a greater steroid production compared with slices. In another series of 6-hr incubations using adrenals from four donors, production of dehydroepiandrosterone sulfate was found to be quite sensitive to architectural disruption, i.e. slices less than minces less than suspensions (0.88 vs. 2.1 vs. 3.0 microg/gm tissue, respectively, P < 0.0001). Alternatively, cortisol and androstenedione production was higher in minces compared with slices or suspensions (25.6 vs. 37.7 vs. 18.7 ng/gm tissue, P < 0.0028, and 254 vs. 709 vs. 456 ng/gm tissue, P < 0.0042, respectively). Production of dehydroepiandrosterone was apparently not significantly affected by the type of tissue preparation (28.2 vs. 22.2 vs. 31.2 ng/gm tissue, P < 0.297, respectively). It is unlikely that generalized tissue disruption alone accounted for the observed differences, as the trends among tissue preparations were not consistent among steroids. We conclude that the type of tissue preparation of fresh adrenal tissue impacts significantly on steroidogenesis in vitro.     

In vitro steroidogenesis by the nonzoned adrenocortical tissue of the skink, Tiliqua rugosa.

Steroid formation by adrenocortical tissue from the shink, Tiliqua rugosa, has been studied using established in vitro techniques. Both in conventional incubations, with timed sampling, and in incubations with dialysis, aldosterone, and corticosterone were major products. From endogenous precursors, and from [¹⁴C]acetate, yields of the two products were of the same order, whereas from [³H]pregnenolone maximal yields of corticosterone were at least tenfold greater than aldosterone. Maximal rates of steroid formation from the radioactive precursors occurred within the first few minutes of incubation, but maximal rates of steroid formation from endogenous precursors occurred significantly later, between 1–2 hr.In incubations with dialysis [¹⁴C]aldosterone was significantly less dialysable than [³H]aldosterone under all conditions, whereas [¹⁴C] and [³H]corticosterone were homogeneous. In contrast, neither aldosterone nor corticosterone formed from endogenous precursors were bound under control conditions, although binding was increased following dexamethasone pretreatment, and decreased following stimulation with Tiliqua pituitary extract (but not Synacthen), with concomitant changes in yields and specific activities.Inter alia the results suggest that products formed from [¹⁴C]acetate and from [³H]pregnenolone may be maintained in separate pools within the tissue, and this accounts for their different metabolic fates. The bound pool, penetrated only by [³H]acetate, yields more aldosterone than the free, and may be termed a “biosynthetic pool.” In addition there exists a “secretory reserve pool.” This is suggested by the difference between rates of steroid secretion from endogenous and added precursors, and also from the changes in dialysibility seen in steroids formed from endogenous precursors under different conditions of stimulation.In both the compartmental arrangement of steroids, and the production of large yields of aldosterone the adrenocortical tissue of Tiliqua shows similarities to the zona glomerulosa, but not the inner zones of the rat adrenal cortex.     

Follicular fluid stimulation of steroidogenesis in immature granulosa cells in vitro.

Granulosa cells from small (1-2 mm) immature porcine follicles were cultured in monolayer in culture media composed of equal parts of culture medium 199 and either (a) fluid from small follicles, (b) fluid from large (6-12 mm) follicles or (c) adult female porcine serum for 6 days, with or without 100 ng LH and/or 2 microgram FSH/ml. Both basal and gonadotrophin-stimulated progesterone secretion were greater in the presence of fluid from large follicles than in serum, for all 6 days. After 4 days of culture, fluid from small follicles enhanced gonadotrophin-stimulated progesterone secretion over that occurring in serum, but to a lesser extent than fluid from large follicles. These studies suggest the presence of a maturation stimulating molecule(s) in follicular fluid which increases in activity or concentration as the follicles enlarge. This factor may be essential for normal granulosa cell maturation in vivo.     

Control of steroidogenesis by the calcium messenger system in human adrenocortical cells

The involvement of the calcium messenger system in the control of steroidogenesis in the rat and bovine adrenal cortex has been studied extensively. However the role of these second messengers in the control of human adrenocortical function is not established. This was therefore studied by incubating collagenase-dispersed human adrenocortical cells with the calcium ionophore A23187 and the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). The effects of the calcium channel blocker verapamil on basal and stimulated steroidogenesis were also studied. Both TPA (1 pmol/l-10 mumol/l) and A23187 (1 nmol/l-10 mumol/l) caused a dose-dependent increase in cortisol, aldosterone and corticosterone production. Verapamil (10 mumol/l) inhibited the increase in aldosterone, corticosterone and cortisol produced in response to ACTH(1-24), potassium, and desacetyl-alpha MSH. Unlike previous results in the rat, these effects were not specific for aldosterone secretion. The results suggest that, as in other species, calcium mobilization and protein kinase C activation have a role in the control of steroidogenesis in the human adrenal cortex. However, in contrast to the rat, these mechanisms appear to be involved in the control of steroidogenesis in both the zona glomerulosa and inner zone cells.     

Site of action of growth hormone on adrenocortical steroidogenesis in rats.

Studies were carried out to define the mechanism of action of growth hormone on adrenocortical steroidogenesis in hypophysectomized female rats. ACTH administration for 7 days increased corticosterone secretion in vivo and corticosterone production by adrenal tissue in vitro. Adrenal mitochondrial and microsomal cytochrome P-450 concentrations as well as the activities of cytochrome P-450-dependent enzymes (cholesterol sidechain cleavage, 11beta-hydroxylase, 21-hydroxylase) were also increased by ACTH. Administration of bovine growth hormone alone to hypophysectomized rats had no effect on any of the parameters evaluated. However, when given in combination with ACTH, growth hormone synergistically enhanced the effects of ACTH on cholesterol sidechain cleavage activity and corticosterone secretion. The magnitude of the pregnenolone-induced difference spectrum in adrenal mitochondria, indicative of cholesterol binding to cytochrome P-450, was also increased by growth hormone, but neither cytochrome P-450 content nor the activities of other steroidogenic enzymes were affected. The results indicate that growth hormone interacts with ACTH to promote corticosterone secretion by increasing the association of cholesterol with adrenal mitochondrial cytochrome P-450, thereby increasing the activity of cholesterol sidechain cleavage, the rate-limiting step in steroidogenesis.     

Immunoglobulins of patients with Cushing's syndrome due to pigmented adrenocortical micronodular dysplasia stimulate in vitro steroidogenesis

The putative stimulation of adrenal steroid production by immunoglobulins (Igs) of five patients with pigmented adrenocortical micronodular dysplasia and clinical Cushing's syndrome was investigated. Ascorbate depletion, a process linked to steroid production, was measured by a cyto-chemical bioassay employing guinea pig adrenal explants in organ culture and exposed to IgG from the patients and normal subjects. We also measured cortisol production by these segments during a 5-h culture period using a RIA. For positive reference values we studied the effects of ACTH-(1-39), ACTH-(1-24), ACTH-(11-24), and ACTH-(18-39) on in vitro ascorbate depletion and cortisol production. Both ACTH-(1-39) and ACTH-(1-24) depleted ascorbate and stimulated cortisol production in adrenal cells. The dose-response kinetics of the peptides were bell-shaped; maximal responses were reached in both instances at 1 fmol/L to 10 pmol/L. In all tests, stimulation of in vitro cortisol production was paralleled by ascorbate depletion. ACTH-(18-39) also stimulated ascorbate depletion and cortisol production, but at one concentration only (100 fmol/L), and TSH and LH had no effect. Protein-A-Sepharose-purified IgG preparations of the five patients stimulated ascorbate depletion and/or cortisol production in a dose-dependent fashion; however, the responses occurred over a narrow concentration range (15-150 micrograms IgG/mL culture fluid). These observations support the hypothesis that the hypercortisolism of the syndrome of pigmented adrenocortical micronodular dysplasia is due to circulating Igs that stimulate adrenal steroidogenesis.     

Transmembrane potentials and steroidogenesis in normal and neoplastic human adrenocortical tissue.

Trans-membrane potentials and steroidogenesis were measured in superfused slices of non-tumor and neoplastic human adrenocortical tissue. Non-tumor tissue was obtained at the time for renal transplant or from tissue removed along with tumors. Non-tumor human adrenocortical tissue had electrophysiological and steroidogenic properties similar to those of the rat and rabbit. In normal medium ACTH stimulated steroidogenesis but had no effect on the membrane potential. In K+-free medium, the cells hyperpolarized, and subsequent addition of ACTH caused depolarization. Trans-membrane potentials of adrenocortical tumors were lower than those of non-tumor cells. Ommission of K+ from the medium caused hyperpolairzation of the tumor cells, but the trans-membrane potentials did not reach the values of hyperpolarized non-tumor cells. ACTH, added to the K+-free medium, caused little or no change in membrane potential of tumor cells except in one case of a virilizing adenoma, which responded very much like non-tumor tissue. Except for the virilizing adenoma, tumor tissue slices produced little or no detectable fluorogenic steroid, even in the presence of large amounts of ACTH or cyclic AMP. The virilizing adenoma responded with increased steroidogensis to ACTH and cyclic AMP.     

Inhibition of adrenocortical steroidogenesis by alpha 2-macroglobulin is caused by associated transforming growth factor beta.

alpha 2-Macroglobulin (alpha 2M) is the major protein secreted by bovine adrenocortical cells in primary culture and its synthesis is stimulated by transforming growth factor beta (TGF beta). We investigated here the effects of alpha 2M on adrenocortical steroidogenesis. We observed that commercial preparations of bovine plasma alpha 2M were able to mimic the inhibitory action of TGF beta on adrenocortical cortisol production, with the same specificity of action directed at the steroid 17 alpha-hydroxylation step. This inhibition was time-dependent and dose-dependent (50% inhibition observed with 2 mg/ml alpha 2M). Acid/ethanol extracts of alpha 2M appeared to retain the full inhibitory activity of alpha 2M. Anti-TGF beta antibodies could reverse the inhibition caused by the acid/ethanol extract but not that caused by native alpha 2M. Taken together, these results indicate that the inhibition of adrenocortical steroidogenesis induced by alpha 2M is caused by associated TGF beta. We estimated that 2 mg of alpha 2M contained approximately 0.1 ng of TGF beta, corresponding to a molar ratio of 1/700,000 between TGF beta and alpha 2M. These results also clearly indicate that the alpha 2M-TGF beta complexes are biologically active on adrenocortical cells, suggesting that these cells possess the enzymatic equipment that can activate the latent alpha 2M-TGF beta complexes.     

Human recombinant activin-A modulates the steroidogenesis of cultured bovine adrenocortical cells.

The effect of human recombinant activin-A on adrenal steroidogenesis was studied in cultured bovine adrenocortical cells. Activin-A significantly reduced cortisol output from ACTH (10nmol/l)-stimulated adrenocortical cells incubated for 24 hours in a dose-dependent manner (10, 100 and 500ng activin-A/ml suppressed cortisol secretion by 19, 33 and 40%), although no significant effect was observed in the case of 3 h incubation. Dehydroepiandrosterone (DHEA) secretion from ACTH-stimulated adrenocortical cells incubated for 24 h was also decreased by the addition of activin-A in a dose-dependent manner. (10, 100 and 500ng activin-A/ml suppressed DHEA secretion by 22, 56 and 58%). These inhibitory effects of activin-A (100ng/ml) on cortisol and DHEA secretion were partially blocked by the addition of follistatin/FSH-Suppressing Protein (200ng/ml). In contrast, activin-A treatment resulted in no significant decrease in aldosterone secretion. There were no significant effects of activin-A on basal secretions of cortisol, DHEA or aldosterone from adrenocortical cells. These results suggest that activin-A has a direct inhibitory effect on ACTH-stimulated bovine adrenocortical steroidogenesis.     

The role of endothelin in the control of adrenocortical function: stimulation of endothelin release by ACTH and the effects of endothelin-1 and endothelin-3 on steroidogenesis in rat and human adrenocortical cells.

The rate of blood flow through the intact adrenal gland is closely linked to steroid hormone secretion, and although the mechanism involved is unknown, it is thought to involve secretory products of the vascular endothelium. In dispersed cell preparations, endothelin-1 and -3 both caused a dose-dependent and highly sensitive increase in steroid secretion by zona glomerulosa and zona fasciculata cells of the rat and human adrenal cortex. In addition, when the perfused rat adrenal was stimulated with ACTH, significant increases in steroid secretion and perfusion medium flow rate were accompanied by significantly increased secretion of immunoreactive endothelin into the adrenal vein. It is proposed that endothelin has a role in mediating the adrenocortical response to ACTH stimulation.     

The inhibition of low density lipoprotein metabolism by transforming growth factor-beta mediates its effects on steroidogenesis in bovine adrenocortical cells in vitro

Transforming growth factor-beta (TGF beta) has a differential effect on the growth and function of bovine adrenocortical cells in vitro. TGF beta inhibits basal as well as ACTH- or angiotensin II-stimulated steroid formation, with no evidence of change in cell growth. The major inhibitory effect of TGF beta occurs at a step before cholesterol formation, since treatment of adrenocortical cells with TGF beta decreased not only delta 4-steroid levels but also delta 5-steroid levels. The addition of cholesterol reverses the suppression of steroidogenesis induced by TGF beta. To determine the mechanism of this inhibition, the effect of TGF beta on low density lipoprotein (LDL) metabolism was investigated. Cells treated with TGF beta showed a significant suppression of [125I]iodohuman LDL ([125I]LDL) binding to the cell surface, followed by decreases in internalization and proteolytic degradation of [125I]LDL. Maximal inhibition of LDL metabolism was observed at a concentration of 1 ng/ml (4 X 10(-11) M) TGF beta. The stimulation of LDL metabolism by ACTH was also inhibited by TGF beta, and the inhibition observed correlated well with the inhibition of steroidogenesis. The inhibitory effect of TGF beta on [125I]LDL binding results from the decrease in the maximal LDL-binding capacity. The stimulation of LDL uptake induced by Bu2cAMP, cholera toxin, forskolin, and Ang II was also decreased by treatment with 1 ng/ml TGF beta. The specificity of this effect is quite high, since the inhibitory effects of TGF beta on LDL metabolism were not observed with either inhibin A or activin, two molecules that have considerable structural homology to TGF beta. We conclude that TGF beta specifically suppresses LDL metabolism in bovine adrenocortical cell cultures and that this step may mediate, at least in part, its role as a potent inhibitor of steroidogenesis.     

Angiotensin stimulation of bovine adrenocortical cell growth.

Factors controlling proliferation of adrenocortical cells have been studied in monolayer cultures of bovine adrenocortical cells. Angiotensin II stimulated cell proliferation and [3H]thymidine incorporation into DNA with a half-maximal effective concentration of 0.96 +/- 0.27 nM. Similar sensitivity to angiotensin III with reduced sensitivity to angiotensin I and tetradecapeptide renin substrate was observed. Although sensitivity to angiotensin II was equivalent to that for fibroblast growth factor (1.5 nM half-maximal effective concentration), maximal effects of angiotensin were less than for fibroblast growth factor and serum. High concentrations of insulin (1-10 micrometer) also stimulated [3H]thymidine incorporation into DNA and cell proliferation. [Sar1,Ile5,Ile8]Angiotensin II, a competitive antagonist of angiotensin II, blocked angiotensin II stimulation of DNA synthesis but did not affect fibroblast growth factor and insulin stimulation of DNA synthesis. Corticotropin (ACTH) blocked the stimulatory effects of both angiotensin II and fibroblast growth factor. The dose-response curves for angiotensin II stimulation of steroidogenesis were similar to those for stimulation of [3H]thymidine incorporation into DNA. Among the seven cell types examined, only adrenocortical cells responded to angiotension II with stimulation of DNA synthesis.     

The effect of oxygen on aldosterone release from bovine adrenocortical cells in vitro: PO2 versus steroidogenesis

Hypoxia decreases plasma aldosterone in vivo without a decrease in PRA, angiotensin II (ANG II), ACTH, or cortisol. The present study evaluated whether this could be due to a direct, specific inhibitory effect on the zona glomerulosa related to the magnitude of the decrease in oxygen (O2). Bovine adrenocortical cells were dispersed with collagenase and studied in vitro within 48 h. Cells were stimulated for 2 h with ANG II (0.1-1000 nM) or (Bu)2cAMP (0.3-3 mM) under oxygen levels ranging from 0 to 100% O2 (PO2 from 66 +/- 4 to 561 +/- 46 torr) vs. a reference gas mixture (21% O2 PO2 approximately 140 torr). Exposure to 123 +/- 8, 110 +/- 12, 100 +/- 16, and 66 +/- 4 torr led to 27%, 30%, 40% and 70% inhibition, respectively, of 3 nM ANG II-stimulated aldosterone secretion as compared to 140 +/- 16 torr (reference). Exposure to hyperoxia (288 +/- 36 to 561 +/- 46 torr) led to a small (10%) increase in ANG II-stimulated aldosterone secretion which was not statistically significant. The P50 (half-maximal PO2) for aldosteronogenesis was approximately 95 torr. The results for other doses of ANG II and for cAMP were similar. The inhibitory effect of low O2 was reversed by returning the cells to reference conditions (140 +/- 16 torr). Cortisol secretion was not significantly affected by changes in oxygen tension. We conclude that small changes in O2 within the physiological range directly and specifically inhibit aldosteronogenesis in a dose-dependent manner with a P50 of approximately 95 torr. Inhibition of cAMP-stimulated aldosterone secretion suggests a postreceptor site of action. This direct, reversible, and specific effect on the zona glomerulosa of the adrenal cortex may account for the dissociation of renin and aldosterone during hypoxia in vivo.     

The effect of melatonin on steroidogenesis by the human ovary in vitro.

Melatonin stimulated steroidogenesis in two compartments of the human ovary in vitro. In a corpus luteum of the menstrual cycle, melatonin increased progesterone synthesis in a dose related manner. Both serotonin and N-acetyl serotonin had no effect on progesterone synthesis. In the ovarian stroma, melatonin stimulated the incorporation of acetate-1-14-C into androstenedione. Binding of radioactive hCG by the corpus luteum was unaffected by melatonin. No specific binding of radioactive melatonin of low specific activity could be detected in homogenates of a human corpus luteum. These observations suggest that melatonin may directly modulate steroidogenesis in the human ovary.     

Prolactin stimulates steroidogenesis by human luteal tissue in vitro

Human luteal tissue recovered from varying stages of the luteal phase was minced and incubated for 3 h and the effect of human chorionic gonadotrophin (hCG), prolactin and hCG + prolactin on progesterone and oestradiol production measured. While hCG generally enhanced both progesterone and oestradiol synthesis, prolactin alone at either 20 or 200 micrograms/l had no significant effect on steroidogenesis. When prolactin was added along with hCG in four of six corpora lutea, however, progesterone production significantly increased and in three of six corpora lutea oestradiol production was increased above that induced by hCG alone. It is concluded that prolactin may play some role in the control of steroidogenesis by the human corpus luteum.     

Studies on oxidative phosphorylation and steroidogenesis by ovarian mitochondria after gonadotropic stimulation.

No differences in oxidative phosphorylation or in the per cent of [4-14C]progesterone were found in ovarian mitochondria of immature rats after treatment with 20 IU of pregnant mare serum gonadotropin (PMSG) iv 30 min before killing. However, treatment of immature rats with 20 IU of PMSG sc 54 h prior to killing decreased the ADP:O ratio and increased the per cent of [4-14C]cholesterol conversion. Electron microscopic studies showed that mitochondria with lamellar cristae were prominent in ovaries of untreated rats, while large pleomorphic mitochondria and mitochondria with tubulovesicular cristae dominated in ovaries of PMSG-treated rats. Ovarian homogenates separated by zonal centrifugation showed three peaks od cytochrome oxidase activity which shifted to the heavier end of the gradient after PMSG treatment. These studies suggest that PMSG treatment influences ovarian mitochondria, possibly by stimulating the synthesis of additional functional components and/or the biogenesis of new mitochondria. Aminoglutethimide addition to bovine luteal mitochondria decreased steroidogenesis by 60% when succinate was used as substrate. However, there was a 16% increase in the ADP:O ratio, apparently due to a decrease in oxygen utilization. When oligomycin was added to luteal mitochondria, there was a 30% decrease in the ACP:O ratio but a 300% increase in [4-14C]cholesterol conversion. Dinitrophenol also decreased mitochondrial steroidogenesis. These results suggest that energy obtained from succinate oxidation can be diverted from phosphorylation to support steroidogenesis.     

The effect of polymyxin B on steroidogenesis from adrenocortical cells

The action of the cyclic peptide polymyxin B (a well known inhibitor of protein kinase C) on adrenal steroid synthesis was examined with Y-1 adrenal tumor cells. Polymyxin B produces a biphasic effect on the stimulation of steroid synthesis by 2 nM ACTH in these cells, with inhibition at low concentrations (less than 10 microM) and a return to control levels at high concentrations (greater than 100 microM). Polymyxin B does not inhibit the stimulatory effect of Bu2cAMP on steroidogenesis. Inhibition of the steroidogenic response to ACTH by a fixed concentration (20 microM) of polymyxin B is overcome by high concentrations of ACTH. Polymyxin B causes a concentration-dependent stimulation of steroid synthesis by Y-1 cells and, over the same concentration range, increases the production of cAMP by these cells. Polymyxin B also partially inhibits the increased production of the cyclic nucleotide produced by ACTH. In addition, polymyxin B inhibits binding of [125I](Phe2,Nle4)ACTH-(1-38) to Y-1 cells. Polymyxin B, like ACTH, promotes rounding of Y-1 cells and partially inhibits rounding produced by ACTH. These effects of polymyxin B are specific to the extent that polymyxins E1 and E2 do not exert similar effects. The actions of polymyxin B are not confined to transformed cells, since responses similar to those seen with Y-1 cells were also observed with cultured rat fasciculata cells. On the other hand, the effect of polymyxin B is specific for adrenal cells, since the cyclic peptide does not influence the steroidogenic response of rat Leydig cells to LH. It is concluded that polymyxin B is a partial agonist of ACTH which is likely to prove useful in studying the molecular basis of the interaction between ACTH and its adrenal receptor.