Rat Adrenocortical Cells Research Articles

Morphological evidence of lysosomal uptake of high-density lipoproteins by rat adrenocortical cells in vitro

The high-density lipoprotein (HDL) pathway in rat adrenocortical cells was studied at the electron microscopic level in vitro via colloidal gold labelling. Steroid hormone assays were performed to confirm that the cells remained intact, viable, responsive to ACTH under the applied conditions, and to reveal the steroidogenic effect of HDL. The gold-labelled HDL particles (HDL-Au) were observed on the surface of the parenchymal cells, often attached to the membranes of the microvilli, but rarely in coated pits and coated vesicles. HDL-Au was accumulated by non-coated vesicles, multivesicular bodies and lysosomes. The lysosomes were identified by means of a non-specific esterase reaction. It is concluded that HDL particles are internalized by both zona glomerulosa and zona fasciculata cells, HDL is required for the enhanced functional activity of these cells in long-term incubation, and the lysosomes are involved in the process.

Characterization of corticotropin receptors in human adrenocortical cells.

[125I-Tyr23,Phe2,Nle4]ACTH-(1-38) ([125I]ACTH analog), which is equipotent with ACTH, was used to characterize ACTH receptors in human adrenocortical cells. Adrenals were obtained from brain-dead patients at the time of renal harvest with permission. Binding of [125I]ACTH analog to human adrenocortical cells was highly specific, rapid, reversible, and saturable. Analysis of the inhibition of binding of [125I]ACTH analog by ACTH was compatible with a single class of binding sites with an apparent dissociation constant (Kd) of 1.6 nM and a mean binding capacity of 3560 sites/cell. Concentration-response curves for cAMP and cortisol production were shifted to the left of the binding curve, with ACTH concentrations required for half-maximal stimulation being 20- and 720-fold less, respectively, than those for binding. Extracellular calcium was essential for binding and stimulation of cAMP production. These results indicate that human adrenocortical cells contain a single class of ACTH receptors which are highly similar in affinity, capacity, and calcium requirement to those of rat adrenocortical cells, but differ from the rat receptors in the concentration of ACTH needed for cAMP generation.

Sterol carrier protein2: further evidence for its role in adrenal steroidogenesis.

Homogeneous rat liver sterol carrier protein (SCP2) has been implicated in adrenal steroidogenesis by studies utilizing as a model system various sub-cellular fractions of rat adrenals. Levels of SCP2 were measured in rat adrenal subcellular fractions and various rat tissues using a highly sensitive radioimmunoassay. The levels of SCP2 in various tissues correlate well with the capacity of each tissue to either synthesize or metabolize cholesterol. The high level of SCP2 in adrenal mitochondria (46% of total tissue SCP2) is consistent with its proposed role of enhancing transfer of cholesterol from the outer to the inner mitochondrial membrane. Neither ACTH nor cycloheximide treatment of rats had a significant effect on SCP2 levels or distribution in the adrenal subcellular fractions. Western blot analysis of adrenal subcellular fractions indicates the presence of a protein of identical molecular weight and at least similar antigenicity as homogeneous rat liver SCP2. In the present studies, intact dispersed rat adrenal fasciculata cells fused with liposomal encapsulated anti-SCP2 IgG showed a 40-65% reduction in their ability to produce corticosterone when stimulated with ACTH. The steroidogenic competence of these anti-SCP2 IgG treated cells can be restored by treatment of the cells with liposomal encapsulated SCP2 prior to ACTH stimulation. These findings provide direct evidence for the involvement of SCP2 in ACTH stimulated steroidogenesis in rat adrenocortical cells, and suggests that SCP2 may not be the putative high turnover "labile protein" involved in acute steroidogenesis.

P-161 - Studies on mechanisms of ACTH action : Effect of chloroquine W-7, cyclohex mide and aminoglutethimide on ACTH-induced corticoidogenesis in rat adrenocortical cells

P-161 - Studies on mechanisms of ACTH action : Effect of chloroquine W-7, cyclohex mide and aminoglutethimide on ACTH-induced corticoidogenesis in rat adrenocortical cells

Corticotropin-releasing factor stimulates the release of adrenocorticotropin from domestic fowl pituitary cells.

The effect of synthetic ovine CRF on ACTH secretion of dispersed, domestic fowl pituitary cells was investigated. Cells preincubated for 2 h, 16 h, or after 48-h culture were incubated briefly with CRF (up to 4 h). ACTH was bioassayed using isolated rat adrenocortical cells; ACTH-(1-24) served as the standard for expressing the data. Results with 16-h preincubated cells were as follows: CRF induced ACTH secretion in a concentration-dependent manner: ED50 and maximal stimulatory concentrations were 1.0 nM and 5.0 nM, respectively. CRF (10 nM) induced significant ACTH secretion within 10 min of incubation; maximal secretion (370% over basal value) was attained at 2 h. Dexamethasone (DEX) inhibited basal and CRF-induced ACTH secretion in a concentration-dependent manner; half-maximal inhibitory and maximal inhibitory concentrations were approximately 10 nM and 1 microM, respectively. In addition, DEX (10 microM) acutely (within 2 h) inhibited maximal CRF-induced ACTH secretion by 46%. 8-Bromo-cAMP (1 mM) also induced ACTH secretion, and DEX inhibited this secretion with a potency equivalent to that for CRF-induced ACTH secretion. In contrast to the effect of CRF, high concentrations (100 nM) of ovine LHRH, TRH, and synthetic human pancreatic GH-releasing factor (1-32) failed to induce significant ACTH secretion, thus suggesting that the effect of CRF was peptide specific. Domestic fowl pituitary cells cultured for 48 h before treatment also responded to CRF but not to any greater extent than that of 16-h preincubated cells. In contrast to 16-h preincubated cells or 48-h cultured cells, 2-h preincubated cells had high basal values of ACTH secretion that may have partially diminished or masked the actions of CRF. These data suggest that 1) CRF is a potent and specific stimulator of ACTH secretion by domestic fowl pituitary cells and 2) 16-h preincubated cells or 48-h cultured cells are amenable for other in vitro investigations on the regulation of avian ACTH secretion.

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

Various lipoxygenase (LO) products of arachidonic acid (AA) have been found to have potent biological activities and modulate physiological processes in various cells including endocrine cells. However, no studies concerning LO products in adrenocortical cells have been reported. The present study was performed to investigate LO products in rat adrenocortical cells and its role in ACTH-stimulated adrenal steroidogenesis. LO metabolites produced in ACTH-stimulated rat adrenocortical cells prelabeled with [ 3H]AA was analyzed by reverse phase and straight phase HPLC and two 5-LO products, 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B 4 (LTB 4) were identified. ACTH-induced 5-HETE and LTB 4 production in adrenal cells was dose dependently inhibited by AA861, a specific inhibitor of 5-LO. AA861 recued ACTH-stimulated corticosteroid production without any change in cyclic AMP formation, while indomethacin did not affect both corticosteroid and cyclic AMP production. Reduced steroidogenesis by AA861 was reversed by the addition of 5-hydroperoxyeicosatetraenoic acid (5-HPETE). Also exogenously added 5-HPETE dose dependently augementd ACTH-stimulated corticosteroid production without any concomitant change in cyclic AMP production. However, 5-HETE and LTB 4 had no such effect. These results indicate that 5-LO pathway is present in rat adrenocortical cells and its metabolites, most likely 5-HPETE, may play an important role in adrenal steroidogenesis.

Effects of heavy metals on corticosteroid production in cultured rat adrenocortical cells

Application of 10 −4 m Zn, 10 −5 m Ba, 10 −6 m Cd, 10 −6 m Hg, and 10 −6 m Mn did not affect the adrenocorticotrophic hormone (ACTH)-induced steroid production in cultured adrenocortical cells. The application of 10 −5 m Pb significantly reduced the ACTH-induced steroid production in cultured cells. However, Pb did not reduce the steroidogenesis induced by dibutyryl cyclic AMP (Dbc-AMP), suggesting that the plasma membrane is the site where Pb interferes with steroid production. The morphological changes induced by the addition of ACTH or Dbc-AMP plus the test metals were similar to those induced by ACTH or Dbc-AMP alone.

Phospholipids, sterol carrier protein 2 and adrenal steroidogenesis

Rat adrenocortical cells and preparations of plasma membrane and mitochondria have been employed to assess the effects of phospholipids and of sterol carrier protein 2 (SCP 2) on specific aspects of adrenal steroidogenesis. With intact cells, liposomal dispersions of cardiolipin caused significant stimulation of corticosterone output, while preparations of phosphatidylcholine, phosphatidylinositol, or the 4'-phosphate and the 4',5'-diphosphate derivatives of phosphatidylinositol were without effect. With the adrenal plasma membrane preparation, none of the added phospholipids affected either sodium fluoride or ACTH-responsive adenylate cyclase activity. With intact mitochondria, only cardiolipin, among the various phospholipids, tested, caused a concentration-dependent stimulation of pregnenolone production. However, even at the highest concentration of cardiolipin tested (500 μM), the stimulatory effect was only half that observed with 0.7 μM SCP 2, and the two effectors were not synergistic. SCP 2 caused a redistribution of cholesterol from mitochondrial outer to inner membranes, while cardiolipin, which is an activator of cytochrome P-450 scc , had no effect on distribution of mitochondrial membrane cholesterol.

Toxaphene: Accumulation in the adrenal cortex and effect on ACTH-stimulated corticosteroid synthesis in the rat

Uptake and distribution of [ 14C]toxaphene was studied in the adrenals of rats using whole-body autoradiography. An accumulation of radioactivity was seen in the adrenal cortex (zona fasciculata) 1–24 h after a single gavage of [ 14C]toxaphene (16 mg/kg b.w.). In in vitro studies toxaphene was found to inhibit ACTH-stimulated corticosterone synthesis in the cultured rat adrenocortical cells (IC 50 2.8 × 10 −5M). Moderate but signficant inhibition ( P < 0.001) of ACTH-stimulated corticosterone synthesis was also observed in the adrenocortical cells isolated from rats after a prolonged exposure (5 weeks) to low levels (1.2 ppm) of toxaphene in feed. The results indicate a direct adrenotoxic effect of toxaphene.

Sources of Extramitochondrial Corticoidogenic Cholesterol in the Adrenal Cortex

Intracellular sources of extramitochondrial corticoidogenic cholesterol in bovine, rat and hamster adrenocortical cells were examined in vitro by comparing the species differences in the effects of various inhibitors on the adrenocorticotropic .hormone (ACTH)-induced corticoidogenesis. The inhibitors were ML-236B (3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor), W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide; calmodulin inhibitor), dichlorvos (O,O-dimethyl-2,2-dichlorovinyl phosphate; organic phosphorylation inhibitor), chloroquine ((7-chloro-4-4-diethylamino-1-methyl-butylamino) quinoline; lysosomal enzyme inhibitor) and cycloheximide (protein synthesis inhibitor). During 2 to 3 hr incubation periods, the ACTH-induced corticoidogenesis was not inhibited by ML-236B (100 μM) in the bovine and rat adrenocortical cells. In the hamster adrenocortical cells, ML-236B (100 μM) did not affect the ACTH-induced corticoidogenesis during the initial 1 hr incubation periods; but thereafter, the ACTH-induced corticoidogenesis during the subsequent 2 hr incubation periods was completely blocked by ML-236B. The ACTH-induced corticoidogenesis was inhibited by W-7 (up to 25 μM) in the bovine and rat adrenocortical cells, but this was not the case in the hamster cells. Chloroquine (up to 400 aM) inhibited the ACTH-induced corticoidogenesis in the adrenocortical cells of three different species, but the hamster adrenal cells were much more vulnerable than the bovine and rat cells. The ACTH-induced corticoidogenesis in the adrenocortical cells of three different species were equally inhibited by cycloheximide (up to 1 mM). It could be deduced from the present data that intracellular sources of corticoidogenic cholesterol during the ACTH-induced corticoidogenesis in vitro are mainly lysosomes and de novo synthetized cholesterol in the hamster cells, and the main sources are lipid droplets and lysosomes in the rat and bovine cells, respectively.

Effect of P-450scc inhibitors on corticosterone production by rat adrenal cells.

Suspensions of rat adrenocortical cells produce corticosterone as the major glucocorticoid. Cholesterol side-chain cleavage, the initial and rate-limiting step in the glucocorticoid biosynthetic pathway, is catalyzed by P-450scc. We have examined the effect of a variety of P-450scc inhibitors on corticosterone production by isolated rat adrenocortical cells. These inhibitors include reversible, noncovalently interacting inhibitors as well as mechanism-based inhibitors which irreversibly inactivate P-450scc in vitro. (20S)-22-nor-22-thiacholesterol and (22R)-22-aminocholesterol cause 50% inhibition of corticosterone production at 4 microM and 30 nM, respectively. Inhibition by these compounds was essentially not time-dependent. (20R)-20-(1-hexynyl)-pregn-5-en-3 beta, 20-diol and (20R)-20-(1,5-hexdiynyl)-pregn-5-en-3 beta, 20-diol at 10 microM inhibited corticosterone production in a time-dependent manner, resulting in 30% inhibition of corticosterone production during a 100-min incubation. (20S)-20-(2-trimethylsilyl ethyl)-pregn-5-en-3 beta, 20-diol inhibited in a strongly time-dependent manner. At 10 microM this compound irreversibly inhibited more than 90% of the side-chain cleavage capacity of the cell during a 40-min incubation. Cells treated with this steroid did not regain their capacity for side-chain cleavage after removal of free steroid. None of the inhibitors described above inhibited production of corticosterone by cells supplied with pregnenolone, the product of the P-450scc reaction. We suggest that the only significant effect of these compounds under these conditions is inhibition of the side-chain cleavage enzyme.

Self-Suppression of Corticosteroidogenesis: Evidence for a Role of Adrenal 5α-Reductase*

Exogenous corticosterone (B), the natural glucocorticoid product of rats, suppressed endogenous B production of isolated rat adrenocortical cells induced by alpha ACTH-(1-24), [9-tryptophan (O-nitrophenylsulfenyl)]ACTH-(1-24) [( Trp (Nps)9]ACTH-(1-24], and cAMP as well as pregnenolone supported-steroidogenesis. This self-suppression occurred within 2 h. It was dependent on the concentration of exogenous B. However, self-suppression did not alter the half-maximal steroidogenic concentration (ED50) of each steroidogenic agent. In addition, exogenous B did not suppress ACTH-induced cAMP production or gross protein synthesis, as measured by leucine incorporation into bulk cellular proteins. These results with isolated cells suggested at least two mechanisms for self-suppression: 1) exogenous B inhibited steroidogenic steps in a noncompetitive manner, and/or 2) exogenous B induced B degradation. In this study we examined the effect of exogenous B on the degradation of B. Accordingly, we measured the adrenal 5 alpha-reductase activity (5 alpha RA) of cell homogenates prepared from treated cells. Isolated adrenocortical cells were incubated for 2 h with alpha ACTH-(1-24), ovine PRL (oPRL), and B. They were then homogenized and assayed for 5 alpha RA, as indicated by the disappearance of exogenous B, as shown by RIA. In addition, the percentage of exogenous tritium-labeled B [( 3H]B) converted to 5 alpha-dihydrocorticosterone (DHB), the principal reduced metabolite of B, was determined by TLC. Isolated adrenocortical cells from intact rats showed insignificant 5 alpha RA and DHB formation when incubated with or without alpha ACTH-(1-24) and with or without oPRL. However, with exogenous B, there was significant 5 alpha RA and DHB formation. oPRL plus B decreased DHB formation. The effects of B and oPRL were more demonstrable with cells from hypophysectomized rats. These cells exhibited high 5 alpha RA and DHB formation; exogenous B increased these values, whereas oPRL acutely reversed the effects of hypophysectomy and exogenous B. In other work avoiding cell homogenization, exogenous B suppressed ACTH-induced B accumulation and increased DHB formation in intact cell suspensions from intact rats and intact male domestic fowl. Furthermore, exogenous B increased the conversion of [3H] pregnenolone to DHB in intact cell suspensions from intact rats, showing that B synthesized de novo as well as exogenous B can be degraded during self-suppression. These data indicate that acute self-suppression of corticosteroidogenesis is at least partly mediated by an increase in 5 alpha RA.

Characterization of high density lipoprotein binding activity in rat adrenocortical cells.

Rat adrenocortical cells take up high density lipoprotein cholesterol for use as steroidogenic substrate. To better understand this unique uptake process, we have first characterized HDL binding. Infusion of human 125I-labeled HDL into rats pretreated with 4-APP demonstrated that the adrenal and ovary accumulate HDL in a saturable fashion in vivo. Subsequent studies using isolated rat adrenocortical cells demonstrated that cellular uptake of HDL is comprised of two events. One event is characterized by reversible membrane binding and is complete by 60 min (t1/2 = 20 min). The second event is marked by irreversible apoprotein accumulation which continues for at least 3 hr. Reversibly bound material exhibits the same apoprotein distribution as unincubated HDL. Irreversible accumulation could not be attributed to internalization or lysosomal accumulation inasmuch as it also occurred with partially purified plasma membranes and was not enhanced by addition of chloroquine. Reversible binding of human HDL3 exhibited a saturable dependence on concentration (Kd = 27 micrograms protein/ml; N = 3.0 X 10(6) sites/cell) similar to that previously reported for rat liver, ovary, and testis. Cell accumulation of HDL decreased by over 80% at 4 degrees C compared to 37 degrees C, did not require calcium, and was not diminished by prior cell treatment with trypsin or pronase. These results indicate that rat adrenocortical cells possess plasma membrane recognition sites for HDL with different properties than those of the LDL receptor. Moreover, adrenal accumulation of HDL apoproteins does not lead to secondary lysosome formation.

Mechanisms of adrenocortical depression during Escherichia coli shock.

The response of the adrenal cortex to corticotropin during sepsis is variable. We have previously demonstrated a significant decrease of corticosterone production by rat adrenocortical cells in response to corticotropin stimulation after incubation with septic shock plasma (SP) as compared with control plasma (CP). We have studied the mechanisms of this depression. The following defects were demonstrated. (1) Cells bound less radioiodinated corticotropin analog after SP treatment (2.9 +/- 0.4 femtomoles/50 micrograms DNA) than after CP treatment (6.4 +/- 0.3 fmole/50 micrograms DNA). (2) Cyclic adenosine monophosphate (cAMP) production was less after SP treatment (59.3 +/- 4 pmole per 10(5) cells per two hours) compared with CP treatment (110.3 +/- 11.3 pmole per 10(5) cells per two hours). (3) Exogenously added dibutyryl cAMP was unable to correct the defect in corticosterone production after SP treatment (4.96 +/- 0.7 micrograms/24 hr) as compared with CP treatment (6.99 +/- 0.5 micrograms/24 hr). Our studies suggest this defect is located in the synthesis of pregnenolone from cholesterol. These mechanisms may be responsible for the low cortisol levels previously observed in humans during septic shock.

Intramitochondrial localization of glutathione-peroxidase (GSH-PO) in the rat adrenocortical cells.

Immunocytochemical localization of glutathione-peroxidase (GSH-PO) in the rat adrenocortical cells was observed not only in cytosol (CYTOSOL GSH-PO) but in mitochondria (MITOCHONDRIAL GSH-PO) by the direct peroxidase labeled antibody method using anti-GSH-PO IgG Fab' fragment. In the untreated rat adrenocortical cells, CYTOSOL GSH-PO was observed mainly in the outer part of the zona fasciculata and zona reticularis. MITOCHONDRIAL GSH-PO was observed mainly in the inner part of zona fasciculata. Biochemical GSH-PO activity in the cytosol and mitochondrial fractions obtained from whole adrenal gland was comparable to the immunocytochemical results. In the hypophysectomized rat adrenocortical cells, markedly atrophic inner part of zona fasciculata showed only CYTOSOL GSH-PO, and biochemical activity of the cytosol GSH-PO was markedly increased. It has been reported by us that this atrophic zone contains accumulated cholesterol esters. By ACTH administration to the hypophysectomized rats, markedly, hypertrophic outer part of zona fasciculata showed increased immunocytochemical localization of MITOCHONDRIAL GSH-PO. And biochemically, mitochondrial GSH-PO activity in this group was higher than that of the hypophysectomized group.These results suggest that the CYTOSOL GSH-PO has its role in the reduction of cholesterol ester hydroperoxides and that the MITOCHONDRIAL GSH-PO in the reduction of sterol hydroperoxides produced during steroidogenesis.

Correlation between intramitochondrial localization of glutathione-peroxidase (mitochondrial GSH-PO) and steroidogenesis in the rat adrenocortical cells.

In order to confirm the relationship between mitochondrial GSH-PO and steroidogenesis in the rat adrenal cortex, we have studied the immunocyto-chemical localization of GSH-PO in the rat adrenocortical cells of Metopiron administration, which blocks the conversion of deoxycorticosterone to corticosterone. In the control rat adrenocortical cells, mitochondrial GSH-PO was mainly localized in lipid-depleted compact cells, of inner fasciculata cells, but not zona glomerulosa and zona reticularis cells. By Metopiron administration, the adrenal cortex was markedly hypertrophic. Immunohistochemically, GSH-PO was observed in outer fasciculata cells and intracellular localization of GSH-PO was mainly observed in cytosol (cytosol GSH-PO). However, mitochondrial GSH-PO was less than that in the control rat adrenocortical cells.Based on our findings, it is strongly suggested that mitochondrial GSH-PO may be a very important enzyme for steridogenesis, especially, corticosterone biosynthesis.

Corticotropin receptors, cyclic AMP and steroidogenesis.

The detection and characterization of the physiologically relevant receptors for corticotropin (ACTH) in rat adrenocortical cells is described. By the use of a radioligand with full biological potency and high specific radioactivity (1800 +/- 75 Ci/mmol), a single class of receptors with an apparent Kd of 1.41 +/- 0.21 nM was detected and the number of sites was estimated to be 3840 +/- 1045 per cell. The binding curve was superimposable on the concentration-response curve for cAMP but dissociated from that for steroidogenesis. These data are best explained by the receptor-reserve model in which occupancy of a small fraction of the receptors is sufficient for inducing maximal steroidogenesis.

Characterization of high density lipoprotein binding activity in rat adrenocortical cells.

Characterization of high density lipoprotein binding activity in rat adrenocortical cells.

Effect of growth hormone and corticotropin on steroidogenesis in cultured rat adrenocortical cells.

Primary cultures of rat adrenocortical cells responded to corticotropin (ACTH; 10 microU/ml) with peak steroid production within 24 h which declined thereafter. In the presence of ACTH and growth hormone (GH; 10 micrograms/ml), steroid production was significantly greater than with ACTH alone and was better maintained over several days. This latter response was not due to changes in cell number or multiplication and required several days to develop. GH also interacted with 10(-6) and 10(-5) M dibutyryl cyclic AMP (dbcAMP) to augment synthesis of corticosterone. At maximal doses of both ACTH and dbcAMP, GH did not have an additional effect on steroid production. In conclusion, GH has a stimulatory effect on steroid production when added in vitro but it is unlike the response seen in vivo in that it is less sensitive, additive rather than synergistic, and without effect on cell growth and multiplication.

The role of serum high density lipoproteins in adrenal steroidogenesis.

The rat adrenal cortex possesses at least two mechanisms for uptake of extracellular lipoprotein cholesterol; one is receptor mediated endocytosis and the second is a non-endocytotic pathway. The latter was revealed by the ability of lipoproteins such as HDL which do not possess apo B or apo E to enhance steroid hormone output. Rat adrenocortical cell uptake of HDL cholesterol is saturable and differs for unesterified cholesterol and cholesterol esters. In addition rat adrenocortical cells possess ACTH regulated HDL binding sites although the relationship of HDL receptor activity to cholesterol uptake remains uncertain. Further elucidation of the "HDL pathway" in the rat adrenal cortex may have biologic significance beyond understanding adrenal function since many non-steroidogenic tissues also possess HDL receptors and in man circulating levels of HDL-cholesterol are a strong inverse risk factor for accelerated atherosclerosis.