Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells. 2. Reversibility of taxol's inhibition of basal and ACTH-induced steroidogenesis is unaccompanied by reversibility of taxol-induced changes in cell ultrastructure

Taxol inhibits the basal and ACTH-stimulated steroidogenesis of cultured mouse adrenocortical tumor cells, presumably by preventing the arrival of cholesterol in mitochondria. In these cells, taxol polymerizes and rearranges microtubules, disperses SER masses, disrupts the Golgi, and impedes the formation of cholesterol-containing lysosomes. However, taxol's alterations in ultrastructure appear likely to permit both a microtubule-based organelle transport proposed to bring mitochondria of unstimulated cells close to alternate sources of cholesterol--the SER and lipid droplets--and postulated ACTH-caused increases in these encounters. Conceivably, taxol may prevent the transfer of cholesterol from the SER and lipid droplets to mitochondria, once the meetings are achieved. To investigate this possibility, we determined the reversibility of taxol's ultrastructural effects and inhibition of steroidogenesis. Primary cultured adrenal tumor cells were incubated for 4 hr with and without ACTH (10 mU/ml). with taxol (50 micrograms/ml), and with ACTH and taxol 50 simultaneously. Some cultures from each set were washed with fresh medium and re-incubated for 1.5 hr. with and without ACTH. Media taken from cultures at the ends of pre- and post-washout incubations were analyzed for the presence of secreted steroids. Sample cultures were fixed for electron microscopy at the ends of both incubations. Data derived from pre-washout incubations confirmed previous reports of taxol's ultrastructural changes and inhibition of steroidogenesis. When cells recovered from taxol in the absence of ACTH, the inhibition of steroidogenesis was completely reversed. In the presence of ACTH, ex-taxol-treated cells demonstrated a "rounding up' and an increased steroid production that are characteristic responses to the hormone. However, in all cases, there was a persistence of taxol's alterations in organelle numbers and arrangements. Our findings establish that the ultrastructural effects of taxol which we recorded cannot prevent mitochondria of unstimulated and ACTH-stimulated adrenal tumor cells from gaining cholesterol. They strengthened the possibility that in pre-washout incubations, taxol allowed organelle motility to bring mitochondria adjacent to cholesterol-containing SER tubules and lipid droplets, but inhibited steroidogenesis by preventing the cholesterol transfer. Taxol might limit the availability of a protein required for the transfer, an effect not visible in our electron micrographs.     

Cinemicrographic observations of cultured adrenocortical tumor cells. Dynamic responses to ACTH and cytochalasin B

ACTH increases the basal steroidogenic activity of cultured adrenocortical tumor cells, whereas moderate-high doses of cytochalasin B (CB) inhibit both basal and ACTH-induced steroidogenesis. Previous ultrastructural studies have revealed that ACTH rearranges microfilaments in these adrenal cells, whereas CB causes microfilaments to aggregate into felt-like masses. It has been postulated that the ACTH effects may facilitate organelle motility and increase organelle interactions that are required for steroid biosynthesis, and that the CB-created "foci" may impede or prevent the organelle meetings. To shed light on these possibilities, we have employed 16 mm cinemicrography of unstimulated adrenal tumor cells and cells incubated for 1-2 h with ACTH (10 mU/ml), or low (10 micrograms/ml), or high (50 micrograms/ml) doses of CB. ACTH caused initial increases in membrane ruffling and a "flurry" of particle (organelle) activity above that seen in unstimulated cells. The stimulated cells then retracted from each other and began their characteristic "rounding up" in response to the hormone. Particles appeared to move towards the nucleus, and in fully-rounded cells were extremely congested. Steroid production rose several fold above basal levels. CB10 produced slight-marked cell convexities, nearly stopped particle motility and inhibited steroid production moderately. CB50 produced an asymmetrical, spidery cell form, stopped membrane ruffling and particle motility and abolished steroidogenesis. After a washout of CB50, particle motility resumed nearly immediately. Our CB data indicate that associations between particles, presumably between mitochondria and various sources of cholesterol, are prerequisite for basal steroidogenesis in the adrenal tumor cells. In ACTH-stimulated cells, increases in steroid output correspond with increased opportunities for particle associations. These opportunities appear to arise directly or indirectly from ACTH effects on microfilaments. The responses of microfilaments to the hormone may be particularly intense in tumorous forms. By these means, the cells may express their differentiated function, although their cytoplasm has a distinctly unspecialized appearance.     

Cholesterol accumulation in adrenocortical mitochondria after ACTH-stimulation

Rat adrenocortical cell suspensions (10(6) cells) were incubated with ACTH (40 nM) in 2 ml of Krebs-Ringer bicarbonate buffer for 90 min. About 42 nmol of corticosterone and 14 nmol of 18-hydroxydeoxycorticosterone were generated and released into the medium. Aminoglutethimide at 50 microM inhibited the steroidogenesis to 16%. Mitochondrial pellets were prepared from the cells incubated in the absence, or in the presence, of ACTH and aminoglutethimide, and cholesterol content was determined. The mitochondria of the cells incubated without the drugs contained 25.2 micrograms cholesterol/mg protein. Cholesterol content increased by 10% in the mitochondria of the ACTH-stimulated cells. The mitochondria of the cells incubated in the presence of both ACTH and aminoglutethimide contained 143% of cholesterol compared to those of the nontreated cells. When rats were subjected to ether stress after aminoglutethimide pretreatment, cholesterol content of the mitochondrial fraction increased to about 200% compared to that of the control rats. These results suggest that a cholesterol pool exists in the adrenocortical mitochondria and that the amount increases during the steroidogenic stimulation of the cells. The mitochondria were fixed with filipin-containing fixative and examined by freeze-fracture electron microscopy. Accumulations of filipin-cholesterol complexes were observed in the inner membrane of the mitochondria as protuberances or pits 25 nm in diameter.     

Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity

The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.     

Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

Summary The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.     

Comparison of the structural features of corticotropin required for stimulation of steroidogenesis and cAMP production in rat and rabbit adrenocortical cells

The steroidogenic activities of ACTH, alpha-MSH, beta-MSH as well as analogs of the hormones have been compared in rat and rabbit adrenocortical cells. ACTH is equally active in both species and the melanotropins have very low steroidogenic potency in either species. The steroidogenic potencies of the peptide analogs are strikingly similar in the two species, suggesting that the structural requirements for eliciting steroidogenesis are the same in rat and rabbit adrenocortical cells. The analog NPS-ACTH has low, comparable steroidogenic activity in both species. NPS-ACTH is a potent antagonist of ACTH-induced cAMP production in rat adrenocortical cells but acts as a weak partial agonist in rabbit adrenocortical cells. These results suggest that steroidogenesis may be mediated by receptors different from those involved in the cAMP response observed at supraphysiological concentrations of ACTH.     

Influence of E. coli endotoxin on ACTH induced adrenal cell steroidogenesis

The effect of endotoxin (lipopolysaccharide from E. coli) on isolated adrenocortical cells was examined. Lipopolysaccharide decreased the ACTH-induced steroidogenesis. This effect was shown by all corticotropin concentrations studied, and the longer the incubation time, the higher the effect produced. The rate of decrease of ACTH-induced steroidogenesis was dependent on the concentration of lipopolysaccharide in the medium. Binding of [125I]ACTH to adrenocortical cells was modified by lipopolysaccharide; this modification was related to a decrease of the ACTH-induced steroidogenesis. This effect supports the hypothesis of a direct interaction between lipopolysaccharide and the cell membrane with a concomitant distortion of the cell surface affecting the ACTH receptor sites of their environment. [14C]Lipopolysaccharide binds to isolated adrenocortical cells. Binding specificity was investigated by competitive experiments in the presence of various types of endotoxins, polypeptide hormones and proteins. Unlabelled lipopolysaccharide from the same bacterial strain and isolated under identical conditions than the labelled lipopolysaccharide exerted the strongest inhibitory activity. Unlabelled lipopolysaccharide of various strains different from that originating the labelled lipopolysaccharide exerted the less displacement. It would imply a certain kind of specificity but the decrease in the binding of lipopolysaccharide produced by ACTH and glucagon suggests the existence of non-specific interactions between lipopolysaccharide and cell membrane.     

A quantitative electron microscopic analysis of the membrana granulosa of rat preovulatory follicles

The ultrastructure of the membrana granulosa (MG) of rat preovulatory follicles was examined using stereological techniques. Organelles studied were nuclei, mitochondria, lipid droplets (LD), lysosomes, and smooth and rough endoplasmic reticulum (SER, RER). The peripheral region of the MG contained the greatest volume of mitochondria, LD and SER, organelles associated with steroidogenesis. The volume of RER, an organelle associated with protein production, was greatest in the cumulus oophorus. These results corroborate previous analyses and demonstrate that the rat MG is composed of discrete subregions.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

ACTH regulation of cholesterol movement in isolated adrenal cells

Confluent bovine adrenal cell primary cultures respond to stimulation by adrenocorticotropin (ACTH) to produce steroids (initially predominantly cortisol and corticosterone) at about one-tenth of the output of similarly stimulated rat adrenal cells. The early events of steroidogenesis, following ACTH stimulation, have been investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-6-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide (AMG), even though cytochrome P-450scc only increases after 12 h. Mitochondrial-free cholesterol levels increased during the same time period (16.5-25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. More prolonged exposure to ACTH prior to addition of AMG caused the elevation in mitochondrial cholesterol to more than double, possibly due to enhanced binding capacity. Early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial-free cholesterol. The maximum rate of cholesterol transport to mitochondria in AMG-blocked cells was consistent with the maximum rate of cellular steroidogenesis. Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial-free cholesterol. Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8) M), for 24 h prior to ACTH addition decreased the rates of ACTH- and cAMP-stimulated steroidogenesis but did not affect the basal rate. In both cases, the effectiveness of TCDD increased with time of exposure to the stimulant. Although cholesterol accumulated in the presence of ACTH and AMG (13-28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13-8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). Since even in the absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h) TCDD pretreatment may increase the presence of a protein(s) that cause this mitochondrial-cholesterol depletion following stimulation by ACTH or cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Golgi apparatus and adrenal function: the effects of monensin on adrenocorticotropic hormone-stimulated steroidogenesis

It has previously been shown that the steroidogenic action of adrenocorticotropic hormone (ACTH) is accompanied by characteristic alterations in cell ultrastructure. These include hypertrophy of the Golgi complex associated with increased vesicle formation and striking elevations of acid phosphatase activity in the Golgi complex and lysosomes. To investigate a possible relationship of these phenomena to steroidogenic function in monolayer cultures of murine adrenal tumor cells, monensin, a carboxylic ionophore which disrupts the ordered structure and transport function of the Golgi complex, was used. Monensin, at a concentration of 1.2 microM, causes massive vacuolization and hypertrophy of the Golgi complex. No effect on mitochondrial structure was seen. Monensin, 0.6-1.2 microM, inhibits both ACTH-stimulated and basal steroidogenesis by approximately 50% in incubations of 4 h or less. Dibutyryl-cAMP-stimulated steroidogenesis was inhibited to a similar degree. Incubations were carried out in serum-free media to eliminate possible effects due to exogenous cholesterol transport into the cell. There were no direct inhibitory effects of monensin on cholesterol side-chain cleavage (SCC) activity in isolated mitochondria. In contrast, mitochondria isolated from cells previously treated with monensin had a reduced capacity for this activity. These experiments suggest that monensin inhibits transport of cholesterol from the Golgi complex to the mitochondrial site of steroidogenesis action or interferes with the transport of key mitochondrial proteins synthesized on cytoplasmic ribosomes.     

Glucocorticoid control of steroidogenesis in isolated rat adrenocortical cells

The role of end-product glucocorticoids in the regulation of corticosteroidogenesis in isolated adrenocortical cells was investigated. Trypsin-isolated cells from male rat adrenal glands were incubated with or without corticotropin (ACTH) and with or without corticosterone. Endogenous corticosterone production was determined by radioimmunoassay at the end of incubation. Cessation of ACTH-induced corticosterone production was apparent after 2-4 h of incubation. The suppression occurred later with lower cell concentrations. Corticosterone production was partially restored after washing the suppressed cells. Supernatant fluid from suppressed cell suspensions also suppressed steroidogenesis of a fresh population of cells. However, the suppressing property of the supernatant fluid was abolished after the removal of corticosterone by charcoal-dextran treatment, suggesting that corticosterone or other steroids caused the suppression. Exogenous corticosterone induced suppression over a wide range of ACTH concentrations, but did not change the half-maximal steroidogenic concentration of ACTH, indicating that the suppression does not change the sensitivity of the cells to ACTH. Suppression occurred within 30-60 min after corticosterone had been added to the incubation medium either at the start of incubation or while steroidogenesis was in progress. Suppression varied directly with the concentration of exogenous corticosterone. These data indicate that glucocorticoids can directly and acutely suppress corticosteroidogenesis and thus control adrenocortical function in concert with other regulators such as ACTH and Ca2+.     

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

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

The effects of taxol, a microtubule-stabilizing drug, on steroidogenic cells

The effects of taxol on steroid production and microtubule polymerization were examined using Y-1 adrenocortical tumor cells, MLTC-1 Leydig tumor cells, and primary cultures of bovine adrenocortical cells. Taxol inhibited the following steroidogenic processes within the Y-1 and MLTC-1 cells: (1) hormonal increase of steroid production, (2) dibutyryl cyclic AMP-increased steroid production, and (3) hormone-stimulated pregnenolone production. The inhibitory action of taxol was concentration dependent and also resulted in an increase in cytoplasmic microtubules. In addition, the inhibitory action of taxol on hormone-stimulated steroid production was reversible. Taxol appeared to inhibit cholesterol movement to the mitochondrial site of cholesterol side-chain cleavage enzyme but did not affect overall protein synthesis. Interestingly, taxol did not affect hormone-stimulated steroid production in bovine adrenocortical cells. This lack of inhibition may correspond to the ultrastructural observation that microtubule bundling after taxol treatment was observed in the tumor cells but not in similarly treated bovine adrenal cells. With this conflicting information between cell types, a direct relationship between taxol treatment and inhibition of steroid production has not been established. However, these results suggest that taxol alters the rate of transport of cholesterol to the cholesterol side-chain cleavage enzyme within the steroidogenic tumor cells.     

Dissection of the signaling mechanism for capsule detachment of lipid droplets in rat adrenocortical cells

In a previous study, we used a monoclonal antibody, A2, to demonstrate the presence of the lipid droplet-specific capsule in adrenocortical cells and the stimulation of steroid secretion with adrenocorticotrophic hormone (ACTH), resulting in the detachment of this capsule from the droplet surface into the cytosol. To investigate the signaling pathway for this event, we tested the role of adenylate cyclase, cAMP, and protein kinases A and C (PKA and PKC) in this response. ACTH-induced decapsulation of lipid droplets was blocked by either adenylate cyclase inhibitor or PKA inhibitor and stimulated by Bt₂cAMP. We conclude that the signaling mechanism involved in lipid droplet decapsulation is the cascade consisting of adenylate cyclase activation, cAMP elevation, and subsequent PKA activation. Furthermore, the cytosolic detached capsular protein was able to relocate to the lipid droplet surface on cessation of ACTH or Bt₂cAMP stimulation. In addition to PKA-mediated decapsulation, inhibition of PKC by calphostin C alone was enough to induce decapsulation, a process that was independent of PKA activity, whereas activation of PKC could prevent Bt₂cAMP-induced decapsulation. A cAMP radioimmunoassay also confirmed that ACTH caused a marked increase in intracellular levels of cAMP, while PMA or calphostin C caused no significant changes. We conclude that PKA and PKC are reciprocally operated to regulate the decapsulation of lipid droplets, the same mechanism adopted in steroidogenesis. A time-course study also indicates that decapsulation of lipid droplets was accompanied by detectable changes in the size and the area of lipid droplets upon the stimulation of Bt₂cAMP or calphostin C, implying a possible coupling between the capsule detachment and steroidogenesis. J. Cell. Biochem. 65:67–74. © 1997 Wiley-Liss, Inc.     

The role of the cytoskeleton in the regulation of steroidogenesis

The slow step in steroid synthesis involves the transport of cholesterol from lipid droplets in the cytoplasm to the first enzyme in the pathway—the cytochrome P450 that converts cholesterol to pregnenolone (P450scc) which is located in the inner mitochondrial membrane. ACTH stimulates this intracellular transport of cholesterol in adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells) and this effect of the trophic hormone is inhibited by cytochalasins, by anti-actin antibodies and DNase I suggesting that the response to ACTH requires a pool of monomeric (G-) actin that can be polymerized to F-actin. Recent studies have shown that lipid droplets and mitochondria of adrenal cells are both attached to intermediate filaments. Moreover ACTH reorganizes the cytoskeleton and changes the shape of the cell. These observations suggest a mechanism for transport of cholesterol that involves reorganization and contraction of actin microfilaments which may, in turn, cause movement of droplets and mitochondria together through their common attachment to intermediate filaments.     

Mechanism of colchicine-induced steroidogenesis in rat adrenocortical cells

Conflicting data for the effects of colchicine on cholesterol transport and steroidogenesis raise the question of the role of microtubules in cholesterol transport from the lipid droplet to mitochondria in steroidogenic cells. In this study, using corticosterone radioimmunoassay and immunofluorescence microscopy, we re-evaluated the effects of colchicine on hormone production and morphological changes of lipid droplets' and studied the signaling pathway involved in colchicine-induced steroidogenesis. Colchicine stimulated steroid production in a dose- and time-dependent manner. The structural integrity of both the microtubules and the lipid droplet capsule was destroyed by colchicine treatment. Disruption of the lipid droplet capsule occurred later than microtubule depolymerization. After cessation of colchicine treatment and a 3 h recovery in fresh medium, capsular protein relocated to the droplet surface before the cytoplasmic microtubule network was re-established. beta-lumicolchicine, an inactive analogue of colchicine, disrupted the capsule and increased hormone production without affecting microtubular structure. Thus, microtubule depolymerization is not required for the increase in steroid production and capsular disruption. To explore the signaling pathway involved in colchicine-induced steroidogenesis, we measured intracellular cAMP levels. Unlike ACTH, colchicine did not increase cAMP levels, suggesting that the cAMP-PKA system is not involved. Colchicine and ACTH had additive effects on corticosterone production, whereas colchicine and PMA did not, implying that part of the PKC signaling mechanism may be involved in colchicine-induced steroidogenesis. Cycloheximide, a protein synthesis inhibitor, completely inhibited colchicine-induced steroidogenesis and capsular disruption. These results demonstrate that the steroid production and lipid droplet capsule detachment induced by colchicine are both protein neosynthesis-dependent and microtubule-independent.     

Inhibition of ACTH action on cultured bovine adrenal cortical cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin through a redistribution of cholesterol

The conversion of cholesterol to cortisol by cultured bovine adrenal cortical cells is stimulated 6-fold by adrenocorticotropin and is limited by the movement of cholesterol to the mitochondria (DiBartolomeis, M.J., and Jefcoate, C.R. (1984) J. Biol. Chem. 259, 10159-10167). Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8)M), for 24 h prior to adrenocorticotropin (ACTH) addition decreased the rate of ACTH-stimulated steroidogenesis but did not affect the basal rate. TCDD was more effective against stimulation at 10(-11) M ACTH (4-fold) than at 10(-7) M ACTH (10%), consistent with an increase in EC50 for ACTH. Stimulation of bovine adrenal cortical cells by cAMP was similarly decreased by TCDD. In both cases the effectiveness of TCDD increased with time of exposure to the stimulant. The transfer of cholesterol to mitochondria in intact cells was quantitated by means of the 2-h accumulation of mitochondrial cholesterol in the presence of aminoglutethimide, an inhibitor of cholesterol side chain cleavage. Although cholesterol accumulated in the presence of ACTH (13 to 28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13 to 8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). In absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h). It is concluded that TCDD pretreatment may increase the presence of a protein(s) that cause mitochondrial cholesterol depletion when the cells are stimulated by ACTH or cAMP. TCDD-enhanced cholesterol efflux from mitochondria diminishes cholesterol side chain cleavage when mitochondrial cholesterol is sufficiently depleted (after 2-4 h).     

Cytological differentiation of the interrenal tissue of the Japanese quail,Coturnix coturnix

Summary As reported for several other avian species there are clearly distinguishable subcapsular (SCZ) and inner (IZ) zones of interrenal tissue in the Japanese quail. The SCZ contains large columnar cells (type I) with rounded nuclei, polymorphic mitochondria with shelf-like cristae, and relatively small numbers of lipid droplets. The IZ contains two and possibly three types of cells. Type II consists of large columnar cells with moderately dense cytoplasm containing large numbers of lipid droplets and many rounded mitochondria with tubular cristae. Smooth endoplasmic reticulum (SER) and Golgi apparatus are well developed; coated vesicles occur in the Golgi area and at the cell surface. Type-III cells occur in IZ and especially in its more peripheral areas. They are columnar cells with strikingly clear cytoplasm (in comparison with type II) containing mitochondria with plate-like cristae and tubular SER. Type-IV cells are sparsely distributed in IZ and occur rarely in SCZ. Type IV may be a degenerating phase of type III.After adenohypophysectomy or section of portal vessels type-I cells atrophy somewhat with a decrease in lipid droplets; type-II cells, also atrophy with conspicuous increase in size and number of lipid droplets, enlargement of mitochondria, and gradual disappearance of SER; type-III cells decrease in number whereas type-IV cells increase.After injection of ACTH, type-I cells enlarge and their mitochondria, SER and Golgi apparatus become more conspicuous; there is a decrease in lipid droplets in type-II cells and a development of SER, polysomes and Golgi apparatus; there is also a decrease in lipid droplets and a development of SER in type-III cells after injection of 2IU ACTH and an almost complete disappearance of lipid droplets after 4IU ACTH; type-IV cells increase in number.The investigation reported herein was supported by Scientific Research Grants from the Ministry of Education of Japan to Professor Mikami; and by grants from the Japan Society for the Promotion of Science, the National Science Foundation (USA), and the Graduate School Fund of the University of Washington to Professor Farner     

Adrenal cells in tissue culture. The effect of papaverine and amytal on steroidogenesis, respiration and replication

The smooth-muscle relaxant papaverine has been shown to be a potent inhibitor of cyclic AMP phosphodiesterase activity (Kukovetz, W. R., and Poch, G. (1970) Naunyn Schmiedebergs Arch. Pharmakol.267, 189). Because of this, papaverine was tested in monolayer cultures of functional mouse adrenal cortex tumor cells for possible stimulatory effects on Steroidogenesis. At 10^?5m, papaverine was found to inhibit ACTH-stimulated steroidogenesis 50% and at 10^?4m, > 95%. This was associated with a > 10-fold increase in [¹⁴C]lactate production from [¹⁴C]glucose and a 50% reduction in ³²P_i, incorporation into macromolecules. These findings were similar to those observed with the barbiturate amytal, an inhibitor of the mitochondrial electron-transport chain at the level of oxidation of NADH (Site I). Papaverine was 100 times more effective than amytal in inhibiting steroidogenesis and 1000 times more effective in initiating an increase in glycolysis. In intact tumor cells and mitochondria isolated from normal rat adrenals, papaverine (10^?4m) completely inhibits oxygen uptake supported by malate or α-ketoglutarate. Oxygen uptake is restored by the addition of succinate, suggesting that, like amytal, papaverine inhibits respiration at Site I.Papaverine does not inhibit NADPH-supported cholesterol side-chain cleavage in bovine adrenal acetone powders or 11β-hydroxylation in normal rat adrenal cortex mitochondria. By contrast, amytal inhibits both these activities at concentrations comparable to that effective in intact adrenal cells, suggesting a direct interaction of amytal with cytochrome P-450. Both papaverine and amytal inhibit incorporation of thymidine into nuclear DNA to an extent far greater than that observed with either maximally stimulating levels of cyclic AMP or high concentrations of ACTH. Succinate does not reverse the inhibitory effects of either papaverine or amytal on thymidine incorporation into DNA. Papaverine increases intracellular cyclic AMP in both resting and ACTH-treated cells. However, the effects of papaverine on steroidogenesis, glycolysis, ATP-P_i exchange, and DNA synthesis in adrenocortical cells are not directly attributable to this action.