Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells. 1. An ultrastructural analysis of cells in which basal and ACTH-induced steroidogenesis was inhibited by taxol

In adrenocortical cells, the first step in the enzymatic processing of cholesterol to steroid end products occurs in the mitochondria. ACTH increases mitochondrial cholesterol and steroidogenesis. In cultured mouse adrenocortical tumor cells, microtubule-based organelle motility may increase the proximity of mitochondria to the SER, lipid droplets and endoscome-derived lysosomes, thereby facilitating the transfer of cholesterol from these organelles to the mitochondrial outer membrane. ACTH may increase opportunities for the transfer by promoting organelle motility and by increasing the number of lysosomes. Taxol, a microtubule polymerizer, inhibits basal and ACTH-induced steroidogenesis in these cells, presumably at the step where mitochondria obtain cholesterol. We examined the ultrastructure of taxol-treated, unstimulated and ACTH-stimulated cells, seeking alterations which conceivably could interefer with the proposed organelle transport and encounters, and thus correlate with taxol's inhibition of steroidogenesis. Primary cultured cells were incubated in serum-containing medium for 4 hr with and without ACTH (10 mU/ml), with 10 micrograms/ml and 50 micrograms/ml of taxol, and with ACTH and taxol 10 or taxol 50 simultaneously. Culture media were analyzed for the presence of secreted steroids at the end of 1, 2, and 4 hr of incubation. At the end of the fourth hour, unstimulated cells and cells treated with ACTH, taxol 50, and both agents simultaneously, were fixed and processed for EM. Taxol inhibited basal and ACTH-induced steroidogenesis in a dose-dependent fashion. In both unstimulated and ACTH-stimulated cells, taxol 50 formed numerous microtubule bundles, but did not markedly change the distribution of mitochondria and lipid droplets. SER tubules, and clusters of Golgi fragments, endosomes, and lysosomes appeared to be translocated towards the cell periphery along some of the microtubules. Taxol permitted an ACTH-induced cell rounding and microfilament rearrangement considered to facilitate organelle motility. Our data indicate that taxol disrupts the formation of lysosomes by these adrenal cells, but it seemed unlikely that taxol's ultrastructural effects could prevent organelle transport proposed to cause meetings between mitochondria and the SER or lipid droplets, or prevent ACTH-caused increases in these encounters. Taxol may instead prevent the transfer of lipid droplet or SER-contained cholesterol to adjacent mitochondria, by a means not detectable 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.     

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.     

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).     

The reversibility of the effects of ACTH and cytochalasin B on the ultrastructure and steroidogenic activity of adrenocortical tumor cells in vitro

We have demonstrated previously that the steroidogenic activity of ACTH on cultured adrenal tumor cells is associated with cell rounding and a rearrangement of microfilaments. Cytochalasin B (CB) also induces cell rounding, but changes the conformation of microfilaments and severely inhibits steroidogenesis. ACTH and CB may have different modes of action on the contractile machinery which are related to their opposing actions on steroidogenesis. To investigate this possibility further, we have examined the reversibility of the morphological and functional effects of these agents. Cultures were incubated for 1 hr, with and without ACTH (10 microU/ml of media), or with CB (50 micrograms/ml), or with both agents simultaneously. After a media wash, the cultures were incubated for 1 hr, with and without ACTH. The steroid production of the cells during pre- and post-washout incubations was determined, and some cultures were fixed for electron microscopy at the end of both incubation periods. The three- to ten-fold increases in steroidogenic activity of ACTH-stimulated cells declined during recovery incubations, but remained well above basal values. These cells nearly reflattened and began to regain stress fibers which had been 'pulled apart'. The 'washed out' ACTH-stimulated cells were often refractory to restimulation. Cells recovering from CB also reflattened. Masses of filamentous felt induced by the drug disappeared from the cytoplasm, lost microvilli reappeared and stress fibers reformed. The 20-50% inhibition of basal steroidogenesis by CB was completely reversed. When ex-CB-treated cells were incubated with ACTH, their morphology and steroid production were typical of acutely stimulated cells. The recovery behavior of cells incubated with ACTH and CB simultaneously reflected the observation that there were cell-specific responses to one agent or the other during initial incubations. The persistence of heightened steroidogenic activity following a washout of ACTH and the rapid reversal of the effects of CB strongly support the concept that regulated actomyosin interactions are an integral part of the steroidogenic process.     

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 role of sterol carrier protein 2 in stimulation of steroidogenesis in rat adrenal mitochondria by adrenal cytosol

Cholesterol side-chain cleavage (CSCC) in isolated rat adrenal mitochondria is enhanced by prior corticotropin (ACTH) stimulation in vivo (8-fold). Part of this stimulation is retained in vitro by addition of cytosol from ACTH-stimulated adrenals to mitochondria from unstimulated rats (2.5- to 6-fold). In vivo cycloheximide (CX) treatment fully inhibits the in vivo response and resolves the in vitro cytosolic stimulation into components: (i) ACTH-sensitive, CX-sensitive; (ii) ACTH-sensitive, CX-insensitive; and (iii) ACTH-insensitive, CX-insensitive. These components contribute approximately equally to stimulation by ACTH cytosol. Components (i) and (iii) most probably correspond to previously identified cytosolic constituents steroidogenesis activator peptide and sterol carrier protein 2 (SCP2). SCP2, as assayed by radioimmunoassay or ability to stimulate 7-dehydrocholesterol reductase, was not elevated in adrenal cytosol or other subcellular fractions by ACTH treatment. Complete removal of SCP2 from cytosol by treatment with anti-SCP2 IgG decreased cytosolic stimulatory activity by an increment that was independent of ACTH or CX treatment. Addition of an amount of SCP2, equivalent to that present in cytosol, restored activity to SCP2-depleted cytosol but had no effect alone or when added with intact cytosol, suggesting the presence of a factor in cytosol that potentiates SCP2 action. Pure hepatic SCP2 stimulated CX mitochondrial CSCC 1.5- to 2-fold (EC50 0.7 microM) but was five times less potent than SCP2 in adrenal cytosol. Two pools of reactive cholesterol were distinguished in these preparations characterized, respectively, by succinate-supported activity and by additional isocitrate-supported activity. ACTH cytosol and SCP2 each stimulated cholesterol availability to a fraction of mitochondrial P450scc that was reduced by succinate but failed to stimulate availability to additional P450scc reduced only by isocitrate.     

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.     

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.     

The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis

The mitochondria, the microsomes and the cystosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.     

Morphological correlates of follicular fluid stimulation of steroidogenesis in immature porcine granulosa cells

Factors in porcine ovarian follicular fluid are known to influence steroidogenesis in cultured ovarian granulosa cells. This study examined whether ultrastructural changes characteristic of normal maturation and/or atresia accompany the steroidogenic alterations. Two and 5 day incubations of immature porcine granulosa cells were performed in media supplemented with either serum or follicular fluids (FFL) from mature follicles. Under these conditions both oestrogen and progesterone secretion were stimulated in FFL supplemented cultures as compared to serum supplemented cultures. Cells in serum exhibited increased size, number and volume of lysosomes and resembled in vivo atretic cells. In comparison, the FFL treated cells had greatly increased steroid output, numerous microvilli and increased size, number and volume of electron dense lipid droplets after 2 days of culture although the differences declined by day 5 of culture. This suggests that mature FFL contains a factor(s) stimulating granulosa maturation while inhibiting ultrastructural correlates of follicular atresia.     

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.     

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.     

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.     

Short-term metabolism of cholesteryl ester from low-density lipoprotein in primary monolayers of bovine adrenal cortical cells

The uptake and metabolism of [14C]cholesteryl ester in bovine LDL to cortisol and to cholesteryl ester was studied in monolayer cultures of bovine adrenal cortical cells over short time periods of up to 8 h. The experiments were designed to determine the intracellular pathway followed by the cholesterol derived from the LDL cholesteryl ester and how this is modified in the short term by the tropic hormone ACTH. The cells were cultured in the presence of mevinolin to remove the contribution of endogenous synthesis of cholesterol for supply of substrate for steroidogenesis. The specific activity of the cortisol secreted by the cells was measured under a variety of conditions. Control incubations showed a relatively steady specific activity in the cortisol secreted over an 8 h period. In the presence of ACTH the specific activity of the cortisol was significantly reduced for the first 2 h of the experiment. This is consistent with dilution of the [14C]cholesterol from the LDL with non-radioactive free cholesterol released from the intracellular stores of cholesteryl ester in the presence of ACTH. The inhibitor of acyl-CoA:cholesterol acyltransferase, Sandoz compound 58-035, increased the specific activity of the secreted cortisol in the absence of ACTH, indicating that much of the incoming cholesterol would normally be esterified but was here diverted to steroidogenesis. In the presence of ACTH this increase was observed only during the first 2 h of the experiment, after which inhibition of acyl-CoA:cholesterol acyltransferase had no effect on the specific activity of the cortisol. The adrenal cells were further fractionated into mitochondrial, lysosomal and microsomal plus cytosol fractions and the appearance of free and esterified cholesterol from the labelled LDL measured in these fractions over a period of up to 8 h. ACTH stimulated the uptake of LDL-cholesteryl ester into the cells and tended to increase the relative amounts of free cholesterol in the cells, consistent with its role in promoting supply of cholesterol for steroidogenesis. These experiments allow the roles of endogenous cholesteryl ester and lipoprotein-derived cholesteryl ester in the bovine adrenal cortical cells to be observed over a short time scale. They show that the cells make a substantial change in the internal flux of cholesterol in a short time after stimulation with ACTH and in these cultures the full expression of the presence of ACTH takes up to 2 h.     

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.     

Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase

The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.     

Structural changes occurring in interrenal tissue of the duck (Anas platyrhynchos) following adenohypophysectomy and treatment in vivo and in vitro with corticotropin

Adrenal glands from ACTH-treated intact ducks and chronically adenohypophysectomized ducks showed clear zonation into a subcapsular zone (SCZ) and an inner zone (IZ). Adenohypophysectomy caused ultrastructural changes in the IZ but not in the SCZ cells. These included increases in lipid droplets, changes in mitochondrial cristae from tubular to shelf-like, and changes in the shape of the nuclei from spherical to crenated. These changes were reversed by treatment with ACTH. Also, cells of the IZ, but not the SCZ, of adrenals from intact birds given ACTH showed more SER, more dense bodies, fewer lipid droplets and more prominent Golgi complexes. IZ cells incubated in buffer containing no ACTH developed mitochondria with shelf-like cristae and numerous opaque granules in the matrix. Exposure to buffer containing ACTH caused the mitochondrial cristae to become tubular and the matrix granules either decreased in number or disappeared. The granules could be extracted by incubating sections with chelating agents. The mitochondria in SCZ cells did not respond structurally to the presence of ACTH in the incubation medium but the matrix granules, like those in IZ cells, responded to the presence of chelating agents.     

A correlated morphological and biochemical study on the rat adrenal steroidogenesis

The ultrastructural and biochemical alterations produced by an hypocholesterolemic drug, 17 alpha-ethinyl estradiol, on the rat adrenal cortex were studied. Male rats aged two months and with approximately 200 g in weight were injected subcutaneously with 10 mg/kg/day of ethinyl estradiol during 9 days; rats injected with 1 ml propylene glycol were used as controls. The animals were sacrificed on the 10th day, and the adrenals from some of them were processed for electron microscopy. The adrenals from the remaining rats were used for measurements of the glands cholesterol and corticosterone, which were also measured in the blood. In estradiol-treated rats the zona fasciculata cells exhibited numerous microvilli, increase in the size of mitochondria and decrease in the number of lipid droplets. The quantitative analysis showed a significant increase of the volumetric density of mitochondria and microvilli and a significant decrease of the lipid droplets in the treated rats, when compared with normal ones. In treated rats, the concentration of cholesterol and corticosterone in the gland and blood were significantly decreased. These data show that hypocholesterolemia produced by estradiol has a remarkable effect on adrenal steroidogenesis, depletes the pool of adrenal cholesteryl esters, and evidences the role of plasma cholesterol in the corticosteroidogenesis.     

Some characteristics of adrenal steroidogenesis and their possible relationships to the action of the adrenocorticotropic hormone.

An attempt to define in quantitative terms the characteristics of the biphasic rate curve for pregnenolone synthesis in cell-free systems from the adrenal using male Sprague-Dawley rats is reported. When adrenocorticotropic hormone (ACTH) was used 2 units of .2 ml of .9% saline were injected ip 15 minutes before killing the rats. The effect of ACTH on adrenal steroidogenesis is in the stimulation of the rate of conversion of cholesterol to pregnenolone. This reaction sequence is thought to occur in the mitochondria. Methods of preparing subcellular fractions are described. Incubation of pregnenolone with mitochondria for 20 minutes at 20 degree C resulted in a 70% disappearance of the pregnenolone. This loss does not occur if the mitochondria are boiled, indicating an enzymatic process. The rate of pregnenolone synthesis characteristically shows a biphasic curve with a rapid primary rate and a slower secondary rate. ACTH administration in vivo increased both rates but the percentage increase was greater for the secondary rate. In addition an increase in the duration of the primary rate resulted. Different explanations are offered for these characteristics. Pregnenolone may act as an inhibitor of its own synthesis from cholesterol but not from 20alpha-hydroxycholesterol. Substances that cause mitochondria to swell may stimulate pregnenolone synthesis. Another theory proposes that the limiting ACTH-sensitive step is the rate at which mitochondrial cholesterol is transported to or binds to the cholesterol side-chain cleavage enzyme. The possible role of an inhibitor in the regulation of steroidogenesis is indicated. Data are consistent with the observation that the transition from the primary rate to the slower secondary rate shows the accumulation of an inhibitory substance. The action of ACTH would then be to modify the structure of the cholesterol side-chain cleavage enzyme so that there is a decreased susceptibility of the enzyme to the inhibitor.