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.     

Steroidogenesis activator polypeptide (SAP) in the rat ovary and testis

A steroidogenesis activator polypeptide (SAP) has previously been identified in the rat adrenal cortex (Pedersen and Brownie, Proc. natn. Acad. Sci. U.S.A. 80 (1983) 1882-1886). This factor apparently facilitates the association of mitochondrial cholesterol with the cholesterol side-chain cleavage cytochrome P-450, a reaction which is generally regarded as rate-controlling in the steroid biosynthetic pathway. The same preparative techniques have now been applied in a search for this material in other rat tissues. Among those investigated, the ovary and testis demonstrate significant concentrations of a factor which is biologically and chromatographically similar to adrenal SAP. In the immature ovary the activator becomes manifest after priming with PMSG and rises dramatically during hCG-stimulated luteinization, an increase which can be blunted with cycloheximide. In the adult rat testis it is increased acutely by treatment with hCG or dibutyryl cAMP and is diminished in response to hypophysectomy or cycloheximide. At approximately equivalent concentrations (10(-7) M), preparations of the activator from the adrenal cortex, the testis, and the superovulated ovary each enhance the activity of cholesterol side-chain cleavage in adrenocortical mitochondria by 5- to 6-fold over basal controls. We conclude that steroidogenic organs share a similar or identical intracellular modulator of cholesterol----pregnenolone conversion which is under pituitary control.     

Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol

Purified sterol carrier protein2 (SCP2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity.     

Inhibition of protohaem ferro-lyase in experimental porphyria. Isolation and partial characterization of a modified porphyrin inhibitor.

Rat adrenal 105,000 g supernatant contains two lipid moieties, 'lipid-I' and 'lipid-II' which contain non-esterified cholesterol and stimulate cholesterol side-chain cleavage in soluble or mitochondrial enzyme systems. Lipid-I contains relatively large low-density heat-stable particles, whereas lipid-II particles are smaller, more dense and heat-labile. Lipid-I and lipid-II can be separated from clear cytosol by ultracentrifugation and gel filtration respectively. Corticotropin plus cycloheximide treatment increases the non-esterified cholesterol concentrations in the lipid fractions, and stimulatory effects of lipids on cholesterol side-chain cleavage appear to correlate with non-esterified cholesterol concentrations therein. On addition of saturating amounts of cholesterol-rich lipid, pregnenolone synthesis and cholesterol binding to cytochrome P-450 are stimulated more in mitochondria from corticotropin-stimulated adrenals than in mitochondria from control or corticotropin-plus cycloheximide-stimulated adrenals. These results support the contention that the corticotropin-induced increase in mitochondrial cholesterol side-chain cleavage involves an increase in cholesterol utilization as well as an increase in cholesterol availability.     

Cholesterol side-chain cleavage activity in the outer and inner zones of the adrenal cortex

Cholesterol side-chain cleavage activity in mitochondria isolated from the outer and inner zones of the guinea pig adrenal cortex was evaluated in order to clarify the role of the zona reticularis in steroidogenesis. It was found that side-chain cleavage activity was three times higher in the outer zone. In addition, ether stress increased side-chain cleavage activity in the outer zone but not the inner zone. The concentration of total and free cholesterol was also found to be higher in the outer zone. However, when exogenous cholesterol was added to mitochondria, there was no enhancement in side-chain cleavage activity in either zone.     

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.     

GTP stimulates pregnenolone generation in isolated rat adrenal mitochondria

Pregnenolone synthesis from cholesterol by adrenal mitochondria isolated from ether-stressed rats exhibits a biphasic time course: upon the addition of a reducing substrate (e.g. malate), a rapid phase of pregnenolone formation occurs during the first 5 min, which has been interpreted as the metabolism of a steroidogenic pool of cholesterol, probably in the inner membrane. A slower rate follows, which is interpreted as translocation of cholesterol into the steroidogenic pool. While a 30-min preincubation of mitochondria with cholesterol alone did not affect the extent of the rapid phase, preincubation with GTP plus cholesterol extended the first phase, resulting in an up to 2-fold increase in pregnenolone synthesis by 20-30 min. The apparent Km for GTP was 0.1-0.4 mM, and stimulation was maximal with preincubation times of 10-30 min, depending upon incubation conditions. Exogenous cholesterol was not required to observe a stimulatory effect, indicating that GTP reorganizes the endogenous mitochondrial cholesterol pools. Nevertheless, stimulation was greater when exogenous cholesterol was provided, consistent with enhanced utilization of both endogenous and exogenous cholesterol. Stimulation by GTP was also seen in mitochondria isolated from cycloheximide-injected/ether-stressed rats, although the activity in these preparations was always lower than that in mitochondria from ether-stressed rats. The stimulation was specific for GTP, since many other nucleotides (e.g. ATP, GDP, and ITP) and GTP analogues (guanosine 5'-O-(3-thiotriphosphate and guanosine 5'-(beta,gamma-imino)triphosphate) had no effect. The GTP-activated state was reversible: after GTP hydrolysis by a mitochondrial GTPase, pregnenolone synthesis returned to the basal level. Sonic disruption of mitochondria abolished the stimulatory effect of GTP. These results suggest that GTP enhances pregnenolone synthesis by promoting the movement of cholesterol to the steroidogenic pool, consistent with a recently proposed general role for GTP in some vectorial transport processes (Bourne, H. R. (1988) Cell 53, 669-671).     

Ultrastructural localization of cytochrome P-450scc in the bovine placentome using protein A-gold technique

We have previously reported that the steroidogenic activity of the bovine placentome is stimulated by a calcium-mediated, cyclic nucleotide-independent mechanism and that this steroidogenesis is limited by the availability of sterol substrate to the side-chain cleavage enzyme. We have recently established that the antibody against bovine adrenal cytochrome P-450 cholesterol side-chain cleavage enzyme (P-450scc) can be used to specifically detect P-450scc in both bovine placentome and corpus luteum. In the present study, we used an immunogold technique to localize the P-450scc in the bovine placentome by electron microscopy. The mononucleate cell of the cotyledon showed both giant and normal-sized mitochondria, with the latter, predominating. Both mitochondrial types found in the mononucleate cells clearly displayed gold particles located on the cristae; in contrast, these particles were absent in the binucleate cells. It is worth noting that giant mitochondria were found exclusively in the placental mononucleate cells in both the fetal and maternal sites but not in the binucleate cells. These findings suggest that the cholesterol side-chain cleavage enzyme is present in bovine cotyledon cells, primarily in mononucleate cells. The variations in P-450scc immunoreactivity among different cells of the placenta are suggestive of different steroidogenetic capacities of the cells.     

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.     

Inhibition of aldosterone synthesis by atrial natriuretic factor

Atrial natriuretic factor (ANF) inhibits basal and stimulated aldosterone synthesis in adrenal glomerulosa cells. ANF probably acts through specific membrane receptors. Alterations in cyclic GMP and cyclic AMP levels do not account for ANF's inhibitory effect. ANF does not block angiotensin II (AngII) receptors nor does it interfere with phosphoinositide metabolism or calcium movements stimulated by adrenal agonists. ANF does not inhibit protein synthesis nor does it work by inhibiting NA+,K+-ATPase or depleting cell potassium. ANF decreases conversion of endogenous cholesterol to pregnenolone, the step stimulated by adrenocorticotropin and AngII. ANF does not affect the conversion of 20-alpha-hydroxycholesterol, which easily penetrates mitochondrial membranes to the site of the cholesterol side-chain cleavage enzyme. These results suggest that ANF inhibits the ability of endogenous cholesterol to reach or interact with the side-chain cleavage enzyme. ANF does not act like a calcium channel-blocking agent. However, ANF is less effective at high-calcium concentrations, which suggests that it may inhibit a step that calcium stimulates. Understanding ANF action will probably require identification of the specific biochemical changes (mediators) that it induces. Parallel efforts to understand how other agents stimulate steroidogenesis (particularly in the areas of protein synthesis, protein phosphorylation, and cholesterol movements) will further this understanding.     

Phospholipids, sterol carrier protein2 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 protein2 (SCP2) 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 microM), the stimulatory effect was only half that observed with 0.7 microM SCP2, and the two effectors were not synergistic. SCP2 caused a redistribution of cholesterol from mitochondrial outer to inner membranes, while cardiolipin, which is an activator of cytochrome P-450scc, had no effect on distribution of mitochondrial membrane cholesterol.     

Interactions between ATP and cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries

ATP stimulated the rate of [4-14C]cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries. The effect of ATP was apparently similar to the stimulatory effect of choriogonadotropin on mitochondrial [4-14C]cholesterol utilization. Enhancement of the rate of steroidogenesis by ATP and choriogonadotropin were not additive. ATP seemed to promote both cholesterol uptake into the inner mitochondrial membrane and the supply of electrons for [4-14C]cholesterol utilization from both endogenous substrate and succinate.     

Electron spin resonance studies of membrane proteins in erythrocytes in myotonic muscular dystrophy.

A goat antibody produced against bovine adrenal ferredoxin has been employed to establish immunochemically the involvement of adrenal ferredoxin in the cholesterol side-chain cleavage reaction catalyzed by mammalian adrenal mitochondria. When added to preparations of bovine adrenocortical mitochondria, this antibody was found to inhibit the conversion of cholesterol to pregnenolone and progesterone, the 11β-hydroxylation of deoxycorticosterone and the NADPH-dependent reduction of cytochrome c. These observations demonstrate that, similar to the NADPH-cytochrome c reductase and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the oxidative cleavage of the cholesterol side-chain catalyzed by bovine adrenocortical mitochondria.The goat antibody to bovine adrenal ferredoxin was also found to interact with the comparable iron-sulfur proteins present in mitochondria prepared from sheep, rat, mouse, cat, dog, guinea pig, rabbit, and human adrenals. The interaction of the antibody with these iron-sulfur proteins resulted in the inhibition of both the cholesterol side-chain cleavage and NADPH-cytochrome c reductase activities catalyzed by these adrenal mitochondria. The NADH-dependent reduction of cytochrome c catalyzed by mammalian adrenal mitochondria was not inhibited by the goat antibody to adrenal ferredoxin. These results demonstrate the immunochemical similarity existing among mammalian adrenal ferredoxins and their involvement in the adrenal cholesterol side-chain cleavage reaction.     

The kinetics of steroidogenesis activator polypeptide in the rat adrenal cortex. Effects of adrenocorticotropin, cyclic adenosine 3':5'-monophosphate, cycloheximide, and circadian rhythm

The behavior of steroidogenesis activator polypeptide (SAP), a recently described modulator of cholesterol side-chain cleavage activity (Pedersen, R. C., and Brownie, A. C. (1987) Science 236, 188-190), was investigated in rat adrenocortical cells using a specific radioimmunoassay. In response to a maximal dose of adrenocorticotropic hormone (ACTH) (1 nM) or of 8-Br-cAMP (1 mM), an increase in intracellular SAP begins rapidly (less than 1 min) and reaches half-maximal and maximal levels (16-fold greater than basal) at 3 and 15 min, respectively. A plateau at this maximal concentration of SAP is then maintained. The levels of intracellular SAP content and of corticosterone output exhibit a similar dose-dependent response to ACTH (EC50 = 25 and 30 pM, respectively). Treatment of ACTH-stimulated cells with cycloheximide reverses the rise in SAP (t1/2 congruent to 5-7 min). In vivo the SAP content of adrenals from quiescent rats is concordant with the circadian rhythm of the pituitary-adrenal axis; at the apex (1800 h), adrenal SAP is 13-fold higher than at the nadir (0800 h), paralleling 2- and 7-fold variations in cholesterol side-chain cleavage activity and serum corticosterone levels, respectively. At both time points, SAP levels rise in response to stress. Of the rat tissues examined, only the major steroid-forming organs (adrenal cortex and gonads) had significant levels of immunoreactive, cAMP-responsive SAP, while cAMP-unresponsive immunoreactivity was also detectable in the thymus, spleen, and brain. Considered together with the biological activity previously demonstrated for SAP in vitro, these data are consistent with its role as a cAMP-dependent, cycloheximide-sensitive modulator of steroid biosynthesis.     

A role for calmodulin in the regulation of steroidogenesis

Two approaches were used to study the possible role of calmodulin in the regulation of steroid synthesis by mouse adrenal tumor cells: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. Trifluoperazine inhibits three steroidogenic responses to both ACTH and dibutyryl cyclic AMP: (a) increase in steroid production, (b) increased transport of cholesterol to mitochondria, and (c) increased side-chain cleavage by mitochondria isolated from cells incubated with ACTH or dibutyryl cyclic AMP. When calmodulin is introduced into the cells via liposomes, steroid synthesis is slightly stimulated. When calmodulin extensively dialyzed against EGTA, this stimulation is abolished. Ca(2+) introduced via liposomes was also without effect. However, when both calmodulin and Ca(2+) are introduced via liposomes (either in separate liposomes or in the same liposomes), steroid synthesis is stimulated. This stimulation does not occur when either anticalmodulin antibodies or EGTA is also present in the liposomes or when trifluoperazine is present in the incubation medium. Calmodulin and Ca(2+) presented together in liposomes to the cells stimulate transport of cholesterol to mitochondria, and side-chain cleavage activity is greater in mitochondria isolated from cells previously fused with liposomes containing calmodulin and Ca(2+) than in mitochondria from cells fused with liposomes containing buffer only. These observations suggest that calmodulin may be involved in regulating the transport of cholesterol to mitochondria, a process which is stimulated by ACTH and dibutyryl cyclic AMP and which may account, at least in part, for the increase in steroid synthesis produced by these agents.     

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.     

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)     

Immunochemical studies on adrenal ferredoxin: Involvement of adrenal ferredoxin-like iron-sulfur proteins in the cholesterol side-chain cleavage reaction of mammalian steroidogenic tissues

The possible immunochemical and functional similarities existing among adrenal ferrdoxin-like iron-sulfur proteins present in the mitochondria of mammalian steroidogenic tissues have been examined by employing a goat antibody produced against homogeneous bovine adrenal ferredoxin. This antibody was found to inhibit the NADPH-cytochrome c reductase and cholesterol side-chain cleavage activities catalyzed by mitochondria prepared from rat adrenals, rat ovaries and the testes of rats which had been treated with human chorionic gonadotropin. No inhibition of the NADH-dependent reduction of cytochrome c catalyzed by these mitochondria was observed in the presence of the anti-adrenal ferredoxin. These results demonstrate that adrenal ferredoxin and the comparable iron-sulfur proteins of ovarian and testicular mitochondria are immunochemically similar and are required for the cholesterol side-chain cleavage reaction occurring in these tissues. Although a precipitin reaction was observed upon double diffusion of the anti-adrenal ferredoxin against human term placental mitochondria, no inhibition of either the NADPH-cytochrome c reductase or the cholesterol side-chain cleavage⁴ activity catalyzed by preparations of these mitochondria was observed in the presence of the antibody. These results indicate that the iron-sulfur protein present in human placental mitochondria at term differs immunochemically from other mammalian adrenal ferredoxin-like iron-sulfur proteins.     

The effects of cytochalasin B and vinblastine on movement of cholesterol in rat adrenal glands.

Vinblastine (antimicrotubular agent) and cytochalasin B (antimicrofilament agent) block the build up of adrenal mitochondrial cholesterol seen in the presence of AMG. ACTH stimulated steroidogenesis is inhibited $\text{in vivo}$ by both agents via a reduction in the transfer of intra-adrenal cholesterol to adrenal mitochondria, resulting in a decrease in the synthesis of adrenal steroids. Both inhibitors also decrease ACTH stimulated formation of cholesterol cytochrome P450_SCC complex in adrenal mitochondria, as determined by difference spectroscopy. The effects of these inhibitors contrast with the actions of protein synthesis inhibitors which decrease cholesterol binding to P450_SCC while increasing mitochondrial cholesterol content.     

Localization of prostaglandin F2 alpha inhibition of lipoprotein use by bovine luteal cells.

Prostaglandin F2 alpha (PGF2 alpha) inhibits lipoprotein-stimulated progesterone production by bovine luteal cells in vitro and the objective of this study was to localize the site of action of PGF2 alpha. Cultured bovine luteal cells were treated with PGF2 alpha for seven days, and then with either lipoproteins or 25-hydroxycholesterol in the presence of aminoglutethimide (which inhibits cholesterol side-chain cleavage) for the final 48 h. The effects of PGF2 alpha on progesterone production, cellular cholesterol content, mitochondrial cholesterol content and cholesterol side-chain cleavage activity were determined. As expected, PGF2 alpha inhibited (P less than 0.05) lipoprotein-stimulated progesterone production. However, PGF2 alpha did not inhibit low-density lipoprotein-stimulated, or high density lipoprotein-stimulated, increases in cellular cholesterol (P less than 0.05) or inhibit lipoprotein-induced increases in mitochondrial cholesterol content (P less than 0.05). Additionally, cholesterol content of mitochondria increased (P less than 0.05) in the presence of PGF2 alpha alone. To determine if the PGF2 alpha-induced inhibition of steroidogenesis occurred at, or after, the side-chain cleavage reaction, we treated cells with the readily diffusable sterol, 25-hydroxycholesterol. Prostaglandin F2 alpha did not inhibit 25-hydroxycholesterol-stimulated progesterone production (P less than 0.05). Prostaglandin F2 alpha may therefore exert its luteolytic effect at a site after cholesterol transport to the mitochondria but before cholesterol side-chain cleavage.