The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.     

Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: effect of Tween20 concentration

Cholesterol side-chain cleavage activities of cytochrome P-450ssc purified from bovine adrenocortical mitochondria were measured for various substrates, including cholesterol, 20[S]-hydroxycholesterol, 22[R]-hydroxycholesterol and 20[R], [R]-dihydroxycholesterol, in the reconstituted enzyme system at various Tween20 concentrations. The side-chain cleavage activity for cholesterol showed more than 10-fold enhancement upon addition of 0.1% Tween20, compared with that without the detergent. Addition of Tween20 did not cause any enhancement of the side-chain cleavage activities for 20[S]-hydroxycholesterol and 22[R]-hydroxycholesterol; rather, it resulted in an inhibition of the activities. The side-chain cleavage activity for 20[R],22[R]-dihydroxycholesterol showed a very high value even without the detergent. As the stimulatory effect of Tween20 was only specific for cholesterol, Tween20 seemed to enhance the rate of access of cholesterol to cytochrome P-450scc. These results are consistent with the suggestion that a transfer of substrate, cholesterol, in mitochondrial inner membrane, to the substrate-binding site of cytochrome P-450scc is the rate-limiting step in the cholesterol side-chain cleavage reaction.     

An early steroidogenic defect in hormone-induced Leydig cell desensitization

To define the nature of the lesion of the early steroidogenic pathway (prior to pregnenolone formation) in gonadotropin-induced desensitization of rat testicular Leydig cells, we evaluated cholesterol side-chain cleavage activity in isolated mitochondria by measurement of pregnenolone synthesis and [14C]isocaproic acid formation from [26-14C]cholesterol. The enzyme activity was shown to be reduced after in vivo treatment with 10 micrograms hCG when compared to that of mitochondria from control animals only when measured in the presence of limiting NADPH concentrations (100 microM). Sonication of mitochondria from control and hCG-treated rats caused complete loss of cholesterol side-chain cleavage activity. When acetone-powdered adrenal cell mitochondria were employed as the source of the enzyme, the addition of sonicated Leydig cell mitochondria from control and hCG-treated animals caused the same differences as those observed with intact Leydig cell mitochondria in the presence of low concentration of NADPH. The Km value of the adrenal enzyme for NADPH incubated with Leydig cell mitochondria increased from 0.111 mM in control to 0.37 mM after hCG, with no changes in Vmax. Moreover, cholesterol side-chain cleavage activity of adrenal mitochondria assayed in the presence of 100 microM cholesterol was progressively inhibited by increasing amounts of acetone powder from Leydig cell mitochondria of control and hCG-treated rats, with ID50 of 500 and 280 micrograms protein, respectively. The inhibiting factor was not a lipid or steroid but a heat-labile protein, with an approximate Stokes radius of 4.8 nm and an isoelectric point of 5.05 +/- 0.23 SD (n = 8). The inhibitory effect was confined to the Leydig cell mitochondrial membrane, and was not related to changes in oxidative phosphorylation. NADPH was not directly oxidized or immobilized by the mitochondrial factor, and this inhibiting substance was not adsorbed on 2',5' ADP-Sepharose 4B. These results have demonstrated that a heat-labile inhibiting protein factor is present in mitochondria from normal Leydig cells and is markedly activated or increased by hCG treatment. This substance that competitively modulates cholesterol side-chain cleavage activity could contribute to the early steroidogenic lesion, and also serve as an endogenous modulator of steroid hormone biosynthesis.     

Purification of cytochrome P-450 from bovine adrenocortical mitochondria

One soluble cytochrome P.450 from bovine adrenocortical mitochondria has been purified to near homogeneity. The purified enzyme catalyses side-chain cleavage of cholesterol and to a much lesser extent 11β-hydroxylation (<13% side-chain cleavage) but shows no 18-hydroxylase activity. The molecular weight of this P.450 is approximately 800,000.     

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.     

Side-chain cleavage of cholesterol sulfate by ovarian mitochondria

Mitochondria isolated from porcine corpora lutea and from the luteinized ovaries of gonadotropin-treated immature rats were found to efficiently cleave the side-chain of cholesterol sulfate to produce 3 beta-hydroxy-5-pregnen-20-one sulfate (pregnenolone sulfate). When mitochondria were preincubated with cholesterol sulfate, the time-course for the side-chain cleavage of cholesterol sulfate was biphasic. With 200 microM cholesterol sulphate, the initial rate of the reaction was the same as that observed for 25-hydroxycholesterol. This rate was not increased when both cholesterol sulfate and 25-hydroxycholesterol were incubated together. The rate of side-chain cleavage by isolated mitochondria supplied with 75 microM cholesterol sulfate as substrate was inhibited by 97% by aminoglutethimide, a specific inhibitor of cytochrome P-450scc. The slow phase of side-chain cleavage of cholesterol sulfate appeared to be limited by the rate of substrate movement to the mitochondrial site of the reaction. Cholesterol sulfate translocation rates were however up to 8 times greater than those observed for cholesterol when equivalent concentrations of the two substrates were added to the mitochondria. We conclude that cholesterol sulfate is a better substrate than cholesterol for side-chain cleavage by isolated mitochondria and that both reactions are catalysed by the same cytochrome P-450scc enzyme.     

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.     

Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells.

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.     

Differences between the regulation of cholesterol side-chain cleavage in Leydig cells from mice and rats

A Leydig cell culture system has been used to study the in vitro modulation by luteinizing hormone (LH) of steroidogenesis in Leydig cells isolated from mice and immature rats. Mouse Leydig cells precultured for 24 h in the presence of increasing concentrations of LH (1 ng-1 microgram/ml) showed a dose-dependent decrease of the maximal LH-stimulated testosterone production. After pretreatment with 1 microgram LH/ml, maximal LH-stimulated testosterone production. After production in the presence of excess 20 alpha-hydroxycholesterol (a cholesterol side-chain cleavage substrate) were reduced to approx. 50% of control values. The possible site of action of LH is probably prior to pregnenolone, because testosterone production in the presence of excess pregnenolone was not affected by the LH pretreatment. Immature rat Leydig cells showed no decrease of maximal steroid production after 24 h culture in the presence of 1 microgram LH/ml. These results indicate that the regulation of the cholesterol side-chain cleavage activity during long-term LH action is different in mouse and rat Leydig cells. The properties of the cholesterol side-chain cleavage enzyme in mouse and rat Leydig cells were further investigated with different hydroxylated cholesterol derivatives as substrates. Steroid production by mouse Leydig cells in the presence of (22R)-22 hydroxycholesterol was similar as in the presence of LH. In contrast, steroidogenesis in rat Leydig cells in the presence of (22R)-22 hydroxycholesterol was at least 10-fold higher than in the presence of LH. It is concluded that the cholesterol side-chain cleaving enzyme in the mouse Leydig cell operates at its maximal capacity during short-term LH stimulation and can be inhibited after long-term LH action, whereas in the rat Leydig cell only a fraction of the potential activity is used during short-term LH stimulation, which is not affected during long-term LH action.     

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.     

Placental cytochrome P450scc (CYP11A1): comparison of catalytic properties between conditions of limiting and saturating adrenodoxin reductase

The mitochondrial side-chain cleavage of cholesterol, catalysed by cytochrome P450scc, is rate-limiting in the synthesis of progesterone by the human placenta. Cytochrome P450scc activity is in turn limited by the concentration of adrenodoxin reductase (AR) in placental mitochondria. In order to better understand which components of the cholesterol side-chain cleavage system are important in the regulation of placental progesterone synthesis, we have examined their effects on P450scc activity with both saturating and limiting concentrations of AR. The present study reveals that decreasing the AR concentration causes a decrease in the K(m) of cytochrome P450scc for cholesterol, facilitating saturation of the enzyme with its substrate. Decreasing AR resulted in P450scc activity becoming less sensitive to changes in P450scc concentration. The adrenodoxin (Adx) concentration in mitochondria from term placentae is near-saturating for P450scc and under these conditions, we found that decreasing AR reduces the K(m) of P450scc for adrenodoxin. Increasing either the cholesterol or P450scc concentration increased the amount of AR required for P450scc to work at half its maximum velocity. A relatively small increase in AR can support considerably higher rates of side-chain cleavage activity when there is a coordinate increase in AR and P450scc concentrations. We conclude from this study that cholesterol is near-saturating for cytochrome P450scc activity in placental mitochondria due to the P450scc displaying a low K(m) for cholesterol resulting from the low and rate-limiting concentration of AR present. This study reveals that it is unlikely that cholesterol or adrenodoxin concentrations are important regulators of placental progesterone synthesis but AR or coordinate changes in AR and P450scc concentrations are likely to be important in its regulation.     

Clomiphene and tamoxifen inhibit the cholesterol side-chain cleavage enzyme activity in hen granulosa cells

A radiochemical assay was utilized to study the inhibitory effects of clomiphene and tamoxifen on the cholesterol side-chain cleavage enzyme activity in a mitochondrial preparation of granulosa cells isolated from mature ovarian follicles of laying hens. At saturating substrate concentrations, both clomiphene and tamoxifen were able to suppress enzyme activity in a dose-related manner (IC50 1.8 X 10(-5) M). Double reciprocal plots of kinetic data show that the inhibition is mixed, exhibiting competitive kinetics at low concentrations, whereas at high concentrations, the inhibition is of a non-competitive nature. The competitive inhibition constants as determined from Dixon plots are 2 X 10(-5) M for clomiphene and 2.3 X 10(-5) M for tamoxifen. It is concluded that, in granulosa cells, clomiphene and tamoxifen directly inhibit the mitochondrial cholesterol side-chain cleavage activity. This inhibition may represent an important aspect of the mode of action of clomiphene and tamoxifen.     

Cholesterol 20, 22-epoxides: no conversion to pregnenolone by adrenal cytochrome P-450SCC.

All of the four 20,22-epoxycholesterols and (E)-20(22)-dehydrocholesterol were chemically synthesized and incubated with purified adrenocortical cytochrome P-450_scc in the presence of an appropriate electron-supplying system. None of these cholesterol derivatives were significantly converted to pregnenolone by the enzyme. A slight inhibition of the side-chain cleavage of radioactive cholesterol was observed by the addition of the cholesterol derivatives, but there occurred no trapping of the radioactivity by these compounds. It may be concluded that the side-chain cleavage of cholesterol by the adrenal cytochrome P-450 does not operate through olefin and epoxide formation as the intermediates.     

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.     

The insulin-like growth factor, somatomedin C, induces the synthesis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in ovarian cells

The actions of insulin and somatomedin C (insulin-like growth factor I) on cholesterol side-chain cleavage activity and the synthesis of cytochrome P-450scc and adrenodoxin were investigated in primary cultures of swine ovarian (granulosa) cells. Nanomolar concentrations of pure human somatomedin C stimulated biosynthesis of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Moreover, in the presence of exogenous sterol substrate for cholesterol side-chain cleavage, somatomedin C significantly enhanced pregnenolone biosynthesis in a time- and dose-dependent manner. This augmentation of functional cholesterol side-chain cleavage activity was accompanied by a dose-dependent (2-16-fold) increase in [35S]methionine incorporation into specific immunoprecipitable cytochrome P-450scc and adrenodoxin. Micromolar concentrations of insulin (but not proinsulin or desoctapeptide) also induced synthesis of cholesterol side-chain cleavage constituents by 4-7-fold. These results demonstrate that an insulin-like growth factor, somatomedin C, exerts discrete differentiating effects on ovarian cells characterized by increased synthesis of immunospecific cytochrome P-450scc and adrenodoxin. Thus, we infer that somatomedin C may serve a critical role in the differentiation of steroidogenic cells in the mammalian ovary.     

Cholesterol sulfate is a naturally occurring inhibitor of steroidogenesis in isolated rat adrenal mitochondria

We previously reported (Lambeth, J. D., Xu, X. X., and Glover, M. (1987) J. Biol. Chem. 262, 9181-9188) that exogenously added cholesterol sulfate inhibits the conversion of cholesterol to pregnenolone in isolated adrenal mitochondria, and does so by affecting intramitochondrial cholesterol movement but not its subsequent metabolism to pregnenolone by cytochrome P-450scc. We now report that a major kinetic component of the inhibition is noncompetitive with respect to cholesterol, consistent with an allosteric effect at a site other than the substrate binding site of cytochrome P-450scc. We now also report that cholesterol sulfate is present as an endogenous compound in preparations of adrenal mitochondria. Its content varied from 0.05 to 0.8 nmol/mg protein. Cholesterol sulfate level correlated inversely with the mitochondrial cholesterol side-chain cleavage activity. Endogenous cholesterol sulfate thus appeared to account for the variable rates of pregnenolone synthesis which were seen in different mitochondrial preparations. Cholesterol sulfate was metabolized to pregnenolone sulfate by a mitochondrial side-chain cleavage system, but proved to be a relatively poor substrate for an extramitochondrial steroid sulfatase activity present in adrenal cortex. Confirming a role as a naturally occurring inhibitor, removal of endogenous mitochondrial cholesterol sulfate by metabolism to pregnenolone sulfate correlated with a 3-fold activation of cholesterol side-chain cleavage. We suggest that cholesterol sulfate functions in steroidogenic tissues to regulate the magnitude of the steroidogenic response.     

Cytochrome P-450 and steroidogenic activities of the human placental mitochondria

The relationship between cytochrome P-450 concentration, cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase/isomerase activity, in different density mitochondrial preparations from human term placenta has been studied. The heavy mitochondrial fraction shows a higher cytochrome P-450 concentration and cholesterol side-chain cleavage activity as compared to the light mitochondrial fraction, it has however lower cytochrome P-450AROM level and 3 beta-hydroxysteroid dehydrogenase/isomerase activity.     

Comparative ability of rat seminiferous tubules, interstitium, and whole testes to utilize cholesterol-1,5-3H as a substrate for testosterone synthesis and the intratesticular distribution of cholesterol side-chain cleavage enzyme.

Homogenates of rat seminiferous tubules, interstitium and intact testis tissues were assessed for their ability to convert cholesterol -1,2-3H to testosterone in vitro. While 3H-testosterone synthesis was observed in incubates of interstitial and whole testis homogenates, no synthesis was detectable in homogenates of seminiferous tubules. To determine whether cholesterol side-chain cleavage enzyme (CSCCE) was deficient or absent in tubules, mitochondria from tubules, interstitium and whole testes were analyzed for CSCCE activity by measuring conversion of cholesterol -26-14C to 14C-isocaproate (+pregnenolone). Interstitial mitochondrial preparations from each of six testes were found to be approximately 200 times more active in CSCCE than the corresponding tubule mitochondria, and 1600-1800 times more active on a specific activity basis. Although caution is required in extrapolation of in vitro data to the in vivo state, these findings suggest rat seminiferous tubules may be incapable of de novo testosterone biosynthesis and that this lack of synthetic ability may be due to a deficiency of CSCCE.     

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.     

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.