Metabolism of (3H) 5 alpha-androstane-3 alpha, 17 beta-diol by cultures of isolated rat sertoli cells and the effect of LH and FSH

Sertoli cells from immature rats metabolized (3H) 5 alpha-androstane-3 alpha, 17 beta-diol to (3H) 5 alpha-androstane-3 alpha, 16 alpha, 17 beta-triol and (3H) 3 alpha-hydroxy-5 alpha-androstan-17-one. This is the first report of 16 alpha-hydroxylation of 5 alpha-reduced androgens in the testis. FSH significantly stimulated 16 alpha-hydroxylation while LH significantly decreased this activity. 3 alpha-Hydroxy-5 alpha-androstan-17-one was the major metabolite formed and its production was significantly increased in the presence of both LH and FSH, although FSH stimulation was significantly more than LH. The possible role of 16 alpha-hydroxylase in androgen metabolism by immature rat Sertoli cells is discussed.     

Identification of a new Sertoli cell metabolite of testosterone: 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol, by HPLC and GC-MS

Testosterone metabolism by isolated rat Sertoli cells cultured in vitro was investigated using HPLC and GC-MS techniques. Monolayer cultures of Sertoli cells (greater than 90% pure and free of Leydig cells) were incubated for 3-day periods with a stable labeled [2,2,4,6,6-d5]testosterone prepared and used in a 1:1 proportion with unlabeled testosterone as the substrate (5 X 10(-7)M). After incubation, the metabolites were extracted from the media and reacted with oestradiol-antibodies. The antibody-bound components were separated on reverse phase HPLC and the fraction corresponding to oestradiol was analyzed by GC-MS in the form of TMS-ether. One of the metabolites whose mass spectrum contained d0 + d5 species was detected and interpreted to be a triol with a mol. wt of 308. Mass spectra data indicated that this testosterone metabolite is one of the sixteen possible isomers of 3,16,17-trihydroxy androstane. This substance was identified based on the Vm value (27.81) closely resembling that of 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol TMS-ether (Vm reported = 27.78) [1] and when compared directly with synthesized compounds [2-3]. Recently we have demonstrated that similar Sertoli cell preparations contain two 16 alpha-hydroxylases by their ability to convert oestradiol to oestriol [4] and 5 alpha-androstane-3 alpha,17 beta-diol to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol [3], where the former conversion is not affected by FSH, the latter is significantly stimulated by the presence of FSH. Presence of this new product represents the first example of testosterone conversion to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol and confirms our previous observation that 16 alpha-hydroxylation of 5 alpha-reduced androgens can occur in the rat testis.     

Effect of cAMP and FSH on the production of estrogens by rat fetal gonads cultured in vitro

The regulation of aromatase activity by cAMP and FSH has been demonstrated in the prepubertal rat testis and ovary, and the question posed whether this regulation was already operative at foetal stages. In the present study, testes and ovaries from 17- to 21-day-old rat foetuses were cultured in vitro in the presence of [3H]-testosterone and in the presence or absence of cAMP or FSH. Oestrone and oestradiol formed from [3H]-testosterone were measured by double isotopic dilution and recrystallization to constant specific activity. Aromatase activity was augmented in both gonads by cAMP, but only in the testis by FSH. Thus, the regulation of aromatase activity by FSH begins earlier in the testis than in the ovary.     

Metabolism of 3H-estradiol-17 beta by cultures of isolated rat Sertoli cells and the effect of FSH: presence of 16 alpha-hydroxylase

The ability of Sertoli cells to metabolize 3H-estradiol-17 beta was investigated utilizing Sertoli cell cultures isolated from 18d rat testes. The Sertoli cells converted estradiol-17 beta to estriol as shown by recrystallization of estriol from samples containing cells and media but not from cell-free control media. The effect of FSH treatment on such metabolism was investigated and was shown to be similar to nontreated samples. This is the first demonstration that 16 alpha-hydroxylase is present in Sertoli cells and that this enzyme activity is not under the influence of FSH.     

Regulation of steroid and steroid sulfate production and aromatase activity in cultured human granulosa-luteal cells

The regulation of the production of steroids and steroid sulfates and the activity of aromatase in human luteinized granulosa cells were investigated. The cells were cultured for 48 h in the presence or absence of hCG and FSH. Basal production of pregnenolone (Pre, 0.3 +/- 0.03 ng/micrograms protein) and progesterone (P, 19.3 +/- 1.7 ng/micrograms protein) were high compared with that of other steroids beyond P in the steroidogenic pathway. The concentration of 17 alpha-hydroxyprogesterone (17-OHP) was lower 0.17 +/- 0.06 ng/micrograms and that of other steroids in the 4-ene and 5-ene pathways and steroid sulfates less than 0.05 ng/micrograms. Both hCG and FSH (100 ng/ml) stimulated the production of Pre and P 3- to 5-fold, but only minimal stimulation of other steroids and steroid sulfates was observed. Aromatase activity of granulosa-luteal cells was measured from the rate of formation of 3H2O from 1 beta-[3H]androstenedione (1 beta[3H]A) after exposing the cells to hCG, FSH or estradiol (E2) for 48 h. Basal aromatase activity was relatively low, but hCG and FSH stimulated aromatase 8- and 4-fold, respectively. The incubation of granulosa-luteal cells with E2 did not affect basal aromatase activity, but E2 augmented FSH-stimulated aromatase 1.4-fold (P less than 0.025). The results suggest that there is low 17 alpha-hydroxylase and steroid sulfokinase activity in human granulosa-luteal cells. Aromatase activity in these cells is regulated by both hCG and FSH, and intra-ovarian estrogens may regulate granulosa cell aromatase activity.     

Steroid metabolism by endometrial and conceptus tissues during early pregnancy and pseudopregnancy in gilts

Endometrial and conceptus tissues were obtained on Days 10.5, 11, 12, 16 and 25 of pregnancy and Day 25 of pseudopregnancy of gilts and incubated for 6 h in Minimal Essential Medium (5 ml) containing 35 ng [3H]progesterone. Metabolism of [3H]progesterone to oestrone, oestradiol and oestriol was determined by gas and high-pressure liquid chromatography and successive recrystallizations with unlabelled standards. Conceptuses collected between Days 10.5 and 12 were spherical, tubular or filamentous and incubated with 500 mg endometrium and [3H]progesterone. Production of oestrone by spherical conceptuses was not detected, but was 44-47 pg/tubular conceptus and 21 pg/filamentous conceptus. A similar trend was observed for oestradiol. Conceptus tissues from Days 16 and 25 (chorion) were most active in producing oestrone (123 and 520 pg/mg tissue, respectively) and oestradiol (277 and 876 pg/mg tissue, respectively). Endometrial oestrogen production was less than that for conceptus tissue for oestrone and oestradiol on Days 16 and 25 of gestation. Coincubations of endometrium and conceptus tissues had lower oestrogen production than conceptus alone. Endometrium from Day 25 of pseudopregnancy metabolized [3H]progesterone to several non-polar metabolites, but no oestrogens were detected. An unidentified phenolic metabolite of [3H]progesterone was detected in higher quantities than either oestrone or oestradiol; 445 to 461 pg/conceptus at the tubular stage. These results indicate temporal changes in the conversion of [3H]progesterone to oestrogens by conceptus and endometrial tissue from pregnant gilts, but not endometrium from pseudopregnant gilts.     

Measurement of testosterone and dehydroepiandrosterone 16alpha-hydroxylase activities by a tritium exchange method.

A new isotopic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group has been developed for the measurement of rat liver testosterone and dehydroepiandrosterone 16alpha-hydroxylase activity. Specifically 16-tritiated substrates were prepared by microbiological (Cylindrocarpon radicicola) transformation of the [16-3H]progesterone and [16-3H]pregnenolone. The incubation medium consists of a phosphate buffer (pH7; 150mM), NADPH (0.1 mM), nicotinamide (10mM) and magnesium chloride (4 mM). Tween 80 (1 mg/ml) is used to solubilize saturating concentrations of [16-3H]testosterone (50 micron) or [16-3H]dehydroepiandrosterone (100 micron). The enzymatically released tritium is recovered in the incubation medium as tritiated water which is distilled under reduced pressure and counted by liquid scintillation. The method is easy to perform, very sensitive (50 pmol of 16alpha-hydroxylated metabolites) and is independent of any further metabolism of the 16alpha-hydroxylated products.     

A rapid and sensitive in vitro assay of 25-hydroxyvitamin D3-1 alpha-hydroxylase and 24-hydroxylase using rat kidney homogenates

A sensitive and rapid in vitro assay of 25-hydroxyvitamin D3 [25-(OH)D3]-1 alpha- and 24-hydroxylase activities was developed using rat kidney homogenates. A potent inhibitor of the enzymes in rat plasma was removed by thoroughly perfusing rats with saline. Kidney homogenates prepared from vitamin D-deficient rats preferentially produced tritiated 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] from 25(OH) [3H]D3. Addition of 10 microliter or more of rat plasma to 3 ml of 10% kidney homogenates suppressed 1 alpha-hydroxylase activity dose-dependently. Thyroparathyroidectomy (TPTX) of vitamin D-deficient rats greatly abolished 1 alpha-hydroxylase activity. Administration of parathyroid hormone to the TPTX rats increased 1 alpha-hydroxylase activity and that of 1 alpha,25(OH)2D3 enhanced 24-hydroxylase markedly. Since this assay is technically simple, rapid and sensitive, it will be useful in studying the regulatory mechanism in the renal metabolism of 25(OH)D3 in mammals.     

Development and characteristics of an oestrogen sulphotransferase in placenta and uterus of the pregnant mouse. Comparison between mouse and rat.

The mouse placenta possesses a soluble oestrogen sulphotransferase activity which increases markedly from at least 12 days of gestation until term. At about 16 days of gestation, a similar activity is found in the uterus. This activity also increases until term and disappears rapidly post partum. The uterine enzyme activity appears to require the presence of the foetal unit for its onset, since unoccupied horns, whether their endometrial stromal cells are differentiated to decidual cells or not, are essentially devoid of it. Uterine cytosols from non-pregnant mice are also inactive in this respect. In late gestation, the uterine sulphotransferase is confined to the decidua basalis, the areas to which the placentas are attached. The sulphotransferase(s) of placenta and uterus has an absolute requirement for 3'-phosphoadenosine 5'-phosphosulphate, and possesses little activity in the absence of exogenous thiol groups. Stimulation is also seen in the presence of Mn2+, Mg2+ or Ca2+. Oestrone and oestradiol, and to a lesser degree oestriol, are substrates for the enzyme(s), whereas testosterone, cortisol and dehydroepiandrosterone are not. Oestrone and oestradiol at higher concentrations (1.0-1.5 microM) completely inhibit the enzyme(s). These enzymes could play a role in altering tissue concentrations of active oestrogens during gestation in the mouse. Oestrogen sulphotransferase activity is low or absent in reproductive tissues of the pregnant rat.     

Change in the relative importance of oestrone and oestradiol synthesis by the rat ovary between fetal and prepubertal stages

Ovaries of rat fetuses at 20 and 21 days and of neonatal rats at 5 and 14 days were cultured in the presence of [3H]testosterone, and the conversion percentages into oestrone (E1) and oestradiol (E2) were determined by double isotopic dilution combined with recrystallization to constant specific activity. Insignificant in the 20- or 21-day-old fetus, oestradiol synthesis increased relative to oestrone synthesis in the 5-day-old neonate (E1/E2 = 3.4) and still more at the stage of 14 days (E1/E2 = 0.78). FSH had no effect on oestrogen synthesis at the 4 stages investigated.     

The effect of tumor necrosis factor/cachectin on follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells

We investigated the effects of tumor necrosis factor (TNF)/cachectin on follicle-stimulating hormone (FSH)-induced aromatase activity in cultured rat granulosa cells using the stereospecific transfer of 3H from [1 beta-3H] androstenedione into 3H2O. TNF (10 pg/ml-10 ng/ml) inhibited FSH (250 ng/ml)-induced aromatase activity in a concentration-dependent manner, and 10 ng/ml of TNF completely abolished the FSH-induced aromatase activity. A time course analysis of the effects of TNF showed that TNF had no effect on induced aromatase activity, but inhibited the further induction of the enzyme by FSH. TNF (10 ng/ml) also inhibited the ability of TGF beta (1 ng/ml) to enhance aromatase activity and increase progesterone synthesis. Thus, TNF is a component of the complex array of proteins that modulate ovarian function and, as such, may play a physiological role in the regulation of the granulosa cell. In view of its association with cachexia, it may also play a pathophysiological role in the suppression of reproductive function during chronic illness.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

The effects of serum oestrogen-binding components on the unbound oestradiol-17 beta fraction in the serum of developing female rats and on inhibition of [3H]oestradiol uptake by uterine tissue in vitro

Total oestradiol concentrations in the serum of young female rats were high and decreased after about 21 days of age. High affinity serum oestradiol-binding components (EBP), however, fell steadily from 5 to 23 days of age while the unbound oestradiol-17 beta fraction, which was low early in development, increased between 21 and 28 days of age. Injection (i.v.) of immature (EBP-rich) oestradiol-free serum into 21-day-old female rats led to a decrease in the unbound oestradiol fraction and an increase in serum FSH concentrations. Incubation of uterine tissue with [3H]oestradiol, with or without the addition of diethylstilboestrol (DES), showed the rate and degree of total and DES-suppressible uptake of [3H]oestradiol to be greatest from buffer, less from adult (EBP-poor) serum and negligible from immature (EBP-rich) serum; moreover, there was a positive correlation between the degree of uptake of [3H]oestradiol and the unbound fraction of [3H]oestradiol in the incubate. It is concluded that, at least in young rats, oestradiol activity depends more on the availability of free oestradiol than on its total plasma concentrations.     

Hormonal regulation of oestrogen and progesterone receptors in cultured bovine endometrial cells.

Changes in the number of progesterone and oestradiol receptors in the endometrium are thought to play a role in the induction of luteolysis. The effect of oestradiol and progesterone on the regulation of their receptors in cultured bovine uterine epithelial and stromal cells was examined to determine the mechanisms involved in this process. Cells were obtained from cows at days 1-3 of the oestrous cycle and were cultured for 4 or 8 days in medium alone (RPMI medium + 5% (v/v) charcoal-dextran stripped newborn calf serum) or with oestradiol, progesterone or oestradiol and progesterone. At the end of culture, receptor binding was measured by saturation analysis. Specific binding of both [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor (6,7-3H) pregn-4-ene-3,20-dione) and [3H]oestradiol to epithelial and stromal cells showed high affinities (Kd = 1.1 x 10(-9) and 6 x 10(-10) mol l-1, respectively, for progesterone receptors; Kd = 5.5 x 10(-9) and 7 x 10(-10) mol l-1, respectively, for oestradiol receptors). In the stromal cells, oestradiol (0.1-10 nmol l-1) increased the number of oestradiol receptors from 0.21 +/- 0.06 to 0.70 +/- 0.058 fmol microgram-1 DNA and the number of progesterone receptors from 1.4 +/- 0.83 to 6.6 +/- 0.70 fmol microgram-1 DNA in a dose-dependent manner after 4 days of culture (P < 0.01). After culture for 8 days, the stimulatory effect of oestradiol increased. Progesterone (50 nmol l-1) had no effect on the number of oestradiol or progesterone receptors (P > 0.05). However, progesterone inhibited the stimulatory effect of oestradiol. In epithelial cells, the lower concentrations of oestradiol (0.1 and 1 nmol l-1) stimulated the number of progesterone receptors (P = 0.05) after 4 days culture, whereas the highest concentration of oestradiol (10 nmol l-1), progesterone (50 nmol l-1) and progesterone (50 nmol l-1) plus oestradiol (1 nmol l-1) had no effect. After culture for 8 days, the stimulatory effect of oestradiol decreased. In contrast to progesterone receptors, the number of oestradiol receptors increased with oestradiol concentration (P < 0.01). These data show that the number of progesterone receptors was higher in the stromal cells than in epithelial cells, whereas the number of oestradiol receptors was higher in the epithelial cells than in stromal cells. Oestradiol upregulates its own receptor and increases the number of progesterone receptors in both cell types in vitro, whereas progesterone has little effect, but inhibits the effects of oestradiol on progesterone receptors.     

Studies on the metabolism of oestrone sulphate. Comparative perfusions of oestrone and oestrone sulphate through isolated rat livers

The metabolism of [4-(14)C]oestrone and of [6,7-(3)H(2)]oestrone sulphate was studied during cyclic perfusion and once-through perfusion of the isolated rat liver. The following results were obtained. 1. As shown by once-through perfusion, the two steroids are metabolized differently during the first passage through the organ. [4-(14)C]Oestrone was taken up by the liver and partly delivered as oestradiol-17beta and oestriol into the medium. After uptake of [6,7-(3)H(2)]oestrone sulphate, only oestrone, liberated by hydrolysis, was delivered into the medium; no oestradiol-17beta or oestriol could be detected in the medium after one passage through the organ. This indicates that intracellular oestrone, which was taken up as such, and oestrone, which derived from intracellular hydrolysis, may be metabolized in different compartments of the liver cell. 2. The results of the cyclic perfusion showed that intracellular oestrone is preferentially conjugated with glucuronic acid, and subsequently excreted into the bile. Intracellular oestrone sulphate is preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate is a better substrate for the 17beta-hydroxy steroid oxidoreductase than is oestrone. 3. Albumin-bound oestrone sulphate acts as a large reservoir, and in contrast with free oestrone is protected from enzyme attack by its strong binding to albumin. 4. Oestrone sulphate is partly converted into the hormonally active oestrone by liver tissue. This suggests that liver not only inactivates oestrogens, but also provides the organism with oestrone, which is subsequently readily taken up by other organs.     

Metabolism of radiolabeled glucose by mouse oocytes and oocyte-cumulus cell complexes.

This study was carried out to examine the metabolism of [1-14C]-, [6-14C]-, and [5-3H]glucose by oocyte-cumulus cell complexes (OCC) and denuded oocytes (DO) and to test the hypothesis that metabolism of glucose through the pentose phosphate pathway is associated with meiotic induction. OCC or DO were cultured in hanging drops suspended from the cap of a microfuge tube, with NaOH serving as a trap to collect released 3H2O or 14CO2. Preliminary experiments established that this culture system supports both spontaneous and ligand-induced meiotic maturation. An initial time course experiment (1.5-6 h) showed that hypoxanthine-treated OCC from eCG-primed animals metabolized glucose principally via glycolysis, with an increase to 2.7-fold in response to FSH. Though more [1-14C]glucose was oxidized than [6-14C]glucose, its metabolism was about two orders of magnitude less than that of [5-3H]glucose. Also, FSH significantly increased oxidation of [1-14C]glucose but not [6-14C]glucose, indicating a preferential activation of the pentose phosphate pathway. Pyrroline carboxylate, an activator of the pentose phosphate pathway, increased the activity of this pathway to over 2-fold but failed to affect glucose oxidation through the tricarboxylic acid cycle. Glycolytic metabolism was increased by 25%. The addition of pyruvate to pyruvate-free medium resulted in significant reduction in the metabolism of all three glucose analogues. In OCC retrieved from hCG-injected, primed mice and cultured under hormone-free conditions, metabolic responses were similar to those in FSH-treated complexes cultured in hypoxanthine. DO metabolized glucose, but at a much reduced rate when compared to OCC. Pyruvate reduced the consumption of all three glucose analogues by DO. Pyrroline carboxylate reduced [5-3H]glucose metabolism by DO but had little effect on [1-14C]- and [6-14C]glucose oxidation. These data demonstrate metabolism of glucose by both DO and OCC, but reveal that cumulus cells are more active than the oocyte in this regard. In addition, induction of maturation by FSH, hCG, or pyrroline carboxylate was accompanied by a significant increase in the oxidation of [1-14C]glucose but not [6-14C]glucose by OCC, supporting a proposed role for the pentose phosphate pathway in meiotic induction.     

Efflux of a suppressive neurotransmitter, GABA, across the blood-brain barrier

In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.     

Gossypol inhibits aromatase activity in cultured porcine granulosa cells

The present study investigated whether gossypol inhibited aromatase activity in cultured porcine granulosa cells. Aromatase activity was assayed by measuring (3)H-H(2)O released from [1beta-(3)H]-androstenedione. First, immature porcine granulosa cells were cultured with various doses of follicle stimulating hormone (FSH, 1 to 1000 ng/ml) for 1 to 5 d to determine optimal culture conditions for aromatase activity assay. Second, porcine granulosa cells were cultured with or without FSH in the presence or absence of gossypol. Gossypol, at 4 muM, significantly inhibited FSH-induced aromatase activity while showing no effect on basal aromatase activity. Gossypol did not inhibit cell proliferation during cell culture. These results suggest that gossypol inhibits aromatase activity by interfering with FSH induction of aromatase in cultured porcine granulosa cells.     

Suitability of primary monolayer cultures of adult rat hepatocytes for studies of cholesterol and bile acid metabolism

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.058 micrograms/mg protein/h. beta-Muricholic acid was synthesized at approximately a 3-fold greater rate than cholic acid in these cultures. Cultured hepatocytes rapidly converted the following intermediates of the bile acid pathway; 7 alpha-hydroxy[7 beta-3H]cholesterol, 7 alpha-hydroxy-4-[6 beta-3H] cholesten-3-one, and 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha, 12 alpha-triol into bile acids. [24-14C]Chenodeoxycholic acid and [3H]ursodeoxycholic acid were rapidly biotransformed to beta-muricholic acid. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity measured in microsomes of cultured hepatocytes decreased during the initial 48 h following plating, but remained relatively constant for the next 72 h. In contrast, cholesterol 7 alpha-hydroxylase activity appeared to decrease during the first 48 h, followed by an increase over the next 48 h. Despite the apparent changes in enzyme activity in vitro, the rate of bile acid synthesis by whole cells during this time period remained constant. It is concluded that primary monolayer cultures of rat hepatocytes can serve as a useful model for studying the interrelationship between cholesterol and bile acid metabolism.     

Interaction of the radiolabelled high-affinity anti-oestrogen [3H]H1285 with the cytoplasmic oestrogen receptor.

The high-affinity triarylethylene anti-oestrogen H1285 [4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between [3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with [3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. [3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. [3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with [3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of [3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for [3H]oestradiol. Because of the extremely slow dissociation of [3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of [3H]H1285-receptor complexes with those of [3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. [3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with [3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of [3H]H1285-receptor complexes and [3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of [3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for [3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor.