Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo

The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in vivo and in vitro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely, 17α-ethynyl estriol, estriol-3-cyclopentyl ether, and 17α-ethynyl estriol-3-cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in vivo. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endometrial cells in primary tissue culture, E₃ has 12% the affinity of estradiol (E₂) for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentrations of E₃(10⁻¹⁰ M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E₃ to be only slightly less effective than E₂. In these endometrial and breast cancer cell systems in vitro, there is no metabolism of E₃ while E₂ is metabolized to estrone.Hence, estriol is an effective estrogen in vitro. In vivo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.     

The uptake of radioactivity by the uterus of the rabbit following the sytemic administration of (3H) estradiol-17beta and (3H) estrone and the identification and subcellular localization of radiometabolites in the uterus

In an effort to determine the relevance of the uterine oxido-reduction of estrogens to their action in the rabbit uterus, the uterine uptake of radioactivity administered subcutaneously as [³h] estradiol-17β or [³H]estrone and the subcellular distribution of radio-metabolites in the uterine tissue were studied. The animals were killed 20 min, 1, 3 and 9 hr after the administration of 0.1 μg tritiated steroid and the relative proportions of radioactive estradiol-17β and estrone in plasma and in ‘cytosol’, ‘mitochondrial/microsomal’ and ‘nuclear’ fractions of the uterine homogenates were studied. Despite the presence of a high proportion of estrone in chloroform extract of plasma, very little was found in the fractions from uterine tissue irrespective of the steroid administered. Highest levels of uterine estrone were found in the ‘mitochondrial/microsomal’ preparation. There was no apparent difference in the pattern of uptake of radioactivity administered as [³H] estradiol-17β or [³H] estrone. The presence of high levels of 17β-hydroxysteroid dehydrogenase activity in the rabbit uterus may be responsible for the apparent difference between these results and those of similar experiments using the rat.     

Receptor and Biological Response to Estriol in the Fetal Uterus of Guinea Pig

Abstract

The binding of ³H-estriol was examined in the fetal uterus of guinea pig. The physico-chemical characteristics of the binding of ³H-estriol to macromolecules are similar to the typical receptor protein for estrogens. Different estrogens (estriol, estradiol, estrone and diethylstilbestrol) compete with this binding but progesterone and testosterone have no effect. The binding affinity has a Kd of 5.5 ± 1.6 ± 10^?10M. By ultra-centrifugation in sucrose gradient, two specific components with sedimentation coefficients of 8 and 45 are found. Competition studies suggest that the same specific binding sites may be present for estriol (E₃) and for estradiol. The s.c. administration of E₃ to the pregnant guinea pig (1 mg/day per kg body weight for 3 days) provokes two biological responses in the fetal uterus: a uterotrophic effect and a significant increase in the progesterone receptor. The increase in the fetal uterine weight is 50–70% in relation to the non-treated animals and the progesterone receptor concentration is 10–14 times higher than in the control animals. These effects are similar (or slightly higher) than in animals primed with equimolecular quantities of estradiol. In contrast, single daily injections of E₃ to newborn guinea pig, results only in weak uterotrophic activity.

It is concluded that estriol is capable of causing a biological response in the uterus during intra-uterine life.     

Solubilization and partial purification of steroid sulfatase from rat liver: characterization of estrone sulfatase.

Steroid sulfatase, a membrane-bound enzyme present in many mammalian tissues, was extracted from rat liver microsomes by treatment with Miranol H2M, a zwitterion detergent, and sonication. It has been purified approximately 33-fold. All steps of the purification, which included salt and solvent fractionation, hydroxylapatite treatment, ion-exchange chromatography, and gel filtration were performed in the presence of Miranol H2M, most of which was removed from the final preparation by gel filtration. The final preparation did not contain any detectable NADPH-cytochrome c reductase or glucose-6-phosphate phophatase activities. According to the elution volume on a Sephadex G-200 column, steroid sulfatase has a molecular weight of approximately 130,000. Polyacrylamide-gel electrophoresis in the presence of Miranol H2M revealed one major protein band which was enzymatically active. Purified steroid sulfatase hydrolyzes all the sulfate esters of estrone, dehydroepiandrosterone, pregnenolone, testosterone, and cholesterol as well as p-nitrophenyl sulfate, the substrate for arylsulfatase C, during the purification. However, estrone sulfatase and arylsulfatase C activities were enriched more than the others. Analysis of kinetic data and the effects of different buffers and of Miranol H2M also suggested that estrone sulfatase and arylsulfatase C are identical but that they are distinct from the other sulfatases. Competitive inhibition studies suggest that estrone sulfatase also catalyzes the hydrolysis of the sulfate esters of other estrogens.     

A comparative study of the effects of cadmium and nickel on liver microsomal drug metabolizing enzymes of guinea-pig in vitro

1. In vitro addition of cadmium chloride (CdCl₂) or nickel chloride (NiCl₂) to an incubation mixture produced a concentration-dependent inhibition of liver microsomal aniline 4-hydroxylase activity of male guinea-pig. The inhibitory effect of CdCl₂ on the enzyme activity was stronger than that of NiCl₂.2. While CdCl₂ also caused a concentration-dependent inhibition of liver microsomal ethylmorphine N-demethylase activity, NiCl₂ increased the enzyme activity between the concentrations 10⁻⁵ and 10⁻³ M and caused a rather abrupt decline at higher concentrations.3. When the liver 10,000 g supematants were preincubated in the presence of metals, metal-induced inhibitions increased as the time of preincubation progressed and attained their maximal rates at about 5 and 15 min for microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase activities, respectively. However, no change was noted by NiCl₂ on liver microsomal ethylmorphine N-demethylase activity as the time of preincubation progressed.4. After preincubations, the concentration-dependent inhibitions produced by metals on liver microsomal drug metabolizing enzyme activities were found to be stronger and in favour of CdCl₂.     

Enzymatic sulfation of bile salts. II. Studies on bile salt sulfotranferase from rat kidney

An enzyme system which catalyzes the transfer of the sulfate group from 3′-phosphoadenosine-5′-phosphosulfate to bile salts has been isolated and characterized from rat kidney. The enzyme is present in the cytosol fraction of kidney cells. It was purified by DEAE-Sephadex A-50, agarose-hexane-adenosine 3′,5′-diphosphate affinity chromatography and isoelectrofocusing electrophoresis. The apparent K_m values of the enzyme are 2 · 10⁻⁶ M for 3′-phosphoadenosine-5′-phosphosulfate, and 4 · 10⁻⁵ M for taurolithocholate. Sulfation occurred with conjugated as well as with unconjugated bile salts. The enzyme reacts with both primary bile salts (cholate, chenodeoxycholate and their conjugates), and secondary bile salts (lithocholate and its conjugates). The rates of reaction in decreasing order are monohydroxylated > dihydroxylated > trihydroxylated and glycoconjugates > tauroconjugates > unconjugates. The enzyme activity is inhibited by p-chloromercuri benzoate and iodoacetate indicating the possible requirement of a sulfydryl group for activity. A molecular weight of 80 000 was estimated by gel filtration techniques which is significantly smaller than the liver enzyme (130 000). The purified enzyme does not react with estrone or dihydroepiandrosterone.     

Estrogen sulfotransferase activity in guinea pig uterus and chorion

An estrogen sulfotransferase (ST) is detectable in high speed supernatants of pregnant guinea-pig uterus and shows maximum activity between about 47 and 55 days of gestation, with a decrease toward term. No appreciable activity was apparent in the non-pregnant state or before at least 43 days of pregnancy. A considerably higher ST activity is present in chorion as early as 30 days of gestation, and this also decreases toward term. The two ST's exhibit similar KM (0.1-0.13 microM with estrone as substrate) and pI (5.8) values, as well as similar specificities. Estradiol-17 beta and estriol are sulfurylated 82 and 6% that of estrone at equimolar concn. Neither p-nitrophenol nor several neutral steroids are substrates for the enzymes. Enzyme activity is poorly expressed in the absence of thiol groups, the presence of monothioglycerol stimulating uterine and chorion enzymes by 5- and 15-fold, respectively. Stimulation is also observed in the presence of Mg2+, Ca2+ or Mn2+. Chromatofocusing on a poly buffer ion-exchanger from pH 7.4 to 4.0 resulted in elution of a sharp peak of enzyme activity, at pH = 5.8, from both tissues provided that the eluting buffer contained thiol groups and 0.25 M sucrose. This single step resulted in at least a 35- to 100-fold increase in specific activity. The partially purified enzyme from chorion exhibited a KM for estrone of 0.13 microM.     

Solubilization and partial purification of steroid sulfatase of human placenta

Steroid sulfatase of human placenta has been solubilized by treatment of the microsomal fraction with an amphoteric surface active agent, Miranol H2M and ultrasound. Criteria of solubility include non-sedimentation of the activity following centrifugation at 160,000 × g, its retention on Sepharose 6B and a single peak of activity after polyacrylamide gel electrophoresis. Enzyme activity was located in the same gel fractions for the two substrates tested; cholesterol sulfate and dehydroisoandrosterone sulfate. The addition of dithiothreitol was found necessary to maintain the stability of the enzyme indicating the presence of sulfhydryl groups in the molecule. A molecular weight of approximately 330,000 has been estimated from the elution volume of the enzyme system on a column of Sepharose 6B. It is believed that this protein represents a sulfatase enzyme complex composed of subunits with different specificities. From kinetic studies, a K_m of 6.2 × 10^?5M for the cleavage of dehydroisoandrosterone sulfate and a K_m of 2 × 10^?6M for the cleavage of cholesterol sulfate have been calculated.     

Effect of using calcium-free Krebs' solution on basal and A23187-stimulated prostaglandin output from the day 15 guinea-pig uterus superfused in vitro

The basal outputs of prostaglandin (PG) F_2α and PGE₂ from the Day 15 guinea-pig uterus superfused in vitro were unaffected by omitting Ca²⁺ from the Krebs' solution. In contrast, this omission of Ca²⁺ reduced the basal output of 6-oxo-PGF_1α (which reflects PGI₂ production) from the uterus by an average of 50%. Spontaneous and A23187-stimulated contractions of, and the stimulation by A23187 of PGF_2α, PGE₂ and 6-oxoPGF_1α outputs from the Day 15 guinea-pig uterus were all abolished by superfusing the tissue with Krebs' solution lacking Ca²⁺. It is concluded that the basal output of 6-oxo-PGF_1α, the occurrence of spontaneous contractions, and the effects of A23187 on PG output and contractility of the Day 15 guinea-pig uterus are dependent on extracellular Ca²⁺. However, the increase in PGF_2α output from the guinea-pig uterus on Day 15 compared to days much earlier in the cycle is apparently not dependent upon extracellular Cat+. The implications of these findings regarding the biochemical mechanisms involved in the increased synthesis of PGF_2α (the uterine luteolytic hormone) by the guinea-pig endometrium during the last one-third of the cycle are discussed.     

Binding characteristics of estrone,estradiol and estriol to the human myometrial estrogen receptor

The human myometrial estrogen receptor in cytosol from pre-menopausal uterine samples has been characterized. At 0° estradiol (K_D 0.38 × 10⁻¹⁰M) has the highest affinity to the receptor followed by estrone (K_D 0.76 × 10⁻¹⁰M) and estriol ((K_D 1.33 × 10⁻¹⁰M). The association rate constant is 2.8 × 10⁵M⁻¹s⁻¹ for estradiol, 2.1 × 10⁵M⁻¹s⁻¹ for estrone and 0.79 × 10⁵M⁻¹s⁻¹ for estriol. The dissociation constants and the association rate constants increase with temperature. The calculated thermodynamic parameters indicate a positive change in entropy for the formation of the estrogen receptor complex.The cytoplasmic estrogen receptor has a sedimentation coefficient of 4 s in low salt sucrose gradients. In buffer containing diisopropylfluorophosphate (DFP) to inhibit proteolytic activity the estrogen receptor complex sediments solely as an 8 s peak if [³H]-estradiol is added to the buffer prior to homogenization and the tissue sample is used immediately after hysterectomy. Estrogen receptor complexes that sediment at 4 s and 8 s are found if [³H]-estradiol is omitted from the homogenization buffer and instead added after the cytosol preparation. Most likely a protease is involved the activity of which is not completely inhibited by DFP.Addition of low concentrations of Cu²⁺ (10 μM) to the cytosol increases the dissociation constant and decreases the estrogen-binding capacity of the receptor. The rate of association is reduced in the presence of 20 μM Cu²⁺. The estrogen receptor complexes do not show any change in their sedimentation profiles in the presence of Cu²⁺.     

Steroid sulfatase activities in normal and cirrhotic livers and plasma levels of estrone sulfate,estrone and estradiol-17β in men

Estrone and dehydroepiandrosterone (DHEA) sulfatases were studied in livers of normal and cirrhotic men. Their Km were 3.2μM and 1.2μM respectively. The musomal sulfatases were solubilized by Miranol H2M and ultrasound. After gel filtration, the soluble material gave a single peak of activity for both substrates with a molecular weight of approximately 330,000. In terms of pmol of product.min^?1 per mg of fresh tissue, the mean (±SD) values of estrone and DHEA sulfatase activities were lower in cirrhotic livers [(n=7) (4.09±2.90 and 0.38±0.20)] than in normal livers [(n=13)(8.29±4.00 and 0.69±0.20)]. The differences were statistically significant: p<0.03 for estrone sulfatase and p<0.01 for DHEA sulfatase. In cirrhotic men, the mean level of plasma estrone is increased whereas that of estrone sulfate is decreased. The variations may be related to the decrease of serum albumin in cirrhotic subjects.     

THE BIOGENESIS OF PRIMARY SEX HORMONES : I. THE FATE OF ESTRINS INJECTED INTO THE RABBIT

1. A method is given for the extraction and fractionation of rabbit urines which frees these urines of inactive chromogens but permits a quantitative recovery of estrone and estriol for the colorimetric determination of these compounds. 2. Estrone and estriol content of rabbit urine extracts can be determined by the concentration of the colored compound they form upon diazotization with sulfanilic acid and by the modified phenolsulfonic acid test of Cohen and Marrian. Estriol can be determined by the specific reaction first described by David. The technique for these tests is presented. 3. Estriol (300 micrograms) injected into rabbits (a) in heat, (b) pregnant, (c) pseudopregnant, (d) hysterectomized in heat, (e) hysterectomized pseudopregnant, (f) ovariectomized, is excreted in the urine as estriol. Rabbit does in the luteal phase (b, c, and e) excrete 3 to 4 times the amount of estriol excreted by females without corpora lutea (a, d, and f). 4. When estrone (300 micrograms) is injected into the same types of rabbit does types a, b, and c excrete both estrone and estriol, type f excretes both estrone and estriol shortly after ovariectomy, but only estrone at 2 months after castration. Hysterectomized animals (types d and e) never excrete estriol after estrone injection. The total urinary estrin (estrone plus estriol) in estrone-injected animals is increased 2 to 3 times in animals in the luteal phase (b, c, and e). 5. It is concluded that the uterus is the site of conversion of estrone to estriol, and that the conversion cannot take place in a uterus completely free of ovarian control (e.g., in long time ovariectomized animals). 6. In neither estrone-injected nor estriol-injected females is all the injected hormone recovered in the urine. The maximum recovery is 66 per cent. When estrone-benzoate (600 micrograms) is injected 94–98 per cent of the hormone is recovered from animals in the luteal phase (types c and e) and about 79 per cent in an ovariectomized female (type f). These data are taken to indicate that luteal secretions give partial protection against destruction to the hormones. 7. The observation that in certain of the urine extracts the hormone titer by bioassay is somewhat higher than the colorimetric titer may indicate that there is a slight conversion of estrone to estradiol, particularly since no equilenin was found in any of the extracts by colorimetric test. 8. The simultaneous injection of 300 micrograms of estrone and 500 micrograms of progesterone 4 days after an initial injection of 300 micrograms of estrone results in: (1) an increased estrin excretion in females in heat, hysterectomized unmated, and ovariectomized, and a slight decrease in the pseudopregnant female; (2) the appearance of estriol in the urine of the long time ovariectomized animal with no urinary estriol in a control ovariectomized animal receiving no progesterone. These findings are taken to prove that the conversion of estrone to estriol occurs in the uterus under the influence of progesterone. Since animals in heat produce small amounts of estriol after estrone injection it is inferred that the ovaries of estrus rabbits produce small amounts of corpus luteum hormone in the absence of formed corpora lutea.     

Carbohydrate and sulfate conjugations of p-nitrophenol by hepatopancreas of Homarus americanus

1. Glucosyltransferase activity is present in hepatopancreas of Homarus americanus. The enzyme appears to have a specific requirement for UDP-glucose, and ADP-, CDP- or GDP-glucose do not substitute for it. The activity is mainly microsomal, exhibits a pH optimum at 7.9–8.1, and its apparent K_m values are 2 mM and 0.3 mM for UDP-glucose and p-nitrophenol respectively. Microsomal glucosyltransferase activity increases with increasing temperature up to 45°.2. Hepatopancreas possesses a very active sulfotransferase which utilizes 3′-phosphoadenosine-5′-phosphosulfate for sulfoconjugation of p-nitrophenol. The activity is associated chiefly with the soluble fraction and amounts to about 16 nmoles/mg protein/30 min.3. No detectable glucuronidation of p-nitrophenol occurred when preparations of hepatopancreas fortified with UDP-glucuronic acid were incubated with p-nitrophenol.     

Conversion,in vitro,of (7n-3H) testosterone to estrone and estradiol-17β and their 3-sulfäte conjugate by the guinea-pig placenta

Different cellular fractions of guinea-pig placenta were incubated in the presence of (7n-³H) testosterone. Microsomal aromatization of ³H-testosterone into estrone and estradiol-17β was demonstrated in the presence of NADPH. The predominance of estrone after incubation with 17β-hydroxylated precursors, (7n-³H) testosterone and (6,7-³H) estradiol-17β, indicate that there is a microsomal 17β-hydroxysterold dehydrogenase activity. In this report, cytosolic sulfurylation of estrogens is demonstrated. This latter activity represents a quite original characteristic of the placental metabolism of estrogens in guinea-pigs. In contrast with the human placenta where there is considerable sulfatase activity, the guinea-pig placenta can sulfurylate estrogens.     

Steroidal control of rat uterine 17β-hydroxysteroid dehydrogenase activity

The interconversion of estradiol-17β and estrone in the rat uterus is due to the action of 17β-hydroxysteroid dehydrogenase. Whole uteri or 800 × g supernatant fractions of the uteri were incubated in the presence of [³H] estradiol-17β and NAD at 37°C for 3 h or 1 h, respectively. In the mature rat uterus the oxidation of estradiol-17β and estrone was dependent on the stage of the estrous cycle, suggesting hormonal control. The 17β-hydroxysteroid dehydrogenase activity was highest at estrus (200 fmol estrone) and lowest at diestrus (80 fmol estrone). An enhancement of activity occurred when adult rats at each stage of the estrous cycle were administered estradiol-17β, while progesterone administration at each stage resulted in decreased enzyme activity. The uterine 17β-hydroxysteroid dehydrogenase activity of estradiol-17β treated ovariectomized rats was time and dose dependent but decreased when progesterone was administered with or without estradiol-17β administration. These results suggest that estradiol-17β caused an increase in enzyme activity that was inhibitable by progesterone in the rat uterus. The increased 17β -hydroxysteroid dehydrogenase activity may reflect a specific response of the rat uterus to estradiol-17β.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg²⁺. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent K_m of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent K_m and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent K_m for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high K_m. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent K_m for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent K_m of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme.     

Collagen proline hydroxylase activity in the uterus of the rat during rapid collagen synthesis in vivo

The activity of collagen proline hydroxylase in the 27,000g supernatant of the uterus was compared in the normal 20-day-old rat and in the adult rat 21 days after ovariectomy. The cofactor requirements of this enzyme were shown to be qualitatively the same as the enzyme from rat liver and skin. The specific activity of collagen proline hydroxylase in the uterus of the immature rat is approximately 250% higher than that of the ovariectomized animal. Although the total protein of the uterus of the ovariectomized rat is much greater, the total activity of this enzyme is 50% higher in the uterus of the immature rat. The daily administration of 5 μg estradiol-17β for 4 consecutive days to either animal results in a significant increase in the activity of collagen proline hydroxylase. Enzyme activity increases significantly 24 hr after the first dose of estradiol-17β and remains elevated in a reproducible pattern throughout the experimental period. Other estrogens including estriol, estrone, diethylstilbestrol, and ethynylestradiol-3-methyl ether also increase significantly the activity of collagen proline hydroxylase in the uterus of the immature rat. The activity of collagen proline hydroxylase was compared in the 27,000g supernatant of uterus of the immature and ovariectomized rat in a dose-response study with estradiol-17β and there appears to be little, if any, difference in total enzyme capacity. These results suggest that the failure of collagen to accumulate in the uterus of the ovariectomized rat administered estradiol-17β is unrelated to a low activity of collagen proline hydroxylase.     

Effects of hydrocortisone, oestradiol and progesterone on A23187-stimulated prostaglandin output from the guinea-pig uterus superfused in vitro

Hydrocortisone (10 μg/ml) had no effect on the basal outputs and A23187-stimulated outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α from the Day 15 guinea-pig uterus superfused . These findings indicate that the high output of PGF_2α from the guinea-pig uterus during the last one-third of the oestrous cycle is not modulated by the adrenal glucocorticoid hormones. Progesterone (10 gmg/ml) had no effect on the A23187-induced increases in PG output from the Day 15 guinea-pig uterus. However, oestradiol (10 gmg/ml but not 1 μg/ml) significantly reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by A23187 from the Day 15 guinea-pig uterus, without affecting basal PG outputs. The increase in uterine tone induced by A23187 in the Day 15 guinea-pig uterus was reduced by 20–50% by oestradiol (10 μg/ml). The addition of oestradiol (10 μg/ml) and progesterone together (10 gmg/ml) produced the same effects on the Day 15 guinea-pig uterus as oestradiol alone. Oestradiol (10 μg/ml) also reduced the A23187-induced increases in PG output from the Day 7 guinea-pig uterus, but did not reduce the increase in uterine tone. Oestradiol (10 gmg/ml) reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by exogenous arachidonic acid from the Day 7 and Day 15 guinea-pig uterus. Previous studies have shown that oestradiol is not a cyclo-oxygenase inhibitor. The present findings suggest that oestradiol, at a relatively high concentration, may interfere with the access of arachidonic acid to the cyclo-oxygenase enzyme. This action of oestradiol may explain its anti-luteolytic action when administered to guinea-pigs in large doses after Day 9 of the cycle.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

Interactions of endogenous and exogenous estrogenic compounds with human placental microsomal cytochrome P-450 (P-450hpm)

Spectral analyses of human placental microsomal cytochrome P-450 (P-450_hpm) revealed that estrone, β-estradiol, and estriol each interacted with the cytochrome to produce Type I difference spectra. β-Estradiol produced the most intense difference spectra and additional studies with this steroid suggested the existence of multiple sites for estrogen binding or of multiple cytochromes in placental microsomes. The magnitude of difference spectra produced by β-estradiol correlated (r = 0.84) with concentrations of P-450_hpm as determined by CO-difference spectra as well as with the intensity of difference spectra produced by androstenedione (r = 0.87). No statistically significant correlations between intensities of β-estradiol-induced binding spectra and placental aryl hydrocarbon hydroxylase activities were observed. The ability of the naturally occurring estrogens to bind to cytochrome P-450 did not appear to be related to their capacity to inhibit the placental aromatization reaction. Diethylstilbestrol, ethinylestradiol, mestranol, progesterone, and pregnenolone all inhibited the placental conversion of androstenedione to estrogens, but these compounds did not produce observable Type I difference spectra. Of the estrogens studied, diethylstilbestrol proved to be the most effective inhibitor of aromatase in vitro causing 99% inhibition at concentrations of 10⁻⁴M. β-Estradiol and ethinylestradiol produced only 60.4 and 81.0% inhibition, respectively, at 10⁻³ M. The results indicated that despite significant binding of endogenous estrogens to P-450_hpm, no evidence for a feedback inhibition of these estrogens on the placental aromatization reaction could be observed.