Ovine placental aromatase: studies of activity levels, kinetic characteristics and effects of aromatase inhibitors

We have measured microsomal steroid aromatase activity in the fetal component of ovine placental cotyledons collected from pregnant ewes between 124 days and 127 days of gestation. Aromatase activity was determined by quantifying the [3H]water by-product when [1 beta-3H(N)] androstenedione was used as substrate. The mean microsomal aromatase activity (+/- SD) was 5.7 +/- 2.2 pmol.min-1.mg protein-1 (n = 12) and was 9% of the aromatase activity of human placental microsomes [mean (+/- SD) of 66.1 +/- 25.0 pmol.min-1.mg protein-1 (n = 7)]. The apparent Km for ovine placental aromatase for androstenedione, at pH 7.4 and 37 degrees C, was 50 nM while the Vmax was 20.6 pmol.min-1.mg protein-1. The respective concentrations effecting 50% inhibition of ovine placental aromatase activity (the I50) for econazole, 4-hydroxyandrostenedione, imazalil, miconazole, ketoconazole and aminoglutethimide were 0.03, 0.05, 0.15, 0.50, 5.0 and 5.5 microM. The order of relative potencies were similar to those obtained for human placental aromatase. Ketoconazole and aminoglutethimide were approx 10 times more potent inhibitors of the sheep enzyme relative to the human. Aromatase activity was not confined to the microsomal fraction of ovine placental tissue but was distributed throughout all the particulate subcellular fractions. The proportionally high activity of the tissue homogenate (1.75 pmol.min-1.mg protein-1) is suggestive that in the last third of pregnancy, aromatase is not rate limiting with regard to placental estrogen production. It would appear, therefore, that the major factor regulating placental estrogen synthesis in ovine pregnancy is the availability of substrate.     

The regulation of ovine placental steroid 17 alpha-hydroxylase and aromatase by glucocorticoid

Parturition in the pregnant sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to estrogen production. This change is believed to be a consequence of the prepartum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha), steroid C-17,20-lyase, and possibly aromatase. We have investigated the activity levels of aromatase and 17 alpha-hydroxylase in placental microsomes in late pregnancy and dexamethasone-induced labor. Over the gestational period of 118-140 days basal levels of placental aromatase were relatively constant [mean value (+/- SD) of 5.6 +/- 1.6 pmol min-1 mg microsomal protein-1 (n = 10)]. Steroid 17 alpha-hydroxylase activity was undetectable [less than 0.5 pmol min-1 mg microsomal protein-1 (n = 7)]. In six animals in labor induced with infusion of dexamethasone into the fetus, placental aromatase activity had a mean value of 14.0 +/- 2.5 pmol min-1 mg protein-1; placental steroid 17 alpha-hydroxylase, measured in four of the animals, had a mean (+/- SD) activity of 319 +/- 58 pmol min-1 mg microsomal protein-1. Immunoblotting of placental microsomal preparations with specific antibodies to cytochrome P-450(17)alpha and NADPH-cytochrome P-450-reductase indicated that the glucocorticoid-induced activity of 17 alpha-hydroxylase was associated with increased content of cytochrome P-450(17)alpha. Northern blotting with a cDNA probe for cytochrome P-450(17)alpha showed that glucocorticoid increased the levels of mRNA for the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and cyclic change of aromatase cytochrome P-450 activity in human uteri

Activities of aromatase cytochrome P-450 in the columnar epithelial region (CE), squamous epithelial region (SE) and connective tissue (CT) of uterine cervix, and endometrium (EM) during the menstrual cycle were determined using [4-14C] and [1 beta-3H]androstenedione. Aromatase activities in the proliferative phase (n = 8) were 15.0 +/- 7.9, 10.9 +/- 10.3, 9.4 +/- 10.6 and 8.0 +/- 7.3 (mean +/- SD) fmol/h/mg protein in CE, SE, CT and EM, respectively, and aromatase activities in the secretory phase (n = 6) were 31.5 +/- 7.6, 19.1 +/- 7.1, 5.6 +/- 4.6 and 6.3 +/- 1.5 fmol/h/mg protein, respectively. The results show that the aromatase activities in these regions in the proliferative phase were not significantly different from each other. On the other hand, the aromatase activity in the secretory phase was significantly higher in CE than in any other regions (P less than 0.05), and significantly higher in SE than in CT or EM (P less than 0.05). There was no significant difference in aromatase activity between CT and EM. By comparison of aromatase activity between these two phases, the activity in CE was significantly higher in the secretory phase than in the proliferative phase (P less than 0.05), but no significant difference was observed in other regions.     

A comparison of methods measuring aromatase activity in human placenta and rat ovary

The single step chromatographic product isolation method using [4-14C]-androstenedione and the tritiated water method using either [1 beta, 2 beta-3H]- or [1 beta-3H]-androstenedione have been used to determine a suitable method to measure the aromatase activity in rat ovarian 1000 g supernatant and human placental microsomes. The single step product isolation method using [4-14C]A4 reveals the presence of four distinct [4-14C]-labelled products in the rat ovary of which only the synthesis of estradiol is markedly inhibited by CGS 16949A, a well established aromatase inhibitor. In the human placenta, the formation of both [4-14C]-estrone and [4-14C]-estradiol is strongly inhibited by CGS 6949A. Therefore, in the rat ovary spurious results are obtained if accumulative radiolabelled product formation is measured without characterisation of the products. The Vmax in the rat ovary using [1 beta, 2 beta-3H]-A4 as a substrate is 13.7 pmol/h/mg compared to 2.9 pmol/h/mg when [1 beta-3H]-A4 is used. In the human placenta, the Vmax is similar using either [1 beta, 2 betat-3H]-A4 or [1 beta-3H]-A4 (1.21 and 1.27 nmol/h/mg, respectively). Consistent results are obtained for the human placenta using either the single step chromatographic product isolation method or the tritiated water method. However, in the rat ovary the more suitable method of the two used to measure the aromatase activity is the tritiated water method employing [1 beta-3H]-A4 as a substrate. Aromatase activity in the rat ovary during estrus cycle was measured using the tritiated water method employing [1 beta-3H]-A4 as a substrate. A peak of aromatase activity at proestrus was seen which returned rapidly to its basal level at estrus. Plasma estradiol concentrations were in parallel with the aromatase activity.     

Studies of the biochemical basis of steroid sulphatase deficiency--III. Phospholipid composition of microsomes from normal and steroid sulphatase deficient placentas

The phospholipid composition has been determined for placental microsomes from 11 normal and eight pregnancies complicated by steroid sulphatase deficiency. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin were found to be the major phospholipids of normal placental microsomes, comprising respectively 41.6 +/- 4.6% (mean +/- SD). 30 +/- 5.7% and 22.5 +/- 4.9% of the total phospholipid content. There was no correlation between the steroid sulphatase activity of the microsomes and the content of any of the three phospholipids. Though their contents were significantly decreased. (P less than 0.001) phosphatidylcholine, phosphatidylethanolamine and sphingomyelin similarly constituted the major portion of the total phospholipids in sulphatase deficient microsomes, representing 36 +/- 4.2%, 34 +/- 6.1% and 22.4 +/- 6.7% respectively. Only the percentage of phosphatidylcholine was significantly different (P less than 0.02) from normal microsomes. The results show that the decreased phospholipid content of steroid sulphatase deficient placental microsomes reflects a lower content of all major classes of phospholipids, particularly phosphatidylcholine.     

Immunocytochemical studies of aromatase in early and full-term human placental tissues: comparison with biochemical assays

An immunocytochemical method for visualizing the aromatase P450 enzyme with a specific monoclonal antibody has been developed for use with unfixed, frozen tissue sections. We compared both monoclonal and polyclonal aromatase-specific antibodies and found that placental aromatase was consistently and exclusively located in the syncytiotrophoblast layer of chorionic villi. The monoclonal antibody had the highest affinity, with negligible associated background stain. Fixation was found to impair stain reaction. Examination of first trimester and term placentae revealed identical immunostaining patterns of similar intensity in 9 of 10 samples. The immunostain reactions of first trimester and term placentae were compared with their respective microsomal aromatase activity, determined simultaneously by both indirect radiometric tritiated water (3H2O) assay, and direct product isolation by HPLC, using [1, 2, 6, 7(-3)H] androstenedione as substrate. The two assays were found to be comparable for enzyme activity estimates of term placental specimens. However, when first trimester specimens were analyzed, the direct-product measurements were significantly larger than the corresponding 3H2O assay results. Nonetheless, biochemical aromatase activity was found to correlate positively with immunostain reaction. Although 17 beta-hydroxysteroid dehydrogenase activity was not directly measured, differences in the estradiol:estrone product ratio (2.49 +/- 0.68 first trimester vs. 0.89 +/- 0.15 term) suggest differential control of this enzyme at the two stages of pregnancy. One first trimester specimen with an atypical, patchy immunostain distribution also had extremely low aromatase activity. The results indicate that both antibodies recognize functional aromatase enzyme and suggest that immunocytochemical detection is a sensitive, qualitative technique for investigating this important steroidogenic enzyme.     

Enzymic studies of sulphatases in tissues of the normal human and in metachromatic leukodystrophy with multiple sulphatase deficiencies: arylsulphatases A, B and C, cerebroside sulphatase, psychosine sulphatase and steroid sulphatases

Abstract— Several sulphatases (arylsulphatases A, B and C, cholesterol sulphatase, dehydroepiandroster-one sulphatase, cerebroside sulphatase and psychosine sulphatase) were deficient in various tissues from two patients with a variant form of metachromatic leukodystrophy. Deficient activities of cerebroside sulphatase and psychosine sulphatase, using physiological substrates, in tissues from metachromatic leukodystrophy with multiple sulphatase deficiencies provided another example that these enzymes may be identical to arylsulphatase A. β-Galactosidase activity was reduced to about 30-50 per cent of normal in brain and liver. Other lysosomal enzyme activities were found to be normal or elevated five to eight times. Arylsulphatase B isolated from the liver of one patient was abnormal, with respect to pi (70) and enzyme kinetics. In mixing experiments with normal enzymes the reduced activities of arylsulphatases A. B and C, cerebroside sulphatase and steroid sulphatases were shown not to be due to the presence of endogenous inhibitors. No arylsulphatase A or B activity in the brain specimen from the patient with multiple sulphatase deficiencies could be detected on isoelectric focussing. In normal brain tissue arylsulphatase A had a pi of 4-6-4-8 while arylsulphatase B had a pi of 7-8 and 8-1. When 4-methylumbelliferyl sulphate was used as a substrate the elution patterns of normal brain and liver arylsulphatase B were more heterogeneous and showed more variation than that when p-nitrocatechol sulphate was used. Arylsulphatase C and steroid sulphatases (cholesterol sulphatase, dehydroepiandrosterone sulphatase and oes-trone sulphatase I were solubilized by the addition of lysolecithin and Triton X-100 and subjected to isoelectric focussing. The pi of cholesterol sulphatase, oestrone sulphatase and arylsulphatase C was 6-8, and the elution patterns of the activities of these enzymes were similar. The pattern of dehydroepiandrosterone sulphatase was more heterogeneous and two major peaks were observed at pi 6 5 and 70. Residual enzyme activities of arylsulphatase C and steroid sulphatases from the brain of the patient with multiple sulphatase activities were not detectable by isoelectric focussing. Simultaneous deficiencies of arylsulphatase C and steroid sulphatases plus isoelectric focussing findings in tissues suggest that these enzymes are closely related in regard to their function. The nature of the genetic defect in metachromatic leukodystrophy with multiple sulphatase deficiencies is discussed.     

Effect of genetically defined oocyte depletion on production of androgens and oestrogens by ovaries of suckling mice

Steroid production and histological features of ovaries were compared either among normal +/+ mice of 3-12 days of age or among 12-day old mutant mice with various degrees of oocyte depletion. Whole ovaries were cultured in the medium containing [3H]progesterone and hCG or 4-androstene-3,17-dione and FSH; amounts of [3H]androgens or oestrogens released from the ovaries were assayed. FSH-responsive aromatase activity was detectable in ovaries of +/+ mice on day 3 after birth (2.6 +/- 0.4 pmol/2 ovaries/48 h), but the activity producing androgens from progesterone, under stimulation of hCG, was not detectable even on day 6 after birth (less than 0.1 pmol/2 ovaries/48 h). The androgen-producing activity appeared on day 9 after birth (1.16 +/- 0.25 pmol/2 ovaries/48 h), when follicles with more than two layers of granulosa cells developed. The ovaries of 12-day old Sl/Slt mice contained a considerable number of follicles with a single layer of granulosa cells, but did not contain any follicles with more than two layers of granulosa cells. The ovaries of Sl/Slt mice possessed aromatase activity (3.3 +/- 0.4 pmol/2 ovaries/48 h) but, not androgen-producing activity (less than 0.1 pmol/2 ovaries/48 h). The present results suggest that development of follicles with more than two layers of granulosa cells may induce the activity producing androgens from progesterone under stimulation of LH in suckling mouse ovaries, though the FSH-responsive aromatase activity is present even in follicles with a single layer of granulosa cells.     

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.     

Direct assay method for guanosine 5'-monophosphate reductase activity.

A sensitive and simple micromethod for the accurate measurement of GMP reductase (EC 1.6.6.8) activity in crude extracts is described. The reaction product of [8-14C]IMP was separated from the substrate [8-14C]GMP by descending chromatography on Whatman DE81 ion-exchange paper. This separation method provides an analysis of the possible interfering reactions, such as the metabolic conversion of the substrate GMP to GDP, GTP, and/or guanosine, and guanine and the loss of the product IMP to inosine, hypoxanthine, and other metabolites. Low blank values (70-90 cpm) were obtained consistently with this assay because the IMP spot moves faster than the GMP spot. The major advantages of this method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of < 10 pmol of IMP production), high reproducibility (< +/- 5%), and capability to measure activity in small samples (9 micrograms protein).     

Uptake of erythrocyte-associated component of blood testosterone and corticosterone to rat brain

To study transport of steroids by erythrocytes, the tissue uptake of erythrocyte-associated testosterone and corticosterone was studied in vivo using a single injection technique into the carotid artery of rats. A brain uptake index (BUI) was calculated by dividing the ratio of [3H]steroid to [14C]butanol (internal reference) in the brain tissue by that in the injection material, and multiplying by 100%. BUIs of testosterone and corticosterone in an erythrocyte suspension were 131 +/- 3% (mean +/- SE, n = 6) and 57.0 +/- 2.7% (n = 6), respectively, which were greater than those in buffer (100 +/- 4%; n = 4, P less than 0.01 and 39.8 +/- 4.6%; n = 4, P less than 0.01, respectively). The erythrocyte accounted for 83.9% and 76.7% of the total testosterone and corticosterone delivered to the tissues, respectively, when calculated on the assumption that the BUIs of steroid in buffer and in the supernatant of an erythrocyte suspension are the same. BUIs of corticosterone in hemolysate and in a suspension of erythrocyte plasma membranes (60.8 +/- 7.0%; n = 4 and 69.5 +/- 3.7%; n = 4, respectively) were also greater than those in buffer (P less than 0.05 and P less than 0.01, respectively). Our results suggest that the erythrocyte-associated component of testosterone and corticosterone are delivered to the tissue of rat brain, and that their membranes may play a major role in their capacity to transport steroids to the tissues.     

The influence of estradiol on cholesterol processing by the corpus luteum

Recent studies from our laboratory have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular cholesterol metabolism including mobilization of cholesteryl esters, stimulation of lipoprotein receptor activity and induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity. To test the functionality of cholesteryl ester turnover per se, we measured the activities of acyl CoA:cholesterol acyltransferase (ACAT) and cholesteryl esterase, the enzymes involved in cholesteryl ester synthesis and hydrolysis, respectively; we also measured de novo synthesis of cholesterol, cholesteryl esters, and steroids. Pregnant rats, hypophysectomized and hysterectomized on Day 12, were treated for 72 h with either estradiol or testosterone, and luteal microsomal and cytosolic fractions were utilized to measure ACAT and cholesteryl esterase activity, respectively. Intact corpora luteal were employed for [14C]acetate incorporation experiments. Basal ACAT activity (expressed as pmol.min-1.CL-1 increased from a mean of 78 +/- 16 in vehicle-treated rats to 119 +/- 18 and 197 +/- 16 in the estradiol- and testosterone-treated rats, respectively. Similarly, total ACAT activity (measured in the presence of exogenous cholesterol) was also increased in estradiol- and testosterone-treated groups. On the other hand, cholesterol esterase activity (expressed either pmol.min-1.CL-1 or pmol.min-1.mg protein-1) was similar in all three groups and comparable to corpora lutea from intact pregnant rats. Hypophysectomy and hysterectomy caused a 50-60% reduction in [14C]acetate incorporation into sterols when compared with intact pregnant rat. Treatment with either estradiol or testosterone not only restored the cholesterol biosynthetic capacity but also enhanced the overall rate of [14C]acetate incorporation into steroids as compared to intact pregnant rats. The major (-80%), newly synthesized steroid was identified as progesterone. In conclusion, the present studies suggest that the major function of luteal estradiol is to induce de novo cholesterol biosynthesis, regulate ACAT activity, and channel available free cholesterol (derived from both endogenous and exogenous sources) for steroidogenesis.     

Testosterone metabolism in the medial basal hypothalamus of the starved male rat

The conversion of testosterone to estradiol by aromatase and to dihydrotestosterone by 5 alpha-reductase was measured in the medial basal hypothalamus of starved and control male rats. Activities of both enzymes were significantly reduced in starved animals. Aromatase activity was 18.2 +/- 2.3 versus 29.8 +/- 5.7 fmol E2/mg protein/90 min (mean +/- SEM, P less than 0.02) and 5 alpha-reductase was 4.95 +/- 0.35 versus 5.96 +/- 0.30 pmol DHT/mg protein/90 min (P less than 0.02) for starved and control animals respectively. The results indicate that hypothalamic metabolism of testosterone is decreased during starvation. Therefore the increased sensitivity of the T-LH feedback described earlier in starved rats [4] cannot be explained by changes in central testosterone metabolism.     

HMG-CoA reductase activity in the microsomal fraction from human placenta in early and term pregnancy

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) mevalonate: NADP oxidoreductase (CoA acylating; EC 1.1.1.34) in microsomes from early- and term-pregnancy placenta has been found to be 24 +/- 2 and 6 +/- 3 pmol/min per mg protein, respectively. Inactivation of the enzyme required the addition of ATP and Mg2+ and was dependent on the time of preincubation. Reactivation of the enzyme was also dependent on the incubation time and prevented by the presence of fluoride--a phosphoprotein phosphatase inhibitor. These data suggest that (despite a low activity) placental HMG-CoA reductase is covalently modulated via the phosphorylation-dephosphorylation system. The conversion of [14C]acetate and [3H]mevalonate into digitonin precipitable placental sterols indicates that the lower reductase activity in term, than in early, placental microsomes is accompanied by a less active conversion of [14C]acetate in this tissue.     

Pseudoaromatase in circulating lymphocytes

A variety of data suggesting a relationship between estrogens and the immune system prompted a study of aromatase activity in blood lymphocytes. A tritiated water aromatase assay detected activity of 1.9 to 25.1 pmol/g protein/h in 14 samples of human lymphocytes. To confirm these results, additional tritiated water and product isolation assays were performed on a large pool of lymphocytes obtained from 4 U of blood. An assay using [1β-³H]androstenedione generated high apparent aromatase activity, 943 pmol/g protein/h, but this activity could not be blocked by the aromatase inhibitor, CGS 16949A. More direct methods of evaluation yielded the following results: (1) PCR demonstrated no aromatase mRNA production in lymphocytes; (2) direct product isolation using [1,2,6,7-³H]androstenedione yielded insignificant production of estrone and estradiol; (3) immunostaining of fixed lymphocyte smears with a polyclonal antibody to aromatase yielded equivocal results. These data suggest the presence of pseudoaromatase in blood lymphocytes. Since circulating lymphocyte pseudoaromatase levels can be correlated with various factors in patients, such as age, menopausal status, and glucose ingestion, further studies of this activity are warranted.     

Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes

Opioid receptors have been characterized in Drosophila neural tissue. [3H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (KD = 8.8 +/- 1.7 nM; Bmax = 2.3 +/- 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [3H]dihydromorphine ([3H]DHM), a mu-selective ligand: KD = 150 +/- 34 nM; Bmax = 3.0 +/- 0.6 pmol/mg of protein. Trypsin or heat treatment (100 degrees C for 15 min) of the Drosophila extract reduced specific [3H]DHM binding by greater than 80%. The rank order of potency of drugs at this site was levorphanol greater than DHM greater than normorphine greater than naloxone much greater than dextrorphan; the mu-specific peptide [D-Ala2,Gly-ol5]-enkephalin and delta-, kappa-, and sigma-ligands were inactive at this site. The other site was labeled by (-)-[3H]ethylketocyclazocine ((-)-[3H]EKC), a kappa-opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (KD = 212 +/- 25 nM; Bmax = 1.9 +/- 0.2 pmol/mg of protein). (-)-[3H]EKC binding could be displaced by kappa-opioids but not by mu-, delta-, or sigma-opioids or by the kappa-peptide dynorphin. Specific binding constituted approximately 70% of total binding at 1 nM and approximately 50% at 800 nM for all three radioligands ([3H]etorphine, [3H]EKC, and [3H]DHM). Specific binding of the delta-ligands [3H][D-Ala2,D-Leu5]-enkephalin and [3H][D-Pen2,D-Pen5]-enkephalin was undetectable in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue androgens and the endocrine autonomy of breast cancer.

To evaluate whether a tumour-directed gradient in androgen levels in fatty tissue can account for the maintenance of intra-tissue oestradiol levels, androstenedione (Adione), dehydroepiandrosterone (DHEA), testosterone (Testo) and androstenediol (Adiol) were assayed in breast tumour tissues and in fatty tissue taken at different distances from the tumour. The concentration of Adione was significantly lower in tumour tissue (5.6 +/- 1.5 pmol/g tissue; mean +/- SEM; n = 14) than in the adjacent fatty tissue (20.4 +/- 2.2; P less than 0.005). Testo, by contrast, occurred in equal concentrations in tumour (0.80 +/- 0.11) and in adjacent fatty tissue (0.70 +/- 0.07). Adione levels tended to be lower after the menopause only in fatty tissue, not in the tumour tissue; for Testo no differences were observed between samples from pre- and postmenopausal patients. Tumour DHEA levels (57 +/- 12 pmol/g tissue) were lower than those in fatty tissue (117 +/- 17; P less than 0.02). As with Adione, fatty tissue DHEA concentrations tended to be higher in pre- than in postmenopausal patients. Adiol showed a similar pattern as Testo. For none of the aromatase substrates nor their precursors a tumour-directed gradient was observed. The concentration of Adione in breast cancer tissue is much lower than the reported Km of the aromatase system for Adione. We have concluded, therefore, that the maintenance of oestradiol concentrations in tumour tissues is not substrate-driven.     

Heparin-modulated binding of pancreatic lipase and uptake of hydrolyzed triglycerides in the intestine

Utilizing small intestine membranes that contain heparin (50 micrograms/mg protein), binding of triglyceride lipase (homogeneous 52 kDa, specific activity, 70 nmol/mg.h) to membranes was shown to be concentration dependent and saturable, and it was characterized by a single dissociation constant (KD = 86 +/- 16 nM) with a maximal binding capacity of 54 +/- 8 pmol/mg of vesicle protein. Specific binding was decreased in a concentration-dependent manner by the addition of exogenous heparin, and binding was virtually eliminated (less than 6% control values) by pretreatment of membranes with bacterial heparinase. Cultured intestinal epithelial cells (CaCo-2), shown to possess membrane-associated heparin, also bound pancreatic triglyceride lipase in a specific and saturable manner, with KD = 77 +/- 12 nM and Bmax = 13.7 +/- 6 pmol/10(6) cells. Soluble heparin not only decreased binding, but it also diminished the enzyme-mediated cellular uptake of [14C]oleate from [14C]triolein by over 75%. Therefore, intestinal heparin, a component of the brush border membrane, localizes pancreatic triglyceride lipase in a receptor-like manner to the plasma membrane to promote the subsequent absorption of fatty acids derived from hydrolyzed triglycerides.     

Activities of steroid metabolic enzymes in secretory endometria from untreated women with Polycystic Ovary Syndrome

Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic pathology related with infertility and recurrent miscarriage. We have previously shown that the endometrium of these patients can exhibit a potentially higher sensitivity to estrogen action, being estrogens important regulators of the cell cycle and tissue homeostasis. The effect of estrogens on tissues depends on their in situ availability, which is in part regulated by the activity of steroid metabolic enzymes within the tissues. Therefore, the objective of the present study was to analyze if the activity and/or expression of steroid metabolic enzymes in endometria from women with PCOS differ from controls. For this purpose, the activity of the enzymes was determined by using radiometric assays and the mRNA levels measured by semi-quantitative RT-PCR. Both assays were assessed in endometria obtained during mid secretory phase from control (CE, n=12) and PCOS women (PCOSE, n=11). For the statistical analyses, Mann-Whitney and Student's t-tests were used to compare CE and PCOSE, considering a p value <0.05 significantly different. The results showed an increase in the sulfatase activity in PCOS respect to control endometria (200+/-28pmol/mg vs. 115+/-13pmol/mgproth; p<0.05), in agreement with the higher mRNA levels found for the enzyme in PCOSE. In addition, a PCOSE exhibited lower activity of sulfotransferase respect to the control group (50+/-21pmol/mg vs. 124+/-10pmol/mgproth; p<0.05), whereas a higher level of 17beta-hydroxysteroid dehydrogenase type 1mRNA was found in PCOSE compared with the control tissues (p<0.05). The activity of 17beta-hydroxysteroid dehydrogenase type 2 and the mRNA levels of sulfotransferase were similar in both groups; meanwhile, the expression of aromatase was undetectable. These data indicate that the sulfatase pathway could play an important role in the local production of estrogens in PCOSE from secretory phase. This potentially higher bioavailability of estrogens in endometria from PCOS women could influence the deregulation of tissue homeostasis that we have previously reported, and could partially explain the poor reproductive performance observed in this group of patients.     

Transport of organic cations by a renal epithelial cell line (OK)

The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane.