The Na+/K+-ATPase reaction of human erythrocytes is not near equilibrium. A 31P-NMR study

We have addressed the question of whether the Na/K+-ATPase in the human erythrocyte is in a state of near-equilibrium by varying the extracellular ratio of Na+ and K+ and following the cytosolic phosphorylation potential by 31P-NMR and by combined enzymatic colorimetric measurements. There was no correlation at room temperature between the extracellular Na+/K+ ratio and the cytosolic phosphorylation potential measured either by NMR or alternative methods. The cytosolic phosphorylation potential measured by NMR was 4100 +/- 1300 (S.E.) M-1 at an extracellular K+ concentration of 5.9 mM (Na+/K+ ratio of 24.3) and 2800 +/- 700 (S.E.) M-1 at 75 mM extracellular K+ (Na+/K+ ratio of 0.99). The chemically determined phosphorylation potential was 6400 +/- 1200 (S.E.) and 5000 +/- 700 (S.E.) M-1 at 5.9 and 75 mM extracellular K+, respectively. Omission of Ca2+ from the buffer solutions did not affect the results. A consistent finding in this study was that the NMR-determined value of ATP was about 10-20% lower than the value determined enzymatically on perchloric acid extracts. The inorganic phosphate (Pi) was fully NMR visible.     

Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.     

Development and quality of porcine embryos in different culture system and embryo-producing methods

In this study, the developmental ability and cellular composition of porcine IVF, parthenote and somatic cell nuclear transfer (SCNT) embryos were evaluated following different in vitro culture systems. Group 1, embryos were cultured in NCSU-23 with 5.55 mM D-glucose (NCSU+) until day 6 on 20% O(2) or 5% O(2) (Group 2). Group 3, embryos were cultured in D-glucose-free NCSU-23 (NCSU-) with 0.17 mM Na pyruvate/2.73 mM Na lactate for 58 h and subsequently cultured in NCSU+ until day 6 (NCSU -/+) on 20% O2 or 5% O(2) (Group 4). IVF blastocysts did not differ significantly with O(2) concentrations, but differed significantly with major energy source (glucose and pyruvate/lactate). In Group 3 and 4 IVF blastocysts, the total cell number and apoptosis rates were not significantly different with different O(2) concentrations. Blastocyst rate, total cell number and apoptosis rate in Groups 3 and 4 parthenote embryos also were not significantly different. Parthenote and SCNT, under the same culture treatment, exhibited significant differences in blastocyst and apoptosis rates (47.5 +/- 16.1 vs. 24.0 +/- 4.0 and 4.9 +/- 9.0 vs. 22.8 +/- 23.3). Apoptosis-generating rate increased in the order parthenote, IVF and then SCNT. In conclusion, in vitro development of porcine embryos was not affected by O(2) concentrations but was affected by major energy source. Even so, the concentration of each major energy source and the timing of its inclusion in culture could accomplish relatively high embryonic development, the apoptosis rate stressed that more work still needs to be done in developing a better defined culture system that could support SCNT embryos equivalent to in vivo preimplantation porcine embryos.     

Parthenogenetic development of in vitro matured porcine oocytes treated with chemical agents

Parthenogenetic activation is a possible way to produce homogeneous embryos with the same ploidy. These embryos could develop to the blastocyst stage during the cultivation. Probably such embryos could be used in other areas of biotechnology. The objectives of the present study were first to assess the ability of strontium-chloride to induce activation and parthenogenetic development in porcine oocytes in comparison with cycloheximide and 6-dimethylaminopurine; second to verify whether the combination of the two treatments improved activation and parthenogenetic development rates. At first, the effects of cycloheximide, 6-dimethylaminopurine and strontium-chloride on oocyte activation and embryonic development were compared. Oocytes from slaughterhouse ovaries were matured for 42h in tissue culture medium (TCM) 199 at 38.5 degrees C, 5% CO(2) in air. Matured oocytes were activated with 10mM strontium-chloride (S), 0.04mM cycloheximide (CX), 2mM 6-dimethylaminopurine (D) for 5h. The activation rate was judged by pronuclear formation of oocytes. Following the activation, oocytes were incubated in NCSU 37 medium for 6 days and in all groups more than 45% of oocytes activated. The activation rate for CX treatment was significantly higher (P<0.05) than for D (57.37+/-4.21% and 48.09+/-3.43%, respectively). In a second experiment in vitro matured porcine oocytes were activated using a combined treatment of strontium-chloride with cycloheximide (SCX) and strontium-chloride combined with 6-dimethylaminopurine (SD). In S and SCX groups more than 50% of oocytes were activated (53.29+/-5.39% and 54.3+/-7.29%, respectively). However a large portion of embryos stopped their development at the two- or four-cell stage. Significantly higher numbers of embryos could reach the eight-cell stage in SD and SCX than for S (7.8+/-1.0%, 7.2+/-4.0% and 3.9+/-3.1%, respectively). Blastocyst formation was only observed in S, CX and SCX. These results show that porcine in vitro matured oocytes can be artificially activated by cycloheximide, 6-dimethylaminopurine and strontium-chloride.     

Binding of S-adenosylhomocysteine to hydroxyindole O-methyltransferase

Mg2+-selective microelectrodes have been used to measure the intracellular free Mg2+ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 micron were backfilled with the Mg2+ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg2+. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18-24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements . In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than -82 mV, the intracellular free Mg2+ concentration was 3.8 +/- 0.41 (S.E.) mM (n = 58) at 22 degrees C. No significant change in the intracellular free Mg2+ was observed following extensive (approx. 6 h) incubation in Mg2+-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg2+]i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na+-free solutions. In depolarized muscle fibers (-23 +/- 2.7 mV) treated with 100 mM K+, the myoplasmic [Mg2+] was 3.7 +/- 0.45 (S.E.) mM, n = 6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg2+. These results point out that [Mg2+]i is not modified under these three different experimental conditions.     

Effect of ethanol on tracheal potassium channels reconstituted into bilayer lipid membranes.

We examined the effect of ethanol on single potassium channels derived from plasma membranes of bovine tracheal smooth muscles. The observed potassium channels had a conductance of 296 +/- 31 pS (mean +/- S.D.) in symmetrical 250 mmol/l KCl solutions, and exhibited a voltage- and Ca2+-dependence similar to BKCa channels. Ethanol at 50, 100 and 200 mM concentrations increased the probability of open potassium channels to 112 +/- 5, 127 +/- 7 and 121 +/- 13% (mean +/- S.E.M.), respectively. It is suggested that increased activity of the BKCa channels by ethanol hyperpolarizes the plasma membrane and thus may contribute to relaxation of tracheal smooth muscle.     

Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation

The present study investigated the effect of lactate/pyruvate supplement in culture medium and of chemical activation after electric stimulus on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were enucleated, reconstructed with fetal fibroblasts, and simultaneously fused/activated using a single pulse of 2.0 kV/cm for 30 microsec. In Experiment 1, reconstructed embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with either 5.5 mM glucose (Group A) or lactate (5.0 mM)/pyruvate (0.5 mM) (Group B). Compared to Group A, cleavage rate (64% vs. 47%) was higher and more blastocysts developed in Group B (17% vs. 6% at Day 6, 21% vs.11% at Day 7). Experiment 2, embryos reconstructed by electric stimulus (2.0 kV/cm for 30 microsec) were subjected to three activation protocols: (1) no chemical activation (Group C), (2) 7.5 microg/ml cytochalasin B treatment at 2 hr after electric stimulus (Group D), and (3) 5 microg/ml 6-dimethylaminopurine (Group E) treatment at 2 hr after electric stimulus. The reconstructed embryos were cultured for 7 days in NCSU-23 medium supplemented with lactate (5.0 mM)/pyruvate (0.5 mM). The rates of blastocyst formation on Day 6 and Day 7 in Group C (17 and 20%, respectively) or Group D (15, 20%, respectively) were higher than in Group E (9 and 12%, respectively). The percentage of two pseudo-pronucleus (PPN) formations in Group D (88%) was significantly higher than in Group C (71%) and Group E (72%). Mean cell numbers of blastocysts in Group D (63.4 +/- 15.8) were higher than in Group C (43.9 +/- 16.5) and Group E (32.9 +/- 17.9), due to increased trophectoderm (TE) cell numbers. Our results indicate that supplementing NCSU-23 medium with lactate/pyruvate and exposure of cytochalasin B after electrical stimulus can improve in vitro developmental competence of porcine SCNT embryos.     

In vivo patterns of circulating steroids in adult male rats. IV. Evidence for rapid oscillations in testosterone in normal and totally parenterally nourished animals.

In vivo patterns of circulating testosterone (T) were investigated in stock fed controls and parenterally nourished (TPN) rats. Rats were sampled at 2 minute intervals for 30 minutes via a jugular cannula. Both groups exhibited a rapid oscillatory T pattern. In the control group, T concentrations at any specific time interval exhibited large differences with coefficients of variation (17-88%). In TPN rats this variation was 34-79%. Moreover, the mean T concentration of all samples obtained during the 30 minute period for each individual animal ranged from 1.3 +/- 0.1 (S.E.M.) to 3.5 +/- 0.3 (S.E.M.) ng/ml in controls and 1.3 +/- 0.1 (S.E.M.) to 2.0 +/- 0.2 (S.E.M.)ng/ml in controls and 1.3 +/- 0.1 (S.E.M.) to 2.0 +/- 0.2 (S.E.M.) ng/ml for TNP rats respectively. The mean coefficient of variation in control animals, however, was twice that of TPN rats, indicating that variation in basal T may be minimized during intravenous feeding. The occurrence of rapid oscillations in T of both stock and parenterally fed animals shows that nutritional regimen does not affect this phenomenon.     

High affinity, calcium-stimulated adenosine triphosphatase activity in the particulate fraction of rat pancreatic acini

Adenosine triphosphatase activity which is Mg2+-dependent and stimulated by submicromolar concentrations of Ca2+ (as Ca . ATP) was identified in the total particulate fraction of rat pancreatic acini. Half-maximal activity (V0.5) is obtained at 100.1 +/- 6 nM Ca . ATP with a Hill coefficient of 2.2 +/- 0.1 (mean +/- S.E.; n = 4). Maximal activity was 75 +/- 19 pmol of Pi released from ATP minute-1 microgram of membrane protein-1 (mean +/- S.E.; n = 7). High affinity Ca2+-ATPase activity was unaffected by ouabain, Na+, K+, La3+, and added calmodulin. Activity was slightly reduced by ruthenium red (0.1 mM) and by oligomycin (80 micrograms/ml) but was reduced almost 50% by the phenothiazine derivative fluphenazine in a dose-related and Ca2+-dependent manner. Hydrolysis of p-nitrophenyl phosphate was 9% of the rate of ATP hydrolysis and was independent of Ca2+ concentration. However, ADP, GTP, UTP, and ITP were hydrolyzed at 76-93% the rate that ATP was hydrolyzed with V0.5 values and Hill coefficients similar to those of Ca . ATP. We conclude that rat pancreatic acini contain an enzyme for active Ca2+ translocation: ATPase activity that is Mg2+-dependent and stimulated by submicromolar concentrations of Ca . ATP. Substrate hydrolysis appears to involve positive cooperative interactions of multiple ligand-binding sites and may be regulated in part by calmodulin.     

Aortic endothelial cells damaged by a nitric oxide donor and protected by flavonoids.

Cultured porcine aortic endothelial cells (PAEC) were exposed to four concentrations (0.00 mM - 5.00 mM) of 3-Morpholino-sydnonimine-hydrochloride (SIN-1, a nitric oxide donor). SIN-1 demonstrated a dose dependent cytotoxicity against PAEC as indicated by the thiobarbituric acid (TBA) assay. Morphologically and biochemically, the presence of selected flavonoids (morin, quercetin, or catechin) was shown to protect the PAEC from SIN-1 toxicity. Protection levels determined from the TBA assay were significant (p<0.05) for all flavonoids, with morin at 72+/-8%. Quercetin and catechin had comparable protective activities of 54+/-6% and 43+/-3%, respectively. This study supports the contention that SIN-1 is cytotoxic to PAEC and that antioxidants such as flavonoids may attenuate such toxicity.     

Induction of adrenocortical special zone in the male possum (Trichosurus vulpecula)

The induction of adrenocortical special zone (S.Z.) by gonadotrophin administration was studied in male brush tailed possums. Castrated males injected with porcine FSH (NIH-FSH-P2) formed well developed S.Z.s, varying in sizes from 5-20% of the gland's volume. Pregnant mare serum gonadotrophin (PMSG) was ineffective. From adrenal homogenates of FSH treated possums incubated with [3H]-progesterone the major conversion products were 5 beta-reduced steroid metabolites (72%) and cortisol (4%). The conversion products from adrenals of saline and PMSG treated males were cortisol and corticosterone (65%). Of the ten untreated castrates, one had a well developed S.Z. and two had S.Z.s at an early stage of development. Significant 5 beta-reduction of [3H]progesterone was only found in one animal. The plasma FSH concentrations were in intact males 317 +/- 41 ng rat FSH ml-1 (mean +/- SEM) and in castrates 769 +/- 64. The possible reasons for the lack of spontaneous S.Z. formation in intact males are discussed.     

Kinetics of 3-O-methyl-D-glucose transport in isolated rat hepatocytes.

3-O-Methyl-D-glucose transport across the plasma membrane of isolated rat hepatocytes was followed for net entry of the sugar into sugar-free cells (zero trans entry), net exit of sugar into sugar-free medium (zero trans exit) and for unidirectional entry and exit fluxes when cells had been equilibrated with sugar in the extracellular medium (equilibrium exchange entry and exit). These measurements were performed at 20 degrees C and pH 7.4 by the use of simple manual methods. Initial rates of transport showed a Michaelis--Menten dependency on the sugar concentration at the cis side of the membrane over the range of concentrations tested (100 microM to 100 mM). Transport was found to be symmetrical with no evidence of substrate stimulation of transport from the trans side of the membrane. Parameters (mean values +/- S.E.M.) of transport were estimated as Vmax. 86.2 +/- 9.7 mmol/litre of cell water per min and Km 18.1 +/- 5.9 mM for exchange entry, Vmax. 78.8 +/- 5.3 mmol/litre of cell water per min and Km 17.6 +/- 3.5 mM for exchange exit, Vmax. 84.1 +/- 8.4 mmol/litre of cell water per min and Km 16.8 +/- 4.6 mM for zero trans exit.     

Catalytic properties of phosphoglucomutase from pea chloroplasts.

Electrophoretically homogeneous phosphoglucomutase (PGM) with specific activity of 3.6 units/mg protein was isolated from pea (Pisum sativum L.) chloroplasts. The molecular mass of this PGM determined by gel-filtration is 125 +/- 4 kD. According to SDS-PAGE, the molecular mass of subunits is 65 +/- 3 kD. The Km for glucose-1-phosphate is 18.0 +/- 0.5 microM, and for glucose-1, 6-diphosphate it is 33 +/- 0.7 microM. At glucose-1-phosphate and glucose-1,6-diphosphate concentrations above 0.5 and 0.2 mM, respectively, substrate inhibition is observed. The enzyme has optimum activity at pH 7.9 and 35 degrees C. Mg2+ activates the PGM. Mn2+ activates the enzyme at concentrations below 0.2 mM, while higher concentrations have an inhibitory effect. The activity of the PGM is affected by 6-phosphogluconate, fructose-6-phosphate, NAD+, ATP, ADP, citrate, and isocitrate.     

Prostacyclin does not change during an oxygen induced increase in pulmonary blood flow in the fetal lamb

The role of prostacyclin in mediating the increase in pulmonary blood flow caused by an increase in oxygen tension in the fetal lamb was investigated. Plasma concentrations of 6-keto-PGF1 alpha, the hydrolysis product of prostacyclin, were measured during an increase in pulmonary blood flow caused by a rise in oxygen tension in eight intrauterine fetal lambs. Fetal oxygen tension was increased by placing the pregnant ewes in a hyperbaric chamber and having them breathe 100% oxygen at three atmospheres absolute pressure. This increased fetal PaO2 from 27 +/- 3 to 60 +/- 6 torr (mean +/- S.E., p less than or equal to 0.0001) and increased the proportion of right ventricular output distributed to the fetal lungs from 6 +/- 2 to 45 +/- 7% (mean +/- S.E., p less than or equal to 0.001). However, the fetal plasma concentration of 6-keto-PGF1 alpha did not change, 186 +/- 26 to 208 +/- 40 pg/ml (mean +/- S.E.). Indomethacin decreased plasma concentrations of 6-keto-PGF1 alpha in each of three fetuses but did not decrease the proportion of right ventricular output distributed to their lungs. The increase in pulmonary blood flow caused by an increase in oxygen tension in the fetal lamb is not associated with an increase in plasma concentrations of 6-keto-PGF1 alpha. Prostacyclin does not appear to be involved in the increase in pulmonary blood flow caused by the increase in oxygen tension at birth.     

Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.

1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated.     

On the mechanism of detergent modification of myosin structure and function

The concentration dependences of the activation of myosin subfragment-1 (S1) Mg-ATPase by the detergents CHAPS and C12E8 were determined at 23 degrees C in 25 mM Tris (pH 7.0), 250 microM EDTA, 5 mM MgCl2, and 100 microM ATP. At detergent concentrations expected to bind hydrophobic S1 surface areas equally, C12E8 caused an 8.5-fold greater increase in activity than CHAPS, which suggests that detergent binding to the surface of S1 is not the mechanism of activation. At detergent concentrations above their critical micelle concentrations, C12E8 was also much more effective than CHAPS, suggesting that micelles are not involved. A series of n-alcohols (which do not form micelles) with from 3 to 10 carbons all increased S1 Mg-ATPase activity as much or more than C12E8. The largest increase (5.7-fold) was caused by n-hexanol. The more hydrophobic alcohols activated S1 at lower concentrations. A linear plot of the alcohol concentration that caused 50% of maximum activity versus the number of carbons in the alcohol, indicated the apparent free energy of binding per CH2-group was -0.60 +/- 0.03 kcal/mol. There were two indications that alcohol binding caused an S1 conformational change. The intrinsic fluorescence increase of S1 during steady-state activity was reduced from 17.5 to 12.8%, and the apparent hydrodynamic rotational mobility of fluorescently labeled S1 was decreased 25% by the present of n-hexanol. The data suggest that S1 activation by C12E8 and by n-alcohols is due to hydrophobic binding to S1 at non-surface sites, which causes an S1 structural change.     

Electrical activation induces reactive oxygen species in porcine embryos

The objectives were to determine factors affecting generation of reactive oxygen species (ROS) in porcine embryos after electrical activation of oocytes, and the effects of an antioxidant and chemical agent on ROS generation. Greater ROS were induced by electrical activation compared to IVF (mean+/-S.E.M., 14.6+/-0.8 vs. 9.2+/-0.4, P<0.05). Furthermore, ROS generation in embryos after electrical activation was significantly increased by higher intensity and longer duration electrical pulses and by higher exogenous Ca(2+) concentrations. Cleavage rate and blastocyst formation rate were not directly related to the level of ROS. Supplementation of the IVC medium with 0.5mM glutathione (GSH) reduced ROS (9.2+/-0.4 vs. 14.7+/-0.9, P<0.05). Treatment with the chemical activation agent, 6-dimethylaminopurine (6-DMAP) for 3h did not induce further ROS generation in combination with electrical activation, but it improved blastocyst formation rate (53.8+/-1.1 vs. 23.7+/-3.5, P<0.05). We concluded that generation of ROS should be considered for optimizing electrical activation and that supplementing an antioxidant or combining electrical and chemical activation induced lower ROS generation in electrically activated porcine embryos.     

Norelgestromin as selective estrogen enzyme modulator in human breast cancer cell lines. Effect on sulfatase activity in comparison to medroxyprogesterone acetate

Human breast cancer tissue contains enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol (E(2)) formation. In this tissue, E(2) can be synthesized by two main pathways: (1) sulfatase-transforms estrogen sulfates into bioactive E(2), and the (2) aromatase-converts androgens into estrogens. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization.In the present study, we demonstrated in T-47D and MCF-7 human breast cancer cells that norelgestromin (NGMN) (a metabolite of norgestimate) is a potent inhibitory agent of the estrone sulfatase activity. After 24h incubation of physiological concentrations of E(1)S (5 x 10(-9)mol/l) the inhibitory effect of NGMN at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 43+/-7, 74+/-4 and 97+/-2%, respectively, in T-47D cells; 25+/-4, 57+/-5 and 96+/-2% respectively, in MCF-7 cells. Comparative studies using medroxyprogesterone acetate (MPA) showed that this progestin also has an inhibitory effect on sulfatase activity, but significantly less intense than that of NGMN. The inhibition for MPA at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 31+/-5, 47+/-3 and 61+/-3%, respectively, for T-47D cells; 6+/-3, 20+/-3 and 63+/-4%, respectively, for MCF-7 cells.In conclusion, the present data show that NGMN is a very potent inhibitory agent for sulfatase activity in the hormone-dependent breast cancer cells, resulting in decreased tissue concentration of E(2). The clinical significance of this finding remains to be elucidated.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.     

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.