In vitro binding of progesterone, cronolone and medroxyprogesterone acetate to uterine progesterone receptors of sheep, rabbit and mouse

1989 ◽  
Vol 1 (3) ◽  
pp. 223 ◽  
Author(s):  
X Zhang ◽  
GM Stone ◽  
BG Miller
Keyword(s):  
Progesterone Receptor ◽  
Medroxyprogesterone Acetate ◽  
Species Differences ◽  
Steroid Receptors ◽  
Specific Binding ◽  
Progesterone Receptors ◽  
Binding Activity ◽  
Biological Responses ◽  
Vitro Binding

Various aspects of the binding of the synthetic progesteongens, cronolone (9 alpha-fluoro-11 beta-hydroxy-17 alpha-acetoxypregn-4-ene-3,20-dione) and medroxyprogesterone acetate (6 alpha-methyl-17 alpha-acetoxypregn-4-ene-3,20-dione, MAP) to uterine cytosol progesterone receptors of the sheep, rabbit and mouse were studied, in an attempt to explain interesting species differences in the biological activity of these steroids. For the sheep, data for binding-site concentration, relative binding affinity (RBA), dissociation constant (Kd) and rates of association and dissociation indicate specific binding of cronolone to the progesterone receptor and these would seem to explain in part the high progestational activity of cronolone in this species. By contrast, with the mouse, there was only a low level of specific binding of cronolone and this appears to explain its inability to maintain pregnancy in this species. Results for the binding activity of cronolone in rabbit uterus were similar to those for the sheep and thus inability of cronolone to maintain pregnancy in the rabbit is not explained by a failure to bind the progesterone receptor. Species differences in binding to the progesterone receptor were also seen with MAP where the RBA, with respect to progesterone, was high in the sheep and rabbit and lower in the mouse. The results, however, do not relate directly to the progestational activity of MAP in these species. Overall, the data indicate that species differences in the binding activity of steroid receptors constitute one factor that causes species-dependent variation in biological responses to progestogens.

scholarly journals Colchicine-binding protein of the liver. Its characterization and relation to microtubules.

1975 ◽  
Vol 66 (3) ◽  
pp. 609-620 ◽  
Author(s):  
C Patzelt ◽  
A Singh ◽  
Y L Marchand ◽  
L Orci ◽  
B Jeanrenaud
Keyword(s):  
High Speed ◽  
Specific Binding ◽  
Electrophoretic Analysis ◽  
Binding Activity ◽  
Low Concentrations ◽  
Electrophoretic Behavior ◽  
High Affinity Binding Site ◽  
High Concentrations

Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(-3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time-dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.

scholarly journals Non-genomic progesterone receptors in the mammalian ovary: some unresolved issues

Reproduction ◽  
2003 ◽  
pp. 3-15 ◽  
Author(s):  
T Bramley
Keyword(s):  
Progesterone Receptor ◽  
Steroid Receptors ◽  
Surface Membrane ◽  
Progesterone Receptors ◽  
Signalling Pathways ◽  
Mediated Effects ◽  
Genomic Effects ◽  
Vitamin D 3 ◽  
Membrane Progesterone Receptor ◽  
Different Tissues

In addition to their well-documented genomic effects, steroid hormones may also exert actions that are: (i) rapid, (ii) insensitive to inhibitors of transcription, (iii) mimicked by steroids coupled to cell membrane-impermeant molecules, and (iv) demonstrable in cells that do not express the classic genomic progesterone receptor (gPR). Such 'non-genomic' effects have been described for all the major classes of steroids (progesterone, oestrogens, androgens and corticoids), as well as for thyroid hormones, retinoids and vitamin D(3). Rapid, membrane-mediated effects of progesterone have been studied most intensively in human spermatozoa and in the Xenopus oocyte. However, similar non-genomic actions of progesterone and other steroids have now been described in a wide variety of different tissues in many species. The first putative membrane steroid receptor to be cloned was that for the pig membrane progesterone receptor (mPR). Subsequently, similar genes were cloned from rats and cattle, and two related mPRs have been described in humans. Despite accumulating evidence for cell-surface membrane actions of steroids, a number of uncertainties remain as to the properties and identity of such 'receptors' and their cellular actions. Furthermore, some rapid steroid effects may be mediated through membrane-associated 'classical' steroid receptors, and steroid receptors may be capable of activating other signalling pathways non-classically. This review focuses on some of these unresolved issues, taking as its model the actions of progesterone in the mammalian ovary.

scholarly journals A phosphorylation site in brain and the delayed neurotoxic effect of some organophosphorus compounds

1969 ◽  
Vol 111 (4) ◽  
pp. 487-495 ◽  
Author(s):  
M K Johnson
Keyword(s):  
Organophosphorus Compounds ◽  
Specific Binding ◽  
Phosphorylation Site ◽  
Test System ◽  
Binding Activity ◽  
Specific Component ◽  
Esterase Inhibitors ◽  
Brain And Spinal Cord

1. It is proposed that part of a neurotoxic dose of di-isopropyl phosphorofluoridate will be covalently bound in vivo to a specific component in the brain and spinal cord as the initial biochemical event in the genesis of the lesion. 2. A test system in vitro was devised that removes many di-isopropyl phosphorofluoridate-binding sites and indicates that the specific component may be a protein present in brain at a concentration comparable with that of the cholinesterases. 3. The site was found to be present and capable of binding di-isopropyl phosphorofluoridate in vitro in brain samples taken from either normal hens or those dosed with organophosphorus esterase inhibitors that are not neurotoxic. 4. Very little of the specific binding activity was found in brain samples from hens pre-dosed with a variety of neurotoxic organophosphorus compounds. 5. A solubilized preparation of the active brain component was obtained, suitable for further purification and study.

OESTROGEN RECEPTORS IN HUMAN UTERINE TISSUE

1971 ◽  
Vol 50 (2) ◽  
pp. 209-229 ◽  
Author(s):  
L. H. EVANS ◽  
R. HÄHNEL
Keyword(s):  
Human Tissue ◽  
Specific Binding ◽  
Binding Activity ◽  
Human Myometrium ◽  
Target Tissue ◽  
Uterine Tissue ◽  
Tissue Slices ◽  
Binding Characteristics ◽  
Significant Difference

SUMMARY The uptake and retention of [3H]oestradiol-17β by human uterine and muscle tissue was studied in vitro by incubation of tissue slices. The binding characteristics of immature rat and adult human tissue were compared. Human endometrium had a greater affinity for tritiated oestradiol than human myometrium. Human uterine tissues bound less [3H]oestradiol-17β than rat uterine horns. The difference in retention between a target and non-target tissue in the human tissue was much smaller than in the rat, although still highly significant. Binding was inhibited by low concentrations of oestradiol-17β, U-11100A and hexoestrol, but was not inhibited by the same concentrations of oestrone, oestriol or oestradiol-17α. Freezing the uterine tissue resulted in a loss of specific binding activity. Binding was shown to vary with different uterine specimens. There was a significant difference in [3H]oestradiol-17β retention between the proliferative and secretory phases of the menstrual cycle. The highest endometrial retention was found in endometrial carcinoma.

scholarly journals Multiinjection Approach for D2 Receptor Binding Quantification in Living Rats using [11C]Raclopride and the β-Microprobe: Crossvalidation with in Vitro Binding Data

2005 ◽  
Vol 25 (11) ◽  
pp. 1517-1527 ◽  
Author(s):  
Gweltas Mauger ◽  
Wadad Saba ◽  
Philippe Hantraye ◽  
Frédéric Dolle ◽  
Christine Coulon ◽  
...  
Keyword(s):  
Arterial Input Function ◽  
Specific Binding ◽  
D2 Receptor ◽  
Correlation Study ◽  
Kinetics Parameters ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Modelling Approach

The purpose of this study was to quantify D2 receptors density and affinity in living rats using [11C]raclopride and to validate the multiinjection modelling approach. To this aim, we used an intracerebral β+-sensitive probe as a highly sensitive system to quantify the radioligand activity using a single three-injection experimental paradigm. The study was divided into three main parts: (i) [11C]raclopride catabolism evaluation without and with cimetidine pretreatment (cytochrome P450 inhibitor); (ii) quantification of kinetics parameters in the striatum, enthorinal cortex, and cerebellum of living rats using a three-compartment model with an arterial input function; (iii) correlation study of in vivo and in vitro binding density and affinity values in the same striatal tissues. (i) raclopride catabolism was very reproducible between individuals; cimetidine pre-treatment resulted in a 30% reduction of raclopride metabolites. (ii) D2 striatal B'max and Kd Vr estimates obtained by compartmental modelling were 19.87 ± 6.45 and 6.2 ± 3.3 nmol/L, respectively. Cerebellum is the best candidate as a reference region with no specific binding detectable in vivo. (iii) When comparing density ( Bmax/ B'max) and affinity ( Kd/ Kd Vr) values in vivo and in vitro for each striatum, a high strict correlation was found ( r2 = 0.90 and 0.72, for density and affinity, respectively). These results validate the multi-injection modelling approach coupled to β-microprobe acquisitions as a mean to provide accurate and separate estimates of dopamine D2-receptor density and affinity, in the living rodent striatum.

Studies on Corticosterone–Receptor Complexes from Mouse Placenta

1974 ◽  
Vol 52 (3) ◽  
pp. 190-195 ◽  
Author(s):  
Ming D. Wong ◽  
A. F. Burton
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Specific Binding ◽  
Receptor Site ◽  
Binding Properties ◽  
Receptor Complexes ◽  
Specific Binding Sites ◽  
Mouse Placenta ◽  
Vitro Binding

The in vitro binding of radioactive steroids to components of mouse placental nuclei and cytoplasm was investigated using Sephadex or charcoal to remove unbound steroid. Specificity was indicated in competition experiments using excess unlabelled competing steroids. Only the active glucocorticoids formed complexes that could be isolated from the nucleus. The binding properties of the cytoplasmic steroid–receptor complex were studied. From the time course of binding the complex was shown to be more stable at 0° than at 37°, and the distribution of receptors in the cytosol appeared to be homogeneous. The complex was labile to heat and to proteolytic digestion but did not appear to be affected by nucleases or sulfhydryl reagents. Kinetic analysis revealed the presence of high affinity specific binding sites with a dissociation constant of 17.5 nM and a receptor site concentration of 0.26 pmol/mg protein. The corticosterone isolated from nuclear complexes and dexamethasone from cytoplasmic complexes were identified by chromatography and by cocrystallization as the unchanged steroid in each case.

Changes in gonadotrophin binding to rat ovaries during sexual maturation

1983 ◽  
Vol 103 (3) ◽  
pp. 413-419 ◽  
Author(s):  
J. Th. J. Uilenbroek ◽  
R. van der Linden
Keyword(s):  
Granulosa Cells ◽  
Sexual Maturation ◽  
Specific Binding ◽  
Interstitial Tissue ◽  
Uterine Weight ◽  
Thecal Cells ◽  
Vitro Binding ◽  
In Vitro Binding Assay ◽  
Ovulatory Follicles

Abstract. Changes in LH and FSH binding to rat ovaries during sexual maturation were measured by in vitro binding assay and autoradiography. Ovaries were obtained from rats at various ages ranging from 5 till 35 days of age. Animals older than 35 days of age were classified in three groups: anoestrus (uterine weight below 60 mg), early pro-oestrus (uterine weight 60–120 mg) and pro-oestrus (uterus distended with fluid). Measurements were made of specific binding of radioiodinated hCG and rFSH to ovarian homogenates (day 5–35) and to granulosa cells isolated from the largest follicles (day 35–pro-oestrus). The earliest age at which specific hCG and FSH binding could be demonstrated was day 11. Thereafter hCG binding to ovarian homogenates, expressed in cpm/μg DNA, increased with age. Specific binding of hCG to granulosa cells increased from 1000 cpm/μg DNA at day 35 to 4000 cpm/μg DNA at pro-oestrus. FSH binding to ovarian homogenates increased till day 21 and remained fairly constant thereafter. Autoradiography revealed the presence of hCG binding to interstitial tissue and thecal cells. hCG binding to granulosa cells was only found in large pre-ovulatory follicles present at early pro-oestrus and pro-oestrus. FSH binding was observed to granulosa cells of all types of follicles, but not to interstitial and thecal cells. The results demonstrate that during sexual maturation FSH binding to granulosa cells remains constant, whereas LH binding to interstitium and theca and ultimately to granulosa cells increases with age suggesting an increased responsiveness of the ovaries to LH.

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