Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [³H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [³H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K _d) of 12 nM, and the binding capacity (B _max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [³H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

Presence of specific binding sites for [3H]sarcophytol-A in cultured human skin fibroblasts

The presence of specific binding sites for [3H]sarcophytol-A in human skin fibroblasts was examined using biochemical and morphological methods. The displacement studies clearly revealed that high (KD = 31.0 nM) and low (KD = 6.05 microM) affinity sites were present in the intact cells. Moreover, autoradiographic studies using light microscopy revealed that the specific binding sites may exist in both the cytoplasm and the nuclei.     

3[H]-Sulpiride labels mesolimbic non-dopaminergic sites that bind antidepressant drugs

3[H]-(-)-Sulpiride and 3[H]-spiperone binding was compared in rat amygdala, nucleus accumbens and striatum, using (+/-)-sulpiride to define specific binding. 3[H]-(-)-Sulpiride bound to twice as many sites in amygdala and nucleus accumbens as 3[H]-spiperone. 3[H]-(-)-Sulpiride binding was directed to these additional sites by using 1 microM spiperone to mask dopaminergic binding. The binding of 3[H]-(-)-sulpiride to these sites was high affinity, reversible, Na+-dependent, but not stereospecific. Metoclopramide, tiapride and antidepressant medications, but not other neuroleptics, ADTN, or serotonin displaced 3[H]-(-)-sulpiride binding to these sites. These data suggest that 3[H]-(-)-sulpiride labels mesolimbic sites other than dopamine receptors which may mediate antidepressant effects.     

Presence of specific binding sites for [3H]sarcophytol-A in cultured human skin fibroblasts

Summary The presence of specific binding sites for [³H]sarcophytol-A in human skin fibroblasts was examined using biochemical and morphological methods. The displacement studies clearly revealed that high (K_D=31.0 nM) and low (K_D=6.05 M) affinity sites were present in the intact cells. Moreover, autoradiographic studies using light microscopy revealed that the specific binding sites may exist in boththe cytoplasm and the nuclei.     

On the binding of steroid sulfates to albumin

3H-Labeled steroid sulfates, sulfate of estrone (E1S) or dehydroepiandrosterone (DHAS), were dialyzed against delipidated human serum albumin or human plasma in the presence of increasing amounts of competing non-labeled sulfates (DHAS or E1S). The apparent equilibrium constants (K) of the tracers did not measurably change at concentrations of the non-radioactive sulfates below 10(-5) mol/l. At higher concentrations, K decreased gradually. The apparent equilibrium constant of 3H-E1S was diminished by plasma in a similar fashion. It may be concluded that albumin possesses one strong, non-specific binding site. This site, however, does not seem to be utilized for the binding of E1S in vivo, because of its preferential occupation by other ligands. This may be true for other steroid sulfates as well, depending on their relative abundance in plasma.     

Comparative study of the 3 beta-hydroxy-delta 5-steroid dehydrogenase activity of the mitochondrial and microsomal fractions of human and cat placentas

Delta 5, 3 beta hydroxysteroid dehydrogenase activity was studied on mitochondrial and microsomal fractions of human and cat placentas. This activity was assessed by measuring the rate of [3H] progesterone formation from [3H] pregnenolone and of [3H] delta 4-androstenedione from [3H] dehydroepiandrosterone (DHA), by a double isotope dilution method. Specific activities measured under initial velocity conditions were similar in both human and cat placentas. However, the kinetics of the reaction with DHA as substrate exhibit a zoological specificity.     

Thiamine-binding activity of Saccharomyces cerevisiae plasma membrane

The specific binding activity to [14C]thiamine was found to be located in hte plasma membrane of Saccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant of Saccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.     

Interaction of epidermal growth factor with human fibroblasts in culture: an autoradiographic study at the ultrastructural level]

The potent mitogen epidermal growth factor (EGF) binds to specific receptors on human fibroblasts. In the present study we have used a quantitative EM autoradiographic approach to visualize the events involved in the binding process. When 125I-EGF is incubated at 4 degrees C for 120 min, labeled EGF primarily localizes to the plasma membrane of the fibroblast but when incubated at 37 degrees C for 120 min., over 2/3 of the labeled material is internalized by the cell. The internalized radioacitivity is primarily localized to lysomes. These studies demonstrate a temperature-dependent internalization of EGF following initial binding to specific plasma membrane receptors.     

Human platelet 5-hydroxytryptamine receptors: binding of [3H]-lysergic acid diethylamide (LSD). Effects of chronic neuroleptic and antidepressant drug administration

Chronic treatment with phenothiazines and thioxanthenes has been found to enhance 5-HT-induced aggregation of human platelets. A method has been developed to study 5-HT2 receptor binding sites on platelets utilising [3H]-LSD and more recently 125I/LSD. Results are presented which suggest that the LSD binding site is indeed the 5-HT2 binding site and that the LSD binding characterises the specific receptor responsible for 5-HT-induced shape change and aggregation. In a group of patients receiving phenothiazines or thioxanthenes, the Bmax of LSD binding was increased. The mean binding affinity was decreased possibly due to a persistence of neuroleptic in the platelet membrane preparation. Analysis showed that this was not the reason why the mean binding capacity was increased. The results show that chronic phenothiazine and thioxanthene delta treatment 'up-regulates' platelet 5-HT2 binding sites and that this may be accompanied by increased sensitivity to platelet aggregation by 5-HT. In normal subjects desipramine treatment increased the Bmax of platelet LSD binding and this was accompanied by an increased prolactin response to tryptophan which is thought to be mediated by central 5-HT function.     

Thiamine-binding activity ofSaccharomyces cerevisiae plasma membrane

Summary The specific binding activity to [¹⁴C]thiamine was found to be located in the plasma membrane ofSaccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant ofSaccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.     

On the binding of steroid sulfates to albumin

Summary ³H-Labeled steroid sulfates, sulfate of estrone (E₁S) or dehydroepiandrosterone (DHAS), were dialyzed against delipidated human serum albumin or human plasma in the presence of increasing amounts of competing non-labeled sulfates (DHAS or E₁S). The apparent equilibrium constants (K) of the tracers did not measuraby change at concentrations of the non-radioactive sulfates below 10^–5 mol/l. At higher concentrations, K decreased gradually. The apparent equilibrium constant of³H-E₁S was diminished by plasma in a similar fashion. It may be concluded that albumin possesses one strong, non-specific binding site. This site, however, does not seem to be utilized for the binding of E₁S in vivo, because of its preferetial occupation by other ligands. This may be true for other steroid sulfates as well, depending on their relative abundance in plasma.Acknowledgment. This investigation received financial support from the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organisation, and from the Ford Foundation.     

Variations au cours de la journée de l'incorporation in vivo de la leucine tritiée dans les protéines du cervelet et du cerveau du jeune rat normal et hypothyro?dien. Daily variations of the in vivo [3H] leucine incorporation into the cerebellar and cerebral proteins of the normal and hypothyroid young rat [(author's transl)

In the normal and hypothyroid 6-day-old rat, the specific radioactivity (RSA) and the relative RSA (ratio of the RSA to the [3H] lecine concentration of the acido soluble phase) of the cerebral and cerebellar proteins, changes during the day synchronally. They show a maximum at 15.00 h and a minimum at 0.300 h. At all stages studied, these values are significantly lower in the hyothyroid animals than in normal ones.     

Variations au cours de la journée de l'incorporation in vivo de la leucine tritiée dans les protéines du cervelet et du cerveau du jeune rat normal et hypothyroïdien

Summary In the normal and hypothyroid 6-day-old rat, the specific radioactivity (RSA) and the relative RSA (ratio of the RSA to the [³H] leucine concentration of the acido soluble phase) of the cerebral and cerebellar proteins, change during the day synchronally. They show a maximum at 15.00 h and a minimum at 03.00 h. At all stages studied, these values are significantly lower in the hypothyroid animals than in normal ones.     

Decrease in [3H]ouabain binding sites in heart and brain from spontaneously hypertensive rats

Summary A decrease in the number of binding sites (B_max) for [³H]ouabain was observed in the heart (37%) and the brain (22%) of spontaneously hypertensive rats (SHR) when compared with age-matched control Wistar Kyoto rats. No variation was detected in the affinity constant (K_D).     

Studies on the serotonin transporter in platelets

[3H]-Imipramine and [3H]-paroxetine label with high affinity a recognition site which is associated with the serotonergic transporter in blood platelets. The pharmacological profile of [3H]-imipramine and [3H]-paroxetine binding is highly correlated with the potency of drugs to inhibit the uptake of serotonin. Dissociation kinetic experiments suggest that the substrate recognition site for serotonin may be different from the modulatory site which is labeled with [3H]-imipramine or [3H]-paroxetine. The existence of an endocoid acting on the imipramine receptor to modulate the serotonin transporter has been proposed by several laboratories. In clinical studies most laboratories have reported a decrease in Bmax of [3H]-imipramine binding in platelets from depressed untreated patients when compared with matched healthy volunteers. The Bmax of [3H]-imipramine binding in platelets appears to be a state-dependent biological marker in depression.     

Visualization of opiate receptors in the locus coeruleus of rats: high resolution radioautography after administration of a tritiated met-enkephalin analog]

Opiate receptor sites were visualized by high resolution radioautography in the locus coeruleus of the Rat following intraventricular injection of the met-enkephalin analogue FK 33-824 tritiated with a high specific activity (3H-FK). After administration of a tracer dose of 3H-FK, more than 75% of the radioactivity detected in the locus coeruleus is specifically bound to opiate receptor sites. These are distributed both between and inside neuronal perikarya. After administration of 3H-FK at high concentration, electron microscope radioautography shows the presence of specific opiate binding sites at the level of axo-somatic and axo-dendritic synaptic junctions. These synaptic binding sites could correspond to receptor sites for endogenous enkephalins.     

Na+ independent binding of [3H] muscimol to a membrane fraction of the brains of normal and dwarf mice

Although their body weights were decreased by about 77% and their brain weights by about 30%, high-affinity [3H] muscimol binding to a cerebral membrane fraction was not altered in hereditary pituitary dwarf mice. Marked changes in the level of pituitary growth-associated hormones do not appear to be associated with a change in cerebral GABA-receptors.     

Binding of matrilysin-1 to human epithelial cells promotes its activity

Matrix metalloproteinase-7 (MMP-7, matrilysin- 1) modulates crucial biological events by processing many epithelial cell surface-associated effectors. We addressed MMP-7 interaction with human epithelial cells and its resulting activity. In human endometrium, a model of controlled tissue remodeling, proMMP-7 was diffusely immunolocalized inside epithelial cells, whereas MMP-7 delineated their entire plasma membrane. Endometrial explants preferentially retained active MMP-7, but not proMMP-7. Endometrial epithelial cells and carcinoma cells from various tissues bound active MMP-7. Endometrial carcinoma-derived Ishikawa cells showed high affinity (K_D of ~2.5 nM) and capacity (~260 000 sites per cell) for MMP-7. MMP-7 binding decreased by extracting membrane sterols or interfering with heparan sulfate proteoglycans, and was abrogated by tissue inhibitors of metalloproteinase-2 (TIMP-2) or synthetic MMP inhibitors. Bound MMP-7 not only remained fully active towards a macromolecular substrate but also became resistant to TIMP-2. We conclude that MMP-7-selective targeting to the plasma membrane of epithelial cells promotes its activity by conferring resistance to TIMP-2. A. Berton, C. Selvais: These authors contributed equally to this work. P. J. Courtoy, E. Marbaix, H. Emonard: These authors contributed equally to the supervision of this work. Received 20 September 2006; received after revision 30 November 2006; accepted 18 January 2007     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

LSD: The distribution of [3H]LSD in the reproductive system of the male rat and placental transfer in the female rat

Summary After administration of [³H]LSD to male rats, radioactivity was present in all tissues of the reproductive system, notably associated with spermatozoa in the epididymis. In addition, in pregnant rats LSD and/or metabolites readily crossed the placental barrier.