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1.
Raj K. Singh Mary F. Ruh Thomas S. Ruh 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,800(1):33-40
In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [3H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [3H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions. 相似文献
2.
The activation by salt or ATP of [3H]estradiol- and [3H]H1285-receptor complexes from rabbit uterus and their binding capacity to DNA-cellulose, phosphocellulose and ATP-Sepharose has been studied. The estrogen-receptor was prepared in 1 mM molybdate which stabilized the receptor; but both salt- and ATP-transformation of estrogen receptors occurred. The binding of molybdate-stabilized cytosol [3H]estradiol-receptor complexes to the various resins revealed that salt-activation by 0.3 M KCl caused the greatest binding (5-6-fold) to DNA-cellulose as compared to other resins. However, 5 mM ATP-dependent activation of receptor-complexes resulted in preferential binding to ATP-Sepharose. Activated cytosol [3H]H1285-receptor complexes bound all the resins to a lesser degree when compared to [3H]estradiol-receptor complexes. Partially purified receptor complexes also showed different resin-binding patterns for salt- and ATP-mediated activation. These findings suggest that salt-activation is different than ATP-activation. Further, the differential magnitude of [3H]estradiol- and [3H]H1285-receptor activation suggests that estrogen-receptor complexes are "fully" activated as compared to "partially" activated antiestrogen-receptor complexes. 相似文献
3.
T W Hutchens R W McNaught T T Yip T Suzuki C M Li P K Besch 《Molecular endocrinology (Baltimore, Md.)》1990,4(2):255-267
A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding. 相似文献
4.
E J Pavlik K Nelson J R van Nagell E S Donaldson M L Walden M B Hanson H Gallion R C Flanigan D E Kenady 《Biochemistry》1985,24(27):8101-8106
Size-exclusion high-performance liquid chromatography was used to characterize the hydrodynamic molecular properties of estrogen receptors complexed with estradiol and the antiestrogen 4-hydroxytamoxifen. Cytoplasmic estrogen receptors complexed with [3H]-4-hydroxytamoxifen did not undergo reductions in hydrodynamic size after exposure to KCl or urea. Nuclear receptors complexed with 4-hydroxytamoxifen eluted as hydrodynamically larger molecules than nuclear receptors complexed with estradiol. Because identical hydrodynamic characterizations were obtained with the covalent ligand [3H]tamoxifen aziridine, these differences in chromatographic behavior are due to differences in ligand-mediated receptor properties and are not the result of ligand dissociation. When estrogen receptors, complexed with either [3H]estradiol or [3H]-4-hydroxytamoxifen, were exposed to trypsin, the receptors complexed with 4-hydroxytamoxifen eluted as larger hydrodynamic forms than receptors complexed with estradiol. These observations are interpreted to indicate that estradiol and 4-hydroxytamoxifen mediate contrasting transitions in the molecular orientation of estrogen receptors. The consequences of the transitions mediated by 4-hydroxytamoxifen appear to be that intermolecular associations become difficult to disrupt with KCl or urea and that the accessibility of trypsin-sensitive proteolytic sites becomes altered. Chromatin fractionation using DNase I and hypotonic Mg2+ solubilization identified a chromatin region that was less readily penetrated by receptors complexed with 4-hydroxytamoxifen than receptors complexed with estradiol.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
5.
The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands. 相似文献
6.
《Neurochemistry international》1986,9(2):287-293
The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS. The [3H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABAA and GABAB receptors. The most potent inhibitors of [3H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [35S]TBPS was partially inhibited by 1.0 × 10−6 M GABA, whilst binding of [3H]flunitrazepam was enhanced by 1.0 × 10−7 M GABA. The pharmacological profile of the [3H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS. 相似文献
7.
[3H]Spiperone ([3H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [3H]tyramine ([3H]PTA), thus indicating that [3H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [3H]PTA binding and [3H]DA uptake processes were markedly decreased, with no effect on [3H]mazindol ([3H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [3H]PTA binding, did not affect the [3H]MAZ binding process. This further supported the suggestion that while [3H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [3H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras 相似文献
8.
D M Ignar-Trowbridge K G Nelson K A Ross T F Washburn K S Korach J A McLachlan 《The Journal of steroid biochemistry and molecular biology》1991,39(1):131-132
The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors. 相似文献
9.
Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [3H]succinic anhydride plus [35S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [3H]succinic anhydride. In contrast, relative labeling by [35S]DABS was similar to [3H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [3H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by14C- and3H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of14C/3H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in14C/3H labeling ratios of core proteins I and II, cytochromec
1, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein. 相似文献
10.
A. Rotondo G. Giannaccini C. Quattrone D. Marazziti C. Martin G. B. Cassano A. Lucacchini 《Neurochemical research》1994,19(10):1295-1300
The serotonin (5-HT) transporter from calf striatum cerebral membranes was solubilized with digitonin and characterized by gel exclusion chromatography. [3H]Imipramine and [3H]paroxetine were utilized as markers for labeling it.3H-imipramine labels a high- and a low-affinity site on striaturn membranes, whereas it binds to a single high-affinity site on the solubilized fraction. [3H]Paroxetine binds with the same affinity to a single site on both membranes and solubilized preparations. After gel exclusion chromatography of the solubilizate both [3H]imipramine and [3H]paroxetine bind on an identical fraction of 205 kDa molecular weight, with a similar maximum number of binding sites (Bmax). Our results suggest that both3H-imipramine and [3H]paroxetine bind to a common site on the 5-HT transporter. 相似文献
11.
The mitochondrial fraction of diethylstilbestrol-treated rat uteri, known to contain an estrogen-induced peroxidase, was able to catalyze the release of 3H2O from either [2-3H]- or [4-3H]estradiol. Hydrogen peroxide added to this system increased the yield of 3H2O but had no effect on mitochondrial preparations from ovariectomized rat uteri having only very low peroxidase activity. The reaction was inhibited by catalase and also occurred with lactoperoxidase in the presence of H2O2 but 2-hydroxyestradiol was not detected in any of these experiments. Under similar conditions, tyrosinase catalyzed the formation of the catechol estrogen with loss of 3H from [2-3H]- or [2,4,6,7-3H]- but not [4-3H]- or [6,7-3H]estradiol. It is proposed that the formation of 3H2O from 3H-labeled estradiol in the estrogen-treated rat uterus may occur by a peroxidative mechanism which does not necessarily result in hydroxylation of the steroid. 相似文献
12.
Benzodiazepine receptors were labeled with [3H] diazepam following intravenous injection in rats. Binding of [3H] diazepam to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [3H] diazepam was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [3H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [3H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain . 相似文献
13.
Kinetic analysis of binding of [3H][N-[2-[4-(2-[O-methyl-3H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide ([3H]WAY100635) to 5-HT1A receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding
experiments revealed that [3H]WAY100635 binds to a single class of receptors with very high apparent affinity (K
D = 87 ± 4 pM, B
max = 15.1 ± 0.2 fmol/mg protein). The binding was almost irreversible, as the dissociation rate constant obtained k
off = (7.8 ± 1.1) × 10−3 min−1, means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25°C. Systematic association
kinetic studies of [3H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive
cooperativity. The affinities of antagonists determined from competition with [3H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of [35S]GTPγS binding probably due to the ligand’s kinetic peculiarities. Thus, [3H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies
is strongly limited. 相似文献
14.
Dr. G. Schleicher W. E. Stumpf U. Drews K. U. Thiedemann M. Sar 《Histochemistry and cell biology》1985,82(5):453-461
Summary The distribution of specific nuclear binding sites for androgens and estrogens in the male accessory sex organs of the mouse
was assessed by autoradiography with3H dihydrotestosterone (3H DHT) and3H estradiol (3H E2).
With3H DHT nuclear labeling differed among the epithelia of the organs. It was high in seminal vesicle and ampullary gland, moderate
in ventral prostate, urethral gland, prostatic excretory ducts and the ampulla ductus deferentis, low in dorsal prostate and
low or absent in coagulation gland. With3H E2, in contrast, epithelial nuclear labeling was high only in coagulation gland, moderate or low in seminal vesicle, low or
absent in ventral and dorsal prostate and absent in ampullary gland and ampulla ductus deferentis. In the lamina propria of
all organs nuclear labeling with3H DHT was generally moderate and existed only in some cells, with the highest number in the ampulla ductus deferentis. With3H E2, nuclear labeling in the lamina propria showed a high intensity in all organs, except in ventral and dorsal prostate which
remained unlabeled. Many labeled cells were found in the deferent duct and its ampulla, while in the other organs only a few
cells showed nuclear labeling with3H E2. In the smooth muscle sheath of all organs, some muscle cells were moderately labeled with3H DHT, but not with3H E2.
The results indicate the presence of nuclear receptors in male accessory sex organs for both dihydrotestosterone and estradiol.
The differential patterns of3H DHT and3H E2 nuclear uptake suggest differential sensitivities of the individual organs and their tissue compartments for androgens and
estrogens.
Supported by PHS grant NSO9914 to W.E.S. and Deutsche Forschungsgemeinschaft Dr94/4 to U.D. The work of Dr. Schleicher and
his stay in Chapel Hill were additionally sponsored by Studienstiftung des Deutschen Volkes and Boehringer-Ingelheim Fonds 相似文献
15.
Abstract— The appearance of γ-[3H]hydroxybutyric acid ([3H]GHB) in rat brain at various times after the intraventricular administration of [3H]GABA was determined. Radioactivity recovered as [3H]GHB was maximal 30 s after [3H]GABA administration and declined exponentially thereafter. From a linear transformation of the disappearance with time of [3H]GHB formed from [3H]GABA, the fractional rate of disappearance and turnover time of GHB were calculated. Administration of amino-oxyacetic acid (50 mg/kg i.p.) 1 h before [3H]GABA, reduced [3H]GHB formation, measured 4 min after [3H]GABA, to 28% of that found in control animals. This strongly suggests that GABA-transaminase catalyzes at least one step in the conversion pathway. [3H]GHB recoverable 4 min after [3H]GABA was unchanged when animals were pretreated with pyrazole (1.25–5.0 mmol/kg), diphenyl-hydantoin (25 and 75 mg/kg), phenobarbital (7.5–60 mg/kg), ethanol (1.25–5.0 g/kg), or morphine (2.5–10 mg/kg). Significantly more [3H]GHB could be recovered at several time points from animals which had been pretreated with 50 mg/kg i.p. of the convulsant 3-mercaptopropionic acid. 相似文献
16.
Calculation of Cell Production from [3H]Thymidine Incorporation with Freshwater Bacteria 总被引:2,自引:0,他引:2
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The conversion factor for the calculation of bacterial production from rates of [3H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20°C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 × 1018 cells mol-1 of thymidine incorporated into the trichloroacetic acid (TCA) precipitate (standard error = 0.29 × 1018; n = 54), when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 × 1018 cells mol-1 of thymidine incorporated into TCA precipitate (standard error = 1.72 × 1018; n = 47). The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20°C than at 15 and 10°C. A detailed examination of the [3H]thymidine concentrations that were needed to achieve maximum labeling in DNA was carried out 6 times during a complete growth cycle. During periods with low generation times and high conversion factors, 15 nM [3H]thymidine was enough for the maximum labeling of the TCA precipitate. This suggests that incorporation of [3H]thymidine into DNA is probably limited by uptake during periods with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 × 1018 cells mol-1 of thymidine incorporated is used. 相似文献
17.
Charles R. George 《Neurochemical research》1982,7(2):205-211
Alpha adrenergic receptor subtypes in rat hippocampal membranes were studied, using [3H]clonidine as the radioactive ligand. On the basis of competitive binding studies, using the selective antagonist-prazosin, WB-4101, and yohimbine, [3H] clonidine appeared to bind to a population of presynaptic sites that are pharmacologically similar to receptors previously classified as alpha2. A computerized model that linearized and produced the best possible fit to the experimental data points indicated that [3H]clonidine binds to a single population of receptors possessing equal affinity for the ligand. Binding data also indicated that rat hippocampus contains significantly fewer [3H]clonidine binding sites than rat cortex. 相似文献
18.
(±)-[3H]Epinephrine and (?)-[3H]norepinephrine bind saturably to calf cerebral cortex membranes under appropriate incubation conditions in a fashion indicating that they label α-noradrenergic receptors. Binding of the two [3H]catecholamines is saturable with dissociation constants of 20–30 nM. Binding is stereoselective with (?)-norepinephrine displaying about twenty times greater affinity than (+)-norepinephrine. The relative potencies of catecholamines in competing for these binding sites parallels their relative pharmacologic effects at α-noradrenergic receptors in numerous tissues. Thus, (?)-epinephrine is 2–3 times more potent than (?)-norepinephrine and 500 times more potent than (?)-isoproterenol. Binding is inhibited by low concentrations of the α-antagonists phentolamine and phenoxybenzamine but not by the β-antagonist propranolol. 相似文献
19.
《Journal of receptor and signal transduction research》2013,33(4-6):363-385
AbstractWe have studied the binding of [3H]-NPY and the newly developed non-peptide Y1 receptor antagonist [3H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y1 receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [3H]-NPY binding was slow, the binding kinetics of [3H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y1 agonists NPY, PYY and [Leu31-Pro34]-NPY completely and potently displaced [3H]-NPY binding, they could only displace 70 to 80 % of the [3H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [3H]-NPY binding sites in CHO-Y1 cells whereas [3H]-BIBP3226 binding parameters remained unchanged. 相似文献
20.
Jin-Jye Feng Fong-Chi Cheng Chun-Hsiung Lin Jiann-Wu Wei Shiaw-Der Yang 《Archives of biochemistry and biophysics》2011,506(1):66
[3H]8-OH-DPAT is a selective ligand for labeling 5-HT1A receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [3H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [125I]RTI-55 and [3H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [3H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [3H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [125I]cyanopindolol, [3H]ketanserin/[3H]mesulergine, [3H]GR-65630, [3H]GR-113808 and [3H]LSD) that specifically labeled 5-HT1, 5-HT2, 5-HT3, 5-HT4 and 5-HT5–7 receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis. 相似文献