共查询到20条相似文献,搜索用时 15 毫秒
1.
Abstract: In the postmortem human brain (27 specimens of frontal cortex, Brodmann area 9), the specific binding of the antagonists [3H]RX 821002 (2-methoxyidazoxan) to α2A-adrenoceptors and that of [3H]idazoxan to l2-imidazo-line sites (a nonadrenoceptor mitochondrial site) were determined in parallel to study the effect of aging (range, 4–89 years) on both brain proteins. The density of α2A-adrenoceptors and age were negatively correlated (r=-0.71; p < 0.001). In contrast, the density of l2-imidazo-line sites was positively correlated with aging (r= 0.59; p < 0.005). The ratio of receptor densities (α2A/l2) also showed a marked negative correlation with age (r=-0.76; p < 0.001). In an age-selected group (range, 10–89 years), the density of monoamine oxidase (MAO)-B sites labeled by [3H]Ro 19–6327 (lazabemide) also showed a positive correlation with age (r= 0.80; p < 0.005). In these subjects, the density of l2-imidazoline sites correlated well with the density of MAO-B sites (r= 0.70; p < 0.005). The ratio of the density of these sites (MAO-B/l2) did not correlate with the age of the subject at death (r=-0.15). In the human frontal cortex, idazoxan displayed very low affinity (Ki= 89 μM) against the binding of [3H]Ro 19–6327 to MAO-B, which discounted a direct interaction of [3H]idazoxan with the active center of the enzyme and indicated that the l2-imidazoline site cannot be identified with MAO-B. However, l2-imidazoline sites and MAO-B show a clear coexpression not only in the human frontal cortex during the process of aging, but also in various brain regions of the human and rat brains. It is suggested that the l2-imidazoline site has a specific location on glial (astrocyte) cells. 相似文献
2.
Luis F. Callado J. Javier Meana Bernardo Grijalba Angel Pazos †Magdalena Sastre †Jesús A. García-Sevilla 《Journal of neurochemistry》1998,70(3):1114-1123
Abstract: The α2A- and α2C-adrenoceptor subtypes were evaluated in postmortem brains from suicides with depression (n = 22), suicides with other diagnoses (n = 12), and controls (n = 26). Membrane assays with the antagonist [3H]RX821002 (2-[3H]methoxyidazoxan) suggested the presence of α2A-adrenoceptors in the frontal cortex and both α2C-adrenoceptors and α2A-adrenoceptors in the caudate. The proportions in caudate were similar in controls (α2A, 86%; α2C, 14%), depressed suicides (α2A, 91%; α2C, 9%), and suicides with other diagnoses (α2A, 88%; α2C, 12%). Autoradiography of [3H]RX821002 binding under α2B/C-adrenoceptor-masking conditions confirmed the similar densities of α2A-adrenoceptors in the cortex, hippocampus, and striatum from controls and suicides. In the frontal cortex of depressed suicides, competition of [3H]RX821002 binding by (?)-adrenaline revealed a greater proportion (61 ± 9%) of α2A-adrenoceptors in the high-affinity conformation for agonists than in controls (39 ± 5%). Simultaneous analysis with the agonists [3H]clonidine and [3H]UK14304 and the antagonist [3H]RX821002 in the same depressed suicides confirmed the enhanced α2A-adrenoceptor density when evaluated by agonist, but not by antagonist, radioligands. The results indicate that depression is associated with a selective increase in the high-affinity conformation of the brain α2A-adrenoceptors. 相似文献
3.
Georges Vauquelin Hilde De Vos Jean-Paul De Backer Guy Ebinger 《Neurochemistry international》1990,17(4)
The antagonist [3H]idazoxan binds with comparable affinity to α2 adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-3H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300–1200 times lower affinity for the non-stereoselective sites. Their affinity for the α2 receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [3H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the α2 receptors (KD = 2.8 nM than [3H]idazoxan (KD = 24 nM) and about equal affinity as [3H]rauwolscine (KD = 3.6 nM).[3H]Rauwolscine binds with comparable affinity to α2 receptors and to 5-HT1A receptors, and competition studies indicate that the Ki value of unlabelled RX 821002 for the 5-HT1A receptors (30 nM) is about one order in magnitude above its Ki value for the α2 receptors (4.1 nM). Labelling of the 5-HT1A receptors by [3H]RX 821002 and by [3H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 μM). Under these conditions, specific binding of [3H]RX 821002 to the α2 receptors represents 84% of total binding (at its KD), as compared to 77% for [3H]rauwolscine and 20% for [3H]idazoxan.[3H]RX 821002 labels the α2 receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37°C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [3H]RX821002. About 60% of the α2 receptors possess high agonist affinity. 相似文献
4.
Abstract: Competition [3H]RX821002 ([3H]2-methoxyidazoxan) binding experiments with α2-adrenoceptor subtype-specific antagonists—BRL 44408 (α2A selective), ARC 239 (α2B selective), and others—were performed to delineate through rigorous computer modeling receptor subtypes in the postmortem human brain. In the hippocampus, hypothalamus, cerebellum, and brainstem the whole population of α2-adrenoceptors appears to belong to the α2A subtype (100%; Bmax = 34–90 fmol/mg of protein). In the frontal cortex, the predominant receptor was the α2A subtype (87%; Bmax = 53 fmol/mg of protein), although a small population of the α2B/C subtype (13%; Bmax = 8 fmol/mg of protein) was also detected. In the caudate nucleus, a mixed population of α2A (64%; Bmax = 9 fmol/mg of protein) and α2B/C (36%; Bmax = 5 fmol/mg of protein) subtypes was detected. In the cortex and caudate and in the presence of ARC 239 (to mask the α2B/C-adrenoceptors), competition experiments with the agonist guanoxabenz clearly modeled the high- and low-affinity states of the α2A subtype. In the presence of ARC 239 and the GTP analogue guanylyl-5′-imidodiphosphate together with NaCl and EDTA (to eliminate the high-affinity α2A-adrenoceptor) guanoxabenz only recognized the low-affinity α2A-adrenoceptor. The results indicate that in the human brain the predominant α2-adrenoceptor is of the α2A subtype and that this functionally relevant receptor subtype is not heterogeneous in nature. 相似文献
5.
Gregory A. Ordway 《Journal of neurochemistry》1995,64(3):1118-1126
Abstract: The binding of [3H]rauwolscine to α2A- (also referred to as α2D-) and α2C-adrenoceptors in homogenates of rat cerebral cortex was measured by exploiting the selectivity of oxymetazoline for α2A-adrenoceptors. Inhibition of [3H]rauwolscine binding by oxymetazoline was modeled best assuming binding to two sites (p < 0.001). Competition curves for oxymetazoline were shifted rightward by the addition of GTP (250 µM) but were still fit best by a two-site model (p < 0.001). A concentration of oxymetazoline was calculated that would optimally antagonize [3H]rauwolscine binding (with GTP present) to oxymetazoline-sensitive α2A-adrenoceptors, minimally inhibiting binding to α2C-adrenoceptors. Subsequently, [3H]rauwolscine binding to α2A- and α2C-adrenoceptors in cortex was examined 3 weeks after destruction of noradrenergic terminals. Binding to α2C-adrenoceptors was increased significantly after treatment with 6-hydroxydopamine (6-OHDA) compared with vehicle-treated controls, whereas binding to α2A-adrenoceptors was unchanged. Pretreatment of rats with desipramine before 6-hydroxydopamine, to protect noradrenergic neurons, resulted in no changes in binding to either α2A- or α2C-adrenoceptors. Thus, α2C-adrenoceptors are regulated by changes in synaptic availability of norepinephrine. α2A-Adrenoceptors are either not regulated by synaptic norepinephrine or are located both post- and presynaptically so that up-regulation of postsynaptic α2A-adrenoceptors is offset by a loss of presynaptic α2A-adrenoceptors. 相似文献
6.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1992,19(8):841-849
On the basis of its established in vitro characteristics, [3H]RX 821002 was evaluated in rats as an in vivo radioligand for central α2-adrenoceptors. Estimates for in vivo binding potential, obtained by compartmental analyses of time-radioactivity data, ranged between 1.9 for hypothalamus and 0.2 for cerebellum, with a regional distribution in brain which was similar to that observed in vitro. Selectivity and specificity of the signal were checked by predosing with either the α2-antagonists, idazoxan or yohimbine, the α2-agonist, clonidine, or the α1-antagonist, prazosin. Pretreatment of the rats with the selective neurotoxin, DSP-4, had no significant effect on [3H]RX 821002 binding, suggesting that the majority of labelled sites were situated post-junctionally. The studies indicate that [3H]RX 821002 can be used experimentally as an in vivo marker for central α2-adrenoceptors. The size and rate of expression of the specific signal encourage the development and assessment of [11C]RX 821002 for clinical PET studies. 相似文献
7.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2009,3(1):66-70
The binding of specific nonselective α1- and α2-adrenoceptor antagonists [3H]prazosine and [3H]RX821002 has been studied on rat cerebral cortex synaptosomal membranes. It is shown that for α1-adrenoceptors the ligand-receptor interaction corresponds to the model assuming the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were: K d= 1.56 ± 0.17 nM, B max = 30.25 ± 1.78 fmol/mg protein, n = 2. The parameters of [3H]RX821002 binding to α2-adrenoceptors were: K d = 1.94 ± 0.08 nM, B max = 12.77 ± 3.17 fmol/mg protein, n = 2. For α2 -adrenoceptors the ligand-receptor interaction corresponded to the same model. For α1 - and α2-adrenoceptor antagonists the dissociation constants (K d) are approximately equal (1.56 ± 0.17 and 1.94 ± 0.08 nM, respectively), but the concentration of α2-adrenoceptors is two times lower than that of α1-adrenoceptors ( 12.77 ± 3.17 and 30.25 ± 1.78 fmol/mg protein, respectively). The efficiency (E = B max/2K d) of the ligand binding to α1-adrenoceptors is 2.3 times higher than that to α2-adrenoceptors (7.46 ± 1.32 and 3.29 ± 0.68 fmol/mg protein/nM, respectively. The data suggest that α1- and α2 -adrenoceptors in rat cerebral cortex exist as dimers. 相似文献
8.
Abstract: The aim of this study was to quantify and compare the turnover of brain α2-adrenoceptors during chronic morphine treatment and after spontaneous morphine withdrawal in rats. The oral administration of increasing doses of morphine (10–90 mg/kg) for 20 days did not alter the specific binding of the agonist [3H]clonidine in the cerebral cortex. However, spontaneous opiate withdrawal (24 h) significantly increased the density of cortical α2-adrenoceptors (Bmax for [3H]clonidine was 21% greater). The recovery of [3H]clonidine binding after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.6 mg/kg) was assessed in naive, morphine-dependent, and morphine-withdrawn rats to study the process of α2-adrenoceptor repopulation and to calculate receptor turnover parameters. The simultaneous analysis of receptor recovery curves revealed that the turnover of brain α2-adrenoceptors in morphine-withdrawn rats was accelerated [appearance rate constant (r) = 21 fmol/mg of protein/day; disappearance rate constant (k) = 0.25 day?1] compared with those in morphine-dependent (r = 13 fmol/mg of protein/day; k = 0.14 day?1) and naive (r = 15 fmol/mg of protein/day; k = 0.16 day?1) rats. Moreover, this analysis also indicated that the increased density of cortical α2-adrenoceptors observed during morphine withdrawal was due to a significantly higher receptor appearance (Δr = 37–57%) and not to a decreased receptor disappearance, which in fact showed also an increase (Δk = 56–79%). It is proposed that the increased rate of α2-adrenoceptor production in the brain of morphine-dependent rats during spontaneous withdrawal is most probably mediated by the overactivity of the adenylyl cyclase/cyclic AMP system induced by opiate addiction. 相似文献
9.
L. A. Nesterova O. V. Boiko B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2016,10(4):278-286
The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α1- and α2-adrenoceptor antagonists [3H]prazosin and [3H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α1- and α2-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [3H]prazosin binding to α1-adrenoceptors were: K d = 1.85 ± 0.16 nM, B max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [3H]RX821002 binding to α2-adrenoceptors were: K d = 1.57 ± 0.27 nM, B max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α1- and α2-adrenoceptors occurred according to the same model. The affinity to [3H]prazosin and the concentration of active α1-adrenoceptors increased by 27% (K d = 1.36 ± 0.03 nM) and 84% (B max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α2-adrenoceptors to [3H]RX821002 decreased by 56% (K d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [3H]prazosin and [3H]RX821002, two pools of receptors were detected with the following parameters: K d1 = 1.13 ± 0.09, K d2 = 6.07 ± 1.06 nM, B m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K d1 = 0.61 ± 0.02, K d2 = 3.41 ± 0.13 nM, B m1 = 1.88 ± 0.028, B m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B max) increased twofold for both ligands. It was suggested that α1- and α2-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α1- and α2- adrenoceptors was revealed, which was manifested in the activating effect on the [3H]prazosin binding parameters, in the inhibitory effect on the [3H]RX821002 binding parameters, and in a change of the general character of binding for both ligands. 相似文献
10.
B. N. Manukhin L. A. Nesterova 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2011,5(4):350-354
Activation and inhibition of muscarinic cholinoceptors by atropine and carbachol are shown to exert allosteric effects on
the binding of specific nonselective α2-adrenoceptor antagonist [3H]RX821002 in rat brain cortex membranes. The ligand-receptor interaction for α2-adrenoceptors corresponded to the model suggesting the presence of one homogeneous pool of receptors with two specific binding
sites. The parameters of the [3H]RX821002 binding were as follows: [3H]RX821002 -K
d = 1.94 ± 0.08 nM, B
max = 13.4 ± 1.8 fmol/mg protein, n = 2. The inhibition of muscarinic cholinoceptors by atropine induced an increase of affinity (K
d = 1.36 ± 0.12 nM) and a decrease of the α2-adrenoceptor density (B
max = 10.18 ± 0.48 fmol/mg protein). The muscarinic cholinoceptor agonist carbachol induced an increase of the affinity (K
d = 1.56 ± 0.05 nM) and quantity of binding sites (B
max = 16.61 ± 0.29 fmol/mg protein). As a result, under the influence of atropine and carbachol, the efficiency of binding (E = B
max/2K
d) increased from 3.50 ± 0.40 to 5.60 ± 0.79 and 6.86 ± 0.20 fmol/mg protein/nM, respectively. The data suggest that α2-adrenoceptors exist in rat brain cortex as homodimers. 相似文献
11.
Abstract: The binding of [3H]dopamine to brain regions of calf, rat, and human was investigated. The calf caudate contained the highest density of [3H]dopamine binding sites, with a Bmax value of 185 fmol/mg protein, whereas rat and human striatum contained one-third this number of sites. The KD values for [3H]dopamine in all tissues were 2–3 nM. Dopaminergic catecholamines (dopamine, apomorphine, 6,7-dihydroxy-2-aminotetralin, and N-propylnorapomorphine) inhibited the binding of [3H]dopamine in all three species, at low concentrations, with IC50 values of 1.5 to 6 nM. Neuroleptics, in contrast, inhibited the binding at high concentrations (with IC50 values of 200 to 40,000 nM). The [3H]dopamine binding sites were saturable, heat-labile, and detectable only in dopamine-rich brain regions; these sites differed from D2 dopamine sites (labeled by [3H]butyrophenone neuroleptics), and from Dl dopamine sites (labeled by [3H]thioxanthene neuroleptics) associated with the dopamine-stimulated adenylate cyclase. We have, therefore, called these high-affinity [3H]dopamine binding sites D3 sites. [3H]Apomorphine and [3H]ADTN also appeared to label D3 sites. These ligands however, were less selective than [3H]dopamine, and labeled sites other than D3 as well. Assay conditions were important in determining the parameters of [3H]dopamine binding. The optimum conditions for selective labeling of the D3 dopaminergic sites, using [3H]dopamine, required the presence of EDTA and ascorbate. 相似文献
12.
The α1 and α6 Subunits Can Coexist in the Same Cerebellar GABAA Receptor Maintaining Their Individual Benzodiazepine-Binding Specificities 总被引:1,自引:1,他引:0
Abstract: Two GABAA receptor subunit-specific antibodies anti-α6 and anti-α1 have been used for elucidating the relationship between the presence of α1 and/or α6 subunits in the cerebellar GABAA receptors and the benzodiazepine-binding specificity. Receptor immunoprecipitation with the subunit-specific antibodies shows that 39% of the cerebellar GABAA receptors have α6, whereas 76% of the receptors have α1 as determined by [3H]muscimol binding. Results show that 42–45% of the receptors having α6 also have α1, whereas 13–15% of the receptors that contain α1 also have α6. The immunoprecipitation results as well as immunopurification and immunoblotting experiments reveal the existence of three types of cerebellar GABAA receptors; i.e., one has both α1 and α6 subunits, a second type has α1 but not α6, and a third type has α6 but not α1 subunits. The results also show that receptors where α1 and α6 subunits coexist have two pharmacologically different benzodiazepine-binding properties, each associated with a different α subunit. The α1 subunit contributes the high-affinity binding of [3H]Ro 15-1788 (flumazenil) and the diazepam-sensitive binding of [3H]Ro 15-4513. The α6 subunit contributes the diazepam-insensitive binding of [3H]Ro 15-4513, but it does not bind [3H]Ro 15-1788 with high affinity. Thus, in the cerebellar α1–α6 GABAA receptors, there is no dominance of the pharmacology of one α subunit over the other. 相似文献
13.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2013,7(4):280-285
The influence of activation and inhibition of serotonin receptors by serotonin (5HT) and miancerin on binding of specific nonselective α2-antagonist [3H]RX821002 in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction for α2-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of the [3H]RX821002 binding to α2-adrenoceptors were as follows: K d = 1.57 ± 0.276 nM, B max = 7.24 ± 1.63 fmol/mg protein, n = 2. In the case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K d1 = 0.82 ± 0.06; K d2 = 2.65 ± 0.22 nM; B m1 = 1.65 ± 0.23; B m2 = 4.20 ± 0.11 fmol/mg protein; n = 2. The affinity of high-affinity receptors increased twofold and the affininty of low-affinity receptors decreased by 69% as compared to control values. The concentration of high-affinity receptors decreased 4.4-fold, and of low-affinity, 1.7-fold. The value of maximal reaction (B max) decreased by 20%. In the case of miancerin-induced inhibition of 5HT-receptors the character of ligand binding also changed; two pools of receptors were detected with the following parameters: K d1 = 0.48 ± 0.09; K d2 = 3.79 ± 0.71 nM; B 1 = 0.63 ± 0.17; B 2 = 4.75 ± 0.21 fmol/mg protein; n = 2. The affinity of high-affinity receptors pool increased by 70% and that of low-affinity receptors decreased by 76% as compared to control values. The concentration of active high-affinity and low-affinity α2-adrenoceptors decreased by 70% and 141%, respectively. The total amount of the receptors (B max) decreased by 26%. The data suggest that α2-adrenoceptors in rat cerebral cortex exist as dimers. Modulatory effects of serotonin and miancerin on specific antagonist binding to α2-adrenoceptors may be accomplished by altering the character and binding parameters of the nonselective α2-antagonist [3H]RX821002. 相似文献
14.
《Journal of receptor and signal transduction research》2013,33(1-4):153-162
AbstractIn the present study, we have provided evidence that [3H] rauwolscine and [3H] idazoxan bind to different sites in rabbit urethra. The [3H] idazoxan capacity and affinity was 215 ± 14 fmol/mg protein and 1.59 ± 0.16 nM while [3H] rauwolscine binding parameters were 45.9 ± 3.4 fmol/mg protein and 2.39 ± 0.27 nM. [3H] idazoxan specific binding was inhibited only by compounds possessing an imidazoli(di)ne or a guanidinium moiety, while [3H] rauwolscine specific binding was inhibited by phenylethanolamines and classical α-antagonists. [3H] idazoxan was inhibited by KCI in a competitive and by MnCI2 in a non-competitive way, while other cations such as Na+, Li+ and Mg2+ did not inhibit [3H] idazoxan binding. Moreover, we investigated the regional distribution of [3H] idazoxan and [3H] rauwolscine along the rabbit urethra using quantitative autoradiography. Analysis of the films revealed a different distribution of these two binding sites on the urethral sections. 相似文献
15.
Fabrice Trovero Denis Hervé Gérard Blanc Jacques Glowinski Jean-Pol Tassin 《Journal of neurochemistry》1992,59(1):331-337
As shown by autoradiography, peripheral injections of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induced a dose-dependent decrease of [3H]SCH 23390 and [3H]prazosin high-affinity binding sites in the rat prefrontal cortex. EEDQ showed similar efficacy in inactivating cortical and striatal dopamine (DA) D1 receptors, whereas prazosin-sensitive alpha 1-adrenergic receptors were more sensitive to the action of the alkylating agent, as for all doses of EEDQ tested (from 0.8 to 3 mg/kg, i.p.), the decrease in cortical [3H]SCH 23390 binding was less pronounced than that of [3H]prazosin. The effects of EEDQ on [3H]SCH 23390 binding and DA-sensitive adenylate cyclase activity were then simultaneously compared in individual rats. In the striatum, whatever the dose of EEDQ used, the decrease of DA-sensitive adenylate cyclase activity was always lower than that of D1 binding sites, suggesting the occurrence of a large proportion of spare D1 receptors. In the prefrontal cortex, a significant increase in DA-sensitive adenylate cyclase activity was observed in rats treated with a low dose of EEDQ (0.8 mg/kg), this effect being associated with a slight reduction in [3H]SCH 23390 binding sites (-20%). Parallel decreases in the enzyme activity and D1 binding sites were observed with higher doses. The EEDQ-induced supersensitivity of DA-sensitive adenylate cyclase did not occur in rats in which the decrease in [3H]prazosin binding sites was higher than 35%.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2011,5(1):85-91
The influence of isoprenaline- and propranolole-induced activation and inhibition of β-adrenoreceptors on the specific nonselective
α2-antagonist [3H]RX821002 binding was studied on rat cerebral cortex subcellular membrane fractions. It was shown that the ligand-receptor
interaction for α2-adrenoreceptors corresponded to the model that assumed the presence of one receptor pool and binding of two ligand molecules
to a receptor dimer. The following parameters were determined for [3H]RX821002 binding to α2-adrenoreceptors: K
d1 = 1.57 ± 0.27 nM, B
max = 7.24 ± 1.63 fmol/mg of protein, n = 2. In the case of isoprenaline-induced activation of β-adrenoreceptors the binding of radiolabeled ligand to α2-adrenoreceptors was described by the same model. The affinity of α2-adrenoreceptors for [3H]RX821002 decreased more than twofold (K
d = 3.55 ± 0.02 nM) and the quantity of active receptors increased by 69% (B
max = 12.24 ± 0.06 fmol/mg of protein). Propranolole changed the model of ligand binding, and two pools of receptors were detected
with the following parameters: K
d1 = 0.61 ± 0.02 nM, K
d2 = 3.41 ± 0.13 nM, B
ml = 1.88 ± 0.028 fmol/mg of protein, B
m2 = 9.27 ± 0.08 fmol/mg of protein, n = 2. The data suggest that α2-adrenoreceptors in subcellular membrane fractions from rat cerebral cortex exist in dimeric form. Isoprenaline and propranolole
exhibit modulating effect on the specific antagonist binding to α2-adrenoreceptors, which results in the inhibition and alteration of [3H]RX821002 binding parameters. 相似文献
17.
GABAA Receptor Subtypes Expressed in Cerebellar Granule Cells: A Developmental Study 总被引:3,自引:1,他引:2
Abstract: The developmental properties of primary rat cerebellar granule cells have been characterised with respect to their expression of GABAA receptor subtypes using both an immunological approach and radioligand binding assays. At day 1 in culture, the GABAA receptor α1 subunit was detectable in immunoblots and increased in level up to day 9. The GABAA receptor α6 subunit was not detectable at day 1; however, at days 3–5, a specific Mr 58,000 anti-α6 1–16 Cys immunoreactive species was present which further increased in level up to 9 days in culture. Similar qualitative results were obtained for the expression of the GABAA receptor α6 subunit in age-matched rat cerebellar membranes. In parallel studies, it was found that although there was an overall increase in [3H]Ro 15–4513 binding sites with days in culture, the relative contributions of diazepam-sensitive and diazepam-in-sensitive [3H]Ro 15–4513 binding changed. A time-dependent enrichment of the diazepam-insensitive binding site up to a maximum of 74% of total [3H]Ro 15–4513 sites was found. This was concomitant with the appearance of the GABAA receptor α6 subunit. These results are in agreement with the pharmacology described for α6βγ2 cloned receptors. They suggest a developmentally regulated expression of the GABAA receptor α6 subunit gene at a time that is correlated in vivo with establishment of neuronal connections. 相似文献
18.
Erik H. F. Wong Douglas W. Bonhaus Irene Wu Eric Stefanich Richard M. Eglen 《Journal of neurochemistry》1993,60(3):921-930
Abstract: RS-42358–197{(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1H-benzo[de]isoquinolin-1-one hydrochloride} displaced the prototypic 5-hydroxytryptamine3 (5-HT3) receptor ligand [3H]quipazine in rat cerebral cortical membranes with an affinity (pKi) of 9.8 ± 0.1, while having weak affinity (pKi < 6.0) in 23 other receptor binding assays. [3H]RS-42358–197 was then utilized to label 5-HT3 receptors in a variety of tissues. [3H]RS-42358–197 labelled high-affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (KD) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (Bmax) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [3H]RS-42358–197 was similar to that labelled with [3H]GR 65630, but was significantly less than that found with [3H]-quipazine. The binding of [3H]RS-42358–197 had a pharmacological profile similar to that of [3H]quipazine, as indicated by the rank order of displacement potencies: RS-42358–197 > (S)-zacopride > tropisetron > (R)-zacopride > ondansetron > MDL72222 > 5-HT. However, differences in 5-HT3 receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1-(m-chlorophenyl)biguanide when displacing [3H]RS-42358-197 binding. [3H]RS-42358–197 also labelled a population (Bmax= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (KD= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5-HT3 receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [3H]RS-42358–197 have detected differences in 5-HT3 receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5-HT3 receptor. 相似文献
19.
A. M. Cesura R. Imhof B. Takacs M. D. Galva G. B. Picotti M. Da Prada 《Journal of neurochemistry》1988,50(4):1037-1043
Abstract: [3H]Ro 16–6491 [N-(2-aminoethyl)-p-chloroben-zamide HCl], a reversible “mechanism-based” inhibitor of monoamine oxidase (MAO) type B, binds selectively and with high affinity to the active site of MAO-B in brain and platelet membranes. Under normal conditions, the binding of [3H]Ro 16–6491 is fully reversible. However, [3H]Ro 16–6491 could be irreversibly bound (covalently) to membranes by the addition of the reducing agent NaBH3CN to the sample and adjusting to pH 4.5 with acetic acid. No irreversible labelling occurred in the absence of NaBH3CN and at neutral pH. The presence of the irreversible MAO-B inhibitor /-deprenyl completely abolished the irreversible labelling of the membranes by [3H]Ro 16–6491. The selective inactivation of MAO-B, e.g., by /-deprenyl prevented the covalent incorporation of [3H]Ro 16–6491 whereas selective inhibition of the MAO-A by clorgyline was without effect. The covalent linkage to membranes of unlabelled Ro 16–6491 and Ro 19–6327 (a selective and reversible MAO-B inhibitor closely related to Ro 16–6491) after the addition of NaBH3CN at pH 4.5 irreversibly inactivated MAO-B activity whereas MAO-A activity was unaffected. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of labelled membranes showed that [3H]Ro 16–6491 was incorporated into a single polypeptide with a molecular mass identical to the one labelled by [3H]pargyline (58 kilodaltons). Our results indicate that the polypeptide that is covalently labelled by [3H]Ro 16–6491 corresponds to one of the two MAO-B subunits. Therefore, [3H]Ro 16–6491 represents a selective probe for affinity labelling of MAO-B and for the investigation of the structural composition of the active site of the enzyme. Whether the reduction with NaBH3CN at pH 4.5 of the [3H]Ro 16–6491-MAO-B complex results in the formation of a stable adduct with the amino acid chain of the MAO-B or with its prosthetic group, FAD, remains to be elucidated. 相似文献
20.
Abstract: Cross-reactions between dopamine D3 and σ receptor ligands were investigated using (±)-7-hydroxy-N,N-di-n-[3H]propyl-2-aminotetralin [(±)-7-OH-[3H]DPAT], a putative D3-selective radioligand, in conjunction with the unlabeled σ ligands 1,3-di(2-tolyl)guanidine (DTG), carbetapentane, and R(?)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane [R(?)-PPAP]. In transfected CCL1.3 mouse fibroblasts expressing the human D3 receptor, neither DTG nor carbetapentane (0.1 µM) displaced (±)-7-OH-[3H]DPAT binding. R(?)-PPAP (0.1 µM) displaced 39.6 ± 1.0% of total (±)-7-OH-[3H]DPAT binding. In striatal and nucleus accumbens homogenates, (±)-7-OH-[3H]DPAT labeled a single site (15–20 fmol/mg of protein) with high (1 nM) affinity. Competition analysis with carbetapentane defined both high- and low-affinity sites in striatal (35 and 65%, respectively) and nucleus accumbens (59 and 41%, respectively) tissue, yet R(?)-PPAP identified two sites in equal proportion. Carbetapentane and R(?)-PPAP (0.1 µM) displaced ~20–50% of total (±)-7-OH-[3H]DPAT binding in striatum, nucleus accumbens, and olfactory tubercle in autoradiographic studies, with the nucleus accumbens shell subregion exhibiting the greatest displacement. To determine directly (+)-7-OH-[3H]DPAT binding to σ receptors, saturation analysis was performed in the cerebellum while masking D3 receptors with 1 µM dopamine. Under these conditions (+)-7-OH-[3H]DPAT labeled σ receptors with an affinity of 24 nM. These results suggest that (a) (±)-7-OH-[3H]DPAT binds D3 receptors with high affinity in rat brain and (b) a significant proportion of (±)-7-OH-[3H]DPAT binding consists of σ1 sites and the percentages of these sites differ among the subregions of the striatum and nucleus accumbens. 相似文献