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1.
The binding of (1)-[3H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P2 fraction from the rat cerebral cortex reached equilibrium within 4 min at 37°C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P2 fractions from the cerebral cortex (Kd:21 nM and 980 nM), striatum (Kd:28 nM and 690 nM), and cerebellum (Kd:22 nM and 833 nM). High affinity Bmax values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity Bmax values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC50:17 nM) and efficaciously (over 90%). Both high affinity and low affinity Bmax values for [3H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by puriffied synaptosomes, crude membranes and P2 fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [3H]vesamicol binding. Isoosmotic replaement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [3H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin.  相似文献   

2.
The role of nucleoside transport in ischemia-reperfusion injury and arrhythmias has been well documented in various animal models using selective blockers. However, clinical application of nucleoside transport inhibitors remains to be demonstrated in humans. It is not known whether human heart has nucleoside transport similar to that of animals. The aim of this study is to pharmacologically identify the presence of nucleoside transport binding sites in the human myocardium compared to animals.Myocardial tissue was obtained from guinea pig left and right ventricle, canine left ventricle, human intraoperative right atrium and human cadaveric right atrium and right and left ventricles. Myocardial preparations were obtained from tissue samples after homogenized and a differential centrifugation.Equilibrium binding assays were performed using [3H]-p-nitrobenzylthioinosine (NBMPR) at room temperature in the presence or absence of non-radioactive NBMPR or other nucleoside transport blockers such as p-nitrobenzylthioguanosine dipyridamole, lidoflazine, papaverin, adenosine and doxorubcine. From saturation curves and inhibition kinetics, we determined the relative maximal binding (Bmax) and dissociation constant (Kd) of [3H]-NBMPR binding of human myocardial preparations.Results demonstrated that the fresh human myocardial preparations have a specific binding site for NBMPR with a Bmax of 283 ± 32 fmol/mg protein and Kd of 0.56 ± 0.12 nM. These values are lower than those obtained from guinea pigs (Bmax = 1440 ± 187 fmol/mg protein and Kd = 0.21 ± 0.03 nM) and canine atrium (Bmax 594 ± 73 fmol/mg protein, and Kd = 1.12 ± 0.22 nM).Displacement kinetics studies revealed the relative potencies (of certain unrelated drugs as follow: p-nitrobenzylthioguanosine > dipyridamole > lidoflazine > pavaverine > Diltazam > adenosine > doxyrubicin. It is concluded that human myocardium contains an active nucleoside transport site which may play a crucial role in post-ischemic reperfusion-mediated injury in a wide spectrum of ischemic syndromes.  相似文献   

3.
[3H]verapamil binding to muscle tubule membrane has the following properties. KD = 27 ± 5 nM and maximum binding capacity Bmax = 50 ± 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [3H]verapamil versus [3H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k1) and dissociation (k?1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [3H]verapamil binding with KD values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [3H]verapamil binding with half-maximum inhibition values (K0.5) between 1 and 5 nM.  相似文献   

4.
The biogenic amine serotonin ( 5‐hydroxytryptamine, 5‐HT) is a neurotransmitter in vertebrates and invertebrates. It acts in regulation and modulation of many physiological and behavioral processes through G‐protein‐coupled receptors. Five 5‐HT receptor subtypes have been reported in Drosophila that share high similarity with mammalian 5‐HT1A, 5‐HT1B, 5‐HT2A, 5‐HT2B, and 5‐HT7 receptors. We isolated a cDNA (Pr5‐HT8) from larval Pieris rapae, which shares relatively low similarity to the known 5‐HT receptor classes. After heterologous expression in HEK293 cells, Pr5‐HT8 mediated increased [Ca2+]i in response to low concentrations (< 10 nM) of 5‐HT. The receptor did not affect [cAMP]i even at high concentrations (> 10 μM) of 5‐HT. Dopamine, octopamine, and tyramine did not influence receptor signaling. Pr5‐HT8 was also activated by various 5‐HT receptor agonists including 5‐methoxytryptamine, (±)‐8‐Hydroxy‐2‐(dipropylamino) tetralin, and 5‐carboxamidotryptamine. Methiothepin, a non‐selective 5‐HT receptor antagonist, activated Pr5‐HT8. WAY 10635, a 5‐HT1A antagonist, but not SB‐269970, SB‐216641, or RS‐127445, inhibited 5‐HT‐induced [Ca2+]i increases. We infer that Pr5‐HT8 represents the first recognized member of a novel 5‐HT receptor class with a unique pharmacological profile. We found orthologs of Pr5‐HT8 in some insect pests and vectors such as beetles and mosquitoes, but not in the genomes of honeybee or parasitoid wasps. This is likely to be an invertebrate‐specific receptor because there were no similar receptors in mammals.

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5.
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   

6.
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   

7.
The effects of activation and inhibition of serotonin receptors by serotonin (5-HT) and mianserin on the specific nonselective α1-antagonist [3H]prazosine binding in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction of α1-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and the binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were as follows: K d =1.85 ± 0.16 nM, B max = 31.1 ± 0.3 fmol/mg protein, n = 2. In 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 = 0.61 ± 0.04, K d2 = 3.82 ± 0.15 nM, B m1 = 6.6 ± 0.7, B m2 = 25.6 ± 0.4 fmol/mg protein, n = 2. The sensitivity of the high-affinity pool increased threefold and the sensitivity of the low-affinity pool decreased twofold as compared to the control. The value of maximal reaction (B max) did not change. In the case of inhibition of 5HT-receptors by mianserin, radioactive ligand is bound to α1-adrenoceptors according to the same model as in the control conditions. The affinity of α1-adrenoceptors to [3H]prazosine decreases twofold and the concentration increases (K d = 3.97 ± 0.12 nM, B max = 40.0 ± 0.5 fmol/mg protein). The data suggest that α1-adrenoceptors in rat cerebral cortex exist as a dimer. The modulatory effects of serotonin and mianserin on the specific binding of [3H]prazosine to α1-adrenoceptors was detected, manifesting itself as changes in the binding parameters and in the general character of ligand-receptor interactions.  相似文献   

8.
Abstract: The binding of radioactive piperidine-4-sulphonic acid ([3H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: KD= 17 ± 7 nM (Bmax= 0.15 ± 0.07 pmol/mg protein) and KD= 237 ± 100 nM (Bmax= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [3H]P4S. The slow binding component (kon= 5.6 × 107 or 8.8 × 107 M-1 min-1, kOff= 0.83 min-1, and KD= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (KD= 11 nM, Bmax= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [3H]P4S appears to involve the same two populations of sites with Bmax values similar to those for [3H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [3H]P4S and [3H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.  相似文献   

9.
The binding sites of 8-[3H]hydroxy-2-(di-n-propylamino)tetralin ([3H]DPAT) were characterized in the retina of goldfish in order to evaluate the selectivity of the ligand for serotonin1A (5HT1A) receptors. Specificity of the binding was performed in the presence of serotonergic and dopaminergic agonists and antagonists. Buspirone, spriroxatrine and 5-methoxy-N,N-dimethyltryptamine were potent inhibitors, followed by propranolol, citalopram, imipramine and desipramine. Serotonin was not a potent inhibitor, and its interaction with the binding sites of [3H]DPAT was complex. Nomifensine displayed an important inhibition, however, other dopamine uptake blockers, such as bupropion and GBR-12909, were less potent. Haloperidol was also a good inhibitor, but the D1 receptor agonist, SKF-38393, the D2 receptor antagonist, sulpiride, and dopamine did not inhibit the binding. GppNHp inhibited the binding in the micromolar range. The analysis of saturation experiments by isotopic dilution, using buspirone to determine nonspecific binding, revealed two sites. The number of binding sites defined by buspirone were higher than the ones defined by nomifesine. The specific binding, using buspirone for definition, was reduced by the intraocular injection of 6-hydroxydopamine. This investigation demonstrates that [3H]DPAT labels 5HT1A receptors in goldfish retina, but also interacts with a non-5HT receptor site. These receptors seem to be localized in dopaminergic neurons.  相似文献   

10.
《Life sciences》1995,57(15):1401-1410
PD 128907 [4a R, 10 b R-(+)-trans- 3, 4, 4a, 10 b - tetrahydro - 4- n-propy12 H,5H-[1] benzopyrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (Ki, 1 nM) versus human D2 receptors (Ki, 1183 nM) and a 10000-fold selectivity versus human D4 receptors (Ki, 7000 nM) using [3H]spiperone as the radioligand in CHO-K1-cells. Studies with [3H]PD 128907, showed saturable, high affinity binding to human D3 receptors expressed in CHO-K1 cells (CHO-K1-D3) with an equilibrium dissociation constant (Kd) of 0.99 nM and a binding density (Bmax) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D2 receptors (CHO-K1-D2). The rank order of potency for inhibition of [3H]PD 128907 binding with reference DA agents was consistent with reported values for D3 receptors. These results indicate that [3H]PD 128907 is a new, highly selective D3 receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D3 receptors.  相似文献   

11.
The nucleus accumbens (NAc) is a crucial forebrain nucleus implicated in reward‐based decision‐making. While NAc neurons are richly innervated by serotonergic fibers, information on the functional role of serotonin 5‐hydroxytryptamine (5‐HT) in the NAc is still sparse. Here, we demonstrate that brief application of 5‐HT or 5‐HT1B receptor agonist CP 93129 induced a long‐term depression (LTD) of glutamatergic transmission in NAc neurons. This LTD was presynaptically mediated and inducible by endogenous 5‐HT. Remarkably, a single cocaine exposure impaired the induction of LTD by 5‐HT or CP 93129. The inhibition was blocked when a selective dopamine D1 receptor antagonist SCH23390 was coadministered with cocaine. Cocaine treatment resulted in increased phosphorylation of presynaptic proteins, rabphilin 3A and synapsin 1, and significantly attenuated CP 93129‐induced decrease in rabphilin 3A and synapsin 1 phosphorylation. Application of cAMP‐dependent protein kinase inhibitor KT5720 caused a prominent synaptic depression in NAc neurons of mice with a history of cocaine exposure. Our results reveal a novel 5‐HT1B receptor‐mediated LTD in the NAc and suggest that cocaine exposure may result in elevated phosphorylation of presynaptic proteins involved in regulating glutamate release, which counteracts the presynaptic depressant effects of 5‐HT1B receptors and thereby impairs the induction of LTD by 5‐HT.  相似文献   

12.
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   

13.
This study has identified specific, stereoselective phenylalkylamine (PAA, (±)- [3H]verapamil) binding sites of low-affinity and high-density in cockroach (Periplaneta americana) nervous system and skeletal muscle membranes. Scatchard transformation of equilibrium binding data revealed a single population of binding sites in both tissues with dissociation constants (Kd) of 273 nM and 377 nM and binding capacities (Bmax) of 23 pmol·mg protein?1 and 37pmol·mg protein?1 for cockroach nervous tissue and skeletal muscle membranes, respectively. The PAA binding site in cockroach nervous tissue membranes was found to be dihydropyridine (DHP)-insensitive, whereas the corresponding site in cockroach skeletal muscle membranes was DHP-sensitive. This property of a DHP-sensitive PAA receptor distinguishes the binding sites identified in cockroach skeletal muscle from those in cockroach nervous tissue and indicates that pharmacologically distinct putative Ca2+ channel subtypes are present in insect nerve and muscle. © 1993 Wiley-Liss, Inc.  相似文献   

14.
Abstract

Homogenates from dog cerebellum were fractionated using sucrose gradient centrifugation. The [3H]inositol 1, 4, 5-trisphosphate binding and the glucose 6-phosphatase activities were found to co-purify. The binding was saturable and had high affinity (Bmax=44 pmol/mg protein, Kd=116 nM). Selective chemical modification was used to examine amino acid residues of the microsomal receptor that might be critical for the binding of inositol trisphophate. Sulfhydryl reagents, p-chloromercuric-phenyl sulfonic acid, eosin 5-maleimide, N-ethyl maleimide and fluorescein 5-maleimide were found to be highly potent inhibitors of the binding with half-maximal inhibition occurring at about 20 µM, 70 µM, 1 mM, and 0.1 mM, respectively. The inhibition was specific since the presence of 10 µM of inositol trisphosphate during the reaction completely protected against the inhibition by these reagents. These results suggest that sulfhydryl group is essential for inositol trisphosphate binding to its receptor.  相似文献   

15.
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   

16.
Binding of [125I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca2+-dependent membrane binding of [125I]calmodulin was found to have a Bmax of 284 pmol/mg protein and an apparent affinity with a Kd of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [125I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10–1 min–1 and a half-time (t1/2) of 1.8 min for the fast component, and a k of 4.8×10–2 min–1 and a t1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10–2 min–1 and a t1/2 of 15.3 min for the fast component, and a k of 5.5×10–3 min–1 and a t1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [125I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (Td) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [125I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10–7–10–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - Kd dissociation constant - Bmax maximum binding - k first-order rate constant - t1/2 half-time - Td transition temperature  相似文献   

17.
Leukotriene C4 (LTC4) has been demonstrated to induce contraction of the smooth muscle cell line DDTIMF2. A partially purified membrane fraction obtained from these cells exhibited a high affinity binding site for LTC4. Binding of [3H]-LTC4 was saturable, specific and reversible with a dissociation constant (Kd) of 21 ± 4 nM. The maximum number of binding sites (Bmax) was 55 ± 5 pmol/mg of protein. Specificity was demonstrated in competition studies in which the Ki of LTC4 against specifically bound [3H] - LTC4 was 12 nM whereas Leukotriene D4 (LTD4) and Leukotriene E4 (LTE4) had a Ki of 38 ± 4 and 4.7 ± 0.5 nM respectively. A previously described antagonist of leukotriene-induced smooth muscle contraction PFL 55712 had a Ki of 23 ± 2 nM as determined by competition binding experiments.  相似文献   

18.
Some of melatonin’s (Mel) well-established physiological effects are mediated via high-affinity cell-membrane receptors belonging to the superfamily of G-protein-coupled receptors. Specific binding of ligand 2-[125I]iodomelatonin, using membrane preparations from osmoregulatory tissues of flounder, rainbow trout and sea bream, together with Mel concentrations in the tissues and plasma were studied. The kidney, gill and small intestine samples were collected during the day and at night. The dissociation constants (K d) and maximal binding densities (B max) were calculated for each tissue at 11:00 and 23:00 h. The binding sites with K d values in the tissues in the picomolar range indicated the high affinity. K d and B max values were tissue- and species-dependent. The GTP analogue [Guanosine 5′-O-(3-thiotriphosphate)] treatment significantly reduced the B max value, indicating that the 2-[125I]iodomelatonin-binding sites are probably coupled to a G-protein. No daily variations in K d and B max values were observed. These are the first studies of the presence of 2-[125I]iodomelatonin-binding sites in the small intestine, kidney tubule and gill of fish. The data strongly suggest new potential targets for Mel action and the influence of Mel on water/ion balance in fish. The intestine seems to be a site of Mel synthesis and/or an active accumulation of the hormone.  相似文献   

19.
[125I]LSD (labeled at the 2 position) has been introduced as the first 125I-labeled ligand for serotonin 5-HT2 (S2) receptors. In the present study we examined the binding of [125I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [125I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75–80% and Scatchard plots of the binding data reveal Kd = 1.1 nM, Bmax = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0°C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC50 values for a variety of compounds show a clear 5-HT2 (S2) serotonergic pattern at this [125I]LSD site. Blockage of this primary 5-HT2 (S2) caudate binding site by 0.3 μM mianserin reveals the presence of a weaker [125I]LSD binding site with a Kd = 9.1 nM, Bmax = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [125I]LSD is a sensitive and selective label for 5-HT2 (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [125I]LSD binding site with mianserin allows the high sensitivity of [125I]LSD to be applied to D2 dopamine receptor studies as well.  相似文献   

20.
《Insect Biochemistry》1990,20(6):557-566
[125I]α-Bungarotoxinisusedasaprobetostudythenicotinic-cholinergicreceptorinmembrane preparations of the cockroach brain. Binding is restricted mainly to particulate fractions of brain homogenates, is time dependent and is saturable above 2 nM with very low non-specific binding. Scatchard analysis indicates that binding is associated with a single affinity site (Kd = 1.09 nM) having a Bmax of 8926 fmol/mg protein which is the highest concentration of binding sites yet reported in insects. Association kinetics are best fit by a mono-exponential model with a kobs = 4.37 × 10−3s−1. Dissociation is best described by a bi-exponential model giving dissociation constants of 1.18 × 10−5 and 9.94 × 10−5s−1. The Kds calculated from kinetic data are 0.029 and 0.25 nM suggesting the possibility of heterogeneous binding sites not detected by saturation studies. Displacement studies indicate that binding follows a nicotinic pharmacology and demonstrate the high affinity of methyllycaconitine and the anthelmintics, morantel and pyrantel. Displacement by neuronal bungarotoxin shows the presence of two distinct binding sites not differentiated by α-bungarotoxin. Autoradiographic studies show α-bungarotoxin to be binding to neuropile regions of the brain, to be displaced from these regions by agents effective in binding studies and demonstrate that the neuronal bungarotoxin binding sites can be regionally localized.  相似文献   

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