共查询到20条相似文献,搜索用时 453 毫秒
1.
Abstract: GTP and GDP decreased the saturable binding of [3 H]baclofen or [3 H]γ-aminobutyric acid ([3 H]GABA) to GABAB but not GABAA receptors whereas GMP displayed negligible activity. This effect was specific to guanyl nucleotides and was not mimicked by high concentrations of ATP. The inhibition of ligand binding was the result of a diminished receptor affinity with no change in receptor number. The use of a complete physiological saline solution rather than Tris buffer plus Ca2+ or Mg2+ increased the potency of GTP at the GABAB receptor. The results are discussed in relation to the effects of GABA and GTP on adenylate cyclase activity in the brain. 相似文献
2.
Abstract: The effect of Zn2+ on t -[3 H]butylbicycloorthobenzoate ([3 H]TBOB) binding to the GABAA receptor complex was studied autoradiographically in rat brain. Zn2+ inhibited [3 H]TBOB binding in a dose-dependent manner at physiological concentrations. Saturation analysis revealed noncompetitive inhibition in various brain regions. The inhibitory effect of Zn2+ had regional heterogeneity; regions showing the greatest inhibition of [3 H]TBOB binding were cortical laminae I–III, most areas of hippocampus, striatum, septum, and cerebellar cortex. Regions with relatively less inhibition of [3 H]TBOB binding included cortical laminae V–VI, thalamus, superior colliculus, inferior colliculus, and central gray matter. The effect of Zn2+ and those of other GABAA ligands, such as benzodiazepines, bicuculline, isoguvacine, and picrotoxin, on [3 H]TBOB binding seemed to be additive. Ni2+ , Cd2+ , and Cu2+ also inhibited [3 H]TBOB binding with a regional heterogeneity similar to that produced by Zn2+ . These results are consistent with Zn2+ acting at the previously detected recognition site on the GABAA receptor complex, distinct from the picrotoxin, GABA, and benzodiazepine sites. The regional heterogeneity of the Zn2+ effect may reflect differential regional distribution of GABAA receptor subtypes among brain regions. Other divalent cations probably act at the Zn2+ binding site. 相似文献
3.
Desensitization of CholecystokininB Receptors in GH3 Cells 总被引:1,自引:0,他引:1
Abstract: Desensitization of the cholecystokinin (CCK) octapeptide (CCK-8)-induced rise in intracellular free calcium concentration ([Ca2+ ],) was characterized in GH3 cells, a pituitary tumor cell line, which are known to possess CCKB receptor subtype. The CCK-8-induced [Ca2+ ], transient was reduced following the initial application of CCK-8. A similar desensitization of the CCK-8-induced response was observed following the first application of thyrotropin-releasing hormone (TRH). By contrast, the TRH- induced response was not desensitized by the preceding application of CCK-8. Desensitization of the CCK-8-induced [Ca2+ ], transient was associated with diminished inositol 1,4,5-trisphosphate formation. The recovery of desensitization of the CCK-8-induced response was delayed by a phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A (100 n M ). The responsiveness to CCK-8 was also reduced by phorbol 12, 13-dibutyrate (PDBu), and this effect of PDBu was completely abolished by preincubation with staurosporine. Staurosporine significantly attenuated the desensitization caused by preincubation with CCK-8, but this effect was too small to attribute the desensitization to the protein kinase C transduction pathway alone. It is likely that desensitization of CCK receptors involves multiple transduction pathways. 相似文献
4.
Abstract: Cations of various size and charge were used as atomic scale probes of D1 and D2 dopamine receptors. Those cations that perturbed the binding of D1 - and D2 -selective dopamine receptor antagonists were identified by screening at 5 m M cation. Pseudo-noble-gas-configuration d-transition metals, such as zinc, exerted a complete inhibition of specific binding, whereas most other cations had little or no effect. The nature of zinc's actions was characterized by measuring the radioligand binding properties of [3 H]SCH-23390 and [3 H]methylspiperone to cloned D1A and D2L dopamine receptors in either the presence or absence of Zn2+ . Zinc exerts a low-affinity, dose-dependent, EDTA-reversible inhibition of the binding of subtype-specific antagonists primarily by decreasing the ligands' affinity for their receptors. The mechanism of zinc inhibition appears to be allosteric modulation of the dopamine receptor proteins because zinc increases the dissociation constant ( K D ) of ligand binding, Schild-type plots of zinc inhibition reach a plateau, and zinc accelerates antagonist dissociation rates. Here we demonstrate the effect of zinc on the binding of D1 - and D2 -selective antagonists to cloned dopamine receptors and show that the inhibition by zinc is through a dose-dependent, reversible, allosteric, two-state modulation of dopamine receptors. 相似文献
5.
Mária Wollemann Judit Farkas Géza Tóth Sándor Benyhe 《Journal of neurochemistry》1994,63(4):1460-1465
Abstract: A tritiated heptapeptide, [3 H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([3 H]Met-enkephalin-Arg6 -Phe7 ), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent K D value of the radioligand calculated from homologous displacement experiments was 3.4 n M , and the maximal number of specific binding sites was 630 fmol/mg of protein. The K D determined from equilibrium saturation binding studies was found to be 3.6 n M . However, the Hill coefficient was far below unity ( n H = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [3 H]Met-enkephalin-Arg6 -Phe7 binding reflects a κ2 - and/or δ-subtype specificity of the heptapeptide. Binding to a κ1 and/or μ site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg6 -Phe7 in frogs cannot be ruled out. The [3 H]Met-enkephalin-Arg6 -Phe7 binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide. 相似文献
6.
Benzodiazepine Treatment Causes Uncoupling of Recombinant GABAA Receptors Expressed in Stably Transfected Cells 总被引:1,自引:1,他引:0
Abstract: GABAA and benzodiazepine receptors are allosterically coupled, and occupation of either receptor site increases the affinity of the other. Chronic exposure of primary neuronal cultures to benzodiazepine agonists reduces these allosteric interactions. Neurons express multiple GABAA receptor subunits, and it has been suggested that uncoupling is due to changes in the subunit composition of the receptor. To determine if uncoupling could be observed with expression of defined subunits, mouse Ltk− cells stably transfected with GABAA receptors (bovine α1, β1, and γ2L subunits) were treated with flunitrazepam (Flu) or clonazepam. The increase in [3 H]Flu binding affinity caused by GABA (GABA shift or coupling) was significantly reduced in cells treated chronically with the benzodiazepines, whereas the K D and B max of [3 H]Flu binding were unaffected. The uncoupling caused by clonazepam treatment occurred rapidly with a t 1/2 of ∼30 min. The EC50 for clonazepam treatment was ∼0.3 µ M , and cotreatment with the benzodiazepine antagonist Ro 15-1788 (5.6 µ M ) prevented the effect of clonazepam. The uncoupling observed in this system was not accompanied by receptor internalization, is unlikely to be due to changes in receptor subunit composition, and probably represents posttranslational changes. The rapid regulation of allosteric coupling by benzodiazepine treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABAA and benzodiazepine receptors as well as benzodiazepine tolerance. 相似文献
7.
Douglas W. Bonhaus Eric Stefanich Dana N. Loury Sherry A. O. Hsu Richard M. Eglen Erik H. F. Wong 《Journal of neurochemistry》1995,65(1):104-110
Abstract: Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine3 (5-HT3 ) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT3 receptors. The affinities of competitive 5-HT3 receptor antagonists were similar regardless of whether the receptors were labeled with [3 H]RS-42358, [3 H]granisetron, or 1-( m -[3 H]chlorophenyl)biguanide ([3 H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [3 H]mCPG. The dissociation of [3 H]mCPG, [3 H]RS-42358, and [3 H]RS-25259, but not [3 H]granisetron, from both cloned and native 5-HT3 receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT3 receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor. 相似文献
8.
Characterization of a Human NK1 Tachykinin Receptor in the Astrocytoma Cell Line U 373 MG 总被引:1,自引:1,他引:0
E. Heuillet J. Ménager V. Fardin O. Flamand M. Bock C. Garret A. Crespo A. M. Fallourd A. Doble 《Journal of neurochemistry》1993,60(3):868-876
Abstract: The human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [3 H]substance P binding sites were present on cell homogenates, whereas [3 H]neurokinin A or [3 H]-senktide binding sites were absent. The binding was saturable and reversible. The binding of [3 H]substance P was inhibited by very low concentrations of [L-Pro9 ]substance P and [Sar9 ,Met(O2 )11 ]substance P; septide was ∼ 1,000-fold less potent. The most potent peptide antagonist was trans -4-hydroxy-1-(1 H -indol-3-ylcarbonyl)-L-prolyl- N -methyl- N -(phenylmethyl)-L-tyrosineamide. The rank order of potency for the nonpeptide antagonists was ( S , S )-CP 96,345 > (±)-CP 96,345 > (±)-2-chlorobenzylquinuclidinone > ( R , R )-CP 96,345 > RP 67580 > RP 68651. In [3 H]-inositol-labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK1 receptor agonists for stimulating inositol phosphate production and for inhibiting [3 H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK1 receptor. 相似文献
9.
B. Kenneth Koe Lorraine A. Lebel Carol B. Fox John E. Macor 《Journal of neurochemistry》1992,58(4):1268-1276
Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT1B receptor than the commonly used 5-HT1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [3 H]CP-96,501, was found to bind with a high affinity ( K D , 0.21 n M ) to a single binding site ( n H , 1.0). The receptor density of this site ( B max , 72 fmol/mg of protein) matched that of the 5-HT1B receptor determined with [3 H]5-HT. Competition curves of 16 serotonergic compounds in [3 H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT1B receptor determined with [3 H]5-HT or [125 I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [3 H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [3 H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [125 I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [3 H]CP-96,501 is a useful agonist radioligand for the 5-HT1B receptor. 相似文献
10.
Paulo F. Santos Maria S. Santos Arsélio P. Carvalho Carlos B. Duarte 《Journal of neurochemistry》1998,71(3):1086-1094
Abstract: We have investigated the effect of endogenous adenosine on the release of [3 H]acetylcholine ([3 H]ACh) in cultured chick amacrine-like neurons. The release of [3 H]ACh evoked by 50 m M KCl was mostly Ca2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist. The effect of adenosine on [3 H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca2+ channels. Ligand binding studies using [3 H]DPCPX confirmed the presence of adenosine A1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca2+ -dependent release of [3 H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A1 receptors. The effect of adenosine on the [3 H]ACh release may be due to a direct inhibition of N-type Ca2+ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins. 相似文献
11.
Identification of Residues in Transmembrane Regions III and VI that Contribute to the Ligand Binding Site of the Serotonin 5-HT6 Receptor 总被引:1,自引:1,他引:0
Frank G. Boess Frederick J. Monsma Jr. Andrew J. Sleight 《Journal of neurochemistry》1998,71(5):2169-2177
Abstract: We have examined the ligand binding site of the serotonin 5-HT6 receptor using site-directed mutagenesis. Replacing the highly conserved Asp106 in transmembrane region III by asparagine eliminated d -[3 H]lysergic acid diethylamide ([3 H]LSD) binding to the mutant receptor transiently expressed in HEK293 cells. The potency of 5-HT and LSD to stimulate adenylyl cyclase was reduced by 3,600- and 500-fold, respectively, suggesting that an ionic interaction between the positively charged amino group of 5-HT and D106 is essential for high-affinity binding and important for receptor activation. In addition, basal cyclic AMP levels in cells expressing this mutant were increased. Mutation of a tryptophan residue one helix turn toward the extracellular side of transmembrane region III (Trp102 ) to phenylalanine produced significant changes in the binding affinity and potency of several ligands, consistent with a role of this residue in the formation of the ligand binding site. The exchange of two neighboring residues in the carboxy-terminal half of transmembrane region VI (Ala287 and Asn288 ) for leucine and serine resulted in a mutant receptor with increased affinities (seven- to 30-fold) for sumatriptan and several ergopeptine ligands. The identification of these interactions will help to improve models of the 5-HT6 receptor ligand binding site. 相似文献
12.
Niels Jensen Martin Hasselblatt Anna-Leena Sirén Lothar Schilling †Martin Schmidt Hannelore Ehrenreich 《Journal of neurochemistry》1998,70(2):473-482
Abstract: Using a whole-cell binding procedure with long incubations at low temperature and subsequent acid stripping, we have characterized an atypical endothelin (ET) receptor in primary rat cortical astrocyte cultures. We found the following: (a) no competition for 125 I-ET-1 binding by the ETA antagonists BQ-123 and LU 135252 or the ETB agonist IRL 1620; (b) weak competition by the ETB antagonist BQ-788 and by the predominant ETB ligand ET-3; (c) potent synergistic competition of ETA and ETB ligands in combination for 125 I-ET-1 binding; (d) potent competition of ET-1 with any of the radioligands used, 125 I-ET-1, 125 I-IRL 1620, and [3 H]BQ-123; (e) lack of competition of IRL 1620 and BQ-123 with the respective other radioligand; (f) shifting of the amount of acid-strippable 125 I-ET-1 binding from 20 to 80% by ETB ligands and to 4% by ETA ligands; and (g) as a control, typical ETA and ETB binding characteristics of the RAT-1 fibroblast and the U373MG astrocytoma cell line, respectively, under our assay conditions. The unusual binding properties of astrocytic ET receptors described in this study appear to be the result of several binding sites in the receptor for different ET ligands or ligand epitopes. 相似文献
13.
Bertil B. Fredholm Karin Lindström Silvio Dionisotti Ennio Ongini 《Journal of neurochemistry》1998,70(3):1210-1216
Abstract: We have characterized the new potent and selective nonxanthine adenosine A2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A2A receptors ([3 H]CGS 21680) or A1 receptors ( N 6 -[3 H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A2A versus A1 receptors ( K i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [3 H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [3 H]SCH 58261 with the following estimated K i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N 6 -cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg2+ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [3 H]CGS 21680. 相似文献
14.
Characterization of Receptors for Cholecystokinin and Related Peptides in Mouse Cerebral Cortex 总被引:9,自引:4,他引:5
Abstract: The characteristics of cholecystokinin (CCK) binding to its receptors in a particulate membrane fraction of mouse cerebral cortex were studied by employing biologically active radioiodinated CCK prepared by conjugation with 125 I-Bolton-Hunter (125 I-BH) reagent. At 24°C binding was rapid, reversible, and linearly related to protein content. Binding was maximal at acidic pH (6.5) and reduced by the presence of monovalent cations. Under physiological conditions (pH 7.4, 118 mM-NaC1, 4.7 mM-KCl) Scatchard plots of CCK binding were linear with a K D value of 1.27 nM and binding capacity of 115 fmol/mg protein. Optimal binding required the presence of both Mg2+ and EGTA, and was inhibited by the addition of micromolar concentrations of Cu2+ (ID50 = 30 μM). The cortical receptor recognized all major forms of CCK, with an order of potency of cholecystokinin octapeptide (CCK8 ) > CCK > cholecystokinin tetrapeptide (CCK4 ). Desulfated cholecystokinin octapeptide (dCCK8 ) had a 10-fold lower affhity than CCK8 . Dibutyryl cyclic GMP, a potent competitive inhibitor of CCK binding to receptors in pancreas, was not a specific inhibitor of CCK binding to brain receptors. These present results support the concept that CCK may function as a regulatory peptide in brain, and that the cortical CCK receptor is different from the receptors mediating the peripheral action of CCK. 相似文献
15.
A. Cagnotto D. Crespi L. Mancini C. Manzoni M. L. Presti M. Gariboldi A. Vezzani T. Mennini 《Journal of neurochemistry》1998,70(2):850-857
Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT1A and 5-HT4 receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[3 H]hydroxy-2-(di- n -propylamino)tetralin ([3 H]8-OH-DPAT) to 5-HT1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT4 receptors, which colocalize with 5-HT1A receptors on hippocampal neurons, were not modified in kindled tissue. [3 H]5-HT uptake and its release as well as the 5-HT1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg−1 8-OH-DPAT or 1,000 µg kg−1 WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability. 相似文献
16.
Abstract: Some data on the concentration range of response and the concentration for half-response (EC50 ) of γ-aminobutyric acid (GABA) for the GABAA receptor are reviewed and compared. An analysis of the 36 CI− flux assay demonstrates that both the EC50 and the slope of a Hill plot depend on the ion influx or efflux assay time. The effects of depletion of the 36 CI− concentration gradient during the assay and of receptor desensitization on the result for a range of assay times are considered. The EC50 can be decreased by orders of magnitude by increasing the assay time. The EC50 measured in a finite time is less than the half-response concentration for the response(s) of the receptor. The extent of this difference depends on the receptor concentration per internal volume. The maximal decrease of EC50 depends on the rate of receptor desensitization. The computer simulations showed that a GABAA receptor with a half-response concentration of 100 μ M GABA can give 36 CI− flux measurements with an EC50 value 100-fold lower. 相似文献
17.
M1 Muscarinic Acetylcholine Receptor in Cultured Rat Neostriatum Regulates Phosphoinositide Hydrolysis 总被引:3,自引:3,他引:0
Abstract: : Muscarinic acetylcholine receptor expression and function in cultured rat neostriatal neurons were examined. All experiments were performed on intact neurons grown in vitro for 12-14 days. The muscarinic antagonist N-[3 H]methylscopolamine ([3 H]NMS) binds to a single site in cultures with a KD of 89 pM and a Bmax of 187 fmol/mg of protein, or 32,000 sites/neuron. Competition studies using [3 H]NMS were performed to determine what receptor sur > types were present. Nonlinear analysis of competition curves was best described with a single binding site for atropine, pirenzepine, and AF-DX 116 {11-[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one}, with Ki values of 0.6, 62, and 758 nM, respectively. These results indicate that the muscarinic receptors present in neostriatal cultures are of the M1 subtype, having high affinity for pirenzepine and low affinity for AF-DX 116. In contrast with antagonists, carbachol displaced [3 H]NMS from two sites with Ki values of 6.5 and 147 μM, with the higher-affinity form predominant (83% of sites). The M1 receptor subtype was linked to phosphoinositide turnover. Carbachol stimulated the formation of phosphoinositides with an EC50 of 37 μM and was antagonized by atropine. At equimolar doses, pirenzepine was more potent than AF-DX 116 at antagonizing the response. 相似文献
18.
Shigeru Hishinuma Akie Naiki Hirofumi Tsuga J. Michael Young 《Journal of neurochemistry》1998,71(6):2626-2633
Abstract: We investigated the regulation by intracellular Ca2+ of agonist-induced sequestration of Gq protein-coupled histamine H1 receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H1 receptors from the cell surface membrane was detected as the loss of [3 H]mepyramine binding sites on intact cells accessible to the hydrophilic H1 -receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [3 H]mepyramine were mirrored by changes in the subcellular distribution of H1 receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H1 receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca2+ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca2+ /calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H1 -receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of Gq protein-coupled receptors is transiently inhibited by Ca2+ /calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation. 相似文献
19.
Anissa Abi-Dargham Marc Laruelle David T. Wong David W. Robertson† Daniel R. Weinberger Joel E. Kleinman 《Journal of neurochemistry》1993,60(2):730-737
Abstract: Binding of [3 H]LY278584, which has been previously shown to label 5-hydroxytryptamine3 (5-HT3 ) receptors in rat cortex, was studied in human brain. Saturation experiments revealed a homogeneous population of saturable binding sites in amygdala ( K D = 3.08 ± 0.67 n M, B max = 11.86 ± 1.87 fmol/mg of protein) as well as in hippocampus, caudate, and putamen. Specific binding was also high in nucleus accumbens and entorhinal cortex. Specific binding was negligible in neocortical areas. Kinetic studies conducted in human hippocampus revealed a K on of 0.025 ± 0.009 n M −1 min−1 and a K off of 0.010 ± 0.002 min−1 . The kinetics of [3 H]LY278584 binding were similar in the caudate. Pharmacological characterization of [3 H]LY278584 specific binding in caudate and amygdala indicated the compound was binding to 5-HT3 receptors. We conclude that 5-HT3 receptors labeled by [3 H]LY278584 are present in both limbic and striatal areas in human brain, suggesting that 5-HT3 receptor antagonists may be able to influence the dopamine system in humans, similarly to their effects in rodent studies. 相似文献
20.
Janine M. Barnes Paul A. Murphy David Kirkhara Jeremy M. Henley 《Journal of neurochemistry》1993,61(5):1685-1691
Abstract— Recent reports have suggested that a major proportion of [3 H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [3 H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [3 H]kainate binding and a 50% reduction in the apparent B max values in both Mg2+ /Na+ and Mg2+ /Na+ -free buffer when assayed at 0°c. The pharmacology of [3 H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known me-tabotropic glutamate receptors, with neither 1 S ,3 R -amino-1,3-cyclopentanedicarboxylic acid (1 S ,3 R -ACPD) nor ibo-tenic acid being effective competitors. The molecular mass of the [3 H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP-γS also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[3 H]cyano-7-ni-troquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [3 H]kainate and 6-[3 H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation. 相似文献