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1.
Abstract: Quantitative autoradiography was used to compare the binding properties of α7-type nicotinic acetylcholine receptors in fetal and adult rat hippocampus. Whereas there were high levels of 125I-α-bungarotoxin (125I-α-BTX) binding throughout fetal hippocampal field CA1, there was a significant decrease in binding site density in the adult. The affinity of 125I-α-BTX binding, as well as α-cobratoxin and nicotine potency to displace 125I-α-BTX, did not change with age. Addition of Ca2+ to the assay buffer did not alter 125I-α-BTX binding, or α-cobratoxin inhibition of 125I-α-BTX binding, although it significantly increased nicotine affinity at both ages. The effect of Ca2+ on agonist affinity was dose-dependent, with an EC50 value of 0.25–0.5 m M . Ca2+ also significantly increased the cooperativity of nicotine displacement curves in stratum oriens of the adult, but not in the fetus. These findings indicate that the properties of hippocampal 125I-α-BTX binding sites are largely similar across age. Ca2+ selectively enhances the affinity of agonist binding, with no change in antagonist binding. This ionic effect may result from potentiation of agonist binding to a desensitized state of the α7 nicotinic acetylcholine receptor and may represent an important neuroprotective mechanism.  相似文献   

2.
All known nicotinic receptor α subunits include a conserved disulfide bond that is essential for function and is a site for labeling via biochemical modification. In an effort to develop a universal ligand for all subtypes of nicotinic receptors, we previously studied the effects of arsenylation with two compounds, ρ-aminophenyldichloroarsine (APA) and bromoacetyl-ρ-aminophenylarsenòxide (BAPA) on nicotinic receptors from Torpedo electroplax. Here we apply these reagents to immunoisolated receptors containing α4, β2, and possibly other subunits from chick brain that bind [3H]cytisine with high affinity (KD∼5 nM). These are distinct from another receptor subtype that also binds [3H]cytisine and [3H]nicotine and can be arsenylated with APA, but instead contains α5,β2, and probably other subunits. Reduction of α4 β2 receptors with dithiothreitol blocked [3H]cytisine binding and this effect was reversed upon reoxidation by dithiobisnitrobenzoic acid. APA or BAPA prevented the dithiobisnitrobenzoic acid reactivation of dithiothreitol-treated receptors with IC50 values of 15 and 70 n M , respectively. However, the antiarsenical dimercaptopropanesulfonic acid restored function to APA- or BAPA- "arsenylated" receptors (EC50∼100 μ M ). APA-treated receptors remained blocked for up to 24 h, but treatment with dimercaptopropanesulfonic acid at any time restored [3H]cytisine binding. APA treatment of reduced receptors protected against irreversible alkylation by Bromoacetyl choline, indicating that arsenylation occurs at least in part in the agonist binding site. Thus, these reagents have similar effects on different nicotinic receptor subtypes from both muscle and nerves.  相似文献   

3.
Abstract: The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick α4β2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the K i values for binding inhibition and EC50 values for agonist-induced α4β2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing α3, α5, β2, and β4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.  相似文献   

4.
Abstract  Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A–C present in α subunits together with loops D–F present in either non-α subunits or homomer-forming α subunits. Although Y151 in loop B has been identified as important in agonist binding, various residues at the 151-site are found among vertebrate and invertebrate nAChR α subunits, such as F151. In Xenopus oocytes expressing Nlα1 or Nlα1Y151F plus rat β2, Y151F mutation was found to significantly change the rate of receptor desensitization and altered the pharmacological properties of acetylcholine, but not imidacloprid, including the decrease of I max, the increase of EC50 (the concentration causing 50% of the maximum response) and the fast time-constant of decay (τf). By comparisons of residue structure, the hydroxyl group in the side chain of Y151 was thought to be important in the interaction between Nlα1/β2 nAChRs and acetylcholine, and the phenyl group to be important between Nlα1/β2 nAChRs and imidacloprid.  相似文献   

5.
6.
Abstract: The effect of dopamine (DA) receptor stimulation on the distribution of γ protein kinase C (γPKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic γPKC (56%) without altering membrane γPKC levels, resulting in decreased total γPKC immunoreactivity. The maximal decrease in cytosolic γPKC occurred at 20 min of incubation and was significantly blocked by the D1 DA antagonist SCH 23390 (10−6 M ) but not by the D2 antagonist sulpiride (10−5 M ). The D1 agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 µ M and 1 n M , respectively. The D2 agonist quinpirole had no effect on γPKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D1-like receptor. DA had no effect on α, δ, or ζPKC isozyme immunoreactivity in the same hippocampal preparations. The DA-induced decrease in cytosolic γPKC immunoreactivity was blocked by the Ca2+-dependent protease inhibitor N -acetyl-Leu-Leu-norleucinal (100 µ M ) and by the inorganic Ca2+ channel blocker Co2+. The data suggest that DA stimulates a D1-like DA receptor, which increases the influx of Ca2+ and activates the Ca2+-dependent proteolysis of γPKC.  相似文献   

7.
Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. Previously, we have identified a nAChR point mutation (Y151S) associated with insecticide resistance in the brown planthopper Nilaparvata lugens . Although this mutation has been identified in two different N. lugens nAChR subunits (Nlα1 and Nlα3) because of difficulties in heterologous expression of Nlα3; its influence on agonist potency has been examined only in Nlα1-containing nAChRs. Here we describe the cloning of a novel nAChR subunit from N. lugens (Nlα8), together with evidence for its co-assembly with Nlα3 in native and recombinant nAChRs. This has, for the first time, enabled the functional effects of the Nlα3Y151S mutation to be examined. The Nlα3Y151S mutation has little effect on agonist potency of acetylcholine but has a dramatic effect on neonicotinoid insecticides (reducing I max values and increasing EC50 values). The apparent affinity of neonicotinoids was higher and the effect of the Y151S mutation on neonicotinoid agonist potency was more profound in Nlα3-containing, rather than Nlα1-containing nAChR. We conclude that Nlα3- and Nlα1-containing nAChRs may be representative of two distinct insect nAChR populations.  相似文献   

8.
Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [32P]Pi-prelabeled rat brain cortical and hippocampal slices. The accumulation of [32P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC50 18 µ M ) was inhibited by the H1 receptor antagonist mepyramine with a K i constant of 0.7 n M and was resistant to H2 and H3 receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC50 44 n M ) was not blocked by BQ-123, a specific antagonist of the ETA receptor. Endothelin-3 and the specific ETB receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC50 values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H1 and ETB receptors, respectively.  相似文献   

9.
Abstract: The effects of extracellular calcium on functional properties of nicotinic receptors from mouse thalamus were investigated. Previous studies have reported that calcium modulates the function of several neuronal nicotinic receptors. A 86Rb+ ion efflux assay was developed to measure nicotinic receptor function from brain tissue, and data indicate that α4β2 receptors may mediate this response. Using the 86Rb+ efflux assay, calcium effects on receptor activation, desensitization induced by high, activating and low, subactivating concentrations of agonist, and recovery from desensitization were examined. Effects of calcium on the kinetics of ligand binding were also investigated. Calcium modulated receptor activation by increasing the maximal response to nicotine in a concentration-dependent manner, without affecting the EC50 of nicotine. Barium, but not magnesium, mimicked the effects of calcium on receptor activation. The increase in receptor activation could not be explained by changes in the ratio of activatable to desensitized receptors as assessed by the kinetics of ligand binding. Desensitization following activation was unaffected by calcium. Calcium, barium, and magnesium, however, increased the potency of nicotine for desensitization induced by exposure to low, subactivating concentrations of nicotine. Recovery from desensitization was not modulated by calcium. These data suggest that calcium modulates various functional aspects of nicotinic receptors from mouse brain and may do so via different mechanisms.  相似文献   

10.
11.
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 36CI 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 36CI 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 36CI flux measurements with an EC50 value 100-fold lower.  相似文献   

12.
α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [125I]-α-bungarotoxin from human α7 nAChRs in the cell line GH4C1 (IC50 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC50 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K d and IC50 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [125I]-ImII(W10Y) revealed specific binding ( K d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC50 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [125I]-ImII(W10Y) only at higher concentrations (IC50≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.  相似文献   

13.
Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro-β-erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro-β-erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro-β-erythroidine by ∼120-fold. IC50 values for blocking α4β4 and α3β4, responding to EC20 concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µ M , respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro-β-erythroidine sensitivity to be distributed throughout the N-terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro-β-erythroidine sensitivity by 4.3-fold.  相似文献   

14.
Abstract: This study examined γ-aminobutyric acidA (GABAA) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the α1 and β2/3 subunit protein expression and [3H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced ( p < 0.05) the maximal GABA-stimulated increase in extracellular acidification rate ( E max) (16.5 ± 1.2% and 11.3 ± 1.0%, 2-day control and treated cells, respectively; 17.4 ± 1.0% and 9.9 ± 0.7%, 7-day control and treated cells, respectively; best-fit E max± SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC50) (1.2 ± 1.7 and 2.3 ± 1.8 µ M , 2-day control and treated cells, respectively; 1.7 ± 1.5 and 1.5 ± 1.5 µ M , 7-day control and treated cells, respectively; best-fit EC50± SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABAA receptor α1 and β2/3 subunit proteins over the initial 2 days, but did not alter [3H]muscimol binding compared with vehicle-treated cells. The results suggest that changes in GABAA receptor subunit protein expression, rather than loss of [3H]muscimol binding sites, underlie the chronic flunitrazepam-mediated desensitisation of GABAA receptor function.  相似文献   

15.
Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca2+-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC50 = 50.1 µ M ) and catecholamines (EC50 = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH2 inhibited carbachol-induced 22Na+ influx via nicotinic receptors (IC50 = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced 45Ca2+ influx via voltage-dependent Ca2+ channels (IC50 = 1.0 µ M ) and catecholamine secretion (IC50 = 1.6 µ M ). It did not alter high K+-induced 45Ca2+ influx via voltage-dependent Ca2+ channels or veratridine-induced 22Na+ influx via voltage-dependent Na+ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca2+-dependent exocytosis in response to nicotinic receptor stimulation.  相似文献   

16.
Disruption of neuronal signaling by soluble β-amyloid has been implicated in deficits in short-term recall in the early stages of Alzheimer's disease. One potential target for β-amyloid is the synapse, with evidence for differential interaction with both pre- and post-synaptic elements. Our previous work revealed an agonist-like action of soluble β-amyloid (pM to nM) on isolated pre-synaptic terminals to increase [Ca2+]i, with apparent involvement of pre-synaptic nicotinic receptors. To directly establish the role of nicotinic receptors in pre-synaptic Ca2+ regulation, we investigated the pre-synaptic action of β-amyloid on terminals isolated from mice harboring either β2 or α7 nicotinic receptor null mutants (knockouts). Average pre-synaptic responses to β-amyloid in hippocampal terminals of α7 knockout mice were unchanged, whereas responses in hippocampal terminals from β2 knockout mice were strongly attenuated. In contrast, pre-synaptic responses to soluble β-amyloid were strongly attenuated in cortical terminals from α7 knockout mice but were moderately attenuated in cortical terminals from β2 knockout mice. The latter responses, having distinct kinetics, were completely blocked by α-bungarotoxin. The use of receptor null mutants thus permitted direct demonstration of the involvement of specific nicotinic receptors in pre-synaptic Ca2+ regulation by soluble β-amyloid, and also indicated differential neuromodulation by β-amyloid of synapses in hippocampus and cortex.  相似文献   

17.
Abstract: The δ-opioid receptor is known to regulate multiple effectors in various tissues. When expressed in human embryonic kidney 293 cells, the cloned δ-opioid receptor inhibited cyclic AMP (cAMP) accumulation in response to the δ-selective agonist [ d -Pen2, d -Pen5]enkephalin. The inhibitory response of [ d -Pen2, d -Pen5]enkephalin was dependent on the expression of the δ-opioid receptor and exhibited an EC50 of 1 n M . The receptor showed ligand selectivity and a pharmacological profile that is appropriate for the δ-opioid subtype. The inhibition was blocked by the opiate antagonist naloxone or by pretreatment of the cells with pertussis toxin. Cotransfection of the δ-opioid receptor with type II adenylyl cyclase and an activated mutant of αs converted the δ-opioid signal from inhibition to stimulation of cAMP accumulation. It is interesting that when transfected into Ltk fibroblasts, the cloned δ-opioid receptor was able to stimulate the formation of inositol phosphates (EC50 = 8 n M ). This response was sensitive to pertussis toxin. The opioid-mediated formation of inositol phosphates exhibited the same ligand selectivity as seen with the inhibition of cAMP accumulation. The ability of the δ-opioid receptor to couple to G proteins other than Gi was also examined. Cotransfection studies revealed that the δ-opioid receptor can utilize Gz to regulate cAMP accumulation and to stimulate the formation of inositol phosphates.  相似文献   

18.
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 [3H]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 [3H]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   

19.
Abstract: Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 n M for α4β2 and 500 n M for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ-32P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA-mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.  相似文献   

20.
Abstract: Four catalytic inhibitors of GABA aminotransferase (gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate) as well as aminooxyacetic acid and valproate were studied for effects on neurochemical assays for GABA synthesis, receptor binding, uptake and metabolism in mouse and rat brain preparations. Gabaculine did not interfere with GABA synthesis as reflected by the activity of glutamate decarboxylase (GAD), it was only a weak inhibitor (IC50= 0.94 mM) of GABA receptor binding sites but was a moderately potent inhibitor of GABA uptake (IC50= 81 μM) and very potent (IC50= 1.8 μM) with respect to inhibition of the GABA-metabolizing enzyme GABA aminotransferase (GABA-T). γ-Acetylenic GABA was a weak inhibitor of GAD and GABA binding (IC50 > 1 mM), but virtually equipotent to inhibit uptake and metabolism of GABA (IC50 560 and 150 μM, respectively). This was very similar to γ-vinyl GABA, except that this drug did not decrease GAD activity. Ethanolamine O -sulphate was found to show virtually no inhibition of GAD and GABA uptake, but was a fairly potent inhibitor of GABA binding (IC50= 67 μM) and in this respect, 500 times more potent than as an inhibitor of GABA-T. Aminooxyacetic acid was a powerful inhibitor of both GAD and GABA-T (IC50 14 and 2.7 μM, respectively), but had very little affinity to receptor and uptake sites for GABA. Valproate showed no effects on GABA neurochemical assays which could be related to anticonvulsant action. The present results suggest that the anticonvulsant properties of the four catalytic inhibitors of GABA-T tested are at least in part mediated through a direct influence on GABA receptors and uptake sites.  相似文献   

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