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1.
Calmodulin (CaM) regulation of cholinergic muscarinic receptor was investigated using synaptic membrane isolated from rat brains and [3H]-QNB as a binding ligand. CaM exerts a biphasic effect on receptor binding showing both a Ca2+-dependent receptor loss and an increase depending on the state of membrane phosphorylation. Calcineurin, a CaM-dependent protein phosphatase, mimicked the stimulatory effect of CaM in a dose-dependent manner. CaM-antagonists, W-7 and TFP reversed the stimulatory effect by CaM. A mechanism of protein phosphorylation and dephosphorylation of the cholinergic muscarinic receptors regulated by CaM-Ca2+ was proposed. 相似文献
2.
Oligomeric potential of the M2 muscarinic cholinergic receptor 总被引:1,自引:0,他引:1
G protein-coupled receptors are known to exist as oligomers. Although such aggregates often are referred to as dimers, there is little direct evidence regarding their oligomeric size. In the present investigation, c-Myc-, FLAG-, and influenza hemagglutinin (HA)-tagged forms of the M2 muscarinic receptor have been coexpressed in Sf9 cells to probe for aggregates larger than a dimer. Immunochromatography, immunoprecipitation, and immunoblotting were carried out with various combinations of antibodies directed against the different epitopes to demonstrate that all three tagged forms of the receptor can be immunopurified within a single complex. Extracts of the M2 muscarinic receptor from Sf9 cells therefore contain aggregates that are at least trimeric, and the levels detected point to the existence of larger complexes. The data also suggest that the oligomers coexist with a sizeable population of monomers. 相似文献
3.
Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system. Specific [3H](-)-quinuclidinyl benzilate binding to the reconstituted receptor was found to be saturable with a Kd (dissociation constant) equal to 48 +/- 4 pM and a Bmax (maximal density of binding sites) equal to 50 +/- 5 fmol/mg of protein. Competitive binding studies indicated that the reconstituted receptors showed stereoselectivity and drug specificity consistent with a high-affinity muscarinic receptor. Agonist binding to the reconstituted receptor was decreased by the addition of guanyl-5'-yl imidodiphosphate. Sixty per cent of the reconstituted receptors were found to be integral membrane proteins. The molecular weight of the reconstituted receptor as determined by sodium dodecyl sulfate-gel electrophoresis was 76,000 +/- 2,000 and was identical to the molecular weight of the muscarinic receptor in the original cardiac membranes. The data indicate that a partially functional, intact muscarinic receptor was reconstituted into human erythrocyte acceptor membranes and that membrane constituents may be required to stabilize the receptor in a high-affinity state for antagonists. 相似文献
4.
Gallamine interacts with an allosteric site on muscarinic acetylcholine receptor complexes in rat brain membranes, thereby slowing the dissociation of a radiolabelled ligand ([3H]N-methylscopolamine) from the receptor complex. This effect involves the elimination of the fast component of the biphasic dissociation curve. The allosteric effect of gallamine is equally prominent in membranes containing predominantly Ml (cerebral cortex) and M2 (brainstem) subtypes of muscarinic receptor. Gallamine's action is not affected by a variety of treatments which influence the conformational state of the receptor as reflected by agonist binding affinity, including treatments with heat,N-ethymaleimide and trypsin. A guanine nucleotide (5-guanylylimidodiphosphate), however, moderates the effects of gallamine on muscarinic receptors in brainstem, but not in cortical, membranes. 相似文献
5.
《Neurochemistry international》1986,9(2):337-347
Transection of the fimbria/fornix, producing a 75% reduction in the activity of the cholinergic marker choline-o-acetyltransferase (CAT EC. 2.3.1.6) in rat hippocampus, did not change the binding characteristics of the non-subtype selective, muscarinic cholinergic receptor antagonist ligand [3H](−)quinuclidinyl benzilate {[3H](−)QNB}. Pirenzepine competition for [3H](−)QNB binding in the hippocampus was best described by a computer derived model assuming two binding sites of high affinity (putative M1 receptors) and low affinity (putative M2 receptors). There was no change in the proportion of high and low affinity pirenzepine binding sites in the hippocampus following cholinergic deafferentation. Thus, these data provide no evidence for a discrete localization of either putative subtype of muscarinic receptor discriminated by pirenzepine restricted to the terminals of CAT containing neurons innervating the rat hippocampus.Chronic scopolamine treatment produced a 48% increase in the Bmax of [3H](−)QNB binding in the hippocampus, but again there was no change in the proportions of the sites discriminated by pirenzepine demonstrating that both putative subtypes were regulated identically. Similarly, carbachol competition for [3H](−)QNB was unaltered following cholinergic deafferentation or chronic scopolamine treatment. Furthermore, similar guanylyl-5′-imidodiphosphate [Gpp(NH)p] modulation of the proportions of high and low affinity carbachol binding sites was found in the hippocampus following transection of the fimbria/fornix or chronic scopolamine treatment. Thus there is no adaptation of receptor-effector coupling following these treatments that is reflected by changes in receptor recognition site characteristics.Carbachol competition for [3H]pirenzepine binding to putative M1 receptors in the hippocampus was biphasic and was also modulated by Gpp(NH)p. In the brainstem, there was a homogeneous population of putative M2 [3H](−)QNB binding sites having low affinity for pirenzepine. Carbachol competition for these binding sites was also biphasic and modulated by guanine nucleotides. Thus, both putative M1 and M2 muscarinic receptors, as defined by high or low affinity for pirenzepine respectively, may mediate their effects in rat brain via a guanine nucleotide regulatory subunit. 相似文献
6.
Garofolo MC Seidler FJ Auman JT Slotkin TA 《American journal of physiology. Regulatory, integrative and comparative physiology》2002,282(5):R1356-R1363
Imbalances of beta-adrenoceptor (beta-AR) and muscarinic ACh receptor (mAChR) input are thought to underlie perinatal cardiovascular abnormalities in conditions such as sudden infant death syndrome. Administration of isoproterenol, a beta(1)/beta(2)-AR agonist, to neonatal rats on postnatal days (PN) 2-5 caused downregulation of cardiac m(2)AChRs and a corresponding decrement in their control of adenylyl cyclase activity. Terbutaline, a beta(2)-selective agonist that crosses the placenta and the blood-brain barrier, was also effective when given either on PN 2-5 or during gestational days 17-20. Terbutaline failed to downregulate brain m(2)AChRs, even though it downregulated beta-ARs; beta-ARs and m(2)AChRs are located on different cell populations in the brain, but they are on the same cells in the heart. Destruction of catecholaminergic neurons with neonatal 6-hydroxydopamine upregulated cardiac but not brain m(2)AChRs. These results suggest that perinatal beta-AR stimulation shifts cardiac receptor production away from the generation of m(2)AChRs so that the development of sympathetic innervation acts as a negative modulator of cholinergic function. Accordingly, tocolytic therapy with beta-AR agonists may compromise the perinatal balance of adrenergic and cholinergic inputs. 相似文献
7.
Sustained agonist stimulation induces an asymmetric down-regulation of brain muscarinic acetylcholine receptor (mAChR): 43±2% in the right and 26±2% in the left cerebral hemisphere, respectively (Ref. 1). In order to determine the possible involvement of endogenous diacylglycerols produced under muscarinic stimulation in the down-regulation phenomenon, here we have studied the effects of synthetic diacylglycerols and a phorbol ester on cells dissociated from rat cerebral cortex. Oleoylacetylglycerol decreased the amount of cell-surface mAChR by 37±2% and 25±2% in right and left cerebral cortex, respectively. Long-term treatment with phorbol dibutyrate also produced internalization of the mAChR (25±1.5% and 33±2% in right and left cortical cells, respectively). These changes occurred without modification of the Kdapp for the selective antagonist pirenzepine. The action of calcium ions was also studied using incubation of cells with the ionophore A23187. No changes were observed in the amount of mAChR detected at the plasma membrane with the ionophore alone, but when used in combination with phorbol dibutyrate and the agonist carbamylcholine a sinergistic decrease in mAChR was apparent. It is concluded that long-term exposure to exogenously added diacyglycerols and phorbol ester significantly reduces the amount of mAChR detected at the plasma membrane and abolishes the asymmetry of the down-regulation phenomenon observed under specific muscarinic stimulation, suggesting that diacylglycerols may be one of the factors responsible for such asymmetry.Abbreviations used A23187
ionophore A23187
- ATRO
atropine
- CARB
carbamoylcholine
- DAG
diacylglycerol
- DMEM
Dulbecco's modified Eagle's medium
- DMSO
dimethylsulfoxide
- HEPES
4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer
- PZ
pirenzepine
- LCC
left cerebral cortex
- mAChR
muscarinic acetylcholine receptor
- OAG
oleoylacetylglycerol
- PDB
phorbol dibutyrate
- RCC
right cerebral cortex 相似文献
8.
9.
《Comparative biochemistry and physiology. C: Comparative pharmacology》1992,101(2):269-272
1. Sphingosine inhibited the binding of [3H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells.2. The inhibition of [3H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites.3. The effect of Sphingosine on the binding was not affected by the chelation of extracellular Ca2+.4. H-7, an inhibitor of protein kinase C, failed to decrease [3H]QNB binding.5. Stearylamine, an analogue of Sphingosine, was as effective as Sphingosine in inhibiting [3H]QNB binding.6. These results suggest that Sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca2+ and protein kinase C. 相似文献
10.
Phosphorylation of chick heart muscarinic cholinergic receptors by the beta-adrenergic receptor kinase 总被引:10,自引:0,他引:10
Previous studies have demonstrated that muscarinic cholinergic receptors (mAChR) become markedly phosphorylated when intact cardiac cells are stimulated with a muscarinic agonist. This process appears to be related to the process of receptor desensitization. However, the mechanism of agonist-induced phosphorylation of mAChR is not known. In situ phosphorylation studies suggested that agonist-induced phosphorylation of mAChR may involve the participation of a receptor-specific kinase and/or require agonist occupancy. These observations regarding phosphorylation and desensitization of mAChR are similar to observations made for beta-adrenergic receptors. Recent studies have indicated that homologous desensitization of beta-adrenergic receptors may be due to the phosphorylation of these receptors by a novel protein kinase that only recognizes the agonist-occupied form of the receptors. As muscarinic receptors are structurally homologous to beta-adrenergic receptors, we have initiated studies to identify the protein kinase responsible for the phosphorylation of muscarinic receptors by determining whether the chick heart muscarinic receptor would serve as a substrate for the beta-adrenergic receptor kinase (beta-AR kinase). We report that the purified and reconstituted chick heart muscarinic receptor serves as an excellent substrate in vitro for the beta-AR kinase. Phosphorylation of mAChR receptors by the beta-AR kinase was only observed in the presence of a muscarinic receptor agonist and was prevented in the presence of antagonist. Both the extent of phosphorylation (3-4 mol of P/mol of receptor) and the phosphoamino acid composition of the mAChR after incubation in vitro with beta-AR kinase were similar to the characteristics of agonist-induced phosphorylation of mAChR in situ.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Tricyclic antidepressants (TADs) were administered (10 mg/kg/day, i.p.) to mice for 2 or 4 weeks. Tolerance to the antimuscarinic effects of these agents was demonstrated by comparing their ability to supress oxotremorine-induced tremors in treated and in control animals. ED50's increased nearly three-fold after four weeks of treatment. CNS muscarinic acetylcholine receptor binding was also examined after 2 to 7 weeks of treatment by measurement of 3H-quinuclidinyl benzilate (QNB) binding. No change was found in either density or affinity of these receptors. The development of tolerance to the antimuscarinic effects of TADs is not due to alteration of either the number or the conformation of central muscarinic receptors. Evidence is presented that this phenomenon may instead be the result of an unidentified mechanism by which the post-synaptic effect of a single receptor-agonist interaction is magnified. 相似文献
12.
蜜蜂复杂的社会行为受到了研究者的广泛关注,了解蜜蜂的学习、记忆、导航、信息传递等行为的神经分子基础,可对探索人类自身的脑科学和复杂社会行为的分子基础提供比较研究信息.本文综述了多巴胺的作用机制及其在蜜蜂行为中的作用,详细介绍了蜜蜂脑部的多巴胺受体,总结了蜜蜂脑部多巴胺水平的影响因子等,最后对进一步研究多巴胺神经通路对蜜蜂行为的作用及机制的前景做一展望. 相似文献
13.
The M(2) muscarinic receptor inhibits the release of acetylcholine from cholinergic fibers in the lungs and elsewhere. In airway parasympathetic neurons, M(2) receptor expression is decreased by viral infections and by interferon-gamma, increasing actylcholine release. Dexamethasone increases M(2) receptor expression, decreasing acetylcholine release. We carried out 5' rapid amplification of cDNA ends beginning with mRNA from human heart and IMR32 human neuroblastoma cells. This demonstrated a 5' UTR of 100 BP, corresponding to two sequences on chromosome 7, separated by a 22.6 kB intron. The splice acceptor site is at -45 relative to the initiating atg. The 3000 BP upstream of 5' RACE product were subcloned into a pGL3 luciferase reporter vector. Deletional constructs were expressed in IMR32 cells. These demonstrated that 412 BP provided full expression of the reporter gene, and suggested a repressor element between -1848 and -1510. 相似文献
14.
15.
The relative muscarinic anticholinergic actions of phenothiazines and related drugs are thought to regulate the propensity of these agents to elicit extrapyramidal side effects, especially those resembling the symptoms of Parkinson's disease. Pimozide, which closely resembles the butyrophenones in its chemical structure and its potent and selective dopamine receptor blockade, differs from the butyrophenones in its relatively low incidence of extrapyramidal side effects. In assays of the binding of 3H-quinuclidinyl benzilate (QNB) to muscarinic sites, pimozide displays a high affinity for these cholinergic receptors, similar to drugs, such as thioridizine and clozapine, which also have a low incidence of extrapyramidal side effects. This observation supports the notion that muscarinic anticholinergic actions can ameliorate the propensity of a drug to elicit extrapyramidal effects. The structure-activity relationships of chlorpromazine metabolites in binding to muscarinic sites in the brain parallels some of their structure-activity relationships as neuroleptic agents. 7-Hydroxychlorpromazine, which has been proposed as an antischizophrenic drug, binds to the muscarinic receptor with a potency similar to that of chlorpromazine itself, suggesting that the incidence of extrapyramidal side effects of 7-hydroxychlorpromazine might be similar to those of chlorpromazine. 相似文献
16.
Children seem more susceptible to increased airway reactivity than adults. Such an age-dependent discrepancy in airway reactivity may involve different airway smooth muscle functions. Therefore, we compared the in vivo and in vitro responsiveness of airway smooth muscles between two age groups of animals. Rats of 6 and 21 weeks old were challenged in vivo with acetylcholine (ACh) infused intravenously and airway resistance (R(aw)) was measured. Tracheal muscle was also isolated and the isometric force developed to ACh or KCl was measured. Furthermore, the level of genes encoding muscarinic receptor subtypes (M(1-3)) and acetylcholinesterase (AChE) expressed in the tracheal muscle was determined by RT-PCR. In results, the basal R(aw) was similar in the two age groups. The R(aw) at each ACh dose was significantly greater in young rats than older rats (p<0.05, n=22-27). Tracheal muscles from young rats were more sensitive to ACh than older rats (p<0.05, n=20-21), while receptor-independent muscle contraction to KCl was greater in older rats (p<0.05, n=10-19). Genes encoding AChE, M(2) and M(3) muscarinic receptors were more highly expressed in the tracheal muscles from young than older rats (p<0.05, n=4-6). In conclusion, airway smooth muscle in young rat is more sensitive to cholinergic stimulation in vivo and in vitro compared to older rats, which may be due to a higher expression of M(2) and M(3) muscarinic receptors in airway smooth muscle. 相似文献
17.
In vitro binding affinities of chlorpromazine, fluphenazine, levomepromazine, perphenazine and some of their metabolites for dopamine D2 receptors, alpha 1- and alpha 2 adrenoceptors in rat brain were previously reported from our laboratories. The present study reports the in vitro binding affinities of the same compounds for muscarinic cholinergic receptors and for histamine H1 receptors in rat brain, using 3H-quinuclidinyl benzilate and 3H-mepyramine as radioligands. Chlorpromazine, levomepromazine, and their metabolites had 5-30 times higher binding affinities for muscarinic cholinergic receptors than fluphenazine, perphenazine and their metabolites. Levomepromazine was the most potent and fluphenazine the least potent of the four drugs in histamine H1 receptor binding. 7-Hydroxy levomepromazine, 3-hydroxy levomepromazine and 7-hydroxy fluphenazine had only 10% of the potency of the parent drug in histamine H1 receptor binding, while the 7-hydroxy-metabolites of chlorpromazine and perphenazine had about 75% of the potency of the parent drug in this binding system. Their histamine H1 receptor binding affinities indicate that metabolites may contribute to the sedative effects of chlorpromazine and levomepromazine. 相似文献
18.
19.
P Govitrapong P Phansuwan-Pujito M Ebadi 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1989,94(1):159-164
1. Using the tritiated muscarinic receptor antagonist, quinuclidinyl benzilate ([3H]QNB) as a ligand, muscarinic cholinergic receptors have been identified and characterized in the pineal glands of cow and swamp buffalo. 2. At 25 degrees C, the specific binding reached equilibrium within 60 min and remained constant for an additional two hours. Furthermore, the specific binding was saturable, reversible and tissue dependent in nature. 3. The kinetic analyses of muscarinic cholinergic receptor sites revealed KD values of 0.423 +/- 0.01 nM and 0.218 +/- 0.01 nM, and Bmax values of 69.75 +/- 20.91 fmol/mg protein and 74.19 +/- 32.73 fmol/mg protein for the cow's- and the swamp buffalo's pineal glands, respectively. 4. The presence of muscarinic cholinergic receptor sites originating from cholinergic innervation of the pineal gland is suggested. 相似文献
20.
The chronic effects of antidepressant treatment on striatal dopaminergic (DA) and muscarinic cholinergic (mACh) receptors of the rat brain have been examined comparatively in this study using 3H- spiroperidol (3H-SPD) and 3H-quinuclidinyl benzilate (3H-QNB) as the respective radioactive ligands. Imipramine and desipramine were used as prototype antidepressants. Although a single administration of imipramine or desipramine did not affect each receptor sensitivity, chronic treatment with each drug caused a supersensitivity of mACh receptor subsequent to DA receptor subsensitivity. Furthermore, it has been suggested that anti-mACh properties of imipramine or desipramine may not necessarily be related to the manifestation of mACh receptor supersensitivity and that sustained DA receptor subsensitivity may play some role in the alterations of mACh receptor sensitivity. 相似文献