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1.
Development of acetylcholine sensitivity during myogenesis 总被引:23,自引:0,他引:23
The development of acetylcholine (ACh) sensitivity during myogenesis has been studied by iontophoretic application of ACh and intracellular recording from myogenic cells from rat forelimbs cultured in vitro. The fine structure of the cells was then examined by electron microscopy. The development of ACh sensitivity is correlated with the appearance of thick and thin filaments and precedes myofibril formation. All myotubes are sensitive to ACh. Myogenic cells arising by cell division in vitro can become sensitive to ACh and construct myofibrils without cell fusion. When cell fusion is inhibited by calcium ion deficiency or when cell division is blocked by FUdR, many mononucleate, striated, ACh-sensitive cells appear in culture. While ACh sensitivity appears at the onset of muscle differentiation, ACh receptors seem to play no role in the early events of myogenesis, as evidenced by the failure of receptor block or of desensitization to interfere with myogenesis. The concurrent appearance of myofilaments and ACh sensitivity is discussed in relation to the early events and control mechanisms of myogenesis. 相似文献
2.
Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses 总被引:45,自引:31,他引:14
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The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft. 相似文献
3.
Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity. 相似文献
4.
Abstract: K+ -evoked acetyl[3 H]choline ([3 H]ACh) release was inhibited in a concentration-dependent manner by apomorphine and the D2 agonist quinpirole in striatal slices prepared from euthyroid and hypothyroid rats. However, there was a significant increase in the maximum inhibition observed with both agonists in the hypothyroid compared with the euthyroid group, which paralleled the increased D2 agonist sensitivity reported for stereotyped behavior. The D2 antagonist raclopride decreased, and the D, antagonist SCH 23390 increased, the inhibition of [3 H]ACh release by apomorphine, confirming an inhibitory role for D2 receptors and an opposing role for D1 receptors. Because there is no difference in D1 or D2 receptor concentration between the euthyroid and hypothyroid groups, it is suggested that thyroid hormone modulation of D2 receptor sensitivity affects a receptor-mediated event. Following intrastriatal injection of pertussis toxin (PTX), apomorphine no longer inhibited [3 H]ACh release. In fact, increased [3 H]- ACh release was observed, an effect reduced by SCH 23390, providing evidence that D1 receptors enhance [3 H]- ACh release, and confirming that a PTX-sensitive G protein mediates the D2 response. As it has been reported that thyroid hormones modulate G protein expression, this mechanism may underlie their effect on dopamine agonist- mediated inhibition of ACh. 相似文献
5.
Acetylcholine sensitivity of innervated and noninnervated Xenopus muscle cells in culture 总被引:3,自引:0,他引:3
The chemosensitivity of Xenopus muscle cells grown in culture to iontophoretically applied acetylcholine (ACh) in the presence or absence of neurons was examined. Muscle cells grown without nerve cells are sensitive to ACh over their entire surface (2.4 mV/pC) with occasional spots of high chemosensitivity (“hot spots”). In cultures containing neural tube cells, the ACh sensitivity of muscle cells increased by approximately 50% regardless of the presence of nerve contacts or functional synapses. A similar increase in the ACh sensitivity was observed in muscle cells cultured in medium conditioned by neural tube cells. The ACh sensitivity of the extrajunctional region in functionally innervated muscle cells was not different from that of noninnervated cells growing in the same cultures. However, the chemosensitivity at the junctional region was about fivefold higher than that of the extrajunctional area. This increase in junctional chemosensitivity may well account for the increase in miniature endplate potential amplitude which has previously been reported to occur during nerve-induced ACh receptor accumulation. 相似文献
6.
Using electrophysiological and quantitative autoradiographic techniques, we studied the kinetics of acetylcholine (ACh) receptor production and incorporation into membranes of muscle fibers developing in culture. These studies were performed by utilizing 125I-labeled α-Bungarotoxin (α-BGT) which binds irreversibly to ACh receptors. α-BGT binding to ACh-sensitive muscle cells in culture correlates well with the level of ACh sensitivity. α-BGT binds to myotubes with two different apparent rates. The slow component of binding is due to the incorporation of new receptors into the membrane at a rate of 90 receptors/μm2 per hour. However, the ACh receptor density increases at a rate of only 35 receptors/μm2 per hour as the result of a concurrent increase in cell surface area. The α-BGT-receptor complexes turn over slowly and the rate of receptor incorporation is not affected by the presence of α-BGT. Inhibition of protein synthesis with cycloheximide depresses receptor incorporation, the percent inhibition increasing with time in cycloheximide. Overnight treatment in actinomycin D has no effect, but inhibition of ATP synthesis with dinitrophenol and iodoacetate or incubation in the cold inhibits the appearance of new ACh receptors. 相似文献
7.
Shui Z Khan IA Haga T Benovic JL Boyett MR 《The Journal of biological chemistry》2001,276(15):11691-11697
Control of the cardiac muscarinic K(+) current (i(K,ACh)) by beta-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K(+) channel, receptor kinase (GRK2), and beta-arrestin 2, desensitization of i(K,ACh) during a 3-min application of 10 micrometer ACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The effect of beta-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of i(K,ACh) in cells transfected with beta-arrestin 2 was significantly slowed (time constant increased from 34 to 232 s). Activation and deactivation of i(K,ACh) on application and wash-off of ACh in cells transfected with beta-arrestin 2 were significantly slowed from 0.9 to 3.1 s (time to half peak i(K,ACh)) and from 6.2 to 13.8 s (time to half-deactivation), respectively. In cells transfected with a constitutively active beta-arrestin 2 mutant, desensitization occurred in the absence of agonist (peak current significantly decreased from 0.4 +/- 0.05 to 0.1 +/- 0.01 nA). We conclude that beta-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K(+) channel. 相似文献
8.
Summary The effects of a water-soluble carbodiimide were examined at the frog neuromuscular junction. Acetylcholine sensitivity was measured using a fluid electrode technique and intracellular recording of miniature end-plate potentials. The carbodiimide blocked synaptic sensitivity by a reversible, curare-like action. Irreversible blockade was also observed, probably due to covalent binding. The conditions of reaction and irreversibility suggest that several different residues may be attacked. The inability of cholinergic antagonists to protect the receptor from attack indicates that nonspecific sites, and not the acetylcholine binding site, are involved. 相似文献
9.
Zanetti E Giaroni C Vanti A Canciani L Giuliani D Lecchini S Frigo G 《Life sciences》2003,73(20):2641-2654
Supersensitivity to muscarinic, kappa- and mu-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals.These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, kappa- and mu-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas kappa- and mu-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed. 相似文献
10.
Experimental evidence has been published from isolated guinea pig muscle in vitro, and from direct ligand binding to receptors from T. californica, indicating that two agonist ions react with the nicotinic receptor by exchanging for one magnesium ion. It is the basis of the ion exchange receptor pair model, in which two acetylcholine ions exchange for one magnesium ion in contact with and between a pair of negatively charged receptor groups about 4 A apart. In the resting state the electrostatic attraction between the negatively charged receptor groups and the Mg2+ ion exerts a binding force. This binding force is opposed by the quantum mechanical repulsions of the electron clouds of the charged groups and ions in contact, together with the mutual repulsion of the pair of receptor oxyanions. When the Mg2+ ion is replaced by two acetylcholine ions the quaternary heads of the latter are positioned so that they form two mutually repelling ACh+ receptor group dipoles. As the Mg2+ ion leaves, its rehydration energy contributes to the sum of the electron cloud repulsions and the ACh+ receptor group dipole repulsions, causing the receptor groups to be forced apart activating the receptor macromolecule. The subsequent decrease in ACh+ concentration results in the reestablishment of the resting state. The coulombic electrostatic energy, the Born repulsion energy, the London attraction energy and the oxyanion ACh+ dipole repulsion energies have been calculated and shown to be consistent with the model. The displacement of the Mg2+ by two ACh+ ions makes several hundred kcals of energy available for receptor group separation and receptor activation. 相似文献
11.
Zwart R van Kleef RG Gotti C Smulders CJ Vijverberg HP 《Journal of neurochemistry》2000,75(6):2492-2500
The effects of the acetylcholinesterase inhibitors physostigmine and tacrine on alpha4beta2 and alpha4beta4 subtypes of neuronal nicotinic acetylcholine (ACh) receptors, expressed in Xenopus laevis oocytes, have been investigated. In voltage-clamp experiments low concentrations of physostigmine and tacrine potentiate ion currents induced by low concentrations of ACh, whereas at high concentrations they inhibit ACh-induced ion currents. These dual effects result in bell-shaped concentration-effect curves. Physostigmine and tacrine, by themselves, do not act as nicotinic receptor againsts. The larger potentiation is observed with 10 microM: physostigmine on alpha4beta4 nicotinic receptors and amounts to 70% at 1 microM: ACh. The mechanism underlying the effects of physostigmine on alpha4beta4 ACh receptors has been investigated in detail. Potentiation of ACh-induced ion current by low concentrations of physostigmine is surmounted at elevated concentrations of ACh, indicating that this is a competitive effect. Conversely, inhibition of ACh-induced ion current by high concentrations of physostigmine is not surmounted at high concentrations of ACh, and this effect appears mainly due to noncompetitive, voltage-dependent ion channel block. Radioligand binding experiments demonstrating displacement of the nicotinic receptor agonist (125)I-epibatidine from its recognition sites on alpha4beta4 ACh receptors by physostigmine confirm that physostigmine is a competitive ligand at these receptors. A two-site equilibrium receptor occupation model, combined with noncompetitive ion channel block, accounts for the dual effects of physostigmine and tacrine on ACh-induced ion currents. It is concluded that these acetylcholinesterase-inhibiting drugs interact with the ACh recognition sites and are coagonists of ACh on alpha4-containing nicotinic ACh receptors. 相似文献
12.
Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 microm at -70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs. 相似文献
13.
Acetylcholine Receptors : Distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity 总被引:28,自引:2,他引:26
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Using 125iodine-labeled α-bungarotoxin (α-BGT-125I) and quantitative radioautography, we have studied the time-course of the change in acetylcholine (ACh) receptor distribution and density occurring in rat diaphragm after denervation. In innervated fibers, ACh receptors are localized at the neuromuscular junction and the extrajunctional receptor density is less than five receptors per square micrometer. The extrajunctional receptor density begins to increase between 2 and 3 days after denervation and increases approximately linearly to 1695 receptors/µm2 at 14 days, subsequently decreasing to 529 receptors/µm2 at 45 days. We have isolated plasma membranes from rat leg muscles at various times after denervation and find that the change in concentration of ACh receptors in the membranes measured by α-BGT-125I binding and scintillation counting follows a time-course similar to the change in ACh receptor density measured radioautographically. Furthermore, we have correlated extrajunctional ACh receptor density measured by radioautography with extrajunctional ACh sensitivity measured by iontophoretic application of ACh and intracellular recording and find that the log of ACh receptor density is related to 0.53 times the log of ACh sensitivity. These results are discussed in terms of the electrophysiological experiments on the ACh receptor and the recent, more biochemical approaches to the study of ACh receptor control and function. 相似文献
14.
Yamanushi TT Shui Z Leach RN Dobrzynski H Claydon TW Boyett MR 《American journal of physiology. Heart and circulatory physiology》2007,292(4):H1737-H1746
In the heart, ACh activates the ACh-activated K(+) current (I(K,ACh)) via the M(2) muscarinic receptor. The relationship between desensitization of I(K,ACh) and internalization of the M(2) receptor has been studied in rat atrial cells. On application of the stable muscarinic agonist carbachol for 2 h, I(K,ACh) declined by approximately 62% with time constants of 1.5 and 26.9 min, whereas approximately 83% of the M(2) receptor was internalized from the cell membrane with time constants of 2.9 and 51.6 min. Transfection of the cells with beta-adrenergic receptor kinase 1 (G protein-receptor kinase 2) and beta-arrestin 2 significantly increased I(K,ACh) desensitization and M(2) receptor internalization during a 3-min application of agonist. Internalized M(2) receptor in cells exposed to carbachol for 2 h was colocalized with clathrin and not caveolin. It is concluded that a G protein-receptor kinase 2- and beta-arrestin 2-dependent internalization of the M(2) receptor into clathrin-coated vesicles could play a major role in I(K,ACh) desensitization. 相似文献
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The forewing stretch receptor (SR) neuron makes monosynaptic connections with wing depressor motoneruons; in this article the pharmacology of its output onto the first baslar motoneuron (BA1) has been investigated. The SR, like other insect afferents that have been studied so far, appears to be cholinergic; transmission was suppressed reversibly by the nicotinic antagonist gallamine (10?4M) and irreversibly by α-bungarotoxin (10?6 M). The choline reuptake blocker hemicholinium-3 (10?4 M) also caused a reversible reduction in the amplitude of SR excitatory postsynaptic potentials (EPSPs) recorded in BA1. The receptor subtype nonselective muscarinic antagonists atropine (10?4 M), scopolamine (10?4 M), and quinuclidinyl benzilate (10?5 M), unlike nicotinic antagonists, caused an augmentation in EPSP amplitude. This effect does not appear to be caused by an increase in sensitivity of the motoneuron to acetylcholine (ACh), since atropine produced a marked reduction rather than an increase in the amplitude of responses to ACh pressure applied to the soma of BA1. Scopolamine only caused a modest reduction in the amplitude of ACh somatic responses. The simplest explanation for these observations is that muscarinic antagonists bring about an increase in EPSP amplitude by blockade of presynaptic autoreceptors that normally down-regulate the release of ACh from SR terminals. The effects of muscarinic receptor subtype-selective antagonists indicate that presynaptic receptors in this preparation may have a pharmacological profile more similar to that of vertebrate M2 receptors than to that of M1 or M2 subtypes. The functional significance of autoreceptors in this preparation are discussed. © 1995 John Wiley & Sons, Inc. 相似文献
18.
乙酰胆碱对大鼠体外抗体生成的影响 总被引:1,自引:1,他引:1
目的:观察不同浓度乙酰胆碱(ACh,10-10~10-5mol/L)对大鼠体外抗体生成的影响,并初步探讨其作用机制,从细胞水平了解乙酰胆碱与免疫功能之间的关系。方法:用体外抗体生成的检测方法,用绵羊红细胞(SRBC)刺激大鼠肠系膜淋巴结B细胞转化成抗体形成细胞(AFC),然后检测其抗体生成量。结果:①10-10~10-7mol/LACh能显著抑制体外抗体生成,其中10-8和10-7mol/LACh的作用较强,而10-6和10-5mol/LACh无明显的抑制作用;②M型胆碱能受体激动剂毛果芸香碱(10-8和10-7mol/L)能明显减弱体外抗体生成,而N型受体激动剂烟碱(10-8和10-7mol/L)没有显著的减弱作用,M型受体拮抗剂阿托品(10-7和10-6mol/L)可完全阻断ACh抑制体外抗体生成的作用;③ACh分别在B细胞用SRBC刺激后3~48h中的6个不同时间与淋巴细胞作用,其抗体生成仍然是减少的。结论:ACh可非浓度依赖性地抑制大鼠的体外抗体生成;此作用可能由B细胞上的M型胆碱能受体介导;且ACh可能主要影响B细胞转化的后期过程。 相似文献
19.
Tamás Rőszer Tamás Józsa A. József Szentmiklósi Gáspár Bánfalvi 《Cell and tissue research》2009,336(2):325-335
Acetylcholine (ACh) is one of the main signals regulating nitric oxide synthase (NOS) expression and nitric oxide (NO) biosynthesis
in mammals. However, few comparative studies have been performed on the role of ACh on NOS activity in non-mammalian animals.
We have therefore studied the cholinergic control of NOS in the snail Helix pomatia and compared the effects of ACh on NO synthesis in the enteric nervous system of the snail and rat. Analyses by the NADPH-diaphorase
reaction, immunocytochemistry, purification with ion-exchange chromatography, Western-blot, and quantitative polymerase chain
reaction have revealed the expression of neuronal NOS in the rat intestine and of a 60-kDa subunit of NOS in the enteric nerve
plexus of H. pomatia. In H. pomatia, quantification of the NO-derived nitrite ions has established that NO formation is confined to the NOS-containing midintestine.
Nitrite production can be elevated by L-arginine but inhibited by Nω-nitro-L-arginine. In rats, ACh moderately elevates nitrite production, whereas ACh, the nicotinic receptor agonists (nicotine,
acetyl thiocholine iodide, metacholine) and the cholinesterase inhibitor eserine reduce enteric nitrite formation in snails.
The nicotinic receptor antagonist tubocurarine also provokes nitrite liberation, whereas the muscarinic receptor agonists
or antagonists have no significant effect in snails. In the presence of EDTA or tetrodotoxin, ACh fails to inhibit nitrite
production. In pharmacological studies, we have found that ACh contracts the midintestinal muscles and, in snails, simultaneously
reduces the antagonistic muscle relaxant effect of L-arginine. Our experiments provide the first evidence for an inhibitory
regulation of neuronal NO synthesis by ACh in an invertebrate species.
This article is dedicated to Dr. Gábor Hollósi on the 50th anniversary of his graduation and being a teacher at the University
of Debrecen. 相似文献
20.
IAA induced a diurnal change in floral response to chillingof Lemna gibba G3 under continuous light, as has been reportedfor ACh. This IAA effect was antagonized by an antiauxin, T-CA.NAA and 2,4-D had no influence on the temperature sensitivityof the duckweed. ACh-like reagents were examined with respectto the sensitivity to their antagonists. Atropine, an ACh-specificantagonist, inhibited the action of IAA. The IAA treatment,however, decreased the ACh content of the plant by ca. 30%.Thus, IAA and ACh are surmised to attack the same or similaraction site(s) independently. (Received July 19, 1978; ) 相似文献