1,721,094 research outputs found
Functional changes of fetal muscle acetylcholine receptor during mouse development
No full textIn developing muscles in vivo and in vitro, the acetylcholine receptor gamma-subunit exists in two splice variants, conferring different single-channel open durations (tau(op)) to reconstituted receptors. In mouse muscles, tau(op) changes around birth possibly as receptors incorporate either variant of gamma-subunit. This might be relevant to the concomitant maturation of muscle innervation
Inhibition of GABA and Glycine Responses by Glutamate in Rat Hippocampal Neurons
No full textCurrents elicited by activation of GABAA, glycine (GLY) and glutamate (GLU) receptors (R) in pyramidal neurons of CA1 region from thin slices of rat hippocampus were studied using the tight-seal whole-cell recording techniques. GLU (100 mM) induced a long-lasting depression of GABA- and GLY-activated currents (IGABA and IGLY) when using standard saline in conjunction with depolarization. The long-lasting depression was not observed: (1) in neurons held at -70 mV during GLU application; (2) in neurons depolarized by current injection but not exposed to GLU; (3) when GLU/depolarization protocol was performed in Ca(2+)-free medium; or (4) by using recording patch-pipettes filled with a medium that tightly controlled cytosolic Ca2+ transients. Sphingosine (10 mM), staurosporine (1 mM) and the specific inhibitor of protein kinase C (PKC(19-36) (200 mM in the patch-pipette solution), blocked the long-lasting depression of IGABA. IGABA was depressed even when the treatment with GLU was performed before patch-clamping the neuron. We conclude that the sustained IGABA and IGLY depression is mediated by cytosolic events triggered by the activation of GLUR
REDUCED ACETYLCHOLINE-INDUCED CHANNEL ACTIVITY IN DYSTROPHIC MOUSE MYOTUBES
No full textSingle channel recording patch-clamp technique was used in the mouse to compare the acetylcholine (ACh)-induced channel behaviour between normal and dystrophic myotubes. While open time and slope conductance were equivalent, ACh-induced channel opening frequency was more than 4-fold reduced in dystrophic compared to normal myotubes. In addition, the steady-state phosphorylation of the ACh receptor (AChR), tested by immunoprecipitation of 32P-labeled cells, indicated that the α-subunit was more heavily phosphorylated in the dystrophic myotubes. We propose that the degree of α-subunit phosphorylation of the AChR, which parallels the reduced AChR-channel opening probability, determines desensitization of the AChR in dystrophic myotubes
Ca2+ permeability of human heteromeric nAChRs expressed by transfection in human cells
No full textThe Ca2+ permeability of the human heteromeric 34, 42 and 44 neuronal nicotinic acetylcholine receptors (nAChRs) was estimated by measuring the fractional Ca2+ current (Pf) flowing through the ligand-activated receptor-channels. Simultaneous recordings of transmembrane currents and fluorescence transients, using the whole-cell patch-clamp technique combined with fura-2 fluorescence microscopy, were performed in transiently transfected human cells. The human 42 nAChR showed a Pf value of 2.6%, while the human 34 nAChR showed a similar Pf value
of 2.7%. Conversely, 44 nAChR exhibited a Pf value (1.5%) significantly smaller than those of both 42 and 34 nAChRs. In test experiments performed in HEK 293 cells stably expressing rat GluR1AMPA receptor subunit,we repeated the determination of Pf, whose value (3.2%) has previously been reported by others using the same fluorescent dye; and we found a very similar Pf value (3.5%). In further test experiments, we found that Pf values of chick 34 (4.4%) and 44 (2.1%) matched those previously reported by us using confocal fluorescence microscopy. Thus, our findings
are consistent with those elsewhere reported even using different experimental procedures, giving a strong support to the following sequence of Ca2+ permeability: h-34 > h-42 > h-44
CA2+ SIGNALING PATHWAYS ACTIVATED BY ACETYLCHOLINE IN MOUSE C2C12 MYOTUBES
No full textIn mouse C2C12 myotubes acetylcholine (ACh) elevates the concentration of myoplasmic Ca2+ ([Ca2+]i) by inducing Ca2+ influx through transmitter-gated and voltage-gated channels, and by mobilizing Ca2+ from internal stores. The relative contribution of each of these ACh-activated sources to the global [Ca2+]i elevation was estimated. We found that Ca2+ entry through voltage- and ACh-gated channels accounts for roughly 80% of the total [Ca2+]i increment, while mobilization from internal caffeine-sensitive and inositoltrisphosphate- (InsP3-) sensitive stores contributes the remaining 20% to the maximal [Ca2+]i increment. Furthermore, we found that ACh-induced mobilization from InsP3-sensitive stores also develops in embryonic chick myotubes. The differential importance of the Ca2+ signalling pathways activated by ACh during myogenesis is discussed
Nicotine modulates the spontaneous synaptic activity in cultured embryonic rat spinal cord Interneurons
No full textThe nicotine-induced modulation of the synaptic activity was studied in cultured spinal cord neurons fromembryonic rats, using the patch-clamp technique, alone or in combination with Ca2 imaging. Morphologically, neurons could be divided into two populations: multipolar nerve cells and bipolar, spindle-shaped neurons. Neurons were predominantly GABAergic, with 70% of bipolar cells and 60%of multipolar cells positive for GABA immunostaining. Nicotine (Nic) did not affect the activity of the spontaneous postsynaptic current (sPSC) in multipolar neurons, whereas bipolar cells responded to Nic applications with an enhancement of both inhibitory and excitatory synaptic activity (threefold for 100 MNic). No change in the mean event amplitude was observed. The increase of sPSC frequency was detectable at 1–10 M Nic, and was prevented by dihydro--erythroidine (DHE) but not by -bungarotoxin. Choline, a selective 7-nAChR agonist, did not mimic the Nic action. Simultaneous treatment with inhibitors of ionotropic glutamate receptors, CNQX (20 M) and AP5 (20 M), completely blocked the excitatory sPSC activity but did not prevent the Nic-induced enhancement of inhibitory sPSCactivity. Tetrodotoxin (1 M) reduced the basal spontaneous activity but did not block the Nic-induced effects on bipolar neurons. In a subset of bipolar neurons (12%) exposed to AP5 and CNQX, Nic activated DHE-sensitive inward currents, associated with an elevation of cytosolic Ca2 ([Ca2]i). Our results provide the first evidence of modulation of both excitatory and inhibitory neurotransmitter release in embryonic spinal cord interneurons by non- 7-containing nicotinic receptors
Interferon-alpha, beta and tumor necrosis factor-alpha enhance the frequency of miniature end-plate potentials at rat neuromuscular junction.
No full textThe effects of the two cytokines, rat interferon-alpha, and human tumor necrosis factor-alpha, were studied at the rat neuromuscular junction by using classical electrophysiological techniques. Both cytokines in a similar way at concentrations of 2,000 and 35,000 U/ml, respectively, increased transiently and with a relatively long delay (15 to 25 min) the frequency of mimiature endplate potentials. The observed effects may be related to complex second messenger mechanisms and contribute to modulation and plasticity of neurotransmission
Inhibition of nicotinic acetylcholine receptors by bicuculline
No full textA study was made on the effects of bicuculline, the classical γ-aminobutyric acid-A receptor antagonist, on heteromeric mouse muscle αβγδ, heteromeric neuronal rat α2β4 and α4β2 and homomeric human α7 nicotinic acetylcholine receptors (nAChRs), expressed in Xenopus oocytes. Bicuculline reduced the ACh-induced currents in a rapid and reversible way, with IC50 values of 34±1.5 μM for mouse muscle αβγδ and 12.4±0.7 and 18±1 μM for rat neuronal α2β4 and α4β2 nAChRs, respectively. Therefore, the three types of heteromeric receptors are inhibited by bicuculline but the neuronal α2β4 and α4β2 receptors were more sensitive than the muscle αβγδ receptor. The Hill coefficients for ACh-current inhibition were close to one for all types of receptors, suggesting a single site of action for bicuculline inhibition of nAChRs. Bicuculline shifted the ACh-dose-current response curve to the right and the maximal current was reduced, a reduction that for the heteromeric receptors was not overcome by high concentrations of ACh. The effect of bicuculline was examined at different membrane potentials, and the ACh-current-membrane potential relationships obtained indicate that the inhibition by bicuculline is voltage-dependent for muscle αβγδ and neuronal α2β4 and α4β2 nAChRs. All these results are consistent with the notion that bicuculline blocks the heteromeric muscle and neuronal nAChRs in a non-competitive way. Studies were also made on the wild type (wt α7) and mutant leu-to-threo (L248T) homomeric human neuronal α7-nAChRs. In sharp contrast to the heteromeric ACh receptors examined, bicuculline blocked in a competitive way the homomeric wt α7-nAChRs, as evidenced by a parallel shift of the bicuculline dose-ACh-current inhibition on raising the ACh concentration. Moreover, similar to the effects of serotonin on wt and mutant α7 ACh receptors, the mutation converted bicuculline from an antagonist into a competitive agonist. All this suggests that bicuculline may serve as a lead molecule to design new anticholinergic substances. © 2001 Published by Elsevier Science Ltd
Ca2+ permeability through rat cloned alpha9-containing nicotinic acetylcholine receptors.
No full textWe investigated the functional properties of rat 9 and 910 nicotinic acetylcholine receptors (nAChRs) expressed by transient transfectionin the rat GH4C1 cell line, using both Ca2+ imaging and whole-cell recording. Acute applications of ACh generated short-delay fast-rising and quick-decaying Ca2+ transients, suppressed in Ca2+-free medium and invariably accompanied by the activation of whole-cell inward currents. The mean amplitude of ACh-induced currents was as small as −16 pA in 9 subunit cDNA-transfected GH4C1 cells (9-GH4C1), while they were much larger (range:−150 to−300 pA) in 910 subunit cDNAs-transfected GH4C1 cells (910-GH4C1). Currents were not activated
by nicotine, were blocked by methyllycaconitine and were ACh concentration-dependent. Because the Ca2+ permeability of 9-containing nAChRs has been estimated in immortalized cochlear UB/OC-2 mouse cells, we also characterized the ACh-induced responses in these cells. Unlike 9- and 910-GH4C1 cells, UB/OC-2 cells responded to ACh with both long-delay methyllycaconitine-insensitive whole-cell currents and long-lasting Ca2+ transients, the latter being detected in the absence of Ca2+ in the extracellular medium and being suppressed by the Ca2+-ATPase inhibitor thapsigargin, known to deplete IP3-sensitive stores. These results indicated the involvement of muscarinic nAChRs
and the lack of functionalACh-gated receptor channels in UB/OC-2 cells. Thus, we measured the fractional Ca2+ current (Pf, i.e. the percentage of total current carried by Ca2+ ions) in 910-GH4C1, obtaining a Pf value of 22±4%; this is the largest value estimated to date for a ligand-gated receptor channel. The physiological role played by Ca2+ entry through 9-containing nAChRs gated by ACh is discussed
Acetylcholine receptor channel properties in rat myotubes exposed to forskolin
No full textRat myotubes exposed to forskolin were studied by patch-clamp technique in cell-attached single channel recording configuration. Channel open time and opening frequency of the main class of acetylcholine receptor- (AChR-) channels (accounting for more than 90% of all unitary events) decreased in the presence of forskolin (20-100 microM). The forskolin-induced action on the AChR function fully developed with a delay of 30-60 minutes from the peak of cytosolic cyclic AMP (cAMPi) concentration. Both cAMP (1 mM), applied intracellularly for 10 min, and dibutyryl cAMP (0.5 mM), applied extracellularly for 90 min, did not accelerate the rate of desensitization of myotubes studied in whole-cell patch-clamp recording configuration. It was concluded that the action of forskolin on AChR-channel function of rat myotubes could be not associated with the cAMPi-dependent phosphorylation of AChR
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