1,720,971 research outputs found
The desensitization of the embryonic mouse muscle acetylcholine receptor depends on the cellular environment.
No full textThe rate of desensitization of nicotinic acetylcholine (ACh) receptor (nAChR), an important characteristic of nAChR function, was studied in myotubes of the mouse C2C12 cell line at different times after fusion, by measuring the decay of ACh-evoked currents (IACh) under various patch-clamp configurations. We observed a progressive slowing of IACh decay rate (half-decay time rose from about 0.5 s to over 5 s) in myotubes of increasing size (i.e. age) under all experimental conditions, except in outside-out patches, when IACh decayed as fast as in the smallest myotubes. Single-channel conductance (about 35 pS) and open time (about 3.5 ms), measured in outside-out and cell-attached patches, were independent of myotube size. In Xenopus oocytes injected with poly(A+)RNA extracted from C2C12 myoblasts or mature myotubes, IACh decay was about 50 times slower than in myotubes. Neither cAMP-dependent nor diacylglycerol-dependent protein kinases, actin nor microtubule polymerization state influenced IACh decay. Our data indicate that the cellular environment, but not readily dialysable cytosolic factors, markedly influences the functional behaviour of nAChR
TNF-ALPHA INCREASES THE FREQUENCY OF SPONTANEOUS MINIATURE SYNAPTIC CURRENTS IN CULTURED RAT HIPPOCAMPAL-NEURONS
No full textTumor necrosis factor-alpha (TNF-alpha) is a cytokine secreted by activated astrocytes and is known to alter evoked synaptic activity in slices of adult rat hippocampus. In this paper we show that TNF-alpha increases the frequency of spontaneous miniature synaptic currents in cultured hippocampal neurons, acting at nanomolar concentrations. In addition, we show that the mRNA for the 55 kDa TNF-alpha receptor (TNF-R1) is detected in embryonic rat hippocampal cultures, as well as in acutely dissected embryonic and adult rat hippocampi. Possible transduction pathways mediating the TNF-alpha effect are discussed
Protein kinase C modulates exogenous acetylcholine current in Xenopus oocytes.
No full textThe modulation of acetylcholine-activated current (IACh) by protein kinase C (PKC) was studied in Xenopus laevis oocytes microinjected with either mRNA extracted from C2C12 myotubes (C2C12 mRNA) or RNAs encoding murine alpha beta gamma delta subunits of the nicotinic ACh receptor (nAChR). Voltage-clamped oocytes were treated for 90 sec with 12-O-tetradecanoylphorbol-13-acetate (TPA, 300 nM), a potent PKC activator. Transient increase in the amplitude and acceleration in the decay of IACh were invariably observed within minutes of TPA application, and were independent of extracellular Ca2+ concentration. Both parameters recovered to control within 20-30 min; then a slight depression of IACh developed. By this time, an initial PKC down regulation was observed. At the peak of TPA-induced potentiation, dose-response relations suggested an increased binding affinity of nAChR for the neurotransmitter. 4 alpha-phorbol 12,13-didecanoate (300 nM), a biologically inactive analogue of TPA, did not affect IACh, while staurosporine (5-10 microM), a potent inhibitor of PKC activity, suppressed the action of TPA on IACh. In oocytes co-injected with C2C12 mRNA and with rat brain mRNA, IACh was potentiated by 5-hydroxy-tryptamine (10 microM), whose receptors are coupled to phosphoinositide hydrolysis. The nAChR-channel activity in cell-attached patches increased when TPA was applied to the oocytes. In 50% of the oocytes examined, a sustained depression of the single channel activity followed. We conclude that in Xenopus oocytes an endogenous PKC system regulates the function of embryonic-type muscle nAChRs
BLOCKAGE OF NICOTINIC ACETYLCHOLINE-RECEPTORS BY 5-HYDROXYTRYPTAMINE
No full textThe action of 5-hydroxytryptamine (5HT) on nicotinic acetylcholine receptor (nAChR) channels was investigated in mouse myotubes, human cloned TE671/RD cells, and Xenopus laevis oocytes. The decay of the ACh-activated whole-cell currents was reversibly accelerated in the presence of 5HT (10(-5) to 10(-3) M), in a dose-dependent manner. 5HT also reduced the size and accelerated the decay of currents elicited by ACh in Xenopus oocytes injected with mRNA extracted from C2 myotubes or Torpedo electroplaques, or oocytes injected with cloned mouse muscle AChR subunit mRNAs. The effect of 5HT was promptly reversed after washout, or by depolarizing the oocyte beyond -10 mV. In patch-clamp recordings from myotubes, bath-application of 5HT did not exert an indirect influence on the ACh-activated channels within the patch membrane. In contrast, when the patch membrane was exposed to 5HT (10(-6) M), ACh unit responses appeared as bursts of short pulses. It is concluded that the regulation of ACh responses by 5HT results from a fast noncompetitive blocking action of nAChR-channels. These results show that ligand-gated channels, activated by their specific neurotransmitter, may be regulated by a different neurotransmitter through a direct action on the receptor molecule
Acetylcholine Induces Voltage-Independent Increase of Cytosolic Calcium in Mouse Myotubes
No full textTWO FORMS OF ACETYLCHOLINE-RECEPTOR GAMMA-SUBUNIT IN MOUSE MUSCLE
No full textNicotinic acetylcholine receptors (nAcChoRs) of skeletal muscle are heterosubunit ligand-gated channels that mediate signal transmission from motor nerves to muscle. While cloning murine nAcChoR subunits, to gain an insight into the receptor diversity across species, we detected two forms of gamma subunits in the myogenic C2C12 cell line. Both forms are functional when expressed in Xenopus oocytes. One gamma subunit [long gamma (gamma 1)] was almost identical to that previously cloned in the murine BC3H-1 tumor cell line. The second form of gamma subunit [short gamma (gamma s)] lacked 156 bp (52 amino acids) in the extracellular N terminus, adjoining the hydrophobic segment M1, which corresponds to the fifth exon of the gamma-subunit gene. The two forms of gamma subunit coexist during myogenesis in vitro and in 17-day embryonic and denervated adult muscle fibers in vivo. However, the gamma s variant was the only form of gamma subunit in newborn muscle. In dissociated muscle fibers of newborn mice, AcCho-evoked channel openings were more prolonged when compared with C2C12 myotubes or denervated adult muscle fibers. The gamma s subunit may, thus, contribute to the structural and functional diversity of nAcChoRs in muscle cells
Identification of a determinant of acetylcholine receptor gating kinetics in the extracellular portion of the gamma subunit
No full textA large body of structure-function studies has identified many of the functional motifs underlying ion permeation through acetylcholine receptor (AChR) channels. The structural basis of channel gating kinetics is, however, incompletely understood. We have previously identified a novel shorter form of the AChR gamma subunit, which lacks the 52 amino acids within the extracellular amino-terminal half, encoded by exon 5. To define the contribution of the missing domain to AChR channel function, we have transiently coexpressed the mouse short gamma subunit [gamma(s)] with alpha, beta and delta subunits in human cells and recorded single-channel currents from the resulting AChRs. Our findings show that replacement of the gamma by the gamma(s) subunit confers a long duration characteristic to AChR channel openings without altering unitary conductance sizes or receptor affinity for the transmitter. We also show that alpha beta gamma(s) delta AChR channels exhibit a peculiar voltage sensitivity characterized by a short opening duration when the membrane potential is hyperpolarized. Together, these findings indicate that the domain in the extracellular amino-terminal half of the gamma subunit that encompasses a conserved disulphide loop and a critical tyrosine residue implicated in receptor oligomerization and insertion at the cell surface is a functional motif that also modulates AChR channel gating kinetics. The results also provide a molecular explanation of the functional diversity exhibited by skeletal muscle AChRs during development
Selective over-expression of mRNA coding for stress protein 90 K Da in human ovarian cancer.
No full textCXC chemokines interleukin-8 (IL-8) and growth-related gene product a (GROα) modulate Purkinje neuron activity in mouse cerebellum
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