The Journal of neuroscience : the official journal of the Society for Neuroscience
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We previously identified a novel, nonselective cation channel in native reactive (type R1) astrocytes (NR1As) from injured rat brain that is regulated by cytoplasmic Ca2+ and ATP (NC(Ca-ATP)) and exhibits sensitivity to block by adenine nucleotides similar to that of sulfonylurea receptor type 1 (SUR1). Here we show that SUR1 is involved in regulation of this channel. NR1As within the site of injury and after isolation exhibited specific binding of FITC-tagged glibenclamide and were immunolabeled with anti-SUR1 antibody, but not with anti-SUR2, anti-Kir6.1 or anti-Kir6.2 antibodies, indicating absence of ATP-sensitive K+ (KATP) channels. ⋯ Several properties previously associated exclusively with SUR1-regulated KATP channels were observed in patch-clamp experiments using Cs+ as the charge carrier: (1) the sulfonylureas, glibenclamide and tolbutamide, inhibited NCCa-ATP channels with EC50 values of 48 nm and 16.1 microm, respectively; (2) inhibition by sulfonylureas was lost after exposure of the intracellular face to trypsin or anti-SUR1 antibody; (3) channel inhibition was caused by a change in kinetics of channel closing, with no change in channel amplitude or open-channel dwell times; and (4) the SUR activator ("KATP channel opener"), diazoxide, activated the NCCa-ATP channel, whereas pinacidil and cromakalin did not. Also, glibenclamide prevented cell blebbing after ATP depletion, whereas blebbing was produced by exposure to diazoxide. Our data indicate that SUR1 is functionally coupled to the pore-forming portion of the NC(Ca-ATP) channel, providing the first demonstration of promiscuity of SUR1 outside of the K+ inward rectifier family of channels.
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The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and activation of p53 precedes apoptosis in many cell types. Controversial reports exist on the role of the transcription factor nuclear factor-kappaB (NF-kappaB) in p53-mediated apoptosis, depending on the cell type and experimental conditions. Therefore, we sought to elucidate the role of NF-kappaB in p53-mediated neuron death. ⋯ It is concluded that a balanced competitive interaction of p53 and NF-kappaB with the transcriptional cofactor p300 exists in neurons. Exposure of neurons to lethal stress activates p53 and disrupts NF-kappaB binding to p300, thereby blocking NF-kappaB-mediated survival signaling. Inhibitors of p53 provide pronounced neuroprotective effects because they block p53-mediated induction of cell death and concomitantly enhance NF-kappaB-induced survival signaling.
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Opioid-receptor activation in cell lines results in phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), which contributes to agonist-induced desensitization of adenylate cyclase signaling. In this study, morphine-induced MAPK modulation was examined in the mouse brain using antibodies against phosphorylated MAPK. Thirty minutes after systemic morphine, MAPK modulation was observed in brain areas associated with analgesia and reward. ⋯ Additionally, a pronounced instatement of morphine-induced MAPK activation was observed in CA3 hippocampal processes. This instatement was observed during expression of tolerance; however, it was not significant during sensitization. In summary, these results provide distinct, region-specific mechanisms for morphine-induced MAPK modulation in the mouse brain and give insight into the brain circuitry involved in acute and adaptive opioid behaviors.
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Nerve injury-induced afferent discharge is thought to elicit spinal sensitization and consequent abnormal pain. Experimental neuropathic pain, however, also depends on central changes, including descending facilitation arising from the rostral ventromedial medulla (RVM) and upregulation of spinal dynorphin. A possible intersection of these influences at the spinal level was explored by measuring evoked, excitatory transmitter release in tissues taken from nerve-injured animals with or without previous manipulation of descending modulatory systems. ⋯ These data demonstrate exaggerated release of excitatory transmitter from primary afferents after injury to peripheral nerves, supporting the likely importance of increased afferent input as a driving force of neuropathic pain. The data also show that modulatory influences of descending facilitation are required for enhanced evoked transmitter release after nerve injury. Thus, convergence of descending modulation, spinal plasticity, and afferent drive in the nerve-injured state reveals a mechanism by which some aspects of nerve injury-induced hyperesthesias may occur.
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Comparative Study
Glycine receptor knock-in mice and hyperekplexia-like phenotypes: comparisons with the null mutant.
Strychnine-sensitive glycine receptors (GlyRs) inhibit neurotransmission in the spinal cord and brainstem. To better define the function of this receptor in vivo, we constructed a point mutation that impairs receptor function in the alpha1-subunit and compared these knock-in mice to oscillator (spdot) mice lacking functional GlyR alpha1-subunits. Mutation of the serine residue at amino acid 267 to glutamine (alpha1S267Q) results in a GlyR with normal glycine potency but decreased maximal currents, as shown by electrophysiological recordings using Xenopus oocytes. ⋯ Homozygous S267Q knock-in mice, like homozygous spdot mice, exhibited seizures and died within 3 weeks of birth. In heterozygous spdot mice, both decreased 3H-strychnine binding and chloride flux were observed; however, neither enhanced acoustic startle responses nor limb clenching were seen. These data demonstrate that a dominant-negative point mutation in GlyR disrupting normal function can produce a more dramatic phenotype than the corresponding recessive null mutation, and provides a new animal model to evaluate GlyR function in vivo.