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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The protooncogene bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that the mechanism of action of Bcl-2 involves antioxidant activity. The involvement of free radicals in ischemia/reperfusion injury to neural cells has led us to investigate the effect of Bcl-2 in a model of delayed neural cell death. We have examined the survival of control and bcl-2 transfectants of a hypothalamic tumor cell line, GT1-7, exposed to potassium cyanide in the absence of glucose (chemical hypoxia/aglycemia). After 30 min of treatment, no loss of viability was evident in control or bcl-2 transfectants; however, Bcl-2-expressing cells were protected from delayed cell death measured following 24–72 h of reoxygenation. Under these conditions, the rate and extent of ATP depletion in response to treatment with cyanide in the absence of glucose and the rate of recovery of ATP during reenergization were similar in control and Bcl-2-expressing cells. Bcl-2-expressing cells were protected from oxidative damage resulting from this treatment, as indicated by significantly lower levels of oxidized lipids. Mitochondrial respiration in control but not Bcl-2-expressing cells was compromised immediately following hypoxic treatment. These results indicate that Bcl-2 can protect neural cells from delayed death resulting from chemical hypoxia and reenergization, and may do so by an antioxidant mechanism. The results thereby provide evidence that Bcl-2 or a Bcl-2 mimetic has potential therapeutic application in the treatment of neuropathologies involving oxidative stress, including focal and global cerebral ischemia.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: We investigated the relationships among N-methyl-d-aspartate, glycine, L-type voltage-dependent calcium channels, and [3H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [3H]PN200-110 ([3H]isradipine) to L-type voltage-dependent calcium channels, that open as a consequence of N-methyl-d-aspartate-induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage-sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N-methyl-d-aspartate-stimulated [3H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N-methyl-d-aspartate-stimulated [3H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N-methyl-d-aspartate-stimulated release in all treatment groups. The enhancement of N-methyl-d-aspartate-stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30-min reperfused animals. These data suggest that glycine may facilitate opening of the voltage-dependent calcium channels activated by N-methyl-d-aspartate and that this facilitation is blocked by the antagonist nitrendipine.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin-sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)-mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK-N-SH human neuroblastoma cells while blocking the IP3-mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) abolished the carbachol-mediated intracellular calcium release without abolishing the carbachol-mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist-induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor-mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Mitochondrial DNA (mtDNA) encodes critical subunit proteins of the oxidative phosphorylation (OXPHOS) complex that generates ATP. This study tested the hypothesis that mitochondrial gene expression in neural cells is regulated by energy demand, as modified via stimulation of cellular sodium transport. Exposure of PC12S cells to the sodium ionophore monensin (250 nm) for 1–6 h caused a 13–60% decrease in cellular ATP (from 15 to 5 nmol per mg protein at 6 h). Levels of mitochondrial DNA-encoded mRNAs (mt-mRNAs) increased significantly (150%) within the first hour of exposure to monensin, and then decreased significantly (50%) at 3–4 h. Levels of mtDNA-encoded 12S rRNA and nuclear DNA-encoded OXPHOS subunit mRNAs were not significantly affected. Exposure of primary cerebellar neuronal cultures to the excitatory amino acid glutamate caused a similar rapid and significant increase followed by a significant decrease in cell mt-mRNA levels. The monensin-induced initial increase in mt-mRNA levels was abolished by pretreatment with actinomycin D or by reducing extracellular sodium ion concentration. The monensin-induced delayed reduction in mt-mRNA levels was accelerated in the presence of actinomycin D, and was accompanied by a 67% reduction in the half-life (from 3.6 to 1.2 h). Exposure of PC12S cells to 2-deoxy-d-glucose significantly decreased cellular ATP levels (from 14.2 to 7.1 nmol per mg protein at 8 h), and increased mt-mRNA levels. These results suggest a physiological transcriptional mechanism of regulation of mitochondrial gene expression by energy demand and a post-transcriptional regulation that is independent of energy status of the cell.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Expression of the protooncogene bcl-2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl-2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl-2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species. To evaluate this proposal, we measured activities of antioxidant enzymes as well as levels of glutathione and pyridine nucleotides in control and bcl-2 transfectants in two different neural cell lines—rat pheochromocytoma PC12 and the hypothalamic GnRH cell line GT1-7. Both neural cell lines overexpressing bcl-2 had elevated total glutathione levels when compared with control transfectants. The ratios of oxidized glutathione to total glutathione in PC12 and GT1-7 cells overexpressing bcl-2 were significantly reduced. In addition, the NAD+/NADH ratio of bcl-2-expressing PC12 and GT1-7 cells was two- to threefold less than that of control cell lines. GT1-7 cells overexpressing bcl-2 had the same level of glutathione peroxidase, catalase, superoxide dismutase, and glutathione reductase activities as control cells. PC12 cells overexpressing bcl-2 had a twofold increase in superoxide dismutase and catalase activity when compared with matched control transfected cells. The levels of glutathione peroxidase and glutathione reductase in PC12 cells overexpressing bcl-2 were similar to those of control cells. These results indicate that the overexpression of bcl-2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes.
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  • 7
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Protein delivery mediated by protein transduction domains (PTD) such as the HIV-1 TAT-PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl-2 anti-death family proteins. A TAT-Bcl-2 construct with the loop domain deleted (TAT-Bcl-2Δloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT-mediated protein internalization in neural cells was also investigated. The purified TAT-Bcl-2Δloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT-Bcl-2Δloop occurs at 37°C but not at 4°C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT-Bcl-2Δloop with cell surface heparan sulfate proteoglycans. TAT-mediated protein delivery in neuronal cells occurs through a lipid raft-dependent endocytotic process, inhibited by the cholesterol-sequestering agent nystatin. Transducible loop deleted Bcl-2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine-induced death. These results support the concept of using protein transduction of Bcl-2 constructs for neuroprotection.
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  • 8
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Calcium-mediated signaling regulates nuclear gene transcription by calcium/cAMP response element binding protein (CREB) via calcium-dependent kinases and phosphatases. This study tested the hypothesis that CREB is also present in mitochondria and subject to dynamic calcium-dependent modulation of its phosphorylation state. Antibodies to CREB and phosphorylated CREB (pCREB) were used to demonstrate the presence of both forms in isolated mitochondria and mitoplasts from rat brain. When energized mitochondria were exposed to increasing concentrations of Ca2+ in the physiological range, pCREB was lost while total CREB remained constant. In the presence of Ru360, an inhibitor of the mitochondrial Ca2+ uptake uniporter, calcium-dependent loss of pCREB levels was attenuated, suggesting that intramitochondrial calcium plays an important role in pCREB dephosphorylation. pCREB dephosphorylation was not, however, inhibited by the phosphatase inhibitors okadaic acid and Tacrolimus. In the absence of Ca2+, CREB phosphorylation was elevated by the addition of ATP to the mitochondrial suspension. Exposure of mitochondria to the pore-forming molecule alamethicin that causes osmotic swelling and release of intermembrane proteins enriched mitochondrial pCREB immunoreactivity. These results further suggest that mitochondrial CREB is located in the matrix or inner membrane and that a kinase and a calcium-dependent phosphatase regulate its phosphorylation state.
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  • 9
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH-linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (ΔΨ) and NAD(P)H redox state. ΔΨ was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to ΔΨ and the level of NAD(P)H reduction at high values of ΔΨ, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p-trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of ΔΨ and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH-dependent substrates is regulated by ΔΨ and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.
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  • 10
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.
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