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T H Murphy

Publications and source records attributed to T H Murphy.

47 records · Page 3Linked to original sources
PubMed1991

Enhanced NAD(P)H:quinone reductase activity prevents glutamate toxicity produced by oxidative stress.

Glutamate toxicity in the N18-RE-105 neuronal cell line results from the inhibition of high-affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one-electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT-diaphorase activity. Using glutamate toxicity in N18-RE-105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t-butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t-butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease.

Antioxidants↗
PubMed1991

Synaptic regulation of immediate early gene expression in primary cultures of cortical neurons.

Neuronal stimulation can rapidly activate several immediate early genes that code for transcription factors. We have used primary cortical cultures to study the regulation of four of these genes, c-fos, c-jun, jun-B, and zif268. Immunocytochemical studies with antibodies to Jun-B, c-Jun, and c-Fos demonstrate intense staining in the nuclei of a subset of cortical neurons in mature cultures (21-25 days in vitro) but not young cultures (3-7 days in vitro). To assess whether this immunoreactivity may be induced by spontaneous synaptic activity that develops with a similar profile, we examined the effects of agents that reduce this synaptic activity. Tetrodotoxin or N-methyl-D-aspartate receptor antagonists suppress basal immunoreactivity to Jun-B and c-Fos, but not c-Jun, indicating that the basal level of c-Jun expression is not dependent on electrical activity. Picrotoxin, an agent that increases synaptic excitation indirectly by blocking inhibitory synaptic currents mediated by gamma-aminobutyric acidA receptors, markedly increases the percentage of neurons displaying immunoreactivity to c-Fos, c-Jun, Jun-B, and Zif268. Northern analysis suggests that the increases in immunostaining induced by picrotoxin are secondary to a rapid increase in mRNA for these proteins. These findings provide evidence for rapid transcriptional regulation of immediate early genes in cortical neurons by synaptic activity.

Animals↗
PubMed1990

Glutamate toxicity in immature cortical neurons precedes development of glutamate receptor currents.

Cationic fluxes resulting from glutamate receptor activity have recently been implicated in neurotoxicity. Immature cortical neurons are insensitive to the toxic effects of glutamate receptor stimulation. However, these neurons are killed by glutamate via a non-receptor-mediated mechanism thought to stem from glutamate's ability to inhibit cystine uptake. To examine the basis for their resistance to receptor-mediated toxicity, we have studied the development of glutamate receptor-mediated inward currents in cortical neurons in culture using the whole-cell voltage-clamp technique. We report that in immature cortical neurons (prepared from day-17 fetal brain and cultured for 1-3 days), N-methyl-D-aspartate, quisqualate, and glutamate are able to evoke only very small inward currents in a low percentage of neurons. After 7 days of culture, greater than 80% of neurons examined exhibited currents activated by these glutamate receptor agonists. Although most neurons expressed glutamate agonist-evoked currents after 7 days in culture, the amplitude of these currents was less than 10% of that observed after 15 days in culture. In contrast to currents activated by glutamate receptor agonists, those activated by gamma-aminobutyric acid reached maximal levels after only 2 days of culture. These results indicate that the delayed development of glutamate receptor-mediated currents accounts for the resistance of immature cortical neurons to glutamate receptor-mediated toxicity.

Animals↗
PubMed1990

Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake.

Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.

Animals↗
PubMed1989

Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress.

Glutamate binds to both excitatory neurotransmitter binding sites and a Cl(-)-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (N18-RE-105) are susceptible to glutamate-induced cytotoxicity. The Cl(-)-dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Lowering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and Ca2+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like N18-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.

Animals↗
PubMed1989

Antioxidants protect against glutamate-induced cytotoxicity in a neuronal cell line.

The effects of reducing agents and antioxidants on L-Glutamate (Glu)-induced cytotoxicity were examined in the N18-RE-105 neuronal cell line. The cytotoxicity by Glu (1 and 10mM) was potentiated by exposure to growth medium containing a low concentration of cystine (5-100 microM), instead of the normal medium containing 200 microM cystine. In contrast, the toxicity was suppressed by increasing the cystine concentration to 500 to 1000 microM. Reducing agents, cysteine (30-1000 microM), dithiothreitol (10-250 microM) and glutathione (GSH, 10-1000 microM) also protected the cells against the cytotoxicity of 10 mM Glu in a concentration-dependent manner. The antioxidants vitamin E (10-100 microM), idebenone (0.1-3 microM) and vinpocetine (10-100 microM) also provided marked protection against the cytotoxicity of Glu (10 mM) or quisqualate (1 mM). Antioxidants also prevented the delayed cell death caused by lowering the concentration of cystine in the medium to 5 microM. Incubation of the cells with 10 mM Glu caused a marked decrease in cellular GSH levels. Although cysteine and dithiothreitol prevented the GSH reduction caused by Glu, antioxidants did not. The cellular levels of oxidants were assessed using 2,7-dichlorofluorescin, a probe that accumulates within cells and is converted to a fluorescent product by oxidation. Glu (10 mM) caused a marked increase in such fluorescence, whereas vitamin E and idebenone reduced markedly the number of fluorescent cells to control levels even added with 10 mM Glu. These results indicate that oxidative stress due to loss of cellular levels of GSH is one mechanism whereby Glu/quisqualate exert cytotoxicity and suggest that centrally active antioxidants may reduce neuronal damage in pathologic conditions associated with excessive Glu release.

Animals↗
PubMed1988

Glutamate cytotoxicity in a neuronal cell line is blocked by membrane depolarization.

To understand better the proximate mechanism involved in the excitotoxic response to L-glutamate (Glu), we have exploited the Glu receptor present in the N18-RE-105 neuroblastoma-embryonic retinal hybrid cell line. These cells undergo lysis dependent on extracellular Ca2+ when exposed to Glu. We now report that the depolarizing action of Glu is not responsible for its cytotoxic effects. Furthermore, depolarization of these cells with elevated K+, ouabain or veratridine does not cause cytotoxicity but rather protects against the cytotoxic effects of Glu. Our results may implicate a role for voltage-sensitive Ca2+ channels (VSCCs) in cytotoxicity, and depolarization-induced inactivation of VSCCs (Nature (Lond.), 316 (1985) 440-443) as a protection against Glu receptor agonists. Our findings demonstrate a clear dissociation between depolarization and the neuronal degeneration caused by Glu.

Animals↗
PubMed1988

Calcium-dependent glutamate cytotoxicity in a neuronal cell line.

Membranes from the neuroblastoma x embryonic retina cell hybrid cell line, N18-RE-105, bind L-[3H]glutamate with a pharmacologic profile consistent with a 'quisqualate-type' brain L-glutamate receptor. We describe here the cytotoxic effect of L-glutamate receptor agonists on intact N18-RE-105 cells. Cytotoxicity was quantitated by measurement of the release of the cytosolic enzyme, lactate dehydrogenase, into the culture medium after addition of L-glutamate and its analogs to the cell culture medium. L-Glutamate (10 mM) and its confirmationally restricted analogs, quisqualate (1 mM) and ibotenate (10 mM), caused cell lysis. In contrast, similar analogs which do not bind to N18-RE-105 cell membranes (kainic acid, N-methyl-D,L-aspartic acid and gamma-aminobutyric acid) were not cytotoxic. L-Glutamate-induced cytotoxicity was eliminated when calcium-free medium was used. Addition of inorganic or organic calcium channel antagonists also reduced the cytotoxicity of L-glutamate, even when 1.8 mM calcium was present in the medium. Cadmium chloride (10 microM) completely blocked L-glutamate toxicity, whereas manganese chloride (150 microM) and lanthanum chloride (25 microM) reduced toxicity by greater than 50%. Dihydropyridine voltage-sensitive calcium channel agonists or antagonists, had little or no significant effect on L-glutamate-induced toxicity. In contrast, the verapamil derivatives, D600 and D888, and the diltiazem derivative, MDL 12,330A reduced L-glutamate toxicity by greater than 50%. These results suggest that a subtype of voltage-sensitive calcium channels is involved in the mechanism of L-glutamate receptor mediated cytotoxicity in this cell line.

Calcium↗
PubMedDate not supplied

Dicarboxylic amino acids block epileptiform activity in hippocampal slice.

Effects of prolonged (5-10 min) continuous perfusion of excitatory amino acids on penicillin (PEN)-evoked epileptiform activity in hippocampal slices were examined with extracellular and intracellular recordings. L-glutamate (GLU), L-aspartate (ASP), quisqualate (QUIS), and N-methyl-D,L-aspartate reversibly depressed multiple (epileptiform) population spikes elicited by PEN (1.7 mM). Intracellularly recorded, PEN-evoked paroxysmal depolarization shifts (PDS) were partially blocked by 1 mM GLU and largely eliminated by 2 mM GLU or ASP. In the presence of PEN, perfusion with both GLU and ASP induced a transient 4 to 6-mV depolarization, usually followed by spontaneous return of membrane potential to control levels. During the amino acid (AA)-induced block of epileptiform activity, there was no significant change in resting membrane potential, input resistance, or the ability to fire action potentials in response to depolarization, indicating that the decreased responsiveness is not a consequence of nonspecific pyramidal cell overdepolarization. The observed depression of epileptiform activity by continued exposure to GLU and its analogues may reflect desensitization or another regulatory mechanism to limit overexcitation.

Action Potentials↗