Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line.

Younkin, Donald P.; Tang, Cha‐Min; Hardy, Marcia; Reddy, Usha R.; Shi, Qing; Pleasure, Samuel J.; Lee, Virginia M.‐Y.; Pleasure, David E

doi:10.1073/pnas.90.6.2174

Cited by 174 publications

(115 citation statements)

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“…For excised outside-out patch recordings and the experiment in Figure 10, hippocampal cell cultures were prepared from prenatal rats (Tang et al, 1991). Recordings of channel events shown in Figure 7 were obtained from a neuron derived from the NT2 human cell line induced to express glutamate receptor channels (Younkin et al, 1993).…”

Section: Methodsmentioning

confidence: 99%

“…At high concentrations, however, multiple channels are invariably activated in excised patches from hippocampal neurons. One way to obtain patches containing only one or two channels is to use a cell line, NT2-N, that expresses glutamate channels nearly identical to hippocampal glutamate channels but in lower density (Younkin et al, 1993). Figure 7 illustrates an NT2-N patch containing two non-NMDA glutamate channels.…”

Section: Whole-cell Current Recordingsmentioning

confidence: 99%

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Benzothiadiazides inhibit rapid glutamate receptor desensitization and enhance glutamatergic synaptic currents

1993

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A distinctive characteristic of the AMPA subset of glutamate receptor channels is their remarkably rapid desensitization. A family of compounds, the benzothiadiazides, is described here that potently inhibit rapid glutamate receptor desensitization. The structure- activity relationships of these compounds are examined and the actions of cyclothiazide (CYZ), the most potent of these compounds, are described in detail. At the macroscopic level CYZ reduced rapid desensitization, enhancing the steady-state and peak current produced by 1 mM quisqualate with EC50 values of 14 and 12 microM, respectively, and shifted the quisqualate peak current concentration-response relation to the left. The slight outward rectification of the steady- state quisqualate current-voltage relationship was reduced by CYZ. At the microscopic level CYZ caused glutamate to induce long bursts of channel openings, and greatly increased the number of repeated openings. At 10 microM CYZ did not have measurable effects on the fast component of deactivation nor did it have statistically significant effects on the distribution of the faster components of glutamate- induced burst duration. In contrast, 10 microM CYZ increased the amplitude and significantly prolonged the duration of the spontaneous miniature EPSCs. The identification and characterization of this new family of gating modifiers may further facilitate the investigation into the mechanisms underlying rapid glutamate receptor desensitization and the physiological roles that it may serve.

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Section: Methodsmentioning

confidence: 99%

Section: Whole-cell Current Recordingsmentioning

confidence: 99%

Benzothiadiazides inhibit rapid glutamate receptor desensitization and enhance glutamatergic synaptic currents

1993

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“…The NT2-N neurons are allowed to grow for 4 wk after last replating, and during this period they develop an extensive network of neuritic processes that can be identified as axons or dendrites. They express several CNS neuronal marker proteins (20), express functional non-NMDA and NMDA receptors (21,22), and contain synaptic vesicle-associated proteins (23).…”

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confidence: 99%

Neuronal Formation of Free Radicals Plays a Minor Role in Hypoxic Cell Death in Human NT2-N Neurons

Almaas¹,

Saugstad²,

Pleasure

et al. 2002

Pediatr Res

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Free radicals are suggested to play an important role in hypoxic-ischemic neuronal death. However, the importance in human disease is not known. Furthermore, whether posthypoxic free radical formation mainly occurs in endothelium and neutrophils, or whether neuronal production is important, is not finally determined. To study this we differentiated human Ntera2 teratocarcinoma cells into postmitotic NT2-N neurons and exposed them to free radicals, hypoxia, or oxygen and glucose deprivation. These cells are devoid of nitric oxide synthase, and we hypothesized that free radicals are important mediators downstream of N-methyl-D-aspartate stimulation. Production of free radicals, evaluated with the fluorescent dyes dihydrorhodamine and 2',7'-dichlorodihydrofluorescein, was significantly higher in neurons deprived of oxygen and glucose after 40 min of reoxygenation than in normoxic cells. The antioxidant trolox, the flavonoid quercetin, thiopental, and the N-methyl-D-aspartateglutamate receptor antagonist MK-801 reduced the formation of free radicals. Treatment with the flavonoid rutin (86 Ϯ 16% of hypoxic cells without drug, p Ͻ 0.01), trolox (86 Ϯ 20%, p Ͻ 0.01), and MK-801 (57 Ϯ 12%, p Ͻ 0.01) reduced lactate dehydrogenase release after 6 h of hypoxia. Trolox, salicylate, and quercetin also significantly reduced lactate dehydrogenase release after 3 h of oxygen and glucose deprivation. The protection offered by these antioxidants was, however, limited compared with the effect of MK-801. We conclude that oxygen and glucose deprivation causes a moderate increase in the formation of free radicals in NT2-N neurons that can be Several experiments have indicated a role for free radicals in the pathogenesis of hypoxic-ischemic cell death. The xanthine oxidase inhibitor and hydroxyl radical scavenger allopurinol reduces hypoxic-ischemic brain damage in rats (1), and van Bel et al. (2) demonstrated beneficial effects of allopurinol in asphyxiated infants. Infarct size and brain edema after focal cerebral ischemia are attenuated in adult transgenic mice overexpressing CuZn SOD (3). However, brain injury after hypoxia-ischemia was exacerbated in a similar newborn mice model (4).During reperfusion after cerebral ischemia in pigs, superoxide anion is generated (5). Production of free radicals after ischemia has been reported from rat brain microvessels (6) and from fetal bovine endothelial cells (7). A substantial part of posthypoxic free radical formation in endothelium has been attributed to xanthine oxidase (7). On the other hand, ScmidElsaesser et al. (8)

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“…Because of recognized inter-relationships between NMDA receptor complexes, cell calcium homeostasis, and mitochondria, we evaluated whether memantine affects mitochondrial function. We studied this under in vitro conditions using the NT2 teratocarcinoma cell line, a neuron-like tumor cell line that expresses critical parts of the NMDA receptor complex [14,15]. We found memantine can influence mitochondrial function, and that at least part (if not all) of this occurs independent of NMDA channel antagonism.…”

Section: Introductionmentioning

confidence: 97%

Effects of memantine on mitochondrial function

McAllister

Ghosh

Berry

et al. 2008

Biochemical Pharmacology

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Because NMDA complex and mitochondrial function are related, we hypothesized memantine would influence mitochondrial function. We addressed this in vitro by studying the effects of chronic and acute memantine exposures on mitochondrial function. For acute exposure experiments, mitochondria were isolated from NT2 cells and assayed for electron transport chain (ETC) enzyme function and peroxide production in buffers containing up to 60 uM memantine. For chronic exposure experiments, NT2 cells were maintained for at least two weeks in medium containing up to 60 uM memantine, following which we assayed cells or their mitochondria for ETC enzyme activities, cytochrome oxidase protein levels, oxidative stress, calcium levels, and mitochondrial DNA levels. The ability of the NMDA receptor antagonist aminophosphonovaleric acid (APV) to modify memantine's mitochondrial effects was evaluated. Acute and chronic memantine similarly affected complex I (increased at high concentrations) and IV (decreased at high concentrations) Vmax activities. APV did not alter the effects of chronic memantine exposure on citrate synthase and complex IV. We detected a lower mitochondrial peroxide production rate with acute exposure, and an increased mitochondrial peroxide production rate with chronic exposure. Micromolar memantine concentrations affect mitochondria, some of these effects are directly mediated, and acute and chronic effects may differ.

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Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line.

Cited by 174 publications

References 50 publications

Benzothiadiazides inhibit rapid glutamate receptor desensitization and enhance glutamatergic synaptic currents

Benzothiadiazides inhibit rapid glutamate receptor desensitization and enhance glutamatergic synaptic currents

Neuronal Formation of Free Radicals Plays a Minor Role in Hypoxic Cell Death in Human NT2-N Neurons

Effects of memantine on mitochondrial function

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