Calcium and excitotoxic neuronal injury.
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Calcium↗
Biomedical subjects
D W Choi
Publications and source records attributed to D W Choi.
Delayed application of aurintricarboxylic acid reduces glutamate-induced cortical neuronal injury.
The non-specific endonuclease inhibitor, aurintricarboxylic acid (ATA), attenuated glutamate-induced destruction of cultured cortical neurons. In part, this protective effect likely reflected the ability of ATA to produce a slowly developing block of N-methyl-D-aspartate receptor-mediated inward whole cell current or increase in intracellular free Ca2+. However, ATA also attenuated a high K(+)-induced increase in intracellular free Ca2+ in the presence of D-amino-phosphonovalerate, suggesting that ATA may have a more general effect on Ca2+ homeostasis. In addition, ATA attenuated glutamate neurotoxicity even if added up to 2 hr after completion of glutamate exposure, a time when glutamate antagonists or lipid peroxidation inhibitors are no longer neuroprotective. Involvement of apoptosis in this excitotoxic death is unlikely, as Southern blotting of genomic DNA revealed no evidence of fragmentation, and death was not prevented by inhibitors of RNA or protein synthesis. Most likely, ATA interferes with some key downstream consequences of excitotoxic glutamate receptor overactivation.
Animals↗
Trolox attenuates cortical neuronal injury induced by iron, ultraviolet light, glucose deprivation, or AMPA.
The vitamin E analog, trolox, protected cultured cortical neurons against damage induced by exposure to either iron ions or ultraviolet (UV) light, consistent with an ability to inhibit free radical-mediated cytotoxicity. Trolox also reduced neuronal death induced by 24 h exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), but not that induced by N-methyl-D-aspartate (NMDA). When combined with the NMDA receptor antagonist dextrorphan, trolox also reduced the neuronal injury induced by glucose deprivation.
Animals↗
AMPA/kainate receptor-mediated damage to NADPH-diaphorase-containing neurons is Ca2+ dependent.
While exposure times of several hours or more are needed for AMPA or kainate to induce widespread cortical neurodegeneration, the small subset of cortical neurons that contain high concentrations of the enzyme, NADPH-diaphorase (NADPH-d(+) neurons) were destroyed by brief (15-30 min) exposures. AMPA or kainate-induced degeneration of NADPH-d(+) neurons was decreased by removal of extracellular Ca2+ and increased by augmentation of extracellular Ca2+. More than 90% of NADPH-d(+) neurons exhibited kainate-activated Co2+ uptake, suggesting that they possess Ca(2+)-permeable AMPA/kainate receptor-gated channels. The heightened vulnerability of NADPH-d(+) neurons to AMPA or kainate toxicity may reflect rapid Ca2+ entry through these Ca(2+)-permeable channels.
Animals↗
The effect of NMDA, AMPA/kainate, and calcium channel antagonists on traumatic cortical neuronal injury in culture.
A traumatic insult was delivered to murine cortical neuronal and glial cell cultures by tearing the cell layer with a stylet in a grid pattern. Consistent with prior observations, neurons adjacent to a tear developed immediate swelling, and then went on to degenerate over the next several hours. Delivery of multiple tears produced enough cell death that measurable levels of lactate dehydrogenase accumulated in the bathing medium 24 h later, correlating well with the extent of cell death as assessed by Trypan blue exclusion and cell counts. 50-75% of this trauma-induced cell death was blocked by the NMDA receptor antagonist MK-801. 10-100 microM CNQX also attenuated neuronal degeneration, but this neuroprotective effect was likely due to attenuation of NMDA receptor-mediated toxicity, since the more specific AMPA/kainate antagonist NBQX was ineffective. CNQX also did not augment the protective effect of MK-801. High concentrations of nimodipine or nifedipine produced modest neuroprotective effects; either dihydropyridine when combined with MK-801 reduced injury more than MK-801 alone. These results suggest that traumatic neuronal death in this in vitro model is mediated in part by excessive activation of NMDA receptors, and in part by mechanisms sensitive to high concentrations of dihydropyridines, but not by AMPA/kainate receptors.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Excitotoxicity, free radicals, and cell membrane changes.
Neuronal injury resulting from glutamate receptor-mediated excitotoxicity has been implicated in a wide spectrum of neurological disease states, including ischemia, central nervous system trauma, and some types of neurodegenerative diseases. Excitotoxicity may interact with other pathophysiological processes to enhance neuronal injury; for example, excess glutamate release due to free radicals generated during the immune response to infection might initiate secondary excitotoxicity, and intracellular pathways that contribute to neuronal destruction may be common to both excitotoxic and nonexcitotoxic injury processes. Defining the contribution of excitotoxicity to neuronal damage in acute zoster infection and post-herpetic neuralgia may provide one means of reducing morbidity from this often debilitating disease.
Animals↗
Cortical neurones with Ca2+ permeable AMPA/kainate channels display distinct receptor immunoreactivity and are GABAergic.
A minority subset of cortical neurones exhibit kainate-activated Co2+ uptake, a marker for AMPA/kainate receptor gated Ca2+-permeable channels. Consistent with enhanced Ca2+ influx through these channels, Co2+-positive neurones are unusually vulnerable to death induced by exposure to either AMPA or kainate. Here we show that Co2+-positive cortical neurones express a distinctive profile of AMPA receptor subunits as determined by immunostaining. Co2+-positive neurones were much less likely to express GluR2/GluR3, and much more likely to express GluR1 or GluR4, than the general cortical neuronal population. Thus expression of AMPA receptors lacking the GluR2 subunit may explain the Co2+ staining, and selective vulnerability to kainate exhibited by Co2+-positive cells. Almost all GABAergic neurones, identified by immunostaining for glutamic acid decarboxylase, were Co2+-positive. The widespread presence of Ca2+-permeable AMPA/kainate receptor-gated channels on cortical GABAergic neurones may have important implications for the fate of cortical inhibition in disease states associated with the excitotoxic overstimulation of glutamate receptors.
Animals↗
Cortical neurones exhibiting kainate-activated Co2+ uptake are selectively vulnerable to AMPA/kainate receptor-mediated toxicity.
Kainate-activated Co2+ uptake, a histochemical method that identifies cells bearing Ca2+-permeable AMPA/kainate receptors, labels approximately 15% of murine cortical neurones in cell culture. While exposure times exceeding several hours were needed for AMPA or kainate to destroy most cultured cortical neurones, the subpopulation exhibiting kainate-activated Co2+ uptake was selectively destroyed after AMPA or kainate exposures of only 10-60 min. No selective loss was seen after exposure to NMDA. Kainate toxicity on Co2+ uptake-positive neurones was dependent on extracellular Ca2+ concentration, and associated with an increase in intracellular free Ca2+ even in the absence of extracellular Na+. These results suggest that a distinct subpopulation of cortical neurones expresses AMPA/kainate receptors linked to Ca2+-permeable channels, and that this characteristic conveys enhanced vulnerability to kainate-induced, Ca2+-mediated, damage.
Animals↗
Dextrorphan inhibits the release of excitatory amino acids during spinal cord ischemia.
The release of excitatory amino acids, particularly glutamate, into the extracellular space plays a causal role in irreversible neuronal damage after central nervous system ischemia. Dextrorphan, a noncompetitive N-methyl-D-aspartate receptor antagonist, has been shown to provide significant protection against cerebral damage after focal ischemia. We investigated the changes in extracellular neurotransmitter amino acid concentrations using in vivo microdialysis in a swine model of spinal cord ischemia. After lumbar laminectomies were performed, all animals underwent left thoracotomy and right atrial-femoral cardiopulmonary bypass with additional aortic arch perfusion. Microdialysis probes were then inserted stereotactically into the lumbar spinal cord. The probes were perfused with artificial cerebrospinal fluid and 15-minute samples were assayed using high-performance liquid chromatography. Group 1 animals (n = 9) underwent aortic clamping distal to the left subclavian and proximal to the renal arteries for 60 minutes. Group 2 animals (n = 7) were treated with dextrorphan before application of aortic clamps, and during aortic occlusion and reperfusion. Five amino acids were studied, including two excitatory neurotransmitters (glutamate and aspartate) and three putative inhibitory neurotransmitters (glycine, gamma-amino-butyric acid, and serine). Somatosensory-evoked potentials and motor-evoked potentials were monitored. Glutamate exhibited a threefold increase in extracellular concentration during normothermic ischemia compared with baseline values and remained elevated until 60 minutes after reperfusion. In animals treated with dextrorphan, glutamate concentrations decreased to one-third of baseline levels before aortic clamping and remained unchanged during ischemia and reperfusion. There was early loss of somatosensory-evoked potentials and motor-evoked potentials during ischemia in group 1 animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Zinc toxicity on cultured cortical neurons: involvement of N-methyl-D-aspartate receptors.
Neuronal injury induced by the excessive release of endogenous Zn2+ at central glutamatergic synapses may contribute to the pathogenesis of epileptic brain damage. We explored the possibility that N-methyl-D-aspartate receptors might be involved in Zn2+ neurotoxicity. Exposure of murine cortical cell cultures to 300-1000 microM concentrations of Zn2+ for 15 min resulted in widespread neuronal degeneration, accompanied by the release of lactate dehydrogenase to the bathing medium. Both non-competitive and competitive N-methyl-D-aspartate antagonists attenuated this degeneration. However, the participation of N-methyl-D-aspartate receptors in Zn2+ neurotoxicity was atypical. Removal of extracellular Ca2+ attenuated N-methyl-D-aspartate neurotoxicity but potentiated Zn2+ neurotoxicity, whereas increasing extracellular Ca2+ potentiated N-methyl-D-aspartate neurotoxicity but attenuated Zn2+ neurotoxicity. Furthermore, the nature of the antagonism of Zn2+ neurotoxicity induced by N-methyl-D-aspartate antagonists was qualitatively different from that seen with other N-methyl-D-aspartate receptor-mediated events. The block of Zn2+ neurotoxicity induced by the non-competitive N-methyl-D-aspartate antagonist MK-801 was better overcome by increasing Zn2+ concentration than the block induced by the competitive antagonists D-aminophosphonovalerate and CGS-19755. We hypothesize that N-methyl-D-aspartate receptor-gated channels contribute to Zn2+ toxicity by providing a route of Zn2+ influx into neurons. Consistent with this idea, intracellular Zn2+ visualized by the fluorescent Zn2+ chelator, N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide, rose during Zn2+ exposure; this rise was increased by N-methyl-D-aspartate and reduced by either N-methyl-D-aspartate antagonists or high Ca2+.2+ in neuronal cell homeostasis.
Animals↗
Basal forebrain cholinergic neurons are selectively vulnerable to AMPA/kainate receptor-mediated neurotoxicity.
We exposed murine basal forebrain neuronal cultures for 24 h to defined concentrations of N-methyl-D-aspartate, kainate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, and assessed the resultant degeneration of the cholinergic neuronal subpopulation, as identified by choline acetyltransferase immunocytochemistry and acetylcholinesterase histochemistry. Cholinergic neurons, representing about 0.5% of the total neuronal population, were atypically vulnerable to excitotoxins. Compared to most basal forebrain neurons, they were more vulnerable to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor-mediated injury and slightly less vulnerable to N-methyl-D-aspartate receptor-mediated injury. The present findings provide quantitative demonstration of a mechanism that preferentially injures basal forebrain cholinergic neurons, and may thus suggest candidate factors pertaining to their loss in disease states like Alzheimer's disease.
Acetylcholine↗
Selective potentiation of NMDA-induced neuronal injury following induction of astrocytic iNOS.
Nitric oxide (NO) produced by the constitutive NO synthase (cNOS) in neurons has been implicated in mediating excitotoxic neuronal death. In our murine cortical cell culture system, NMDA neurotoxicity was not blocked by addition of the NOS inhibitors, NG-nitro-L-arginine or aminoguanidine. However, following activation of inducible NOS in astrocytes by interleukin-1 beta plus interferon-gamma, NMDA but not kainate neurotoxicity was markedly potentiated. This selective potentiation of NMDA neurotoxicity was blocked by NOS inhibition or antioxidants (superoxide dismutase/catalase or Tempol) and could be mimicked by NO generators (SIN-1 or SNAP) or the oxygen radical generator, pyragallol. These results raise the possibility that NO production by astrocytes may contribute to NMDA receptor-mediated neuronal death, perhaps through interaction with oxygen radicals.
Amino Acid Oxidoreductases↗
Glutamate receptors and the induction of excitotoxic neuronal death.
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Animals↗
Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen-glucose deprivation or excitatory amino acids.
We examined the effect of moderate hypothermia (30 degrees C) on neuronal injury in murine cortical cell cultures. Lowering the temperature during and after a period of oxygen-glucose deprivation reduced both the release of glutamate to the bathing medium and accompanying neuronal degeneration. Hypothermia immediately after brief exposure to high concentrations of NMDA or glutamate also reduced the resulting neuronal degeneration. This protective effect was not eliminated when MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione were added immediately after washout of the exogenously added excitotoxin, suggesting that it was mediated by actions additional to reduction of endogenous late glutamate release. Hypothermia applied only during exposure to NMDA or glutamate, whether brief or prolonged, did not reduce subsequent cytosolic calcium accumulation or neuronal degeneration, suggesting that the postsynaptic induction of NMDA receptor-mediated excitotoxicity is not sensitive to temperature reduction. However, hypothermia during prolonged S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate exposure did reduce neuronal degeneration.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Dipyridamole increases oxygen-glucose deprivation-induced injury in cortical cell culture.
BACKGROUND AND PURPOSE: Adenosine transport inhibitors attenuate ischemic central neuronal damage in vivo, but the locus of this protective action is presently unknown. To help address the question of whether adenosine transport inhibitors have a protective effect directly on brain parenchyma, we tested the effect of the adenosine transport inhibitor dipyridamole on neuronal loss induced by oxygen-glucose deprivation in vitro. METHODS: Murine cortical cultures were exposed to combined oxygen and glucose deprivation, N-methyl-D-aspartate, or kainate. The extracellular concentrations of glutamate and adenosine were measured by high-performance liquid chromatography; neuronal cell death was assessed by morphological examination and measurement of lactate dehydrogenase release. RESULTS: Cultures exposed to oxygen-glucose deprivation for 30 to 75 minutes exhibited an insult-dependent increase in extracellular adenosine, followed shortly by an increase in extracellular glutamate and 24 hours later by neuronal death. Addition of the A1 receptor antagonist 8-cyclopentyltheophylline during oxygen-glucose deprivation enhanced both glutamate release and neuronal damage. Addition of 10 mumol/L dipyridamole decreased extracellular adenosine and also enhanced extracellular glutamate and neuronal death. In contrast, dipyridamole increased the levels of extracellular adenosine stimulated by N-methyl-D-aspartate or kainate. CONCLUSIONS: These results are consistent with the idea that endogenous adenosine has a neuroprotective effect directly on cortical cells exposed to oxygen-glucose deprivation. However, inhibition of adenosine transport with dipyridamole was surprisingly not an effective strategy for enhancing this protective effect. The beneficial effects of adenosine transport inhibitors observed in vivo may be mediated indirectly--for example, by effects on the vasculature.
Adenosine↗
Interferon-gamma and interleukin-1 beta induce nitric oxide formation from primary mouse astrocytes.
An inducible form of nitric oxide synthase (iNOS) capable of producing large quantities of nitric oxide (NO) exists in some cell types. We demonstrate by immunoprecipitation and nitrite formation that interleukin-1 beta (IL1 beta) plus interferon-gamma (INF gamma) induce the expression of nitric oxide synthase in primary cultures of murine cortical astrocytes. This induction is time and dose dependent, and inhibited by the NOS inhibitor NG-nitro-L-arginine and the protein synthesis inhibitor cycloheximide.
Amino Acid Oxidoreductases↗
Nitric oxide: foe or friend to the injured brain?
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Animals↗
Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl-D-aspartate neurotoxicity.
We examined the role of nitric oxide in N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in rat and mouse primary cortical cell cultures. In rat and mouse cultures, the NO synthase inhibitor, NG-Nitro-L-arginine, blocked cGMP formation but not neuronal cell death following a 5-10 min exposure to 300-500 microM NMDA. NG-Monomethyl-L-arginine was also unable to prevent neuronal death. In contrast, the non-competitive NMDA receptor antagonist, dextrorphan, prevented both cGMP formation and cell death. While other data suggest that the synthesis of nitric oxide can mediate NMDA receptor-mediated neurotoxicity, present results suggest that such synthesis is not necessarily required.
Animals↗