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Title: Inhibition of N-methyl-D-aspartate receptors increases paraoxon-induced apoptosis in cultured neurons

Abstract

Organophosphorus (OP) compounds, used as insecticides and chemical warfare agents, are potent neurotoxins. We examined the neurotoxic effect of paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), an organophosphate compound, and the role of NMDA receptors as a mechanism of action in cultured cerebellar granule cells. Paraoxon is neurotoxic to cultured rat cerebellar granule cells in a time- and concentration-dependent manner. Cerebellar granule cells are less sensitive to the neurotoxic effects of paraoxon on day in vitro (DIV) 4 than neurons treated on DIV 8. Surprisingly, the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, enhances paraoxon-mediated neurotoxicity suggesting that NMDA receptors may play a protective role. Pretreatment with a subtoxic concentration of N-methyl-D-aspartate (NMDA) [100 {mu}M] protects about 40% of the vulnerable neurons that would otherwise die from paraoxon-induced neurotoxicity. Moreover, addition of a neuroprotective concentration of NMDA 3 h after treatment with paraoxon provides the same level of protection. Because paraoxon-mediated neuronal cell death is time-dependent, we hypothesized that apoptosis may be involved. Paraoxon increases apoptosis about 10-fold compared to basal levels. The broad-spectrum caspase inhibitor (Boc-D-FMK) and the caspase-9-specific inhibitor (Z-LEHD-FMK) protect against paraoxon-mediated apoptosis, paraoxon-stimulated caspase-3 activity and neuronal cell death. MK-801 increases, whereas NMDA blocks paraoxon-induced apoptosis and paraoxon-stimulated caspase-3 activity. Thesemore » results suggest that activation of NMDA receptors protect neurons against paraoxon-induced neurotoxicity by blocking apoptosis initiated by paraoxon.« less

Authors:
 [1];  [1];  [1];  [2]
  1. Department of Neurology, Uniformed Services University of the Health Sciences, Building A, Room 1036, 4301 Jones Bridge Road, Bethesda, MD 20814 (United States)
  2. Department of Neurology, Uniformed Services University of the Health Sciences, Building A, Room 1036, 4301 Jones Bridge Road, Bethesda, MD 20814 (United States). E-mail: amarini@usuhs.mil
Publication Date:
OSTI Identifier:
20722006
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 208; Journal Issue: 1; Other Information: DOI: 10.1016/j.taap.2005.01.018; PII: S0041-008X(05)00038-4; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ACETYLCHOLINE; APOPTOSIS; IN VITRO; INHIBITION; INSECTICIDES; NERVE CELLS; PHOSPHATES; RATS; RECEPTORS; SAFETY; TIME DEPENDENCE

Citation Formats

Wu Xuan, Tian Feng, Okagaki, Peter, and Marini, Ann M. Inhibition of N-methyl-D-aspartate receptors increases paraoxon-induced apoptosis in cultured neurons. United States: N. p., 2005. Web. doi:10.1016/j.taap.2005.01.018.
Wu Xuan, Tian Feng, Okagaki, Peter, & Marini, Ann M. Inhibition of N-methyl-D-aspartate receptors increases paraoxon-induced apoptosis in cultured neurons. United States. doi:10.1016/j.taap.2005.01.018.
Wu Xuan, Tian Feng, Okagaki, Peter, and Marini, Ann M. 2005. "Inhibition of N-methyl-D-aspartate receptors increases paraoxon-induced apoptosis in cultured neurons". United States. doi:10.1016/j.taap.2005.01.018.
@article{osti_20722006,
title = {Inhibition of N-methyl-D-aspartate receptors increases paraoxon-induced apoptosis in cultured neurons},
author = {Wu Xuan and Tian Feng and Okagaki, Peter and Marini, Ann M.},
abstractNote = {Organophosphorus (OP) compounds, used as insecticides and chemical warfare agents, are potent neurotoxins. We examined the neurotoxic effect of paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), an organophosphate compound, and the role of NMDA receptors as a mechanism of action in cultured cerebellar granule cells. Paraoxon is neurotoxic to cultured rat cerebellar granule cells in a time- and concentration-dependent manner. Cerebellar granule cells are less sensitive to the neurotoxic effects of paraoxon on day in vitro (DIV) 4 than neurons treated on DIV 8. Surprisingly, the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, enhances paraoxon-mediated neurotoxicity suggesting that NMDA receptors may play a protective role. Pretreatment with a subtoxic concentration of N-methyl-D-aspartate (NMDA) [100 {mu}M] protects about 40% of the vulnerable neurons that would otherwise die from paraoxon-induced neurotoxicity. Moreover, addition of a neuroprotective concentration of NMDA 3 h after treatment with paraoxon provides the same level of protection. Because paraoxon-mediated neuronal cell death is time-dependent, we hypothesized that apoptosis may be involved. Paraoxon increases apoptosis about 10-fold compared to basal levels. The broad-spectrum caspase inhibitor (Boc-D-FMK) and the caspase-9-specific inhibitor (Z-LEHD-FMK) protect against paraoxon-mediated apoptosis, paraoxon-stimulated caspase-3 activity and neuronal cell death. MK-801 increases, whereas NMDA blocks paraoxon-induced apoptosis and paraoxon-stimulated caspase-3 activity. These results suggest that activation of NMDA receptors protect neurons against paraoxon-induced neurotoxicity by blocking apoptosis initiated by paraoxon.},
doi = {10.1016/j.taap.2005.01.018},
journal = {Toxicology and Applied Pharmacology},
number = 1,
volume = 208,
place = {United States},
year = 2005,
month =
}
  • Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL,more » EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC{sub 50} values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k{sub inact}/K{sub I}) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC{sub 50} values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1 and CES2 respectively. Together, the results define the kinetics of inhibition of three important hydrolytic enzymes by activated metabolites of widely used agrochemicals. -- Highlights: ► IC{sub 50} values and bimolecular rate constants (k{sub inact}/K{sub I}) of human recombinant CES1, CES2, and MGL proteins and chlorpyrifos oxon, paraoxon and methyl paraoxon were determined. ► The IC{sub 50} values for the oxons with CES1, CES2, and MGL followed the rank order: chlorpyrifos oxon > paraoxon > methyl paraoxon. ► The order of reactivity for the oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. ► Chlorpyrifos oxon was less reactive with MGL than with either CES1 or CES2.« less
  • Recent data suggest that anesthetic drugs cause neurodegeneration during development. Ketamine is frequently used in infants and toddlers for elective surgeries. The purpose of this study is to determine whether glycogen synthase kinase-3 (GSK-3) is involved in ketamine-induced apoptosis. Ketamine increased apoptotic cell death with morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation. In addition, insulin growth factor-1 completely blocked the ketamine-induced apoptotic cell death. Ketamine decreased Akt phosphorylation. GSK-3 is known as a downstream target of Akt. The selective inhibitors of GSK-3 prevented the ketamine-induced apoptosis. Moreover, caspase-3 activation was accompanied by the ketamine-induced cellmore » death and inhibited by the GSK-3 inhibitors. These results suggest that activation of GSK-3 is involved in ketamine-induced apoptosis in rat cortical neurons.« less
  • The inhibition of N-methyl-D-aspartate (NMDA)-induced ({sup 3}H)norepinephrine (({sup 3}H)NE) release by a putrescine analog was studied. We report that arcaine, diguanidinobutane, a putative competitive polyamine antagonist, completely and noncompetitively antagonized NMDA-induced ({sup 3}H)NE release from rat hippocampal minces with an IC50 value of 102 microM. Arcaine did not alter kainate- or potassium-induced ({sup 3}H)NE release suggesting a specific effect on NMDA-mediated responses. Spermidine did not alter NMDA-induced ({sup 3}H)NE release, nor did it reverse the effect of arcaine when introduced in a normal physiologic superfusion buffer. However, spermidine reversed the effect of arcaine when superfusing with buffer that contained 5%more » (v/v) of the organic solvent dimethylsulfoxide. This finding suggests that the polyamine site may be located at the intracellular surface of the cell membrane. Our results provide the first evidence for polyamine modulation of the NMDA receptor ionophore complex in a functional physiologic system.« less
  • Ro 15-4513 (ethyl-8-azido-5, 6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5{alpha}), (1,4) benzodiazepine-3-carboxylate) is reported to be a selective ethanol antagonist in biochemical and behavioral studies. The effect of chronic ethanol treatment on the binding of ({sup 3}H)Ro 15-4513 was investigated in cultured spinal cord neurons, which are shown to possess all the elements of GABA benzodiazepine receptor complex. Chronic ethanol treatment (50 mM for 6 hr, 12 hr, 18 hr, 3 days, and 5{sub 3} days) produced an increase in the specific binding of ({sub 3}H)Ro 15-4513. The increase in binding in these neurons was due to an increase in the number (B{sub max}) ofmore » receptor sites. This effect was specific for Ro 15-4513, since identical ethanol treatment did not alter the binding of benzodiazepine antagonist ({sup 3}H)Ro 15-1788 or agonist ({sup 3}H)flunitrazepam or inverse agonist ({sup 3}H)methyl-{beta}-carboline-3-carboxylate. Similar results have been reported following chronic ethanol treatment to rats. These results suggest that the Ro 15-4513 binding sites on the oligomeric GABA receptor complex are altered following chronic ethanol administration, and support the notion of a unique role of Ro 15-4513 as an ethanol antagonist.« less
  • In the presence of 1.2 mM Mg2+, glycine (30-100 microM) inhibited (3H)dopamine ((3H)DA) release stimulated by N-methyl-D-aspartate (NMDA), in fetal rat mesencephalic cell cultures. Strychnine (1 microM) blocked the inhibitory effect of 100 microM glycine, indicating an action via strychnine-sensitive inhibitory glycine receptors. A higher concentration of strychnine (100 microM), by itself, inhibited NMDA-evoked (3H)DA release in the presence or absence of Mg2+. Spontaneous (3H)DA release and (3H)DA release stimulated by kainate and quisqualate were unaffected by glycine (less than or equal to 100 microM) or strychnine (less than or equal to 100 microM), indicating that glycine and strychnine modulatorymore » effects are only associated with the NMDA receptor subtype. (3H)DA release evoked by K+ (56 mM) was unaffected by glycine (less than or equal to 100 microM) but was attenuated by a high concentration of strychnine (100 microM). In the absence of exogenous Mg2+, glycine (30-100 microM) potentiated NMDA-evoked (3H)DA release by a strychnine-insensitive mechanism. A selective antagonist of the NMDA-associated glycine receptor, 7-chlorokynurenate (10 microM), attenuated NMDA-evoked (3H)DA release in the absence of Mg2+. The effect of 10 microM 7-chlorokynurenate was overcome by 1 microM glycine. Also, when tested in the presence of 1.2 nM Mg2+ and 1 microM strychnine, 100 microM 7-chlorokynurenate inhibited NMDA-evoked (3H)DA release, and this antagonism was overcome by 30 to 100 microM glycine. These results indicate that two distinct glycine receptors modulate NMDA-stimulated (3H)DA release from mesencephalic cells in culture. Manipulation of extracellular Mg2+ permits the differentiation of a strychnine-sensitive glycine response (inhibition of NMDA-evoked (3H)DA release) from a strychnine-insensitive glycine response.« less