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Colin L Willis - One of the best experts on this subject based on the ideXlab platform.
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methylation of the nmda receptor agonist l trans 2 3 pyrrolidine dicarboxylate enhanced excitotoxic potency and selectivity
Toxicology and Applied Pharmacology, 1997Co-Authors: Colin L Willis, Danielle L Dauenhauer, John Michael Humphrey, Richard A Chamberlin, Amy L Buller, Daniel T Monaghan, Richard J BridgesAbstract:This study investigated the excitotoxic properties of a novel series of NMDA analogues in which a methyl group was introduced to the 5-position of the pyrrolidine ring ofl-trans-2,3-PDC, a previously identified NMDA receptor agonist. While all of these compounds induced NMDA-receptor-mediated injury, methylation increasedin vivoexcitotoxic potency 1000-fold. Injections (1 μl) in rat dorsal hippocampus ofcis- andtrans-5-methyl-l-trans-2,3-PDC (0.1 nmol) induced 50–70% neuronal damage to areas CA1 and CA4, comparable to that induced by 100 nmol ofl-trans-2,3-PDC. Further,cis- andtrans-methylated analogues induced distinct patterns of hippocampal pathology consistent with differential excitotoxic vulnerability of neurons expressing NMDA receptors. Neuronal damage produced by the 5-methyl-l-trans-2,3-PDCs could be blocked by coadministration of MK-801 (3 mg/kg ip), but not NBQX (25 nmol). Biochemical and physiological assays confirmed the action of the analogues as NMDA agonists, but did not provide an explanation for differences in excitotoxic potency between the methylated and nonmethylated 2,3-PDCs. For example, the activity of the compounds as inhibitors of3H-glutamate binding (IC50 values: 0.4, 1.4, and 1.2 μmforcis-5-methyl-,trans-5-methyl-, andl-trans-2,3-PDC, respectively), agonists at NR1A/NR2B receptors (EC50values: 5, 49, and 16 μmforcis-5-methyl-,trans-5-methyl-, andl-trans-2,3-PDC, respectively), andin vitroExcitotoxins in cortical cultures varied only two- to fivefold as a consequence of methylation. Potential roles of NMDA receptor subtypes and transport in these effects are discussed. As potent and selective NMDA Excitotoxins,cis- andtrans-5-methyl-l-trans-2,3-PDC will be of value studying excitotoxic mechanisms, NMDA-receptor-mediated pathology, and NMDA receptor heterogeneity.
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l trans 2 3 pyrrolidine dicarboxylate characterization of a novel Excitotoxin
Neuropharmacology, 1996Co-Authors: Colin L Willis, John Michael Humphrey, Hans P Koch, T Lakely, L Ralsto, C A Ake, S Shim, M Kadri, A R Chamberli, Richard J IdgesAbstract:This study investigated the in vitro and in vivo excitotoxic properties of a novel conformationally constrained analogue of L-trans-2,3-pyrrolidine dicarboxylate (L-trans-2,3-PDC). When tested for excitotoxic activity in rat cortical cultures, L-trans-2,3-PDC mimicked the action of NMDA in both acute (30 min) and chronic (24 hr) exposure paradigms. This neurotoxicity was attenuated by co-addition of MK-801 (10 μM). Microinjections of L-trans-2,3-PDC into the dorsal hippocampus of male rats also induced a selective pattern of pathology indicative of an NMDA receptor Excitotoxin. In contrast to the equipotency observed in vitro, 100 nmol of L-trans-2,3-PDC were needed to produce cellular damage comparable to that induced by 25 nmol of NMDA. Consistent with an action at NMDA receptors, L-trans-2,3-PDC-induced damage could be significantly reduced by co-administration of MK-801 (3 mg/kg i.p.), but not by NBQX (25 nmol). In radioligand binding assays L-trans-2,3-PDC inhibited the binding of 3H-L-glutamate to NMDA receptors (IC50 1 μM), although it also exhibited some cross reactivity with KA and AMPA receptors. L-trans-2,3-PDC was also identified as a competitive inhibitor (Ki = 33 μM) of 3H-D-aspartate uptake into rat forebrain synaptosomes. In contrast to the action of a transporter substrate, such as L-glutamate, L-trans-2,3-PDC did not exchange with 3H-D-aspartate that had been previously loaded into the synaptosomes. Copyright © 1996 Elsevier Science Ltd.
Richard J Idges - One of the best experts on this subject based on the ideXlab platform.
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l trans 2 3 pyrrolidine dicarboxylate characterization of a novel Excitotoxin
Neuropharmacology, 1996Co-Authors: Colin L Willis, John Michael Humphrey, Hans P Koch, T Lakely, L Ralsto, C A Ake, S Shim, M Kadri, A R Chamberli, Richard J IdgesAbstract:This study investigated the in vitro and in vivo excitotoxic properties of a novel conformationally constrained analogue of L-trans-2,3-pyrrolidine dicarboxylate (L-trans-2,3-PDC). When tested for excitotoxic activity in rat cortical cultures, L-trans-2,3-PDC mimicked the action of NMDA in both acute (30 min) and chronic (24 hr) exposure paradigms. This neurotoxicity was attenuated by co-addition of MK-801 (10 μM). Microinjections of L-trans-2,3-PDC into the dorsal hippocampus of male rats also induced a selective pattern of pathology indicative of an NMDA receptor Excitotoxin. In contrast to the equipotency observed in vitro, 100 nmol of L-trans-2,3-PDC were needed to produce cellular damage comparable to that induced by 25 nmol of NMDA. Consistent with an action at NMDA receptors, L-trans-2,3-PDC-induced damage could be significantly reduced by co-administration of MK-801 (3 mg/kg i.p.), but not by NBQX (25 nmol). In radioligand binding assays L-trans-2,3-PDC inhibited the binding of 3H-L-glutamate to NMDA receptors (IC50 1 μM), although it also exhibited some cross reactivity with KA and AMPA receptors. L-trans-2,3-PDC was also identified as a competitive inhibitor (Ki = 33 μM) of 3H-D-aspartate uptake into rat forebrain synaptosomes. In contrast to the action of a transporter substrate, such as L-glutamate, L-trans-2,3-PDC did not exchange with 3H-D-aspartate that had been previously loaded into the synaptosomes. Copyright © 1996 Elsevier Science Ltd.
John Michael Humphrey - One of the best experts on this subject based on the ideXlab platform.
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methylation of the nmda receptor agonist l trans 2 3 pyrrolidine dicarboxylate enhanced excitotoxic potency and selectivity
Toxicology and Applied Pharmacology, 1997Co-Authors: Colin L Willis, Danielle L Dauenhauer, John Michael Humphrey, Richard A Chamberlin, Amy L Buller, Daniel T Monaghan, Richard J BridgesAbstract:This study investigated the excitotoxic properties of a novel series of NMDA analogues in which a methyl group was introduced to the 5-position of the pyrrolidine ring ofl-trans-2,3-PDC, a previously identified NMDA receptor agonist. While all of these compounds induced NMDA-receptor-mediated injury, methylation increasedin vivoexcitotoxic potency 1000-fold. Injections (1 μl) in rat dorsal hippocampus ofcis- andtrans-5-methyl-l-trans-2,3-PDC (0.1 nmol) induced 50–70% neuronal damage to areas CA1 and CA4, comparable to that induced by 100 nmol ofl-trans-2,3-PDC. Further,cis- andtrans-methylated analogues induced distinct patterns of hippocampal pathology consistent with differential excitotoxic vulnerability of neurons expressing NMDA receptors. Neuronal damage produced by the 5-methyl-l-trans-2,3-PDCs could be blocked by coadministration of MK-801 (3 mg/kg ip), but not NBQX (25 nmol). Biochemical and physiological assays confirmed the action of the analogues as NMDA agonists, but did not provide an explanation for differences in excitotoxic potency between the methylated and nonmethylated 2,3-PDCs. For example, the activity of the compounds as inhibitors of3H-glutamate binding (IC50 values: 0.4, 1.4, and 1.2 μmforcis-5-methyl-,trans-5-methyl-, andl-trans-2,3-PDC, respectively), agonists at NR1A/NR2B receptors (EC50values: 5, 49, and 16 μmforcis-5-methyl-,trans-5-methyl-, andl-trans-2,3-PDC, respectively), andin vitroExcitotoxins in cortical cultures varied only two- to fivefold as a consequence of methylation. Potential roles of NMDA receptor subtypes and transport in these effects are discussed. As potent and selective NMDA Excitotoxins,cis- andtrans-5-methyl-l-trans-2,3-PDC will be of value studying excitotoxic mechanisms, NMDA-receptor-mediated pathology, and NMDA receptor heterogeneity.
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l trans 2 3 pyrrolidine dicarboxylate characterization of a novel Excitotoxin
Neuropharmacology, 1996Co-Authors: Colin L Willis, John Michael Humphrey, Hans P Koch, T Lakely, L Ralsto, C A Ake, S Shim, M Kadri, A R Chamberli, Richard J IdgesAbstract:This study investigated the in vitro and in vivo excitotoxic properties of a novel conformationally constrained analogue of L-trans-2,3-pyrrolidine dicarboxylate (L-trans-2,3-PDC). When tested for excitotoxic activity in rat cortical cultures, L-trans-2,3-PDC mimicked the action of NMDA in both acute (30 min) and chronic (24 hr) exposure paradigms. This neurotoxicity was attenuated by co-addition of MK-801 (10 μM). Microinjections of L-trans-2,3-PDC into the dorsal hippocampus of male rats also induced a selective pattern of pathology indicative of an NMDA receptor Excitotoxin. In contrast to the equipotency observed in vitro, 100 nmol of L-trans-2,3-PDC were needed to produce cellular damage comparable to that induced by 25 nmol of NMDA. Consistent with an action at NMDA receptors, L-trans-2,3-PDC-induced damage could be significantly reduced by co-administration of MK-801 (3 mg/kg i.p.), but not by NBQX (25 nmol). In radioligand binding assays L-trans-2,3-PDC inhibited the binding of 3H-L-glutamate to NMDA receptors (IC50 1 μM), although it also exhibited some cross reactivity with KA and AMPA receptors. L-trans-2,3-PDC was also identified as a competitive inhibitor (Ki = 33 μM) of 3H-D-aspartate uptake into rat forebrain synaptosomes. In contrast to the action of a transporter substrate, such as L-glutamate, L-trans-2,3-PDC did not exchange with 3H-D-aspartate that had been previously loaded into the synaptosomes. Copyright © 1996 Elsevier Science Ltd.
Carmelo Romano - One of the best experts on this subject based on the ideXlab platform.
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susceptibilities to and mechanisms of excitotoxic cell death of adult mouse inner retinal neurons in dissociated culture
Investigative Ophthalmology & Visual Science, 2004Co-Authors: Akemichi Baba, Carmelo Romano, Toshio MatsudaAbstract:PURPOSE. To explore the susceptibilities of adult retinal neurons in dissociated culture to treatments with excitotoxic agonists and the mechanisms of the resultant retinal cell death. METHODS. C57B6 mice were used. Retinas were removed, dissociated, plated on a polylysine/laminin substrate, and maintained in vitro for 5 to 7 days. Excitotoxic agonists (glutamate, N-methyl-D-aspartate [NMDA], or kainic acid [KA]) were added for 30 minutes or 24 hours, sometimes in the presence of modified extracellular ion concentrations or potential blocking agents. The next day, cells were fixed and immunocytochemically stained to identify ganglion and amacrine cells. Surviving cells were counted. RESULTS. Ganglion cells from adult mouse retinas were much less susceptible to excitotoxic death than those prepared from neonatal retinas. Adult amacrine cells were killed by KA, NMDA, or glutamate. Experiments with selective blockers demonstrated that KA killed through AMPA (-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, whereas NMDA and glutamate exerted toxicity through a combination of AMPA and NMDA receptors. The KA-induced death of amacrine cells was not mediated by chloride ions. Removal of extracellular sodium, however, completely prevented the amacrine cell death, and removal of extracellular calcium prevented approximately 70% of the death. The path of calcium entry was investigated. Experiments with selective blockers indicated that the lethal calcium entry was via reverse operation of a sodium-calcium exchanger. CONCLUSIONS. There is a profound developmental regulation in the sensitivity of retina ganglion cells to excitotoxic insults. Excessive intracellular sodium and calcium are the proximal causes of amacrine cell death. The pathologic calcium entry is dependent on the sodium overload, which then drives a sodium‐calcium exchanger to take up calcium. (Invest Ophthalmol Vis Sci. 2004;45:4576‐4582) DOI:10.1167/iovs.04-0166
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delayed excitotoxic neurodegeneration induced by excitatory amino acid agonists in isolated retina
Journal of Neurochemistry, 2002Co-Authors: Carmelo Romano, M T Price, John W OlneyAbstract:: Evidence from in vitro studies suggests that excitotoxic neuronal degeneration can occur by either an acute or delayed mechanism. Studies of the acute mechanism in isolated chick embryo retina using histological methods indicate that this process is rapidly triggered by activation of glutamate receptors of either the N-methyl-d-aspartate (NMDA) or non-NMDA subtypes. The delayed mechanism, studied primarily in cortical and hippocampal cell cultures prepared from embryonic rodent brain, requires activation of NMDA receptors. In these cell culture systems, stimulation of non-NMDA receptors does not rapidly trigger delayed neuronal degeneration, or does so only indirectly, via activation of NMDA receptors secondary to glutamate release. To provide a more valid basis for comparison of these two mechanisms, we have modified the isolated chick embryo retina model to permit studies of delayed as well as acute excitotoxic neurodegeneration. Retinas maintained for 24 h exhibited no morphological or biochemical signs of damage. Retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium at various times after exposure to agonists and normalized to total LDH in each retina. Glutamate exposure (1 mM, 30 min) did not result in LDH release by the end of the exposure period, but LDH was released over the following 24 h. Briefer periods also led to substantial LDH release. Incubation in the presence of NMDA, or the non-NMDA agonists kainate (KA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), led rapidly to delayed LDH release. NMDA and AMPA were more potent than glutamate, but high concentrations of glutamate led to more LDH release than high concentrations of these agonists. KA was a powerful Excitotoxin, providing more LDH release than glutamate, NMDA, or AMPA at every concentration tested. The delayed LDH release induced by glutamate involved activation of both NMDA and non-NMDA receptors, as a combination of receptor-selective antagonists was necessary to provide complete blockade. These results indicate that glutamate, NMDA, AMPA, and KA all cause delayed as well as acute excitotoxic damage in the retina. It is interesting that brief exposure to the non-NMDA receptor agonists, in relatively low concentrations, led to delayed LDH release. This is different than in other in vitro models of delayed excitotoxic neurodegeneration.
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ca2 independent excitotoxic neurodegeneration in isolated retina an intact neural net a role for cl and inhibitory transmitters
Molecular Pharmacology, 1998Co-Authors: Quan Chen, Todd Almli, Peter D. Lukasiewicz, John W Olney, Carmelo RomanoAbstract:Rapidly triggered excitotoxic cell death is widely thought to be due to excessive influx of extracellular Ca2+, primarily through the N -methyl-d-aspartate subtype of glutamate receptor. By devising conditions that permit the maintenance of isolated retina in the absence of Ca2+, it has become technically feasible to test the dependence of excitotoxic neurodegeneration in this intact neural system on extracellular Ca2+. Using biochemical, Ca2+ imaging, and electrophysiological techniques, we found that (1) rapidly triggered excitotoxic cell death in this system occurs independently of both extracellular Ca2+ and increases in intracellular Ca2+; (2) this cell death is highly dependent on extracellular Cl−; and (3) lethal Cl− entry occurs by multiple paths, but a significant fraction occurs through pathologically activated γ-aminobutyric acid and glycine receptors. These results emphasize the importance of Ca2+-independent mechanisms and the role that local transmitter circuitry plays in excitotoxic cell death.
Richard J Bridges - One of the best experts on this subject based on the ideXlab platform.
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methylation of the nmda receptor agonist l trans 2 3 pyrrolidine dicarboxylate enhanced excitotoxic potency and selectivity
Toxicology and Applied Pharmacology, 1997Co-Authors: Colin L Willis, Danielle L Dauenhauer, John Michael Humphrey, Richard A Chamberlin, Amy L Buller, Daniel T Monaghan, Richard J BridgesAbstract:This study investigated the excitotoxic properties of a novel series of NMDA analogues in which a methyl group was introduced to the 5-position of the pyrrolidine ring ofl-trans-2,3-PDC, a previously identified NMDA receptor agonist. While all of these compounds induced NMDA-receptor-mediated injury, methylation increasedin vivoexcitotoxic potency 1000-fold. Injections (1 μl) in rat dorsal hippocampus ofcis- andtrans-5-methyl-l-trans-2,3-PDC (0.1 nmol) induced 50–70% neuronal damage to areas CA1 and CA4, comparable to that induced by 100 nmol ofl-trans-2,3-PDC. Further,cis- andtrans-methylated analogues induced distinct patterns of hippocampal pathology consistent with differential excitotoxic vulnerability of neurons expressing NMDA receptors. Neuronal damage produced by the 5-methyl-l-trans-2,3-PDCs could be blocked by coadministration of MK-801 (3 mg/kg ip), but not NBQX (25 nmol). Biochemical and physiological assays confirmed the action of the analogues as NMDA agonists, but did not provide an explanation for differences in excitotoxic potency between the methylated and nonmethylated 2,3-PDCs. For example, the activity of the compounds as inhibitors of3H-glutamate binding (IC50 values: 0.4, 1.4, and 1.2 μmforcis-5-methyl-,trans-5-methyl-, andl-trans-2,3-PDC, respectively), agonists at NR1A/NR2B receptors (EC50values: 5, 49, and 16 μmforcis-5-methyl-,trans-5-methyl-, andl-trans-2,3-PDC, respectively), andin vitroExcitotoxins in cortical cultures varied only two- to fivefold as a consequence of methylation. Potential roles of NMDA receptor subtypes and transport in these effects are discussed. As potent and selective NMDA Excitotoxins,cis- andtrans-5-methyl-l-trans-2,3-PDC will be of value studying excitotoxic mechanisms, NMDA-receptor-mediated pathology, and NMDA receptor heterogeneity.