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John E. Casida - One of the best experts on this subject based on the ideXlab platform.
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Cellular function of Neuropathy Target Esterase in lysophosphatidylcholine action
Toxicology and applied pharmacology, 2008Co-Authors: Sarah C. Vose, Kazutoshi Fujioka, Alexander G. Gulevich, Amy Y. Lin, Nina Holland, John E. CasidaAbstract:Neuropathy Target Esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary Target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.
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evidence that mouse brain Neuropathy Target Esterase is a lysophospholipase
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Gary B. Quistad, Christopher J. Winrow, Carrolee Barlow, Susan E Sparks, John E. CasidaAbstract:Abstract Neuropathy Target Esterase (NTE) is inhibited by several organophosphorus (OP) pesticides, chemical warfare agents, lubricants, and plasticizers, leading to OP-induced delayed Neuropathy in people (>30,000 cases of human paralysis) and hens (the best animal model for this demyelinating disease). The active site region of NTE as a recombinant protein preferentially hydrolyzes lysolecithin, suggesting that this enzyme may be a type of lysophospholipase (LysoPLA) with lysolecithin as its physiological substrate. This hypothesis is tested here in mouse brain by replacing the phenyl valerate substrate of the standard NTE assay with lysolecithin for an “NTE-LysoPLA” assay with four important findings. First, NTE-LysoPLA activity, as the NTE activity, is 41–45% lower in Nte-haploinsufficient transgenic mice than in their wild-type littermates. Second, the potency of six delayed neurotoxicants or toxicants as in vitro inhibitors varies from IC50 0.02 to 13,000 nM and is essentially the same for NTE-LysoPLA and NTE (r2 = 0.98). Third, the same six delayed toxicants administered i.p. to mice at multiple doses inhibit brain NTE-LysoPLA and NTE to the same extent (r2 = 0.90). Finally, their in vivo inhibition of brain NTE-LysoPLA generally correlates with delayed toxicity. Therefore, OP-induced delayed toxicity in mice, and possibly the hyperactivity associated with NTE deficiency, may be due to NTE-LysoPLA inhibition, leading to localized accumulation of lysolecithin, a known demyelinating agent and receptor-mediated signal transducer. This mouse model has some features in common with OP-induced delayed Neuropathy in hens and people but differs in the neuropathological signs and apparently the requirement for NTE aging.
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Loss of Neuropathy Target Esterase in mice links organophosphate exposure to hyperactivity
Nature Genetics, 2003Co-Authors: Christopher J. Winrow, Matthew L. Hemming, Duane M. Allen, Gary B. Quistad, John E. Casida, Carrolee BarlowAbstract:Neuropathy Target Esterase (NTE) is involved in neural development and is the Target for neurodegeneration induced by selected organophosphorus pesticides and chemical warfare agents. We generated mice with disruptions in Nte , the gene encoding NTE. Nte ^ −/− mice die after embryonic day 8, and Nte ^ +/− mice have lower activity of Nte in the brain and higher mortality when exposed to the Nte-inhibiting compound ethyl octylphosphonofluoridate (EOPF) than do wild-type mice. Nte ^ +/− and wild-type mice treated with 1 mg per kg of body weight of EOPF have elevated motor activity, showing that even minor reduction of Nte activity leads to hyperactivity. These studies show that genetic or chemical reduction of Nte activity results in a neurological phenotype of hyperactivity in mammals and indicate that EOPF toxicity occurs directly through inhibition of Nte without the requirement for Nte gain of function or aging.
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Localization of [3H]octylphosphonyl-labeled Neuropathy Target Esterase by chicken nervous tissue autoradiography
Neuroscience letters, 1999Co-Authors: Michihiro Kamijima, John E. CasidaAbstract:Abstract Neuropathy Target Esterase (NTE) undergoes phosphorylation and aging as the initial steps in organophosphorus (OP)-induced delayed Neuropathy (OPIDN). Localization of NTE is an important step in characterizing the mechanism of OPIDN. Earlier histochemical immunoreactivity or Esterase assays localized NTE in areas of the brain and spinal cord rich in neuronal cell bodies and in the dorsal root ganglion. We use a more direct and quantitative autoradiographic approach of forming phosphorylated and aged [3H]octylphosphonyl-NTE on treatment with the highly potent [octyl–3H]octyl-4H-1,3,2-benzodioxaphosphorin 2-oxide to determine NTE as the labeling site resistant to the non-neuropathic paraoxon and sensitive to the neuropathic mipafox. NTE is observed in the cerebral cortical layer, some layers of the optic tectum, the gray matter of the spinal cord and the sensory neurons of the dorsal root ganglion to a higher extent than in adjacent areas.
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Organophosphorus Neuropathy Target Esterase inhibitors selectively block outgrowth of neurite-like and cell processes in cultured cells
Toxicology letters, 1998Co-Authors: John E. CasidaAbstract:This study compares two direct-acting Neuropathy Target Esterase (NTE) inhibitors (mipafox and 2-octyl-4H-1,3,2-benzodioxophosphorin 2-oxide (OBDPO)), a metabolic precursor to an NTE inhibitor (tri-o-cresyl phosphate or TOCP) and a potent acetylcholinEsterase inhibitor (chlorpyrifos oxon or CPO) for their effects on outgrowth of neurite-like and cell processes and on viability in differentiated cultured cells (rat adrenal pheochromocytoma (PC-12) and brain glial tumor (C6)). The direct-acting NTE inhibitors block process outgrowth by 50% or more at 50–100 μM for OBDPO and 100–200 μM for mipafox, well below their cytotoxic levels (EC50 values, 445–474 μM for OBDPO and 1021–1613 μM for mipafox). In contrast, the effects on process development for TOCP and CPO parallel their cytotoxicity. These findings suggest that inhibition of neurite-like and cell process outgrowth by OBDPO and mipafox may be associated with NTE inhibition.
Paul Glynn - One of the best experts on this subject based on the ideXlab platform.
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Neuropathy Target Esterase (NTE/PNPLA6) and organophosphorus compound-induced delayed neurotoxicity (OPIDN).
Advances in neurotoxicology, 2020Co-Authors: Rudy J Richardson, Robert B Hufnagel, John K. Fink, Paul Glynn, Galina F. Makhaeva, Sanjeeva J WijeyesakereAbstract:Abstract Systemic inhibition of Neuropathy Target Esterase (NTE) with certain organophosphorus (OP) compounds produces OP compound-induced delayed neurotoxicity (OPIDN), a distal degeneration of axons in the central nervous system (CNS) and peripheral nervous system (PNS), thereby providing a powerful model for studying a spectrum of neurodegenerative diseases. Axonopathies are important medical entities in their own right, but in addition, illnesses once considered primary neuronopathies are now thought to begin with axonal degeneration. These disorders include Alzheimer's disease, Parkinson's disease, and motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Moreover, conditional knockout of NTE in the mouse CNS produces vacuolation and other degenerative changes in large neurons in the hippocampus, thalamus, and cerebellum, along with degeneration and swelling of axons in ascending and descending spinal cord tracts. In humans, NTE mutations cause a variety of neurodegenerative conditions resulting in a range of deficits including spastic paraplegia and blindness. Mutations in the Drosophila NTE orthologue SwissCheese (SWS) produce neurodegeneration characterized by vacuolization that can be partially rescued by expression of wild-type human NTE, suggesting a potential therapeutic approach for certain human neurological disorders. This chapter defines NTE and OPIDN, presents an overview of OP compounds, provides a rationale for NTE research, and traces the history of discovery of NTE and its relationship to OPIDN. It then briefly describes subsequent studies of NTE, including practical applications of the assay; aspects of its domain structure, subcellular localization, and tissue expression; abnormalities associated with NTE mutations, knockdown, and conventional or conditional knockout; and hypothetical models to help guide future research on elucidating the role of NTE in OPIDN.
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Neuropathy Target Esterase nte pnpla6 and organophosphorus compound induced delayed neurotoxicity opidn
Advances in neurotoxicology, 2020Co-Authors: Rudy J Richardson, G F Makhaeva, Robert B Hufnagel, John K. Fink, Paul Glynn, Sanjeeva J WijeyesakereAbstract:Abstract Systemic inhibition of Neuropathy Target Esterase (NTE) with certain organophosphorus (OP) compounds produces OP compound-induced delayed neurotoxicity (OPIDN), a distal degeneration of axons in the central nervous system (CNS) and peripheral nervous system (PNS), thereby providing a powerful model for studying a spectrum of neurodegenerative diseases. Axonopathies are important medical entities in their own right, but in addition, illnesses once considered primary neuronopathies are now thought to begin with axonal degeneration. These disorders include Alzheimer's disease, Parkinson's disease, and motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Moreover, conditional knockout of NTE in the mouse CNS produces vacuolation and other degenerative changes in large neurons in the hippocampus, thalamus, and cerebellum, along with degeneration and swelling of axons in ascending and descending spinal cord tracts. In humans, NTE mutations cause a variety of neurodegenerative conditions resulting in a range of deficits including spastic paraplegia and blindness. Mutations in the Drosophila NTE orthologue SwissCheese (SWS) produce neurodegeneration characterized by vacuolization that can be partially rescued by expression of wild-type human NTE, suggesting a potential therapeutic approach for certain human neurological disorders. This chapter defines NTE and OPIDN, presents an overview of OP compounds, provides a rationale for NTE research, and traces the history of discovery of NTE and its relationship to OPIDN. It then briefly describes subsequent studies of NTE, including practical applications of the assay; aspects of its domain structure, subcellular localization, and tissue expression; abnormalities associated with NTE mutations, knockdown, and conventional or conditional knockout; and hypothetical models to help guide future research on elucidating the role of NTE in OPIDN.
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Neuropathy Target Esterase Is Required for Adult Vertebrate Axon Maintenance
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2009Co-Authors: David J. Read, Moses V. Chao, John Cavanagh, Paul GlynnAbstract:The enzyme Neuropathy Target Esterase (NTE) is present in neurons and deacylates the major membrane phospholipid, phosphatidylcholine (PtdCho). Mutation of the NTE gene or poisoning by neuropathic organophosphates—chemical inhibitors of NTE—causes distal degeneration of long spinal axons in humans. However, analogous neuropathological changes have not been reported in nestin-cre:NTEfl/fl mice with NTE-deficient neural tissue. Furthermore, altered PtdCho homeostasis has not been detected in NTE-deficient vertebrates. Here, we describe distal degeneration of the longest spinal axons in ∼3-week-old nestin-cre:NTEfl/fl mice and in adult C57BL/6J mice after acute dosing with a neuropathic organophosphate: in both groups early degenerative lesions were followed by swellings comprising accumulated axoplasmic material. In mice dosed acutely with organophosphate, maximal numbers of lesions, in the longest spinal sensory axon tract, were attained within days and were preceded by a transient rise in neural PtdCho. In nestin-cre:NTEfl/fl mice, sustained elevation of PtdCho over many months was accompanied by progressive degeneration and massive swelling of axons in sensory and motor spinal tracts and by increasing hindlimb dysfunction. Axonal lesion distribution closely resembled that in hereditary spastic paraplegia (HSP). The importance of defective membrane trafficking in HSP and the association of NTE with the endoplasmic reticulum—the starting point for the constitutive secretory pathway and transport of neuronal materials into axons—prompted investigation for a role of NTE in secretion. Cultured NTE-deficient neurons displayed modestly impaired secretion, consistent with neuronal viability and damage in vivo initially restricted to distal parts of the longest axons.
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Axonal degeneration and Neuropathy Target Esterase.
Arhiv za higijenu rada i toksikologiju, 2007Co-Authors: Paul GlynnAbstract:This brief review summarizes recent observations which suggest a possible mechanism for organophosphate-induced delayed Neuropathy (OPIDN). Neuropathy Target Esterase (NTE) has been shown to deacylate endoplasmic reticulum (ER) membrane phosphatidylcholine (PtdCho). Raised levels of PtdCho are present in the brains of Swiss cheese/NTE mutant Drosophila together with abnormal membrane structures, axonal and dendritic degeneration and neural cell loss. Similar vacuolated pathology is found in the brains of mice with brain-specific deletion of the NTE gene and, in old age, these mice show clinical and histopathological features of Neuropathy resembling those in wild-type mice chronically dosed with tri-ortho-cresylphosphate. It is suggested that OPIDN results from the loss of NTE's phospholipase activity which in turn causes ER malfunction and perturbation of axonal transport and glial-axonal interactions.
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Neuropathy Target Esterase and phospholipid deacylation.
Biochimica et biophysica acta, 2005Co-Authors: Paul GlynnAbstract:Abstract Certain organophosphates react with the active site serine residue of Neuropathy Target Esterase (NTE) and cause axonal degeneration and paralysis. Cloning of NTE revealed the presence of homologues in eukaryotes from yeast to man and that the protein has both a catalytic and a regulatory domain. The latter contains sequences similar to the regulatory subunit of protein kinase A, suggesting that NTE may bind cyclic AMP. NTE is tethered via an amino-terminal transmembrane segment to the cytoplasmic face of the endoplasmic reticulum. Unlike wild-type yeast, mutants lacking NTE activity cannot deacylate CDP–choline pathway-synthesized phosphatidylcholine (PtdCho) to glycerophosphocholine (GroPCho) and fatty acids. In cultured mammalian cells, GroPCho levels rise and fall, respectively, in response to experimental over-expression, and inhibition, of NTE. A complex of PtdCho and Sec14p, a yeast phospholipid-binding protein, both inhibits the rate-limiting step in PtdCho synthesis and enhances deacylation of PtdCho by NTE. While yeast can maintain PtdCho homeostasis in the absence of NTE, certain post-mitotic metazoan cells may not be able to, and some NTE-null animals have deleterious phenotypes. NTE is not required for cell division in the early mammalian embryo or in larval and pupal forms of Drosophila, but is essential for placenta formation and survival of neurons in the adult. In vertebrates, the relative importance of NTE and calcium-independent phospholipase A2 for homeostatic PtdCho deacylation in particular cell types, possible interactions of NTE with Sec14p homologues and cyclic AMP, and whether deranged phospholipid metabolism underlies organophosphate-induced Neuropathy are areas which require further investigation.
Ding-xin Long - One of the best experts on this subject based on the ideXlab platform.
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Effect of Inhibition of Neuropathy Target Esterase in Mouse Nervous Tissues In Vitro
2016Co-Authors: On Phosphatidylcholine, Lysophosphatidylcholine Homeostasis, Wei-yuan Hou, Ding-xin LongAbstract:Neuropathy Target Esterase has been shown to be a lysophospholipase in mouse. The authors investigate the effect of Neuropathy Target Esterase inhibition in mouse nervous tissues in vitro on the homeostasis of phosphatidylcholine and lysophosphatidylcholine by treating the homogenates with tri-ortho-cresyl phos-phate, paraoxon, paraoxon plus mipafox, and phenyl-methylsulfonyl fluoride. The activity of Neuropathy Target Esterase is significantly inhibited by phenyl-methylsulfonyl fluoride and paraoxon plus mipafox but not by paraoxon alone. Tri-ortho-cresyl phosphate slightly but significantly inhibits Neuropathy Target Esterase activity in brain. The levels of phosphatidyl-choline and lysophosphatidylcholine in all 3 nervous tissues are not obviously altered after treatment with tri-ortho-cresyl phosphate, paraoxon, or paraoxon plus mipafox. However, phosphatidylcholine and lysophos-phatidylcholine levels are clearly enhanced by phenyl-methylsulfonyl fluoride. It is concluded that inhibition of Neuropathy Target Esterase in mouse nervous tissues is not enough to disrupt the homeostasis of phosphati-dylcholine and lysophosphatidylcholine and that the upregulation by phenylmethylsulfonyl fluoride may be the consequence of combined inhibition of Neuropathy Target Esterase and other phospholipases
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Neuropathy Target Esterase Is Degraded by the Ubiquitin-Proteasome Pathway with ARA54 as the Ubiquitin Ligase.
Biochemistry, 2015Co-Authors: Ding-xin Long, Pan Wang, Ying-jian Sun, Rui ChenAbstract:Neuropathy Target Esterase (NTE) is an endoplasmic reticulum membrane-associated phospholipase B, which is essential for embryonic and nervous system development. However, the regulation of NTE at the protein level had not been thoroughly investigated. Our previous study showed that NTE was degraded not only by the macroautophagy-lysosome pathway but also by the ubiquitin-proteasome pathway. Here we further reveal that androgen receptor-associated protein 54 (ARA54) regulated the ubiquitin-proteasome degradation of NTE. We find that deletion of the regulatory domain of NTE, which possesses a putative destruction box and thus is essential for its degradation by the proteasome, prevented its degradation by the proteasome. In addition, we demonstrate that ARA54, which has a RING finger domain and E3 ligase activity, interacts directly with NTE. Overexpression of ARA54 downregulates the protein level of NTE, and knockdown of ARA54 inhibits the degradation of NTE. The mutation in the RING domain of ARA54 blocks the degradation of NTE by ARA54, which indicates that the RING domain is essential for ARA54's E3 activity. These findings suggest that ARA54 acts as the ubiquitin ligase to regulate the ubiquitin-proteasome degradation of NTE.
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regulation of Neuropathy Target Esterase by the camp protein kinase a signal
Pharmacological Research, 2010Co-Authors: Jia-xiang Chen, Ding-xin Long, Wei-yuan HouAbstract:As a phospholipase B, Neuropathy Target Esterase (NTE) is responsible for the conversion of phosphatidylcholine (PC) to glycerophosphocholine (GPC). We examined the role of cAMP in the regulation of NTE in mammalian cells. Endogenous NTE activity was increased by cAMP-elevating chemicals, including dibutyryl cAMP, forskolin and forskolin plus 1-isobutyl-3-methylxanthine (IBMX), but decreased by the adenyl cyclase inhibitor SQ22536 which can reduce intracellular cAMP levels. Exogenous GFP-tagged NTE activity was not affected by changes in intracellular cAMP. NTE protein levels were up-regulated by the cAMP-elevating reagents and down-regulated by the inhibitor. The effect of the adenyl cyclase activator forskolin on NTE protein and mRNA levels was blocked by pretreatment with the protein kinase A (PKA) activity inhibitor H89. In addition, we found that changes in GPC, but not PC, levels were correlated with cAMP induced changes in NTE activity. These results are the first evidence that cAMP/PKA signals regulate NTE expression and GPC content in mammalian cells.
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Regulation of Neuropathy Target Esterase by the cAMP/protein kinase A signal.
Pharmacological research, 2010Co-Authors: Jia-xiang Chen, Ding-xin Long, Wei-yuan HouAbstract:As a phospholipase B, Neuropathy Target Esterase (NTE) is responsible for the conversion of phosphatidylcholine (PC) to glycerophosphocholine (GPC). We examined the role of cAMP in the regulation of NTE in mammalian cells. Endogenous NTE activity was increased by cAMP-elevating chemicals, including dibutyryl cAMP, forskolin and forskolin plus 1-isobutyl-3-methylxanthine (IBMX), but decreased by the adenyl cyclase inhibitor SQ22536 which can reduce intracellular cAMP levels. Exogenous GFP-tagged NTE activity was not affected by changes in intracellular cAMP. NTE protein levels were up-regulated by the cAMP-elevating reagents and down-regulated by the inhibitor. The effect of the adenyl cyclase activator forskolin on NTE protein and mRNA levels was blocked by pretreatment with the protein kinase A (PKA) activity inhibitor H89. In addition, we found that changes in GPC, but not PC, levels were correlated with cAMP induced changes in NTE activity. These results are the first evidence that cAMP/PKA signals regulate NTE expression and GPC content in mammalian cells.
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The role of cell cycle-dependent Neuropathy Target Esterase in cell proliferation.
Molecular biology reports, 2010Co-Authors: Ping-an Chang, Yu-ying Chen, Wen-zhen Qin, Ding-xin LongAbstract:Neuropathy Target Esterase (NTE) is a novel phospholipase B and plays a role in phospholipid homeostasis. Although over-expression of NTE inhibits cell division, the role of NTE in cell proliferation is still unknown. In the current study, we firstly used synchronous HeLa cells to study the expression profile of NTE during the cell cycle. NTE protein and activity are regulated during the cell cycle with highest level at G1 and lowest at G2/M phase. However, NTE mRNA levels are constant during the cell cycle. The role of NTE in cell proliferation was investigated by short hairpin RNA (shRNA) to suppress the expression of NTE. Knockdown of NTE significant down-regulated of NTE expression and reduced the glycerophosphocholine level. However, suppression of NTE did not affect phosphatidylcholine content or cell cycle progression. In addition, NTE was demonstrated to be degraded by the ubiquitin-proteasome pathway. These results suggested for the first time that NTE is a cell cycle-dependent protein, but is not essential for cell proliferation, and the ubiquitin-mediated proteolysis may be involved in the regulation of NTE during the cell cycle.
Eugenio Vilanova - One of the best experts on this subject based on the ideXlab platform.
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Expression of Neuropathy Target Esterase in mouse embryonic stem cells during differentiation
Archives of Toxicology, 2010Co-Authors: David Pamies, Eugenio Vilanova, Juan Antonio Reig, Miguel A SogorbAbstract:Neuropathy Target Esterase (NTE) was initially identified as the primary Target Esterase of some organophosphorus compounds that cause delayed Neuropathy. Some studies in vivo suggest that this protein may also perform a function in embryonic development and therefore also in cell differentiation. The aim of this work was to characterize embryonic stem cells (ESC) as cellular model before to approach to the role of NTE in embryotoxicity processes through mechanistic studies. Mouse D3 ESC in monolayer expressed an NTE activity of 23 nmol phenol/min/mg of protein, while mouse R1 ESC showed a specific NTE activity 3 times higher than D3. An increased expression of gene Pnpla6 (that codifies for NTE) was seen during differentiation in both the D3 cells in monolayer and embryonic bodies (EBS). The maximums of the Pnpla6 expression were reached after 30 h and 5 days of differentiation in monolayer and EBS cultures, respectively. This peak of the Pnpla6 expression correlated with the peak of the NTE enzymatic activity in D3 monolayers. NTE activity and Pnpla6 expression returned to basal levels after 48 h (in monolayer cultures) and 10 days (in EBS) of differentiation, respectively. The changes in the Pnpla6 expression did not correlate with changes noted in the expression of two endoderm, two ectoderm and one neuroectoderm gene markers. In conclusion, this manuscript reports about NTE expression in ESC and its variation during first stages of differentiation. Nevertheless, the role of this activity and the meaning of the variations detected during differentiation must be further studied.
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inhibition and aging of Neuropathy Target Esterase by the stereoisomers of a phosphoramidate related to methamidophos
Toxicology Letters, 1997Co-Authors: Miguel A Sogorb, Nuria Diazalejo, Maria De La Cruz Pellin, Eugenio VilanovaAbstract:Abstract Discrepancies in the aging reaction between Neuropathy Target Esterase (NTE) inhibited in vitro and in vivo by racemic mixtures of O-alkyl O-2,5-dichlorophenyl phosphoramidates have been observed. It suggested the existence of differences in the interactions (inhibition and aging) between NTE and each stereoisomers of the above mentioned compounds. In order to verify this hypothesis, stereoisomers of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) were isolated by chiral column chromatography, followed by the evaluation of NTE inhibition and aging for each stereoisomers. The loss of reactivation capacity by KF was used as criterion of aging. The stereoisomer S-(−)-HDCP inhibited hen brain NTE with an I50 of 7.6 nM for 30 min of incubation, this being similar to the value obtained for the racemic mixture (I50=6.2 nM), and much lower than that recorded for R-(+)-HDCP (I50=191 nM). NTE inhibited by HDCP racemic mixture and the stereoisomer S-(−)-HDCP was reactivated by KF after 20 h of incubation at 37°C. The NTE inhibited by R-(+)-HDCP could not be fully reactivated after inhibition.
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Inhibition and aging of Neuropathy Target Esterase by the stereoisomers of a phosphoramidate related to methamidophos.
Toxicology letters, 1997Co-Authors: Miguel A Sogorb, M.c. Pellin, N Díaz-alejo, Eugenio VilanovaAbstract:Discrepancies in the aging reaction between Neuropathy Target Esterase (NTE) inhibited in vitro and in vivo by racemic mixtures of O-alkyl O-2,5-dichlorophenyl phosphoramidates have been observed. It suggested the existence of differences in the interactions (inhibition and aging) between NTE and each stereoisomers of the above mentioned compounds. In order to verify this hypothesis, stereoisomers of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) were isolated by chiral column chromatography, followed by the evaluation of NTE inhibition and aging for each stereoisomers. The loss of reactivation capacity by KF was used as criterion of aging. The stereoisomer S-(-)-HDCP inhibited hen brain NTE with an I50 of 7.6 nM for 30 min of incubation, this being similar to the value obtained for the racemic mixture (I50 = 6.2 nM), and much lower than that recorded for R-(+)-HDCP (I50 = 191 nM). NTE inhibited by HDCP racemic mixture and the stereoisomer S-(-)-HDCP was reactivated by KF after 20 h of incubation at 37 degrees C. The NTE inhibited by R-(+)-HDCP could not be fully reactivated after inhibition.
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Properties of partly preinhibited hen brain Neuropathy Target Esterase
Chemico-biological interactions, 1993Co-Authors: J.l. Vicedo, Victoria Carrera, José Barril, Eugenio VilanovaAbstract:Abstract NTE inhibitors cause different toxicological consequences (protection, induction or potentiation/promotion of Neuropathy) depending on the order of dosing. These effects might be explained in terms of several phosphorylable sites with ‘allosteric irreversible’ behaviour. Brain Neuropathy Target Esterase (NTE) has been preinhibited with phenylmethylsulphonyl fluoride (PMSF) (0, 5, 10, 15, 30 and 60 μM) or with diisopropylphoshoro fluoridate (DFP) (0, 0.2, 0.5, and 1 μM) at 37°C for 30 min. After washing by centrifugation, tissues were then reinhibited with a range of PMSF (0 to 80 μM) or DFP (0 to 1 μM) concentrations. The slopes of the inhibition curves (log % activity vs. concentration) of pretreated tissues were identical to those of the non-pretreated tissues, with non-distinguishable I 50 values. It is concluded that allosteric effects are not likely to be involved in membrane-bound NTE of hen brain.
Marcello Lotti - One of the best experts on this subject based on the ideXlab platform.
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The relevance of inhibitor-substrate interactions when measuring Neuropathy Target Esterase inhibition.
Archives of toxicology, 2000Co-Authors: Angelo Moretto, Milan Jokanović, Marcello LottiAbstract:Neuropathy Target Esterase (NTE), thought to be the Target for organophosphate polyNeuropathy, is operationally defined as that neural phenyl valerate Esterase resistant to paraoxon (40 μM) and sensitive to mipafox (50 μM; 20 min, pH 8.0, 37°C). The time course of inhibition of particulate paraoxon pretreated Esterases by mipafox showed that the lines indicating the rate of inhibition did not pass through the log 100% activity when extrapolated at zero time. Slopes of inhibition of NTE were not linearly related to the concentration of mipafox. Kinetic parameters derived from Wilkinson type plots were: K a=49–199 μM, k +2=0.24–0.64 min−1 and k a=3.1–5.0 mM−1 m−1. When mipafox was removed (either by dilution or centrifugation) before the addition of phenyl valerate intercepts below 100% disappeared. We confirm that the formation of Michaelis complex between NTE and mipafox is not prevented by phenyl valerate and that inhibition proceeds after addition of phenyl valerate. We compared inhibitions obtained with experiments by using the traditional method (sequential incubation with inhibitors and phenyl valerate) to those obtained with a method where mipafox is removed before the addition of substrate. When calculating fixed-time 50% inhibitory concentrations (IC50s) of some inhibitors for NTE, the longer the hydrolysis time, the lower were the IC50s. Therefore, the inhibitory potency of certain NTE inhibitors, is accurately assessed only when calculating second-order rate constants (k a).
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Phenyl Valerate Esterases Other than Neuropathy Target Esterase and the Promotion of Organophosphate PolyNeuropathy
Chemical Research in Toxicology, 1997Co-Authors: Dejan Milatovic, Angelo Moretto, Khaled A. Osman, Marcello LottiAbstract:Certain Esterase inhibitors (such as phenylmethanesulfonyl fluoride, PMSF) enhance the clinical and morphological signs of organophosphate-induced delayed polyNeuropathy (OPIDP) in hens. This is called promotion of OPIDP. The Target of promotion is unknown, but it is likely to be different from Neuropathy Target Esterase (NTE), the Target of OPIDP. NTE is a neural phenyl valerate (PV) Esterase, operationally defined by selective inhibition with organophosphates. This study was aimed to ascertain whether the Target for promotion is a PV Esterase other than NTE. Brain and sciatic nerve PV Esterases of hens were incubated with diisopropylphosphorofluoridate (DFP; 5 μM) or N,N-diisopropyl phosphorodiamidofluoridate (mipafox; 50 μM) to inhibit NTE and other Esterases thought not to be relevant to promotion. Remaining activities, quantitatively similar after either inhibition, were titrated with PMSF (up to 500 μM) and analysis of time course of inhibition showed first-order kinetics. Mipafox (50 μM)-resistant ...
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Phenyl Valerate Esterases Other than Neuropathy Target Esterase and the Promotion of Organophosphate PolyNeuropathy
Chemical research in toxicology, 1997Co-Authors: Dejan Milatovic, Angelo Moretto, Khaled A. Osman, Marcello LottiAbstract:Certain Esterase inhibitors (such as phenylmethanesulfonyl fluoride, PMSF) enhance the clinical and morphological signs of organophosphate-induced delayed polyNeuropathy (OPIDP) in hens. This is called promotion of OPIDP. The Target of promotion is unknown, but it is likely to be different from Neuropathy Target Esterase (NTE), the Target of OPIDP, NTE is a neural phenyl valerate (PV) Esterase, operationally defined by selective inhibition with organophosphates. This study was aimed to ascertain whether the Target for promotion is a PV Esterase other than NTE. Brain and sciatic nerve PV Esterases of hens were incubated with diisopropylphosphorofluoridate (DFP; 5 microM) or N,N-diisopropyl phosphorodiamidofluoridate (mipafox; 50 microM) to inhibit NTE and other Esterases thought not to be relevant to promotion. Remaining activities, quantitatively similar after either inhibition, were titrated with PMSF (up to 500 microM) and analysis of time course of inhibition showed first-order kinetics. Mipafox (50 microM)-resistant PMSF (500 microM)-sensitive activity (about 80% of mipafox-resistant ones) was tested both in vitro and in vivo with several inhibitors. No correlation was found between inhibition of mipafox-resistant PMSF-sensitive activity and the capability of several inhibitors to promote OPIDP. We conclude that the Target of promotion is unlikely to be a PV Esterase resistant to mipafox (50 microM).
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interaction of methamidophos with hen and human acetylcholinEsterase and Neuropathy Target Esterase
Archives of Toxicology, 1991Co-Authors: Mariaemilia Bertolazzi, Stefano Caroldi, Angelo Moretto, Marcello LottiAbstract:Methamidophos causes acute cholinergic toxicity in several species, including man, and organophosphate-induced delayed polyNeuropathy which has been reported in man but not in the hen. AcetylcholinEsterase (AChE) and Neuropathy Target Esterase (NTE) are thought to be the molecular Targets of acute and delayed toxicity, respectively. The rate constants of inhibition (ka) and reactivation (k+3) of human and hen brain AChE and NTE by methamidophos resolved optical isomers are here reported. NTE inhibition was progressive and irreversible. Human and hen NTE ka (M−1·m−1) ford-(+) methamidophos was 88 and 59, respectively, and forl-(−) methamidophos 3.2 and 3.0, respectively. AChE spontaneously reactivates after inhibition.d-(+) methamidophos 10−3·ka (M−1·m−1) for human and hen AChE was 0.24 and 0.13; 103·k+3 (m−1) was 0.83 and 0.69, respectively,l-(−) Methamidophos 10−3·ka (M−1·m−1) for human and hen AChE was 5.7 and 2.8, whereas 103 · k+3 (m−1) was 6.50 and 1.52, respectively.l-(−)-Inhibited AChE reactivated to about 60% for human and 30% for hen enzymes, respectively.d-(+)-Inhibited AChE reactivated to about 10–20% for both species. Maximal reactivation occurred within 4–6 h when a plateau was reached. The larger and faster reactivation of human AChE inhibited in vitro byl-(−) methamidophos suggests that a corresponding effect might be possible in vivo and therefore explain, in part, the relatively higher susceptibility of man to delayed polyNeuropathy induced by racemic methamidophos which occurs, however, with doses always causing severe cholinergic toxicity.