RESUMO
Diesel exhaust particles are major constituents of ambient air pollution, and are associated with respiratory and cardiovascular diseases and lung cancer. The organic part of the particles is heterogenic and complex, and seems to be responsible for many of the adverse effects. Increased formation of ROS is often connected to the adverse effects. We have therefore investigated the effect of an organic extract of diesel exhaust particles on the reactive oxygen species (ROS) status in human neutrophil granulocytes and rat alveolar macrophages in vitro. ROS formation were studied by three different assays namely the use of DCFH-DA, lucigenin and luminol. The organic extract increased ROS assayed with DCFH-DA, but it decreased the amount of ROS in cells stimulated by PMA in all three assays. The identities of the ROS affected were further studied in cell free systems. The cell free studies confirmed that the extract had scavenging effects against superoxide, hypochlorite and to a smaller extent against peroxynitrite, but not against the hydroxyl radical and nitric oxide. ROS take part in the intracellular signalling pathways as well as in the defence against invading microorganisms, and the possible effects of interference of the redox status in the cells are discussed.
Assuntos
Fluoresceínas/química , Pneumopatias/induzido quimicamente , Macrófagos Alveolares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Material Particulado/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Emissões de Veículos/toxicidade , Acridinas/química , Adulto , Animais , Fluorometria , Humanos , Radical Hidroxila/metabolismo , Ácido Hipocloroso/metabolismo , Luminol/química , Macrófagos Alveolares/metabolismo , Masculino , Neutrófilos/metabolismo , Óxido Nítrico/metabolismo , Material Particulado/química , Material Particulado/metabolismo , Ácido Peroxinitroso/metabolismo , Ratos , Ratos Endogâmicos WKY , Superóxidos/metabolismoRESUMO
Penitrem A is a fungal neurotoxin that recurrently causes intoxication in animals, and occasionally also in humans. We have previously reported that penitrem A induced the production of reactive oxygen species (ROS) in rat cerebellar granule cells, opening for a new mechanism of action for the neurotoxin. The aim of this study was to examine the potential of penitrem A to induce ROS production in isolated human neutrophil granulocytes, and to study possible mechanisms involved. Penitrem A significantly increased the production of ROS in human neutrophils at concentrations as low as 0.25µM (40% increase over basal levels), as measured with the DCF fluorescence assay. The EC50 determined for the production of ROS by penitrem A was 3.8µM. The maximal increase in ROS production was approximately 330% over basal levels at a concentration of 12.5µM. ROS formation was significantly inhibited by the antioxidant vitamin E (50µM), the intracellular Ca+2 chelator BAPTA-AM (5µM), the mitogen activated protein kinase kinase (MEK) 1/2 and 5 inhibitor U0126 (1 and 10µM), the p38 mitogen activated protein kinase (MAPK) inhibitor SB203580 (1µM), the c-Jun amino-terminal kinase (JNK) inhibitor SP600125 (10µM), and the calcineurin inhibitors FK-506 and cyclosporine A (1.5 and 0.5µM, respectively). These finding suggest that penitrem A is able to induce an increase in ROS production in neutrophils via the activation of several MAPK-signalling pathways. We suggest that this increase may partly explain the pathophysiology generated by penitrem A neuromycotoxicosis in both humans and animals.
Assuntos
Micotoxinas/toxicidade , Neurotoxinas/toxicidade , Neutrófilos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Antracenos/farmacologia , Antioxidantes/farmacologia , Calcineurina/farmacologia , Linhagem Celular , Ciclosporina/farmacologia , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Humanos , Imidazóis/farmacologia , Concentração Inibidora 50 , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neutrófilos/metabolismo , Piridinas/farmacologia , Tacrolimo/farmacologia , Vitamina E/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
(+/-)-2-Chloropropionic acid (2-CPA) is a neurotoxic compound which kills cerebellar granule cells in vivo, and makes cerebellar granule cells in vitro produce reactive oxygen species (ROS). We have studied the effect of 2-CPA on ROS formation in human neutrophil granulocytes in vitro. We found an increased formation of ROS after 2-CPA exposure using three different methods; the fluorescent probe DCFH-DA and the chemiluminescent probes lucigenin and luminol. Four different inhibitors of ROS formation were tested on the cells in combination with 2-CPA to characterize the signalling pathways. The spin-trap s-PBN, the ERK1/2 inhibitor U0126 and the antioxidant Vitamin E inhibited the 2-CPA-induced ROS formation completely, while the mitochondrial transition permeability pore blocker cyclosporine A inhibited the ROS formation partly. We also found that 2-CPA induced an increased nitric oxide production in the cells by using the Griess reagent. The level of reduced glutathione, measured with the DTNB assay, was decreased after exposure to high concentrations of 2-CPA. Western blotting analysis showed that 2-CPA exposure led to an elevated phosphorylation of ERK MAP kinase. This phosphorylation was inhibited by U0126. Based on these experiments it seems like the mechanisms for 2-CPA induced toxicity involves ROS formation and is similar in neutrophil granulocytes as earlier shown in cerebellar granule cells. This also implies that 2-CPA may be immunotoxic.
Assuntos
Granulócitos/metabolismo , Neutrófilos/metabolismo , Propionatos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Acridinas , Western Blotting , Separação Celular , Ácido Ditionitrobenzoico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fluoresceínas , Corantes Fluorescentes , Glutationa/metabolismo , Granulócitos/efeitos dos fármacos , Humanos , Hidrocarbonetos Clorados , Indicadores e Reagentes , L-Lactato Desidrogenase/metabolismo , Luminescência , Luminol , Neutrófilos/efeitos dos fármacos , Nitratos/metabolismo , Nitritos/metabolismo , Propionatos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Reagentes de Sulfidrila , Regulação para Cima/efeitos dos fármacosRESUMO
Exposure to polychlorinated biphenyls (PCBs) and brominated flame-retardants (BFRs) in human, primates, and rodents is accompanied by neurobehavioral changes. These involve adverse effects on both memory and learning and motor activity. There are also adverse effects observed on the endocrine and immune system. This review is restricted to our laboratory's recent findings of effects of these compounds on the nervous system and some molecular effects on the immune system. In the nervous system, data showed that PCBs and BFRs produce an effect on neurotransmitter transport mechanisms, in particular the neurotransmitter dopamine. It was demonstrated that this might explain the loss of dopamine in the brain seen after exposure to PCB. Further, it may explain the behavior of dopamine in preparations in vitro from brain tissue after exposure to PCB. Recently it was also reported that PCB and some BFRs induce formation of reactive oxygen species (ROS) in neurons. ROS act as messengers in the nervous system and may also be involved in cell death. In the case of PCB exposure, a correlation between ROS formation and death of neurons was found. In the immune system it was shown that PCBs and some of the BFRs induce formation of ROS in neutrophils (granulocytes). This takes place primarily through phosphorylation and subsequent activation of the NADPH oxidase. This production of ROS may have an adverse effect on the immune system.
Assuntos
Retardadores de Chama/toxicidade , Hidrocarbonetos Bromados/toxicidade , Bifenilos Policlorados/toxicidade , Animais , Cálcio/metabolismo , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Éteres Fenílicos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Postembedding immunocytochemistry was used to localize aspartate, glutamate, gamma-aminobutyric acid (GABA), and glutamine in hippocampus and striatum during normo- and hypoglycemia in rat. In both brain regions, hypoglycemia caused aspartatelike immunoreactivity to increase. In hippocampus, this increase was evident particularly in the terminals of known excitatory afferents-in GABA-ergic neurons and myelinated axons. Aspartate was enriched along with glutamate in nerve terminals forming asymmetric synapses on spines and with GABA in terminals forming symmetric synapses on granule and pyramidal cell bodies. In both types of terminal, aspartate was associated with clusters of synaptic vesicles. Glutamate and glutamine immunolabeling were markedly reduced in all tissue elements in both brain regions, but less in the terminals than in the dendrosomatic compartments of excitatory neurons. In glial cells, glutamine labeling showed only slight attenuation. The level of GABA immunolabeling did not change significantly during hypoglycemia. The results support the view that glutamate and glutamine are used as energy substrates in hypoglycemia. Under these conditions both excitatory and inhibitory terminals are enriched with aspartate, which may be released from these nerve endings and thus contribute to the pattern of neuronal death characteristic of hypoglycemia.
Assuntos
Aminoácidos/metabolismo , Corpo Estriado/metabolismo , Hipocampo/metabolismo , Hipoglicemia/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Animais , Ácido Aspártico/metabolismo , Corpo Estriado/patologia , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Hipocampo/patologia , Hipoglicemia/patologia , Imuno-Histoquímica , Insulina , Microscopia Imunoeletrônica , Terminações Nervosas/metabolismo , Terminações Nervosas/ultraestrutura , Neurônios/patologia , Neurônios/ultraestrutura , Ratos , Ratos Wistar , Valores de Referência , Ácido gama-Aminobutírico/metabolismoRESUMO
Glial-neuronal interchange of amino acids was studied by 13C nuclear magnetic resonance spectroscopy of brain extracts from fluoroacetate-treated mice that received [1,2-(13)C]acetate and [1-(13)C]glucose simultaneously. [13C]Acetate was found to be a specific marker for glial metabolism even with the large doses necessary for nuclear magnetic resonance spectroscopy. Fluoroacetate, 100 mg/kg, blocked the glial, but not the neuronal tricarboxylic acid cycles as seen from the 13C labeling of glutamine, glutamate, and gamma-aminobutyric acid. Glutamine, but not citrate, was the only glial metabolite that could account for the transfer of 13C from glia to neurons. Massive glial uptake of transmitter glutamate was indicated by the labeling of glutamine from [1-(13)C]glucose in fluoroacetate-treated mice. The C-3/C-4 enrichment ratio, which indicates the degree of cycling of label, was higher in glutamine than in glutamate in the presence of fluoroacetate, suggesting that transmitter glutamate (which was converted to glutamine after release) is associated with a tricarboxylic acid cycle that turns more rapidly than the overall cerebral tricarboxylic acid cycle.
Assuntos
Aminoácidos/metabolismo , Fluoracetatos/farmacologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/metabolismo , Acetatos/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Sangue/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Ácido Cítrico/metabolismo , Feminino , Glucose/metabolismo , Glutamina/metabolismo , Ácido Láctico/metabolismo , Camundongos , Camundongos EndogâmicosRESUMO
Suckling rats were infected intranasally with the temperature-sensitive mutant G41 strain of vesicular stomatitis virus. The rats survived but demonstrated lifelong learning deficits in the Morris maze and impaired exploratory behaviour in the open field test. When examined at 18 months of age they had a severe loss of neurons in the medial and dorsal raphe nuclei in the brain stem and reduced levels of serotonin and its metabolite 5-hydroxyindole acetic acid in the cerebral neocortex and hippocampus. The levels of noradrenaline, dopamine, homovanillic acid, 3,4-dihydroxyphenylacetic acid, choline acetyltransferase and glutamate decarboxylase were largely unaffected. The permanent disturbance in brain serotonin metabolism did not cause any histological changes in the cerebral cortex. Thus there were no neurofibrillary tangles or amyloid plaques as has been reported as a late effect of chemically induced lesion to the cholinergic system in the rat brain. It is concluded that the brain serotonergic system is especially vulnerable to an episode of virus attack along olfactory pathways and that the neurochemical and behavioural alterations caused by such an episode persist during a major part of the animal's life span.
Assuntos
Envelhecimento/fisiologia , Comportamento Animal/fisiologia , Química Encefálica/fisiologia , Serotonina/metabolismo , Estomatite/metabolismo , Vírus da Estomatite Vesicular Indiana , Viroses/metabolismo , Envelhecimento/patologia , Envelhecimento/psicologia , Animais , Animais Lactentes/fisiologia , Encéfalo/patologia , Catecolaminas/metabolismo , Cognição/fisiologia , Comportamento Exploratório/fisiologia , Deficiências da Aprendizagem/fisiopatologia , Neurotransmissores/metabolismo , Ratos , Ratos Endogâmicos , Estomatite/patologia , Estomatite/psicologia , Viroses/patologia , Viroses/psicologiaRESUMO
The topography of cholinergic and substance P containing habenulo-interpeduncular projections has been studied in the rat. The research has been carried out by combining choline acetyltransferase and substance P immunohistochemistry to experimental lesions and biochemical assays in microdissected brain areas. In addition, computer-assisted image analysis has been performed in order to obtain quantification of immunohistochemical data. The results show that cholinergic and substance P containing neurons have a different localization in the medial habenula and project to essentially different areas of the interpeduncular nucleus. Cholinergic neurons are crowded in the ventral two-thirds of the medial habenula while substance P containing cells are exclusively localized in the dorsal part of the nucleus. In most parts of the interpeduncular nucleus, choline acetyltransferase and substance P containing fibres and terminals are similarly segregated and no overlapping is apparent except for the rostralmost and the caudalmost ends of the nucleus. Cholinergic activity is largely concentrated in the central core of the nucleus, while substance P is preferentially localized in the peripheral subnuclei of the interpeduncular nucleus. In addition, both substance P and choline acetyltransferase levels show peculiar regional variations along the rostrocaudal axis of the interpeduncular nucleus. The results of experimental lesions demonstrate that the substance P projection carried by each fasciculus retroflexus is prevailingly ipsilateral in the rostral part of the interpeduncular nucleus and becomes progressively bilateral as far as more caudal regions of the nucleus are reached. By contrast, the cholinergic projections carried by each fasciculus retroflexus intermingle more rapidly and only show a slight ipsilateral dominance in the interpeduncular nucleus. The results of the study are discussed with reference to previous anatomical and neurochemical data which, in several instances, had given rise to discrepant interpretations.
Assuntos
Fibras Colinérgicas/enzimologia , Diencéfalo/análise , Mesencéfalo/análise , Substância P/análise , Animais , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/efeitos dos fármacos , Diencéfalo/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Histocitoquímica , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Ácido Caínico/farmacologia , Masculino , Mesencéfalo/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Substância P/metabolismoRESUMO
Dysfunction of subcortical serotoninergic neurons has been implicated in some behaviour disturbances. The serotoninergic neurons in the dorsal and median raphe project widely in the brain. They innervate the olfactory bulbs and can be targets for exogenous agents attacking the olfactory epithelium and bulbs. We report here an injury to the serotoninergic neurons after intranasal infection in 12-day-old rats with a temperature-sensitive mutant of vesicular stomatitis virus. The brain infection was focal and transient. Viral antigens could no longer be detected 13-15 days after infection. In spite of this the animals, as adults, had a severe serotonin depletion in the cerebral cortex and hippocampus, and showed abnormal locomotor and explorative behaviour as well as learning deficits. The neocortex was histologically intact and parameters related to other neurotransmitters such as dopamine, noradrenaline, GABA and acetylcholine showed no marked changes. A relatively selective damage to serotoninergic nuclei as a result of virus neuroinvasion through a natural portal of entry, may constitute a new pathogenetic mechanism for cortical dysfunction and behavioural deficits.
Assuntos
Encéfalo/fisiopatologia , Cognição , Atividade Motora , Serotonina/metabolismo , Viroses/psicologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Encéfalo/metabolismo , Colina O-Acetiltransferase/metabolismo , Dopamina/metabolismo , Feminino , Glutamato Descarboxilase/metabolismo , Ácido Homovanílico/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Mutação , Norepinefrina/metabolismo , Ratos , Ratos Endogâmicos , Vírus da Estomatite Vesicular Indiana/genética , Viroses/fisiopatologiaRESUMO
Different methods for measuring GABA turnover in rat brain were compared. One method was based on the irreversible inhibition of GABA transaminase (EC 2.6.1.19) by microinjection of gamma-vinyl-GABA into neostriatum of rat. The accumulation of GABA was almost linear for 4 hr. The GABA turnover in control animals was estimated to be 25.8 +/- 1.1 nmole/mg protein/hr. Another method was based on the post mortal increase in GABA level in an 8 min interval after decapitation. This method gave a GABA turnover of 54.3 +/- 4.8 nmole/mg protein/hr in neostriatum. The methods were compared with respect to their ability to detect the effect of high doses of diazepam and morphine on the turnover rate of GABA. The GABA transaminase inhibition method resulted in a 27% and a 17% decrease in GABA turnover for diazepam and morphine respectively. The post mortem method did not detect any change in GABA turnover after administration of these drugs. Hypoglycemia leads to a decrease in GABA turnover of 17% with the GABA transaminase inhibition method and a 43% decrease in GABA turnover with the post mortem method. The advantages and limitations of the methods for estimating GABA turnover changes during drug exposure is discussed, and are compared with results from a third method based on steady state kinetics extracted from the literature.
Assuntos
Corpo Estriado/metabolismo , Ácido gama-Aminobutírico/metabolismo , 4-Aminobutirato Transaminase/antagonistas & inibidores , Animais , Corpo Estriado/efeitos dos fármacos , Diazepam/farmacologia , Técnicas In Vitro , Insulina/farmacologia , Cinética , Masculino , Métodos , Morfina/farmacologia , Ratos , Ratos EndogâmicosRESUMO
This study investigated the effects of C7 and C9 aliphatic (n-heptane, n-nonane), naphthenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) and aromatic (toluene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat brain synaptosome fraction. Methyl mercury (MeHg) was included as a positive control. Exposure of the synaptosomes to the hydrocarbons produced a concentration-dependent linear increase in the formation of the fluorescence of 2',7'-dichlorofluorescein (DCF) as a measure of the production of ROS and RNS. Formation of RNS was demonstrated by preincubation of the synaptosome fraction with the neuronal nitric oxide synthase (nNOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME), which reduced the MeHg and TMCH-stimulated fluorescence by 51% and 65%, respectively. The naphthenic hydrocarbon TMCH showed the strongest potential for ROS and RNS formation in rat brain synaptosomes, followed by TMB, toluene, n-nonane, n-heptane, and methylcyclohexane, respectively. TMCH was selected for mechanistic studies of the formation of ROS. Both MeHg and TMCH induced an increase in intracellular calcium concentration [Ca(2+)]i as measured with Fura-2. Blockade of voltage-dependent Ca(2+) channels with lanthanum prior to stimulation with MeHg and TMCH led to a reduction in the ROS/RNS formation of 72% and 70%, respectively. Furthermore, addition of cyclosporin A (CSA), a blocker of the mitochondrial permeability transition pore (MTP), lowered both the MeHg and TMCH-elevated DCF fluorescence by 72% and 59%. Preincubation of the synaptosome fraction with the protein tyrosine kinase inhibitor genistein lowered the MeHg and TMCH-stimulated fluorescence by 85% and 91%, respectively. Addition of the extracellular signal-regulated protein kinase (MEK)-1 and -2 inhibitor U0126 reduced the fluorescence stimulated by MeHg and TMCH by 62% and 63%. Furthermore, the protein kinase C inhibitor bisindolylmaleimide reduced the fluorescence stimulated by MeHg and TMCH by 52% and 56%. The compound 1-(6-[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122), which inhibits phospholipase C, was shown to decrease the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, respectively. The phospholipase A2 (PLA2) inhibitor 7,7-dimethyl eicosadienoic acid (DEDA) reduced fluorescence in response to MeHg and TMCH by 49% and 54%. Simultaneous addition of L-NAME, CSA, and DEDA to the synaptosome fraction totally abolished the DCF fluorescence. In conclusion, C7 and C9 aliphatic, naphthenic, and aromatic hydrocarbons stimulated formation of ROS and RNS in rat brain synaptosomes. The naphthenic hydrocarbon TMCH stimulated formation of ROS and RNS in the synaptosomes through Ca(2+)-dependent activation of PLA2 and nNOS, and through increased transition permeability of the MTP. Exposure of humans to the naphthenic hydrocarbon TMCH may stimulate formation of free radicals in the brain, which may be a key factor leading to neurotoxicity.
Assuntos
Hidrocarbonetos Aromáticos/farmacologia , Canais Iônicos , Óxido Nítrico Sintase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sinaptossomos/efeitos dos fármacos , Alcanos/farmacologia , Animais , Derivados de Benzeno/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cálcio/metabolismo , Ácidos Carboxílicos/química , Cicloexanos/farmacologia , Ácidos Graxos/química , Heptanos/farmacologia , Técnicas In Vitro , Masculino , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A2 , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Wistar , Sinaptossomos/metabolismo , Tolueno/farmacologia , Fosfolipases Tipo C/metabolismoRESUMO
The carboxylesterase activity in both plasma and liver of guinea-pig were separated into three main peaks by chromatofocusing. Two of the three plasma enzymes were retained by affinity chromatography on Affi-Gel Blue (100-200 mesh). Isoelectric points determined by chromatofocusing or isoelectrofocusing were pI 6.1, pI 5.2 and pI 4.0 for the plasma enzymes, and pI 5.7, pI 5.2 and pI 4.5 for the liver enzymes. The effect of selective esterase inhibitors, soman, physostigmine (cholinesterase inhibitor) and bis-4-nitrophenyl phosphate (carboxylesterase inhibitor), suggested that the three enzymes in both tissues may be regarded as carboxylesterases. However, the pI 5.7 carboxylesterase was partially inhibited by physostigmine, and the pI 4.5 carboxylesterase was almost not affected by bis-4-nitrophenyl phosphate. The ratio between the activities towards 4-nitrophenyl butyrate and methyl butyrate differed among the carboxylesterases in both tissues. All three carboxylesterases in plasma were partially reactivated by diacetylmonoxime after soman inhibition in vitro, but to a different extent. The soman inhibited liver carboxylesterases were not reactivated by diacetylmonoxime.
Assuntos
Butanonas/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Diacetil/farmacologia , Reativadores Enzimáticos/farmacologia , Fígado/enzimologia , Animais , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/sangue , Cromatografia de Afinidade , Diacetil/análogos & derivados , Feminino , Cobaias , Técnicas In Vitro , Focalização Isoelétrica , Ponto Isoelétrico , Fígado/efeitos dos fármacos , Nitrofenóis/farmacologia , Compostos Organofosforados/farmacologia , Fisostigmina/farmacologia , Soman/farmacologiaRESUMO
The in vitro exposure of rat bronchial smooth muscle to the cholinesterase inhibitor soman (O-[1,2,2-trimethylpropyl]-methyl-phosphonofluoridate) potentiated the rapid and concentration dependent increase in the contraction induced by acetylcholine (ACh). There was a substantial increase in the response to ACh when soman was present in concentrations from 10 nM to 1 microM which correspond to a 65-100% inhibition of acetylcholinesterase (AChE). The apparent affinity (pD2) to ACh increased from 3.7 to 6.7 without any change in intrinsic activity (alpha) in this concentration interval. In contrast, soman did not alter the apparent affinity or intrinsic activity of carbachol, which supports the suggestion that the effect of soman is entirely due to its anticholinesterase activity. Soman by itself induced contraction which begun at 1-10 nM. This may be explained from its anticholinesterase activity and the subsequent increase in the synaptic concentration of spontaneously released ACh. The effect of soman on inhibition of cholinesterase and carboxylesterases have also been examined. The results demonstrate that low concentrations of soman induces contraction of the airway smooth muscle.
Assuntos
Brônquios/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , Soman/farmacologia , Acetilcolina/farmacologia , Acetilcolinesterase/metabolismo , Animais , Brônquios/efeitos dos fármacos , Carbacol/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Colinesterases/metabolismo , Estimulação Elétrica , Cinética , Masculino , Músculo Liso/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
Rat liver cells were separated into parenchymal cells (PC), Kupffer cells (KC) and endothelial cells (EC). The distribution of carboxylesterases (EC 3.1.1.1) between these cell types was investigated by PAGE and chromatogenic substrate staining, and compared with the results for total liver preparation and individual isoenzymes isolated by chromatofocusing. All of the liver carboxylesterase isoenzymes could be detected in the PC, whereas in both KC and EC only those with isoelectric point (pI) 6.4/6.2 could be detected. Use of carboxylesterase inhibitors like bis-(4-nitrophenyl)phosphate and paraoxon, and organophosphorus compound hydrolase inhibitors like 4-hydroxymercuribenzoate and EDTA confirmed that these esterases were of the carboxylesterase type.
Assuntos
Hidrolases de Éster Carboxílico/análise , Isoenzimas/análise , Fígado/enzimologia , Animais , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Eletroforese em Gel de Poliacrilamida , Endotélio/enzimologia , Focalização Isoelétrica , Isoenzimas/antagonistas & inibidores , Células de Kupffer/enzimologia , Masculino , Monoacilglicerol Lipases/análise , Ratos , Ratos EndogâmicosRESUMO
The different isoenzymes of carboxylesterase (CarbE) from guinea pig liver, lung and plasma were separated by gel filtration and chromatofocusing. The isoenzymes were characterized by inhibition with several different organophosphorus compounds. The bimolecular rate constants showed the same tendency of decreased inhibition for all of the isoenzymes in the order; paraoxon greater than soman greater than diisopropylphosphofluoridate (DFP) greater than bis(p-nitrophenyl)phosphate. With two exceptions the inhibition constants for the different isoenzymes differed little. Subcutaneous and intraperitoneal administration of DFP and paraoxon rapidly inhibited the CarbE activity in guinea pig plasma. Much higher doses were necessary to obtain a marked inhibition in lung and liver. About 25% of CarbE activity in lung was resistant to paraoxon and DFP inhibition. Gel filtration of lung homogenate after treatment with the organophosphorus compounds showed that the CarbE activity of the medium molecular mass fractions was inhibited only weakly. This could be due to reduced accessibility to some of the lung CarbE isoenzymes.
Assuntos
Hidrolases de Éster Carboxílico/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Compostos Organofosforados/toxicidade , Animais , Hidrolases de Éster Carboxílico/sangue , Hidrolases de Éster Carboxílico/isolamento & purificação , Relação Dose-Resposta a Droga , Cobaias , Injeções Intraperitoneais , Injeções Subcutâneas , Focalização Isoelétrica , Isoenzimas/sangue , Isoenzimas/isolamento & purificação , Isoflurofato/administração & dosagem , Isoflurofato/toxicidade , Fígado/enzimologia , Pulmão/enzimologia , Masculino , Compostos Organofosforados/administração & dosagem , Paraoxon/administração & dosagem , Paraoxon/toxicidadeRESUMO
The irreversible acetylcholinesterase inhibitor soman (O-[1,2,2-trimethylpropyl]-methyl-phosphonofluoridate) induced contraction of guinea-pig primary bronchial smooth muscle. The apparent affinity (ED50) of acetylcholine (ACh) was altered from control value of 12 microM to 0.3 microM following exposure of the bronchial smooth muscle to 14 microM soman for 15 min in vitro. The ED50 of the cholinergic agonist carbachol was not changed even when the acetylcholinesterase (AChE) activity was inhibited completely. The intrinsic activity (alpha) of ACh and carbachol was not significantly changed after exposure to soman for 15 min. The results demonstrate that the effect of soman is only due to its anticholinesterase activity. Furthermore, the contraction induced by histamine was not altered by concentrations of soman which increase the cholinergic stimulation. This indicates that histamine does not induce contraction of bronchial smooth muscle in guinea pig through the release of ACh or by modulation of muscarinic receptors. Furthermore, soman also inhibited the carboxylesterase activity in the primary bronchi. In respiratory tissue this group of enzymes may have a major protective function, due to their ability to bind several organophosphorus compounds. Compared to studies performed on other species, this study shows that guinea-pig bronchi are very sensitive to the AChE-inhibitor soman. Therefore, exposure to very low concentrations of AChE-inhibitors may induce contraction of bronchial smooth muscle.
Assuntos
Brônquios/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Soman/farmacologia , Acetilcolinesterase/metabolismo , Animais , Butirilcolinesterase/metabolismo , Carbacol/farmacologia , Carboxilesterase , Hidrolases de Éster Carboxílico/metabolismo , Estimulação Elétrica , Cobaias , Histamina/farmacologia , Técnicas In Vitro , MasculinoRESUMO
A radiochemical assay for carboxylesterase based on the substrate methyl[1-14C]butyrate is described. The blank value corresponds to 1.04 micrograms (liver)-1.44 mg (plasma) of tissues with the highest and lowest activity respectively, which constitute the sensitivity of the method. The hydrolysis of methyl butyrate and 4-nitrophenyl butyrate by plasma, liver, lung, heart, diaphragm, cerebrum, kidney and duodenum of rat have been compared. The results showed that the two substrates were hydrolysed preferentially by two different groups of the enzyme. The effect of selective esterase inhibitors showed that both groups can be characterized as carboxylesterase, because bis-4-nitrophenyl phosphate inhibits the hydrolysis of both substrates, physostigmine has only a slight effect and EDTA is no inhibitor. The exception is the enzyme in the duodenum which is inhibited by all three inhibitors. The effect of phenobarbital induction and soman treatment on enzyme activity towards the two substrates were similar. Sex difference in the plasma activity towards methyl butyrate, but not 4-nitrophenyl butyrate, indicates that the group of carboxylesterase preferentially hydrolyzing 4-nitrophenyl butyrate may be the most important for the detoxification of soman.
Assuntos
Butiratos/metabolismo , Hidrolases de Éster Carboxílico/análise , Fenilbutiratos/metabolismo , Animais , Radioisótopos de Carbono , Estabilidade de Medicamentos , Feminino , Hidrólise , Masculino , Fenobarbital/farmacologia , Ratos , Ratos Endogâmicos , Fatores Sexuais , Soman/farmacologiaRESUMO
Acute and sub-acute inhalation exposure of rats to the organophosphorus compound soman (O-[1,2,2-trimethylpropyl]-methylphosphonofluoridate) reduced the contraction of the bronchial smooth muscle induced by cholinergic stimulation. Acute exposure to 8.51 mg/m3 of soman for 45 min (total dose of 383 mg X min/m3) inhibited the acetylcholinesterase (AChE) activity of the bronchial smooth muscle by 85% and reduced the contraction induced by ACh and carbachol by 70% and 80% respectively. In spite of the extensive inhibition of AChE and reduction in the contraction following cholinergic stimulation, there was no alteration of the binding capacity (Bmax) or the equilibrium dissociation constant (Kd) to [3H]-quinuclidinyl benzilate ([3H]-QNB) in the rat bronchi following such an acute exposure. After sub-acute exposure (40 hr) to 0.45-0.63 mg/m3 of soman (total dose of 1080-1519 mg X min/m3) there was a reduction in AChE-activity of 94% and in the contraction of the bronchial smooth muscle induced by ACh and carbachol of 70%. Furthermore, also a reduction of the binding capacity to [3H]-QNB of approximately 40% was observed. Following exposure to soman by both acute and sub-acute inhalation exposure there was an increase in the apparent affinity (pD2) to ACh in the bronchial smooth muscle, due to the extensive inhibition of the AChE-activity. Inhalation of soman also induced a substantial inhibition of the AChE-activity in the lung (86%), but somewhat smaller inhibition in the hippocampus (70%) and almost no inhibition in the neostriatum (19%). Moreover, it was only in the lung where sub-acute exposure to soman produced a reduction of the binding capacity to [3H]-QNB and the reduction was approximately 50%. The results therefore show that after sub-acute inhalation of a relatively low concentration of the AChE-inhibitor soman, alterations in the number of cholinergic receptors are only observed in the peripheral cholinergic nervous system.
Assuntos
Receptores Muscarínicos/efeitos dos fármacos , Soman/toxicidade , Acetilcolina/farmacologia , Acetilcolinesterase/análise , Administração por Inalação , Animais , Carbacol/farmacologia , Pulmão/efeitos dos fármacos , Masculino , Contração Muscular/efeitos dos fármacos , Quinuclidinil Benzilato/metabolismo , Ratos , Ratos Endogâmicos , Receptores Muscarínicos/análiseRESUMO
The specificity of the vesicular L-glutamate carrier was characterized using dyes with biphenyl and amino- and sulphonic acid substituted naphthyl groups, structurally similar to the specific vesicular L-glutamate inhibitor Evans Blue. The dye Trypan Blue was the most potent inhibitor; the IC50 value was determined to be 49 nM. Naphthol Blue Black, Reactive Blue 2, Benzopurpurin 4B, Ponceau SS, Direct Blue 71 and Acid red 114 were also highly potent inhibitors with IC50 values from 330 to 1670 nM (series 1). The dyes were competitive inhibitors of vesicular glutamate uptake, and acted therefore on the glutamate transporter. Their IC50 values for the vesicular uptake of gamma-aminobutyric acid (GABA) were all higher than 20 microM. They had no effect on synaptosomal uptake of glutamate. Furthermore, we have also found several other dyes with IC50 values for the vesicular uptake of glutamate ranging between 1 and 30 microM and for gamma-aminobutyric acid higher than 50 microM (series 2). The most potent inhibitor Trypan Blue contains a biphenyl group, linked by azo groups to side chains containing sulphonic, amino and/or hydroxyl groups coupled to a naphthalene ring system. Trypan Blue and Evans Blue are by molecular mechanics, shown to have planar structures with conjugated double bonds throughout the structure. The other dyes, which were less effective, had phenyl and/or naphthalene groups linked by an azo group. We have also tested a series of amino and/or hydroxyl naphthalene di-/sulphonic acids that correspond to the side chains of the most potent dyes, but they had no effect on glutamate nor on gamma-aminobutyric acid uptake. We conclude that the inhibitory action of these compounds is strictly dependent of the complete molecule.
Assuntos
Corantes/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Relação Dose-Resposta a Droga , Azul Evans/farmacologia , Masculino , Conformação Molecular , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Azul Tripano/farmacologiaRESUMO
Human and rat blood hydrolysed T-2 toxin along two different pathways giving HT-2 toxin and neosolaniol as primary metabolites, respectively. Neosolaniol represents a metabolic pathway different from that obtained by liver. Rat erythrocytes formed neosolaniol as a primary metabolite whereas white blood cells hydrolysed T-2 toxin to HT-2 toxin. Human erythrocytes formed both HT-2 toxin and neosolaniol whereas all human white cells produced only HT-2 as the primary metabolite. The enzymes responsible for hydrolysis of T-2 toxin to HT-2 toxin in white blood cells and T-2 toxin to neosolaniol in red blood cells were all identified as carboxylesterases by use of specific inhibitors. The ratio between trichothecene hydrolysis and 4-nitrophenyl butyrate hydrolysis varied among the different cell fractions indicating that specific isoenzymes are involved.