Accumulation Of ACh Research Articles

Mechanistic insights into phenanthrene-triggered oxidative stress-associated neurotoxicity, genotoxicity, and behavioral disturbances toward the brandling worm (Eisenia fetida) brain: The need for an ecotoxicological evaluation

In this study, earthworm (Eisenia fetida) brain was chosen as targeted receptors to probe the mechanisms of oxidative stress-related neurotoxicity, genotoxicity, and behavioral disturbances triggered by PHE. Results showed that PHE stress can initiate significant amounts of ROS, thus triggering oxidative stress in E. fetida brain. These effects were accompanied by a significant increase of damage to macromolecules DNA and lipids, resulting in severe oxidative effects. PHE exposure can induce AChE inhibition by ROS-induced injury and the accumulation of excess ACh at the nicotinic post-synaptic membrane, thus inducing aggravated neurological dysfunction and neurotoxicity of E. fetida through an oxidative stress pathway. Moreover, the burrowing behavior of earthworms was disturbed by oxidative stress-induced neurotoxicity after exposure to PHE. Furthermore, the abnormal mRNA expression profiles of oxidative stress- and neurotoxicity-related genes in worm brain were induced by PHE stress. The IBR results suggested that E. fetida brain was suffered more serious damage caused by PHE under higher doses and long-term exposure. Taken together, PHE exposure can trigger oxidative stress-mediated neurotoxicity and genotoxicity in worm brain and behavioral disorder through ROS-induced damage. This study is of great significance to evaluate the harmful effects of PHE and its mechanisms on soil ecological health.

Ecotoxicological evaluation of oxidative stress-mediated neurotoxic effects, genetic toxicity, behavioral disorders, and the corresponding mechanisms induced by fluorene-contaminated soil targeted to earthworm (Eisenia fetida) brain

Fluorene is a commonly identified PAH pollutant in soil and exhibits various worrisome hazardous effects to soil organisms. Currently, the toxicity profiles of fluorene on earthworm brain are rare, and the mechanisms and their corresponding pathways involved in fluorene-triggered neurotoxicity, genotoxicity, and behavior changes have not been reported hitherto. Herein, earthworm (Eisenia fetida) brain was chosen as targeted receptor to explore the neurotoxic effects, genetic toxicity, behavioral disorders, and related mechanisms caused by fluorene-induced oxidative stress pathways. The results showed excess fluorene initiated the release of excessive quantities of ROS in earthworm brain, which have caused oxidative stress and accompanied by serious oxidative effects, including LPO (lipid peroxidation) and DNA injury. To minimize the damage effects, the antioxidant defense mechanisms (antioxidant enzymes and non-enzymatic antioxidants) were activated, and entailed a decrease of the antioxidant capacity in E. fetida brain, which, in turn, causes further ROS-induced ROS release. Exposure of fluorene induced the abnormal mRNA expression of genes relevant to oxidative stress (e.g., GST, SOD, CAT, GPx, MT, and Hsp70) and neurotoxicity (e.g., H02, C04, D06, and E08) in E. fetida brain. Specifically, fluorene can bind directly to AChE, destroying the conformation of this protein, and even affecting its physiological functions. This occurrence caused the inhibition of AChE activity and excess ACh accumulation at the nicotinic post-synaptic membrane, finally triggering neurotoxicity by activation of pathways related to oxidative stress. Moreover, the avoidance responses and burrowing behavior were obviously disturbed by oxidative stress-induced neurotoxicity after exposure to fluorene. The results form IBR suggested more severe poisoning effects to E. fetida brain initiated by high-dose and long-term exposure of fluorene. Among, oxidative stress injury and genotoxic potential are more sensitive endpoint than others. Collectively, fluorene stress can provoke potential neurotoxicity, genotoxicity, and behavioral disturbances targeted to E. fetida brain through the ROS-mediated pathways involving oxidative stress. These findings are of great significance to estimate the detrimental effects of fluorene and the corresponding mechanisms on soil eco-safety.

Phytochemical Analysis and Screening of Acetylcholinesterase and Carbonic Anhydrase I and II Isoenzymes Inhibitory Effect of Heptaptera triquetra (Vent.) Tutin Root

Alzheimer's disease (AD) is characterized by progressive memory loss, deterioration of other cognitive functions, and inability to perform activities of daily living. Inhibiting the AChE enzyme causes Ach accumulation in cholinergic synapses and is expected to increase cognitive functions. Carbonic anhydrase enzymes (CAs) are ubiquitous in all living organisms. They have crucial physiological and pathological roles. CA inhibitors bind to catalytic zinc ion in the active site of CA isoenzymes and block their activity. The clinical use of CAIs had been established as antiglaucoma, anticonvulsant agents, diuretics, and anti-obesity drugs, in the management of mountain sickness, gastric and duodenal ulcers, neurological disorders, osteoporosis, and tumors. To evaluate the bioactive profile of dichloromethane extract prepared from Heptaptera triquetra root, isolation studies, AChE, and hCA I and II inhibitory activities were performed. According to isolation studies, one fatty acid, coniferyl palmitate (1); four sesquiterpene coumarins, umbelliprenin (2), badrakemin acetate (4), colladonin (5), karatavicinol (6); and two sterols, stigmasterol (3a), β-sitosterol (3b) were isolated. The dichloromethane extract and all isolated compounds showed high potency against all enzymes (except badrakemin acetate for AChE) when compared to standards. Umbelliprenin (2) with IC50 value of 31.500 nM against hCA I, colladonin (5) with IC50 value of 36.473 nM against hCA II and stigmasterol (3a), and β-sitosterol (3b) mixture with IC50 value 9.000 nM against AChE demonstrated the best activity.

Chlorpyrifos induces cell proliferation in MCF-7 and MDA-MB-231 cells, through cholinergic and Wnt/β-catenin signaling disruption, AChE-R upregulation and oxidative stress generation after single and repeated treatment

Chlorpyrifos (CPF) biocide, is associated with breast cancer. The processes underlying this association have not been elucidated to date. CPF increases MCF-7 and MDA-MB-231 cell proliferation after acute and long-term treatment, partially through KIAA1363 overexpression and aryl-hydrocarbon receptor activation but also through estrogen receptor-alpha activation after 24 h exposure in MCF-7 cells, suggesting other mechanisms may be involved. CPF induces reactive oxygen species (ROS) generation, acetylcholine accumulation, and overexpression of acetylcholinesterase-R/S (AChE-R/S) variants, while it also alters the Wnt/β-catenin pathway, both in vitro and in vivo, in processes different from cancer. These latter mechanisms are also linked to cell proliferation and could mediate this effect induced by CPF. Our results show that CPF (0.01–100 μM), following one-day and fourteen-days treatment, respectively, induced ROS generation and lipid peroxidation, and acetylcholine accumulation due to AChE inhibition, Wnt/β-catenin up- or downregulation depending on the CPF treatment concentration, and AChE-R and AChE-S overexpression, with the latter being mediated through GSK-3β activity alteration. Finally, CPF promoted cell division through ACh and ROS accumulation, AChE-R overexpression, and Wnt/β-catenin signaling disruption. Our results provide novel information on the effect of CPF on human breast cancer cell lines that may help to explain its involvement in breast cancer.

Integrated pathway-based and network-based analysis of GC-MS rice metabolomics data under diazinon stress to infer affected biological pathways

Diazinon insecticide is widely applied in rice (Oryza sativa L.) fields in Iran. However, concerns are now being raised about its potential adverse impacts on rice. In this study, a time-course metabolic change in rice plants was investigated after diazinon treatment using gas chromatography-mass spectrometry (GC-MS) and subsequently three different methods, MetaboAnalyst, MetaboNetwork, and analysis of reporter reactions, as a potential multivariate method were used to find the underlying changes in metabolism with stronger evidence in order to link differentially expressed metabolites to biological pathways. Results clearly showed the similarity of acetylcholinesterase (AChE) of rice plants to that of animals in terms of its inhibitability by diazinon and emphasized that subsequent accumulation of AChE mainly affects the metabolism of osmolites and tricarboxylic acid intermediates subsequent accumulation of ACh mainly affects the metabolism of osmolites and TCA intermediates.

An acetylcholinesterase inhibitor, eserine, induces long-term depression at CA3-CA1 synapses in the hippocampus of adult rats.

Studies in humans and rodents support a role for muscarinic ACh receptor (mAChR) and nicotinic AChR in learning and memory, and both regulate hippocampal synaptic plasticity using complex and often times opposing mechanisms. Acetylcholinesterase (AChE) inhibitors are commonly prescribed to enhance cholinergic signaling in Alzheimer's disease in hopes of rescuing cognitive function, caused, in part, by degeneration of cholinergic innervation to the hippocampus and cortex. Unfortunately, therapeutic efficacy is moderate and inconsistent, perhaps due to unanticipated mechanisms. M1 mAChRs bidirectionally control synaptic strength at CA3-CA1 synapses; weak pharmacological activation using carbachol (CCh) facilitates potentiation, whereas strong agonism induces muscarinic long-term depression (mLTD) via an ERK-dependent mechanism. Here, we tested the prediction that accumulation of extracellular ACh via inhibition of AChE is sufficient to induce LTD at CA3-CA1 synapses in hippocampal slices from adult rats. Although AChE inhibition with eserine induces LTD, it unexpectedly does not share properties with mLTD induced by CCh, as reported previously. Eserine-LTD was prevented by the M3 mAChR-preferring antagonist 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), and pharmacological inhibition of MEK was completely ineffective. Additionally, pharmacological inhibition of p38 MAPK prevents mLTD but has no effect on eserine-LTD. Finally, long-term expression of eserine-LTD is partially dependent on a decrease in presynaptic release probability, likely caused by tonic activation of mAChRs by the sustained increase in extracellular ACh. Thus these findings extend current literature by showing that pharmacological AChE inhibition causes a prolonged decrease in presynaptic glutamate release at CA3-CA1 synapses, in addition to inducing a likely postsynaptic form of LTD.

Non‐quantal release of acetylcholine in rat atrial myocardium is inhibited by noradrenaline

In the mammalian myocardium, ACh, which is the main neurotransmitter of cardiac parasympathetic postganglionic fibres, can be released via both quantal (vesicular) and non-quantal (non-vesicular) mechanisms of secretion. Non-quantal release is continuous and independent of vagus activity and exocytosis of ACh-containing vesicles. During the incubation of myocardium in the presence of acetylcholinesterase (AChE) inhibitors, non-quantal ACh release leads to accumulation of ACh in the myocardium and cholinergic effects, which are proportional to the intensity of non-quantal secretion. The aim of the present study was to reveal whether non-quantal release of ACh can be modulated by another major cardioregulator, noradrenaline, or whether it represents uncontrolled leakage of ACh from cholinergic fibres. Cholinergic changes of electrical activity induced by the AChE inhibitor paraoxon (5 × 10(-6) M) in isolated rat right atrial preparations were determined by means of a standard microlectrode technique and used as a measure of the intensity of non-quantal release. Noradrenaline (10(-7) and 10(-6) M) substantially suppressed, but did not abolish, effects of paraoxon via stimulation of α-adrenoceptors, because all experiments were conducted in the presence of the β-blocker propranolol (5 × 10(-6) M). A blocker of ganglionic transmission, hexamethonium bromide (10(-4) M), failed to alter the inhibitory effect of noradrenaline, indicating that only non-quantal ACh release is suppressed by this neurotransmitter. The effects of noradrenaline could be reduced by the α2-antagonist yohimbine (10(-6) M). However, both the α1-agonist phenylephrine (10(-6) M) and the α2-agonist clonidine (10(-6) M) significantly inhibited the cholinergic effects of paraoxon, indicating the possible involvement of both α-adrenoceptor subtypes in mediation of the adrenergic inhibition of non-quantal ACh release. Thus, cardiac non-quantal ACh release can be negatively regulated by noradrenaline, providing another facet of sympathetic-parasympathetic interaction in the heart.

Muscarinic receptor activation in the lumbosacral spinal cord ameliorates bladder irritation in rat cystitis models

To investigate whether activation of spinal cholinergic pathways affects bladder activity in rats with chemical cystitis induced by acetic acid (AA) and cyclophosphamide (CYP). The effects of intrathecal (i.t.) application of neostigmine as an acetylcholine esterase (AChE) inhibitor, oxotremorine-M (OXO-M) as a muscarinic ACh receptor (mAChR) agonist or epibatidine as a nicotinic AChR (nACHR) agonist with or without atropine as a mAChR antagonist or mecamylamine (MEC) as a nAChR antagonist at the level of L6-S1 spinal cord on C-fibre mediated bladder irritation were examined by using continuous-infusion cystometry (0.1 mL/min) in urethane-anaesthetized female Spraque-Dawley rats. Bladder irritation was induced by intravesical AA (0.25%) or pretreatment with intraperitoneal injection of CYP (150 mg/kg) 24 h before cystometry. In control rats during intravesical saline, the effects of above drugs on the bladder activity were also examined. The intercontraction intervals (ICI) in AA- and CYP-treated rats were significantly shorter than those in control rats. Neostigmine (i.t.) significantly increased ICI dose-dependently without changing maximum voiding pressure in all groups. The mean (sem) maximal percentage increases in ICI after i.t. neostigmine as compared with the pretreatment value in control, AA- and CYP-treated rats were 93.2 (18.5)%, 117.5 (20.2)% and 200.7 (19.6)%, respectively. The percentage increases of ICI in the CYP-treated group were significantly (P < 0.05) higher than those in the AA-treated or control groups. In all groups, pretreatment with atropine, but not MEC, almost completely antagonized the inhibitory effects of neostigmine. OXO-M produced almost the same effects as that of neostigmine in all groups. Conversely, i.t. epibatidine decreased the ICI in all groups and these excitatory effects were completely antagonized by pretreatment with MEC and significantly inhibited by pretreatment with MK-801(noncompetitive n-methyl-d-aspartate receptor antagonist). These results indicate that accumulation of ACh by AChE inhibition in the spinal cord can ameliorate frequent urination in chemical cystitis via mAChRs, but not nAChRs.

Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+) measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, omega-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of approximately 50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.

Targeting acetylcholinesterase to the neuromuscular synapse

The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the specialized synaptic basal lamina at the neuromuscular junction (NMJ). This enzyme form consists of both catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail. We have previously shown that catalytic subunits are assembled in the rough endoplasmic reticulum and that after approximately 90 min a subset of these tetramers assemble with collagenic tail subunits in the Golgi apparatus. In muscle, blocking ER to Golgi transport with Brefeldin A prevents the appearance of ColQ-AChE, consistent with assembly of asymmetric forms in the Golgi. Moreover, newly synthesized and assembled ColQ-AChE associates with perlecan intracellularly and can only be co-immunoprecipitated with anti-perlecan antibodies 90 min after the first appearance of catalytic subunits. Once assembled, the ColQ-AChE/perlecan complex is externalized where it co-localizes with other components of the NMJ including dystroglycan, rapsyn, laminin and MuSK. These clusters tend to form over the nuclei that are expressing the components, suggesting local vectorial transport to the cell surface, and may form a primary scaffold that in turn can capture other molecular constituents of the neuromuscular synapse. While most AChE clusters on quail myotubes are devoid of acetylcholine receptors, treatment of the culture with recombinant agrin results in a rapid translocation of receptors to the AChE clusters in less than 4 h. It remains to be determined if MuSK is localized to the clusters. In vivo, AChE transcripts and enzyme are more highly expressed at the NMJs, implying higher rates of AChE translation and assembly in the synaptic regions, and hence more ColQ-AChE for localized export. We have previously shown that binding sites for ColQ-AChE are concentrated at sites of nerve-muscle contact where they colocalize with AChR and perlecan. ColQ-AChE binds directly to perlecan using solid phase microtiter plate assay, the Biacore assay, and co-immunoprecipitations. Moreover, perlecan binds to dystroglycan at the NMJ. In perlecan or dystroglycan null mice there is no accumulation of AChE at the NMJ, supporting the hypothesis that this heparan sulfate proteoglycan is an essential component of the ColQ-AChE localization mechanism. Together, these studies suggest a model of synaptic development whereby AChE can be targeted to and clustered on the muscle membrane together with dystroglycan and perlecan to form scaffolds to which AChR can be clustered through activation of the MuSK receptor. At mature synapses ColQ-AChE is secreted directly into the synaptic cleft where it binds to the heparan sulfate proteoglycan perlecan as well as potentially other molecules including MuSK, as was recently reported.

Fenvalerate induced changes in the Ach and associated AchE activity in different tissues of fish Cirrhinus mrigala (Hamilton) under lethal and sub-lethal exposure period

The disruption of acetylcholinesterase activity in freshwater fish Cirrhinus mrigala (Hamilton) is demonstrated in the present study using acetylcholine chloride as substrate. Fish, C. mrigala on exposure to lethal and sub-lethal concentration of synthetic pyrethroid insecticide, fenvalerate showed inhibition in the activity of the AchE suggesting decrease in the cholinergic transmission and consequent accumulation of Ach in the tissues, namely, gill, muscle, liver and brain. This might have lead to behavioural changes and create widespread disturbance in the normal physiology, ultimately causing the death of the fish. Residue analysis using gas–liquid chromatography technique (GLC) revealed that highest quantity of fenvalerate accumulated in gill followed by liver and muscle under lethal concentration, whereas in sub-lethal concentration muscle accumulated highest concentration followed by gill and liver. The results suggest that in biomonitoring programme, AchE can be a good diagnostic tool for fenvalerate toxicity.

Evidence for epidermal acetylcholine accumulation after oxidative stress by H2O2 and possible implications

Acetylcholine (Ach) has been shown to be synthesized de novo and degraded in the human skin (1), while the presence of acetylcholinesterase (AchE), the degrading enzyme, was first mentioned in 1989 (2). It is now accepted that H2O2‐related oxidative stress is a major player in the development/acceleration of vitiligo (3). This oxidative stress affects the recycling of the essential cofactor (6R)‐l‐erythro 5,6,7,8 tetrahydrobiopterin (6BH4) via H2O2‐mediated oxidation of Trp and Met residues in the structure of pterin‐4a carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) (3). Only very recently, it was recognized that AchE is also affected and deactivated by 10−3 m H2O2 causing the accumulation of Ach in the epidermis of patients with vitiligo (4). One of the implications of oxidative stress in vitiligo includes pruritus, which was for a long time attributed to the presence of epidermal H2O2 in the 10−3 m range. In this context, a role for Ach has been suggested in association with pruritus, and therefore, we would like to suggest that Ach accumulation caused by H2O2‐mediated deactivation of AchE may well initiate pruritus (5). The Ach accumulation can also explain the earlier documented decreased sweating in patients with vitiligo (6).

Role of protein kinase C in the release of [ 3H]acetylcholine from myenteric plexus treated with vesamicol

The present experiments investigated the release of [ 3H]acetylcholine ([ 3H]ACh) from the guinea pig myenteric plexus treated with 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a drug that impairs ACh accumulation by synaptic vesicles. Ouabain, an Na +-K + ATPase inhibitor, released [ 3H]ACh synthesised in the presence of (−)-vesamicol, while electrical field stimulation or KCl depolarisation were not effective to release the transmitter in this condition. The effect of ouabain was Ca 2+-dependent and in the presence of (−)-vesamicol it was blocked by calphostin C, an inhibitor of protein kinase C (PKC). In addition, stimulation of kinase C activity by a phorbol ester, but not by its inactive isomer, prevented (−)-vesamicol from interfering with the release of [ 3H]ACh in electrically-stimulated myenteric plexus, similar to the effect of ouabain. We conclude that release of [ 3H]ACh induced by ouabain in the presence of (−)-vesamicol depends on PKC activation.

Target and non-target effects of anticholinesterase pesticides in fish

In fish, the inhibition of the enzyme, acety1cholinesterase (AChE), has a far-reaching effect as evidenced by two major non-target effects, namely, thyroid and gonadal dysfunction in Channa punctatus, mediated by ACh accumulation. Organification of tyrosine is stimulated and TSH-mediated release of the hormone is inhibited. A depressed AChE function could be related to low kidney iodide peroxidase activity and thyroxine in the blood of fish exposed to Elsan and Furadan. In the analysis of results obtained from studies on gonadal function it was obvious that reproductive efficiency was compromised in fish exposed to Elsan, metacid-50 and carbaryl. Channa punctatus exposed to 211 ppb of Elsan over a 90-day period covering the spawning season resulted in significant reduction in the ovarian weight. This suggested that even at non-lethal levels of organophosphate exposure there is a low degree of ovarian recrudescence. Pesticides, such as metacid-50 and carbaryl, below mortality levels of exposure, demonstrated reduced pituitary and serum gonadotropin levels accompanied by inhibited activity of the hypothalamic gonadotropin releasing hormone. It is surmised that these hormonal effects are mediated via the ACh-ACNE system.

Tonic activation of locus coeruleus neurons by systemic or intracoerulear microinjection of an irreversible acetylcholinesterase inhibitor: Increased discharge rate and induction of c-fos

Recent studies in this laboratory have demonstrated that intramuscular injection of the irreversible acetylcholinesterase (AChE) inhibitor, soman (pinacolylmethylphosphonofluoridate), produces a rapid (1–2 h) and profound depletion (70% of control) of norepinephrine (NE) in the olfactory bulb and forebrain. NE is decreased only in convulsing animals. As NE-containing locus coeruleus (LC) neurons provide the only NE input to the olfactory bulb and the major NE innervation of the forebrain, the reduction of NE suggests that soman may cause tonic activation of LC neurons leading to rapid depletion of NE. Activation of LC may result from: (i) facilitation of cholinergic transmission in LC; (ii) soman-induced activation of excitatory inputs to LC; or (iii) generalized activation of LC neurons due to seizures. The present experiments were designed to assess these alternatives. We examined whether LC neuronal activity, c-fos expression, and AChE staining are altered after peripheral (systemic) or direct intracoerulear injection of soman in anesthetized rats. Both modes of soman administration rapidly and potently increase the spontaneous discharge rate of LC neurons. This activation was associated with a desynchronization of the electroencephalogram, but not with seizures. The discharge of LC neurons remained elevated at all postsoman intervals examined (up to 2 h) and was rapidly and completely reversed by systemic injection of the muscarinic receptor antagonist scopolamine hydrochloride, but not by the nicotinic receptor antagonist mecamylamine. Both systemic and intracoerulear soman administration completely inhibited AChE staining in LC and rapidly induced the expression of c-fos in LC neurons. These results demonstrate that soman potently and tonically activates LC neurons. This effect appears to be mediated by direct inhibition of AChE in LC leading to a rapid accumulation of ACh. Unhydrolyzed ACh tonically activates LC neurons via muscarinic receptors. Soman-induced activation of LC neurons does not require seizures. We conclude that depletion of forebrain and olfactory bulb NE after systemic administration of soman results from tonic hypercholinergic stimulation of LC.

Organophosphorus and Carbamate Insecticide Poisoning in Large Animals

Organophosphorus and carbamate insecticides are used commonly in agriculture to control pests of crops and animals. These compounds are toxic and livestock poisoning has occurred from mistaken addition of unused insecticide to feeds and animal access to improperly disposed of materials or "empty" containers. The morbidity rate approximates a third of animals exposed and about half of those affected die. Organophosphorus and carbamate insecticides generate their toxic effects by bonding to and inhibiting ChE enzymes (most importantly, AChE), which are responsible for breaking down the neurotransmitter ACh. The accumulation of ACh results in uninhibited impulse transmission at the cholinergic endings, including autonomic preganglionic junctions, certain neurons in the central nervous system, and motor nerves to the skeletal muscles. This eventually leads to fatigue of end organs; death usually is a result of respiratory failure. The "delayed neuropathy syndrome" is caused by some OP insecticides, but the pathophysiology of this syndrome does not involve the inhibition of ChE and accumulation of neurotransmitter. Over 100 anticholinesterase insecticides are in use. Variations in toxicity exist among these compounds, but larger diversities exist in the likelihood of poisoning because of differences in formulations, solvents, and conditions of use. Young animals usually are more sensitive than adults. Some of these agents are among the most toxic of manmade chemicals. These insecticides generally do not accumulate in tissues. The "chronic" effects depend on accrued physiological changes from frequent exposure to nonlethal doses. Clinical signs occur within minutes to a few hours and include the muscarinic signs of salivation, excessive lacrimation, frequent urination, and diarrhea. Concurrent nicotinic effects include muscular tremors followed by weakness and paralysis. Topical exposure results in similar poisoning but the onset of clinical signs may be delayed. Certain pour-on insecticides may cause a delayed (more than a week) syndrome in some breeds. Diagnosis of OP or carbamate insecticide poisoning is made by (1) interpretation of clinical signs and history that are consistent with this poisoning, (2) detection of the chemical compound in stomach or rumen contents and tissues, and (3) demonstration of the adverse biological effect, ChE inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of peripheral cholinergic nerves by glutamate indicates a new peripheral glutamate receptor

The bronchial smooth muscle of the rat was examined for contractile responses to excitatory amino acids. The nerve-mediated contraction induced by electrical field stimulation was enhanced by exogenous L-glutamate (L-Glu). The apparent affinity (ED 50) of L-Glu was 3.5 ± 0.1 mM. Both tetrodotoxin and hemicholinium-3 completely abolished the electrical field-induced contraction and therefore the potentiation by L-Glu, which indicates that L-Glu has a prejunctional effect. Concentrations of L-Glu higher than 22 mM inhibited the electrical field-induced contractions and enhanced the tonus of the smooth muscle by postjunctional stimulation. The ED 50 of exogenous ACh was not altered by L-Glu. High concentrations (62 mM) of L-Glu increased the intrinsic activity (α) of ACh, indicating a postjunctional potentiation of ACh-induced contractions. L-Glu did not inhibit the activity of acetylcholinesterase, therefore the postjunctional potentiation was not due to ACh accumulation. Inhibition of the electrical field-induced contraction was seen with high concentrations of D-Glu, L-aspartate (L-Asp), L-α-amino adipate and ibotenate. Neither glutamate diethyl ester nor 2-amino-5-phosphonovalerate had any inhibitory effects on the L-Glu- and L-Asp-induced alterations of the electrical field-stimulated contraction or on the L-Glu-enhanced tonus of the bronchial smooth muscle. Kainate, N-methyl-D-aspartate, quisqualate and N-acetyl-aspartyl-glutamate had only minor transient potentiating effects on the electrical field-induced contraction. The results provide evidence for a L-Glu receptor in rat bronchi that has a different specificity for glutamate agonists and antagonists than the L-Glu receptor described in the CNS. The receptor seems to be located prejunctionally and enhances nerve-mediated responses and thereby stimulates the bronchial smooth muscle to contract. The possible involvement of this type of receptor in the ‘Chinese restaurant syndrome’ is discussed.

Molecular forms and localization of acetylcholinesterase and nonspecific cholinesterase in regenerating skeletal muscles.

Molecular forms and histochemical localization of acetylcholinesterase and nonspecific cholinesterase were analysed in muscle regenerates obtained from rat EDL and soleus muscles after ischaemic-toxic degeneration and irreversible inhibition of preexistent enzymes. Regenerating myotubes and myofibres produce the 16S AChE form in the absence of innervation. The 10S AChE form prevails over 4S form with maturation into striated fibres. Although the patterns of AChE molecular forms in normal EDL and soleus muscles differ significantly no such differences were observed in noninnervated regenerates from both muscles. Two types of focal accumulation of AChE appear on the sarcolemma of regenerating muscles: first, in places of former motor endplates and, second, in extra-junctional regions. The 4S form of nonspecific cholinesterase is prevailing in regenerating myotubes whereas its asymmetric forms or focal accumulations could not be identified reliably. The satellite cells which survive after muscle degeneration probably originate from some type of late myoblasts and transmit the information concerning the ability to synthesize the asymmetric AChE forms and to focally accumulate AChE to regenerating muscle cells. Synaptic basal lamina from former motor endplates may locally induce AChE accumulations in regenerating muscles.

Extrasynaptic accumulations of acetylcholinesterase in the rat sternocleidomastoid muscle after neonatal denervation. Light and electron microscopic localization and molecular forms.

Denervated neonatal rat sternocleidomastoid muscle has decreased levels of total AChE when compared to control muscle. Denervated versus control values of total muscle AChE present a three-phase curve in function of time after denervation. There is a rapid initial fall 0-3 days after denervation, an increase during about 2 weeks, then again a decrease in total AChE. Thus, there is a transitory net accumulation of AChE after the initial fall of activity in denervated developing muscle. Extrasynaptic areas of high AChE activity develop between 1 and 2 weeks after denervation and remain visible up to 1 month after denervation before vanishing. An electron microscope study shows that these accumulations are internal to the muscle fiber, close to a limited number of muscle nuclei and associated to the sarcoplasmic reticulum and nuclear envelope, but not to the T-tubule system. As found in adult rat muscle, the initial fall in AChE affects first the 16 S AChE form, and soon after, the 4 S and 10 S AChE forms. A main difference with adult muscle is the sudden increase and predominance over other forms of 10 S AChE 2 weeks after denervation at birth. Later, the decrease in AChE affects 16 S and 4 S AChE before 10 S AChE. The regions rich in extrasynaptic sites of AChE accumulation possess a very high proportion of 10 S AChE. Thus, the mechanisms of biosynthesis, intracellular transport and/or secretion of AChE may be very different in young, developing muscle compared to adult muscle.

Effect of sympathectomy on axoplasmic transport of selected enzymes including MAO and other mitochondrial enzymes.

Abstract— Axoplasmic transport in guanethidine sympathectomized and control rats was investigated by monitoring the accumulations of various enzyme activities proximal to a ligature placed on the sciatic nerve. Sympathectomy affected the accumulations of three different mitochondrial enzymes quite differently: the accumulation of monoamine oxidase (MAO, EC 1.4.3.4) activity was inhibited 65% or more, that of hexokinase (HK, EC 2.7.1.1) activity was only inhibited 26%, while accumulation of glutamic dehydrogenase (GDH, EC 1.4.1.3) activity was unaffected by Sympathectomy. Accumulation of AChE (EC 3.1.1.7) activity was depressed 40%, but accumulations of the activities of the lysosomal enzyme, acid phosphatase (acid P'tase, EC 3.1.3.2), and of the cytosolic enzyme, choline acetyltransferase (CAT, EC 2.3.1.6) were unchanged.Despite impressive inhibition of MAO accumulation, the intrinsic activity of this enzyme in sciatic nerve was unaffected by Sympathectomy. The existence in nerve of isozymes of MAO was demonstrated using the inhibitors clorgyline and deprenyl. Transported MAO activity was almost entirely type A; intrinsic activity was 2/3 type A and 1/3 type B.The differential response of the accumulations of the three mitochondrial enzyme activities measured was interpreted to indicate the existence, within neurons, of mitochondria with different enzyme complements.