Total Acetylcholinesterase Activity Research Articles

Developmental neurotoxicity of triphenyl phosphate in zebrafish larvae

Triphenyl phosphate (TPhP), a typical organophosphate ester, is frequently detected in the environment and biota samples. It has been implicated as a neurotoxin as its structure is similar to neurotoxic organophosphate pesticides. The purpose of the present study was to investigate its potential developmental neurotoxicity in fish by using zebrafish larvae as a model. Zebrafish (Danio rerio) embryos were exposed to 0.8, 4, 20 and 100 μg/L of TPhP from 2 until 144 h post-fertilization. TPhP was found to have high bioconcentrations in zebrafish larvae after exposure. Further, it significantly reduced locomotor activity as well as the heart rate at the 100 μg/L concentration. TPhP exposure significantly altered the content of the neurotransmitters γ-aminobutyric and histamine. Downregulation of the genes related to central nervous system development (e.g., α1-tubulin, mbp, syn2a, shha, and elavl3) as well as the corresponding proteins (e.g., α1-tubulin, mbp, and syn2a) was observed, but the gap-43 protein was found to upregulated. Finally, marked inhibition of total acetylcholinesterase activity, which is considered as a biomarker of neurotoxicant exposure, was also observed in the larvae. Our results indicate that exposure to environmentally relevant concentrations of TPhP can affect different parameters related to center nervous system development, and thus contribute to developmental neurotoxicity in early developing zebrafish larvae.

In situ induced metal-enhanced fluorescence: A new strategy for biosensing the total acetylcholinesterase activity in sub-microliter human whole blood

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities (i.e., total AChE) in human blood are biomarkers for theranostic monitoring of organophosphate neurotoxin-poisoned patients. We developed an ultra-sensitive method to detect the total AChE activity in sub-microliter human whole blood based on in situ induced metal-enhanced fluorescence (MEF). Both AChE and BChE can catalyze the hydrolysis of the acetylthiocholine (ATCh) substrate and produce positively-charged thiocholine (TCh). TCh can reverse the negatively-charged surface of core–shell Ag@SiO2 nanoparticles (NPs). The negatively-charged fluorescent dye (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS) is then confined to the surface of Ag@SiO2 NPs and generates an enhanced fluorescence signal in situ. Changes in the surface charge of Ag@SiO2 NPs are monitored by Zeta potential, and the MEF effect is confirmed by the measurements of fluorescence time decay. AChE activity has a dynamic range of 0U/mL to 0.005U/mL and a detection limit of 0.05mU/mL. The total AChE activity in the sub-microliter human whole blood could be determined; the results were further validated. Therefore, combining the AChE catalytic reaction with MEF provides a simple, ultra-sensitive, and cost-effective “in situ MEF” approach to determine the total AChE activity in human whole blood sample down to sub-microliters without matrix interferences. The strategy also allows potential usage in other tissues and other fields.

Toxicological attributes of plant chemicals and their biochemical impacts on cholinesterase and protein levels in relation with conventional insecticides against mosquito larvae of Karachi city

In order to control mosquito, different chemicals were applied against the larvae of Culex fatigans (Diptera Culicidae). The tested chemicals were Lambda cyhalothrin, chlorpyrifos, Azadirachta indica (Spindales meliaceae) neem extract, and Sueda monoica extract. All chemicals were found to have significant efficacy against Culex fatigans mosquito larvae. In terms of efficacy, the biochemical effects after treatment with such chemicals were found to vary. Total protein levels and acetylcholinesterase (AChE) activity were the parameters examined. Chlorpyrifos, Lambda cyhalothrin, and neem elevated AChE activity levels whereas the plant extract S. monoica decreased enzymic activity. Total protein levels were decreased by Lambda cyhalothrin and chlorpyrifos whereas protein was increased by neem extract and S. monoica.

Differential effects of developmental hypo- and hyperthyroidism on acetylcholinesterase and butyrylcholinesterase activity in the spinal cord of developing postnatal rat pups

The plasticity and vulnerability of the rat spinal cord (SC) during postnatal development has been less investigated compared to other CNS structures. In this study, we determined the effects of thyroid hormonal (TH) deficiency and excess on postnatal growth and neurochemical development of the rat SC. The growth as well as the specific and total activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes of the SC were determined in hypo- and hyperthyroid rat pups at postnatal (P) days P1, P5, P10 and P21 (weaning), and were compared to age-matched untreated normal controls. AChE is a cholinergic synaptic enzyme while BuChE is a metabolic enzyme mainly found in glial cells and neurovascular cells. The SC is rich in somatic motor, autonomic cholinergic neurons and associated interneurons. Daily subcutaneous injection of pups with thyroxine (T4) and administration of antithyroid goitrogen propylthiouracil (PTU) in the litter's drinking water were used to induce hyper- and hypothyroidism, respectively. Enzyme assays were carried out spectrophotometrically at the above-mentioned ages, using SC homogenates with acetylthiocholine-chloride as the substrate, together with specific cholinesterase inhibitors, which specifically target AChE and BuChE. SC weights were significantly lower at P10 and P21 in hypothyroid pups but unchanged in the hyperthyroid ones. Hypothyroidism significantly reduced both specific and total AChE activity in SC of P10 and P21 rat pups, while having no effects on the BuChE activity, although total BuChE activity was decreased due to reduced total tissue weight. In contrast both specific and total AChE activities were markedly and significantly increased (>100%) in the P10 and P21 hyperthyroid pups. However, BuChE specific activity was unaffected by this treatment. The results indicate that hypothyroid condition significantly reduces, while hyperthyroidism increases, the postnatal development of cholinergic synapses, thereby influencing the functional development of this major sensory and motor structure. However, the neurochemical development of glia and other non-neuronal cells, where BuChE is mainly localized, is comparatively unaffected in these abnormal developmental conditions.

Therapeutic effects of multi-dose activated charcoal on the acute dichlorvos poisoning in rats

Objective To assess the therapeutic effects of activated charcoal on the acute dichlorvos poisoning in rats. Method Thirty male clean grade Wistar rats were randomly (random number) divided into three groups: control group (group A, n = 10), single dose activated charcoal group (group B, n = 10) and multi-dose activated charcoal (group C, n=10). The rats of group A were suffered from 35 mg/kg dichlorvos exposure by oral without activated charcoal and senna. The rats of group B received 35 mg/kg dichlorvos exposure by oral with 175 mg/kg activated charcoal given immediately after dichlorvos exposure and 35 mg/kg senna given half an hour later. In the group C, 35 mg/kg dichlorvos was given to rats by oral with 175 mg/kg activated charcoal given immediately after dichlorvos exposure and 35 mg/kg senna given half an hour later and then every four hours. Blood samples were collected from the carotid artery at different intervals after exposure. DDVP concentration and total blood acetyl-cholinesterase activity were detected. Differences in serum DDVP concentration, Cmax, AUC (0→∞ ), MRT and acetylcholinesterase among three groups were calculated by using ANOVA. Results Serum DDVP levels in single dose group and in multi-dose group were significantly different from those in control group (P 0.05). Another four hours later, no differences in acetylcholinesterase were found a-mong three groups (P > 0.05). Conclusions The peak concentrations of dichlorvos in blood are lower in group B and group C, and the blood acetylcholinesterase inhibition is quelled by activated charcoal. Therefore, the effects of multi - dose of activated charcoal is better than that of single dose of activated charcoal. Key words: Dichlorvos; Activated carbon; Organophosphate poisoning; Oral intaken; LC-MS; Rat; Toxicoki-netics; Statistical moment model

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.

Dissociation of Transcription, Translation, and Assembly of Collagen-tailed Acetylcholinesterase in Skeletal Muscle

The synaptic form of acetylcholinesterase (AChE) in skeletal muscle ColQ-AChE derives from two separate genes encoding the catalytic and the non-catalytic collagenic tail (ColQ) subunits, respectively. ColQ-AChE expression is regulated by muscle activity; however, how this regulation takes place in skeletal muscle remains poorly understood. In this study, we overexpressed or knocked down ColQ expression in skeletal muscle and found that the level of this non-catalytic component by itself was sufficient to change the levels of total AChE activity by promoting assembly of higher order oligomeric forms including the collagen-tailed forms. These results initially suggested that ColQ could be limiting in the assembly of synaptic ColQ-AChE during development and differentiation. We then determined the levels of ColQ protein and ColQ mRNA during primary quail muscle cell development and differentiation in culture (QMCs) and as a function of muscle activity. Surprisingly, we found dissociation between transcription and translation of the non-catalytic subunit from its assembly into ColQ-AChE. Furthermore, we found that the vast majority of the steady state ColQ molecules in mature quail muscle cultures are not assembled into ColQ-AChE, suggesting that they are either rapidly degraded or have alternative function(s).

Correlation of Therapeutic Effect of Obidoxime and Dosing Time in the Acute Intoxication by Chlorfenvinphos in Rats

The ability of obidoxime with atropine and diazepam mixture to reactivate acetylcholinesterase inhibited by the organophosphorus compound chlorfenvinphos was compared in the central nervous system and peripheral tissues of rats. The animals were intoxicated with chlorfenvinphos (6 mg/kg, p.o.) and treated immediately, 24 and 48 hrs later with obidoxime (50 mg/kg, i.p.), atropine (10 mg/kg, i.p.), and diazepam (10 mg/kg, i.p.) in a single dose, or in various combinations (with 2-3 drugs) simultaneously. Total tissue acetylcholinesterase activities were monitored at 2, 72, and 168 hrs after intoxication. Enzyme activity was determined using Ellman's colorimetric method. The results of the present study show that obidoxime administered separately and jointly with atropine and diazepam 24 hrs after intoxication was effective on reactivation of chlorfenvinphos-inhibited acetylcholinesterase in the central nervous system and in the peripheral tissues. However, the application of obidoxime alone or in combination with atropine and diazepam 48 hrs after chlorfenvinphos intoxication caused an increased unfavourable effect in rats. The results obtained also indicate an unfavourable interaction of obidoxime with diazepam in the course of chlorfenvinphos poisoning, when antidotes were administered immediately, 24 and 48 hrs after intoxication.

Effects of estrogen on intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors and on acetylcholinesterase activity in rat hippocampus

The aim of the present study was to investigate the effects of estrogen lack and estrogen replacement on the production of total [ 3H]inositol phosphate ([ 3H]IP) induced by the activation of muscarinic acetylcholine receptors (mAChRs) and on the mechanisms for inactivation of acetylcholine. Hippocampi were obtained from rats in proestrus (PE), ovariectomized for 15 days (C15), ovariectomized for 15 days and then treated with 17β-estradiol for 7 days (E7) and ovariectomized and immediately treated with 17β-estradiol for 21 days (E21). Ovariectomy did not change the basal level of total [ 3H]IP in the hippocampus. 17β-Estradiol replacement (E7 and E21) reduced the basal level of total [ 3H]IP. In all experimental groups, carbachol (CCh) caused a concentration-dependent rise in total [ 3H]IP. The maximum effect was reached with 10 −4 M CCh. The response to 10 −4 M CCh in the hippocampi from C15 and E7 rats was twofold higher than in hippocampi from PE and E21 animals and was blocked by pirenzepine, but not by methoctramine. Ovariectomy or 17β-estradiol treatment for 7 days did not change neither the total acetylcholinesterase (AChE) activity nor the relative amount of mono- and dimeric G 1/G 2 and tetrameric G 4 globular forms. Conversely, hormonal treatment for 21 days induced an increase in AChE activity of G 1/G 2 and G 4 forms, indicating that 17β-estradiol stimulates both synthesis and assembly of AChE molecular forms. The present results suggest that the duration and/or a critical period with regard to the initiation of estrogen therapy are important to regulate the function of mAChRs and AChE activity in female rat hippocampus.

Variation of Musca domestica L. acetylcholinesterase in Danish housefly populations

Anti-cholinesterase resistance is in many cases caused by modified acetylcholinesterase (MACE). A comparison was made of toxicological data and AChE activity gathered from 21 field populations and nine laboratory strains of houseflies, Musca domestica L., to elucidate the best way of generating data to provide advice for management strategies and gathering information for resistance risk assessment on the organophosphates azamethiphos and dimethoate and the carbamate methomyl, which have been the primary insecticides used against adult houseflies in Denmark. Cluster analysis was performed and > 2000 houseflies were assigned to one of three phenotypes based on total acetylcholinesterase activity as well as inhibition by azamethiphos, methomyl or omethoate. A cluster, i.e. a phenotype, with high total AChE activity and high sensitivity to azamethiphos and less sensitivity to inhibition by methomyl and omethoate was shown to be linked to methomyl resistance. It was not possible to define any clusters that could be linked to azamethiphos or dimethoate resistance. The five mutations V180L, G262A, G262V, F327Y and G365A causing anticholinesterase resistance in houseflies were all identified in the Danish housefly strains. The data are very heterogeneous, and a correlation of molecular genetic background and resistance of phenotypes is not obvious with the available data.

Peripheral cholinergic disturbances in Alzheimer's disease

The most pronounced neurochemical abnormality in Alzheimer's disease (AD) is cholinergic dysfunction in the central nervous system. Peripheral tissues may also be affected, however, including blood. The present study undertook to determine the activity of acetylcholinesterase (AChE) and its molecular forms in erythrocytes, lymphocytes and platelets of normal elderly subjects and probable AD cases. These samples contained dimeric globular (G2), tetrameric globular (G4) and asymmetric (A12) AChE forms, but no globular monomeric (G1) enzyme. In both lymphocytes and platelets, the major AChE molecular form was G2 (approximately 80%), with G4 and A12 forms accounting for nearly equal portions of the remainder. Total AChE activities and measured sedimentation coefficients were similar in the control and AD samples (from patients with mild and moderately severe cognitive deficiency). However, the groups differed significantly in the proportion of certain AChE molecular forms. Thus, as compared with controls, the amount of A12 AChE in the AD samples was increased 148 and 161% in lymphocytes and platelets, respectively. Genotyping for apolipoprotein E (ApoE) and the butyrylcholinesterase K (BCHE-K) variant, carried out using the polymerase chain reaction, showed that AD patients carried the ApoE4 allele at a significantly higher frequency than the controls. On the other hand there were no significant group differences in the occurrence of the BCHE-K variant and no synergism between ApoE alleles and the BCHE-K variant in our Hungarian AD population.

Sex-Linked Altered Acetylcholinesterase Resistance to Carbamate Insecticides in Adults of the Oriental Fruit Moth, Grapholita molesta (Lepidoptera: Tortricidae)

Insecticide resistance continues to be a major problem in the production of many important horticultural and field crops. The inheritance of resistance to a carbamate insecticide was measured in adults of a resistant population of the Oriental fruit moth (OFM), Grapholita molesta (Busck), a destructive pest of peach in northeastern North America. The inheritance of variations in inhibition of acetylcholinesterase in susceptible and resistant OFM adults was determined and quantitative measurements were made of total acetylcholinesterase and nonspecific esterase activity. Resistance and acetylcholinesterase inhibition were linked to the sex chromosome and were shown to be highly correlated (r2 > 0.91), suggesting that the mutant acetylcholinesterase was the basis for most of the observed resistance in adults. Levels of nonspecific esterase activity were 2.0- to 4.8-fold higher in resistant than in susceptible OFM and twice as high in males as in females, but there was no evidence that this resistance factor was sex linked. Results of bioassays of susceptible, resistant, and F1 hybrid OFM indicated that resistance appeared to be incompletely recessive in all F1 males and in females of the (R♀ × S♂) F1 progenies. In contrast, resistance appeared to be intermediate in daughters of resistant males mated with susceptible females (S♀ × R♂). Because of the lack of effective alternative control strategies, the identification of actual or potential resistance mechanisms and its genetic basis in actual pests are needed to maximize and predict the likelihood of success in resistance management programs.

Short- and long-term influences of calcitonin gene-related peptide on the synthesis of acetylcholinesterase in mammalian myotubes.

The present study analyses the short- (15 min - 2 h) and long-term (24 - 48 h) influences of calcitonin gene-related peptide (CGRP) on acetylcholinesterase (AChE) expression in the rat cultured skeletal muscle and the signal transduction events underlying CGRP actions. To assess the effect of CGRP on AChE synthesis, myotubes were pre-exposed to the irreversible AChE inhibitor diisopropyl fluorophosphate (DFP) and treated with CGRP or forskolin, an adenylyl cyclase (AC) activator. Treatment of myotubes with 1 - 100 nM CGRP for 2 h increased by up to 42% the synthesis of catalytically active AChE with a parallel increase in the intracellular cyclic AMP. The stimulation of AChE synthesis induced by CGRP was mimicked by direct activation of AC with 3 - 30 microM forskolin. In contrast, pre-treatment of cultures with 100 nM CGRP for 20 h reduced by 37% the subsequent synthesis of AChE, resulting in a 15% decrease in total AChE activity after 48 h CGRP treatment. Moreover, 24 h treatment of myotubes with 100 nM CGRP reduced by 54% the accumulation of cyclic AMP induced by a subsequent CGRP treatment. These findings indicate that, in skeletal muscle cells, CGRP modulates the AChE expression in a time-dependent manner, initially stimulating the enzyme synthesis through a cyclic AMP-dependent mechanism. The decreased AChE synthesis observed after long-term CGRP treatment suggests that CGRP signalling system is subject to desensitization or down-regulation, that might function as an important adaptative mechanism of the muscle fibre in response to long-term changes in neuromuscular transmission.

Insulin Secretion and Acetylcholinesterase Activity in Monosodium L-Glutamate-Induced Obese Mice

Objective: Pancreatic islets isolated from mice treated neonatally with monosodium L-glutamate (MSG) were used to study insulin secretion. Material and Methods: Total acetylcholinesterase (AchE) activity of tissue extract was measured as a cholinergic activity marker. Obesity recorded in 90-day-old MSG mice (OM) by Lee index reached 366.40 ± 1.70, compared to control mice (CM) 324.40 ± 1.10 (p < 0.0001). Glucose 5.6 mM induced insulin secretion of 36 ± 5 pg/15 min from islets of CM and 86 ± 13 from OM (p < 0.001). When glucose was raised to 16.7 mM, islets from OM secreted 1,271 ± 215 and 1,017 ± 112 pg/30 min to CM. AchE activity of pancreas from OM was 0.64 ± 0.02 nmol of substrate hydrolyzed/min/mg of tissue and 0.52 ± 0.01 to CM (p < 0.0001). Liver of obese animals also presented increase of AchE activity. Results: These indicate that OM insulin oversecretion in low glucose may be attributed, at least in part, to an enhancement of parasympathetic tonus.

A Simple Method for Isolation of a Soluble Acetylcholinesterase from Rat Brain

ABSTRACTA soluble impure acetylcholinesterase (AChE), (EC 3.1.1.7) was isolated from rat brain. The enzyme showed tendency to form aggregates in a medium of low ionic strength that leaded to a decrease of the acetylcholinesterase activity. After an increase of the protein concentration of the obtained soluble enzyme, was observed a decrease or absolute loss of the activity which regenerated after diluting with water. Moreover detachment of the enzyme from the membrane (solubilization) leaded up to raising of the total AChE activity as well as the activity of the residual membrane bound AChE. Possible reason might be the transition of the enzyme to more active form with lower degree of aggregation. The soluble AChE was further purified by ion exchange chromatography (FPLC), but the obtained enzyme was not stable enough and rapidly lost its activity in a high ionic strength environment.

Age-related changes in activity of Fischer 344 rat brain acetylcholinesterase molecular forms.

Total acetylcholinesterase (AChE) and the molecular forms of the enzyme from six brain regions were compared in young adult (6 mo) and aged (24 mo) Fischer 344 rats. Total AChE activity was significantly reduced in aged striatum (48.7%), parietal cortex (39%), cerebellum (30.2%), and medulla/pons (23.1%). Forebrain of aged rats showed nonsignificant reduction of AChE (18.4%), but olfactory bulbs exhibited no differences in aged rats. The ratio of G4/G1 molecular forms, as isolated on sucrose density gradients, was unaltered in all aged rat brain tissues examined. These results indicate that aged rats exhibit reduced brain AChE, but there is no evidence for selective effects on individual molecular forms.

Anthelmintic efficacy of flemingia vestita (fabaceae): Genistein-induced alterations in the esterase activity in the cestode,raillietina echinobothrida

To ascertain the anthelmintic efficacy ofFlemingia vestita (an indigenous leguminous plant of Meghalaya, having putative anthelmintic usage), its crude root-tuber peel extract and active chemical principle, genistein, were testedin vitro with reference to esterase activity in the fowl tapeworm,Raillietina echinobothrida. With the localization of non-specific esterases (NSE) and cholinesterase (ChE), the organization of the cholinergic components of the nervous system in toto could be visualized in the cestodeo The specific ChE in the parasite is acetylcholinesterase (AChE). Both NSE and ChE were found in close association with the central and peripheral nervous components, besides being present in the tegument and muscular parts of the terminal male genitalia. The whole tissue homogenate of the parasite also showed a high AChE activity. After exposure to the crude peel extract (50 mg/ml of the incubation medium) and to genistein (0.5 mg/ml), a pronounced decline in the visible stain intensity in the cholinergic components of the nervous system and in the tegument was noticeable, indicating extremely reduced activity of NSE and ChE in these sites. The total AChE activity was also reduced to 4907% and 56–77%, following treatment with the peel extract and genistein, respectively. The reference drug, praziquantel (0.01 mg/ml) also caused reduction in the enzyme activity, somewhat at par with the genistein treatment. Genistein appears to have a transtegumental mode of action. Alteration in the AChE activity points towards acetylcholine, an inhibitory neurotransmitter in cestodes, as the potential target of action.

Mechanical stimulation increases expression of acetylcholinesterase in cultured myotubes.

We tested the hypothesis that acetylcholinesterase (AChE) expression in skeletal muscle cells is increased by passive mechanical stimulation. To this end, primary cultures of myotubes were subjected to repeated cycles of stretch-relaxation for 5 min, 30 min, 3 h, and 24 h, using the Flexercell FX-2000 strain unit. Although mechanical stimulation did not affect AChE expression at early time points, it led to a significant increase (42%; P < 0.05) in total AChE activity at 24 h. This increase reflected a general elevation in the activity of all AChE molecular forms as opposed to a preferential increase in a specific form. Tetrodotoxin (TTX) treatment did not prevent the increase in AChE expression, whereas nifedipine partially blocked it. These changes in enzyme expression were accompanied by increases in the levels of AChE mRNA, suggesting the involvement of pretranslational regulatory mechanisms. Together, these results illustrate that, in addition to neural activation and trophic factors, passive mechanical forces modulate expression of AChE in skeletal muscle cells. Because TTX did not prevent the increase in AChE expression, it appears that the effects of mechanical stimulation are independent of electrical activity, which further indicates the use of an alternate signaling pathway.

Regulation of cholinesterase gene expression affects neuronal differentiation as revealed by transfection studies on reaggregating embryonic chicken retinal cells.

In the embryonic chicken neuroepithelium, butyrylcholinesterase (BChE) as a proliferation marker and then acetylcholinesterase (AChE) as a differentiation marker are expressed in a mutually exclusive manner. These and other data indicate a coregulation of cholinesterase expression, and also possible roles of cholinesterases during neurogenesis. Here, both aspects are investigated by two independent transfection protocols of dissociated retina cells of the 6-day-old chick embryo in reaggregation culture, both protocols leading to efficient overexpression of AChE protein. The effect of the overexpressed AChE protein on the re-establishment of retina-like three-dimensional networks (so-called retinospheroids) was studied. In a first approach, we transfected retinospheroids with a pSVK3 expression vector into which a cDNA construct encoding the entire rabbit AChE gene had been inserted in sense orientation. As detected at the mRNA level, rabbit AChE was heterologously overexpressed in chicken retinospheroids. Remarkably, this was accompanied by a strong increase in endogenous chicken AChE protein, while the total AChE activity was only slightly increased. This increase was due to chicken enzyme, as shown by species-specific inhibition studies using fasciculin. Clearly, total AChE activity is regulated post-translationally. As an alternative method of AChE overexpression, transfection of spheroids was performed with an antisense-5'-BChE vector, which not only resulted in the down-regulation of BChE expression, but also strongly increased chicken AChE transcripts, protein and enzyme activity. Histologically, a higher concentration of AChE protein (as a consequence of either AChE overexpression or BChE suppression) was associated with an advanced degree of tissue differentiation, as detected by immunostaining for the cytoskeletal protein vimentin.

Chronic enhancement of neuromuscular activity increases acetylcholinesterase gene expression in skeletal muscle.

We determined levels of mRNA encoding acetylcholinesterase (AChE) in muscles of rats subjected to chronic enhancement of neuromuscular activation. After 8 wk of voluntary wheel running, extensor digitorum longus (EDL) muscles displayed a 72% increase in total AChE activity as a result of a selective threefold increase in the G4 content. Soleus muscles, on the other hand, exhibited a 30% decrease in A12 while displaying a small (33%) increase in total AChE activity. These enzymatic adaptations were paralleled by increases in the levels of AChE mRNAs in both EDL (32%; P < 0.03) and soleus (42%; P < 0.02) muscles. In addition, compensatory hypertrophy of the plantaris muscle increased total AChE activity by 75%. This change was reflected by an elevation in all AChE molecular forms with A12 (89%) and A8 (179%) showing the most prominent increases. Similar to exercise-trained muscles, hypertrophied plantaris muscles displayed an increase in AChE transcripts (25%; P < 0.04). These results indicate that increases in neuromuscular activity modulate expression of the AChE gene in vivo and suggest the involvement of pretranslational regulatory mechanisms in the adaptive response of AChE to enhanced neuromuscular activation.