Membrane-bound Acetylcholinesterase Research Articles

Assessment of neuromuscular dysfunction during poisoning by organophosphorus compounds

Dysfunction of respiratory muscles is a life-threatening complication in poisoning by organophosphorus compounds (OPs). It is both of central and peripheral origin due to impaired cholinergic signalling upon inhibition of acetylcholinesterase (AChE). The dysfunction at neuromuscular synapses is not amenable to anticholinergics and remains a therapeutic challenge. Thus, a clear understanding of the distinct mechanisms occurring at neuromuscular synapses is decisive for the development and improvement of therapeutic strategies, particularly with nerve agent poisoning, where clinical studies are prevented by ethical considerations. Using red blood cell AChE, the kinetics of OP induced inhibition, aging, and spontaneous and oxime-induced reactivation have been elucidated. In a dynamically working in vitro model with real-time determination of membrane-bound AChE, it was shown that the kinetic constants derived from erythrocyte AChE are comparable to muscle AChE in a given species. To assess, whether kinetic considerations of AChE activity are relevant for the neuromuscular function, organotypic spinal cord–skeletal muscle cocultures have been established. In this model neostigmine and VX affected neuromuscular transmission as anticipated from their known actions on AChE. Also oxime-induced restoration of the neuromuscular transmission was observed. These findings were confirmed by functional studies on diaphragm muscles of various species with determination of muscle force generation upon phrenic nerve or indirect electrical field stimulation techniques. Investigations with human intercostal muscles are in progress to assess the conditions in human tissue. The results obtained with paraoxon favourably correlate with data from clinical findings of parathion-poisoned patients where the correlation of neuromuscular transmission with the activity of erythrocyte AChE could be established. In conclusion, a variety of methods are available to follow the microscopic reactions occurring at the synaptic level. Due to the lack of clinical data with different OPs, e.g. nerve agents, well designed animal experiments, reflecting the human situation as close as possible, are indispensable for the development of new drugs against the deleterious OP effects.

Elevated Cholinesterase Activity in Patients with TIA and Other Thrombotic Disorders.

Abstract 2991Poster Board II-967 Background.The natural function of ACTH in blood is not well defined. However elevated plasma level was reported in patients with chronic inflammation, suggesting as one of inflammatory markers. Erythrocytes possess membrane-bound acetylcholinesterase (AChE) while plasma contains a less specific cholinesterase (ChE). We reported at a previous meeting [Blood 2008, 112(11) Abst #3849] that AChE activity of RBC-derived microparticles (RMP) is 6 times higher than those in PMP. In this study we measured plasma ACTH levels in several groups and encountered unexpectedly high plasma ChE activities in patients with TIA and other thrombosis.Methods. (i) Patient population.A series of n=108 consenting patients were recruited sequentially having various disorders For purpose of analysis, they were divided in 2 main groups: n=53 with thrombosis (TBS), F/M 27/26; and n=55 non-TBS, F/M 24/31. Thrombosis group consists of venous and arterial thrombosis including those with TIA. Non TBS group consists of anemias, thrombocytopenias including ITP, MDS. (ii) Assaywas essentially by Ellman's method. In our system, milli-absorbance units/min (mA/min) x 0.065 = umols substrate cleaved/min. Values reported here are in unitsof mA/min per mL plasma. Normal controls (NC, n=14) had a cutoff of 3000 mA/min per mL plasma, = mean +2SD. (iii) Sample handling.Platelet-poor plasma (PPP) was prepared by centrifuging 10 min at 1800 xg, then frozen in aliquots. For assay, it was diluted 1:20 with saline, then 5 uL and 10 uL were used in 96-well microtiter plate. Results:(i) Significantly higher ChE activity was observed in thrombosis (TBS) patients compared to non-TBS. Mean value ±SD in TBS was 2928 ±773, and in Non-TBS was 1897 ±713 (units as above). This difference was significant, p<0.001. (ii) When analyzed elevated levels of ACTH above 2SD of normal controls (n=26) between 2 groups, the TBS group had elevated ChE (>3000) in 30% of patients, while the Non-TBS had elevated ChE in 7.8%. (iii) Further analysis among thrombosis group revealed that the subgroup with neurological TBS (TIA, minor strokes) had the most consistent elevations of ACTH; 10 of15 with TIA group (66%) had ChE activities >3000. Summary/ Discussion.Although ChE and AChE in blood has long been studied, to our knowledge, this is the first report to show a relation between plasma ChE and thrombosis. Its elevation is very pronounce in thrombosis of the CNS, manifesting as TIA. Further study is warranted to elucidate how ChE is related to AChE of the CNS and on red cells. Disclosures:No relevant conflicts of interest to declare.

Comparison of the oxime-induced reactivation of rhesus monkey, swine and guinea pig erythrocyte acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

Recently, a dynamically working in vitro model with real-time determination of membrane-bound human acetylcholinesterase (AChE) activity was shown to be a versatile model to investigate oxime-induced reactivation kinetics of organophosphate- (OP) inhibited enzyme. In this assay, AChE was immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. Subsequently, AChE activity was continuously analyzed in a flow-through detector. Now, it was an intriguing question whether this model could be used with erythrocyte AChE from other species in order to investigate kinetic interactions in the absence of annoying side reactions. Rhesus monkey, swine and guinea pig erythrocytes were a stable and highly reproducible enzyme source. Then, the model was applied to the reactivation of sarin- and paraoxon-inhibited AChE by obidoxime or HI 6 and it could be shown that the derived reactivation rate constants were in good agreement to previous results obtained from experiments with a static model. Hence, this dynamic model offers the possibility to investigate highly reproducible interactions between AChE, OP and oximes with human and animal AChE.

Acetylcholine‐induced seizure‐like activity and modified cholinergic gene expression in chronically epileptic rats

The entorhinal cortex (EC) plays an important role in temporal lobe epilepsy. Under normal conditions, the enriched cholinergic innervation of the EC modulates local synchronized oscillatory activity; however, its role in epilepsy is unknown. Enhanced neuronal activation has been shown to induce transcriptional changes of key cholinergic genes and thus alter cholinergic responses. To examine cholinergic modulations in epileptic tissue we studied molecular and electrophysiological cholinergic responses in the EC of chronically epileptic rats following exposure to pilocarpine or kainic acid. We confirmed that while the total activity of the acetylcholine (ACh)-hydrolysing enzyme, acetylcholinesterase (AChE) was not altered, epileptic rats showed alternative splicing of AChE pre-mRNA transcripts, accompanied by a shift from membrane-bound AChE tetramers to soluble monomers. This was associated with increased sensitivity to ACh application: thus, in control rats, ACh (10-100 microm) induced slow (< 1Hz), periodic events confined to the EC; however, in epileptic rats, ACh evoked seconds-long seizure-like events with initial appearance in the EC, and frequent propagation to neighbouring cortical regions. ACh-induced seizure-like events could be completely blocked by the non-specific muscarinic antagonist, atropine, and were partially blocked by the muscarinic-1 receptor antagonist, pirenzepine; but were not affected by the non-specific nicotinic antagonist, mecamylamine. Epileptic rats presented reduced transcript levels of muscarinic receptors with no evidence of mRNA editing or altered mRNA levels for nicotinic ACh receptors. Our findings suggest that altered cholinergic modulation may initiate seizure events in the epileptic temporal cortex.

Identical kinetics of human erythrocyte and muscle acetylcholinesterase with respect to carbamate pre-treatment, residual activity upon soman challenge and spontaneous reactivation after withdrawal of the inhibitors

The efficacy of oxime treatment in soman poisoning is limited due to rapid aging of inhibited acetylcholinesterase (AChE). Pre-treatment with carbamates was shown to improve antidotal treatment substantially. Recently, by using a dynamically working in vitro model with real-time determination of membrane-bound AChE activity, we were able to demonstrate that pre-inhibition of human erythrocyte AChE with pyridostigmine or physostigmine resulted in a markedly higher residual AChE activity after inhibition by soman or paraoxon than in the absence of reversible inhibitors. The purpose of the present study was to compare the effect of carbamate pre-treatment and soman challenge with human erythrocyte and muscle homogenate AChE. Both enzyme sources were immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. AChE activity was continuously analyzed in a flow-through detector. Pre-inhibition of AChE with pyridostigmine or physostigmine resulted in a concentration-dependent increase in carbamylation, residual activity after soman inhibition and fraction of decarb-amylation AChE after discontinuation of the inhibitors without differences between human erythrocyte and muscle AChE. This data support the view that human erythrocyte AChE is an adequate surrogate marker for synaptic AChE in OP poisoning.

Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex ®-GS, 0.22 μm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax ® and Potter–Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5 h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 μM) or HI 6 (30 μM) of sarin-inhibited human muscle acetylcholinesterase (0.142 ± 0.004 min −1 and 0.166 ± 0.008 min −1, respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker.

Alterations in kinetic and thermotropic properties of cerebral membrane-bound acetylcholineesterase during thioacetamide-induced hepatic encephalopathy: Correlation with membrane lipid changes

Acetylcholinesterase (AchE) is an important peripheral membrane-bound enzyme, crucial for cholinergic neurotransmission. Changes in AchE activity, kinetic and thermotropic properties were studied in hepatic encephalopathy (HE) associated with acute liver failure induced experimentally by the administration of the hepatotoxin thioacetamide (TAA). Activity of AchE decreased significantly following TAA administration. AchE from cerebral cortex membrane isolates of TAA-treated rats also showed a decrease in Vmax and an increase in Km. Arrhenius plots revealed considerable changes in the thermotropic behavior of AchE from membrane isolates obtained from TAA-treated rats as evident from the decreased transition temperature. A positive correlation was observed between changes in membrane cholesterol ( r 2 = 0.987), sphingomyelin ( r 2 = 0.99) levels and AchE activity, thus indicating that membrane lipid changes could underlie the observed changes in kinetic and thermotropic properties of membrane-bound AchE during TAA-induced HE.

Development of a dynamic model for real-time determination of membrane-bound acetylcholinesterase activity upon perfusion with inhibitors and reactivators

Quantitative predictions of the course of acetylcholinesterase (AChE) activity, following interference of inhibitors and reactivators, are usually obscured by the time-dependent changes of all reaction partners. To mimic these dynamics we developed an in vitro model. Immobilized human erythrocyte ghosts in a bioreactor were continuously perfused while AChE activity was monitored by a modified Ellman method. The perfusion system consisted of two HPLC pumps with integrated quaternary low-pressure gradient formers that were programmed by a computer using commercial HPLC software. The combined eluates passed a particle filter (Millex ®-GS, 0.22 μm) containing a thin layer of erythrocytes that was immersed in a temperature-controlled water bath. The effluent passed a flow cell in a UV–vis detector, the signal of which was digitized, written to disc and calculated with curve fitting programs. AChE activity decreased by 3.4% within 2.5 h. The day-to-day variation of the freshly prepared bioreactor using the same enzyme source was ±3.3%. Residual activity of 0.2% marked the limit of quantification. Following perfusion with paraoxon, pseudo first-order rate constants of inhibition were established that did not differ from results obtained in conventional assays. The same holds true for reactivation with obidoxime. The set-up presented allows freely programmable time-dependent changes of up to eight solvents to mimic pharmacokinetic profiles without accumulation of products. Due to some hysteresis in the system, reaction half-lives should be >3 min and concentration changes in critical compounds should exceed half-lives of 5 min. Otherwise, the system offers much flexibility and operates with high precision.

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

Acetylcholinesterase is an enzyme whose best-known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X-100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X-100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C-terminal anti-acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N-terminal anti-acetylcholinesterase IgG antibody revealed a double band around 66-70 kDa. Using the Ellman's method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane-bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system.

Modification of substrate inhibition of synaptosomal acetylcholinesterase by cardiotoxins.

Different types of cardiotoxin (I-V and n) were isolated and purified from the venom of the Taiwan cobra (Naja naja atra). The effects of these cardiotoxins were studied on membrane-bound acetylcholinesterase, which was isolated from a sheep's brain cortex. The results showed that cardiotoxins I-III, V, and n activated the enzyme by modification of substrate inhibition, but cardiotoxin IV's reaction was different. The inhibition and activation of acetylcholinesterase were linked to the functions of the hydrophobicity index, presence of a cationic cluster, and the accessible arginine residue. Our results indicate that Cardiotoxins have neither a cationic cluster nor an arginine residue in their surface area of loop I; therefore, in contrast to fasciculin, cardiotoxins are attached by loop II to the peripheral site of the enzyme. As a result, fasciculin seems to stabilize nonfunctional conformation, but cardiotoxins seem to stabilize the functional conformation of the enzyme. Based on our experimental and theoretical findings, similar secondary and tertiary structures of cardiotoxins and fasciculin seem to have an opposite function once they interact with acetylcholinesterase.

Synaptic plasma membrane-bound acetylcholinesterase activity is not affected by docosahexaenoic acid-induced decrease in membrane order

We investigated the effect of administration of docosahexaenoic acid (C22:6, n-3; 300 mg/kg .day, for 12 weeks) on the degree of membrane order and membrane-bound acetylcholinesterase activity of the cerebral cortex synaptic plasma membrane in male Wistar rats. Docosahexaenoic acid levels in the synaptic plasma membrane increased significantly by 16% over levels in control rats concomitant with an increase in the molar ratio of docosahexaenoic acid to arachidonic acid. Synaptic plasma membrane order, assessed by 1,6-diphenyl-1,3,5-hexatriene, which measures order of the bulk internal hydrophobic lipid core, decreased significantly in the docosahexaenoic acid-fed rats. Lateral mobility of both global and annular lipids measured by pyrene also increased. Acetylcholinesterase activity of the synaptic plasma membrane was unaffected, and synaptic plasma membrane phospholipid contents increased in the docosahexaenoic acid-fed rats, with a concomitant decrease in the cholesterol/phospholipid molar ratio. Lipid peroxide and reactive oxygen species, indicators of tissue oxidative stress, decreased in both the cerebral cortex synaptosome and homogenate of the docosahexaenoic acid-fed rats. Arrhenius plot showed a break point in acetylcholinesterase activity at 22 °C and 24 °C in plasma membranes from docosahexaenoic acid-fed and control rats, respectively. The present experiment indicates that chronic administration of docosahexaenoic acid does not affect synaptic acetylcholinesterase activity and evoke oxidative stress, although it increases the disorder of the global and annular lipids of rat synaptic plasma membranes.

Palladium and platinum ions interfere with the measurement of erythrocyte vesiculation by inhibiting the acetylcholinesterase activity of the released spectrin-depleted microvesicles

Palladium (Pd 2+) and platinum (Pt 2+) ions were found to inhibit erythrocyte membrane-bound acetylcholinesterase (AChE) with Ki values of 6.0 and 6.5 μg/ml, respectively. Lineweaver–Burke plots revealed that the inhibition of erythrocyte AChE by both metal ions was competitive in nature. Binding studies using alkaline phosphatase as a reporting enzyme confirmed that both metal ions indeed did bind to the enzyme molecules. In the process of red cell vesiculation, membrane-bound AChE is shed along with vesicles. The measurement of AChE activities in the medium containing vesiculated RBC could potentially be served as an index of vesiculation. Inhibition of AChE activities by both metal ions can thus constitute a potential source of error in vesiculation measurment. To illustrate these effects, a simulated vesiculation system, using green tea polyphenol in the presence (25 μg/ml) or absence of Pd 2+ ion was simultaneously examined by the electronmicrography and the AChE method. We observed vesiculation under the experimental condition in Pd 2+-free controls that was associated with a time-dependent increase in AChE activity were barely detected in the Pd 2+-spiked specimen because of the masking effect exerted by the metal ions themselves.

Muscle histopathology in schizophrenia

Acetylcholinesterase plays a major role in neuromuscular transmission and is regulated by neuromuscular activity. Since fast-twitch motor units are recruited with increased motor demand, we examined acetylcholinesterase regulation in rat leg muscles following treadmill training. Total acetylcholinesterase and specifically the membrane-bound tetramer increased in exercised fast-, but not slow-twitch muscles, while other isoforms remained unchanged. Synaptic acetylcholinesterase increased markedly in neuromuscular junctions of trained fibers, without concomitant changes in synaptic acetylcholine receptor, thus elevating synaptic acetylcholinesterase/receptor ratios. Electron microscopy showed that acetylcholinesterase increased in postjunctional folds and primary cleft, where it was added adjacent to the postsynaptic muscle membrane. Thus, although the primary acetylcholinesterase at the neuromuscular junction is the collagen-tailed asymmetric isoform associated with synaptic basal lamina, physiological demands such as strenuous exercise, or potentially pathological conditions, can selectively recruit the membrane-bound acetylcholinesterase tetramer to the synapse for optimal synaptic transmission.

Effect of exogenous melatonin on ethanol-induced changes in Na(+),K(+)- and Ca(2+)-ATPase activities in rat synaptosomes.

In the present study, the effects of acute ethanol (EtOH) toxicity and of exogenous melatonin (MLT) on this toxicity were examined by measuring membrane-bound ATPases and acetylcholinesterase activities in rat synaptosomal membranes. The concentrations of plasma alpha-tocopherol and adrenal ascorbic acid (AA) were also measured. Synaptosomal Na(+),K(+)-ATPase and Ca(2+)-ATPase activities were significantly depressed in acute EtOH-intoxicated rats compared with controls, while acetylcholinesterase activity remained unaltered. Pretreatment with MLT (10 mg/kg) prior to acute EtOH administration prevented EtOH-induced inhibition of ATPases. Adrenal AA and plasma alpha-tocopherol levels were also depressed regardless of MLT pretreatment. MLT treatment alone affected neither membrane-bound enzyme activities nor tissue and blood levels of vitamins C and E. It is concluded that acute EtOH intoxication disturbs neural transport functions through the membrane-bound ATPase activity depression. Reduced AA and alpha-tocopherol levels may contribute to the neurodegenerative effects of EtOH. However, pretreatment with a high dose of MLT before EtOH administration may be beneficial to prevent EtOH-induced toxicity on ATPase-mediated neural transport functions.

Effect of Altered Dietary n-6-to-n-3 Fatty Acid Ratio on Erythrocyte Lipid Composition and Membrane-Bound Enzymes.

Three experimental diets with varied n-6-to-n-3 fatty acid ratios (120, 40 and 8) were prepared by a suitable blending of safflower oil containing 72.5% linoleic (18:2 n-6) acid and non-detectable levels of alpha-linolenic (18:3 n-3) acid, and soybean oil having 56.1% linoleic (18:2 n-6) acid and 7.9% alpha-linolenic (18:3 n-3) acid. These diets were fed to weanling female Wistar/NIN (inbred) rats for 16 wk to assess the impact of altered dietary n-6-to-n-3 fatty acid ratio on erythrocyte membrane (EMS) cholesterol, phospholipids, fatty acid composition and activities of membrane-bound enzymes such as Na+,K+-ATPase, Ca2+, Mg2+-ATPase and acetylcholinesterase. Activities of total and ouabain-sensitive-ATPases were significantly higher in the erythrocyte membranes of rats fed diets with a n-6-to-n-3 fatty acid ratio of 40 compared to other groups, whereas the erythrocyte membrane-bound acetylcholinesterase was significantly different among the three groups. The highest and lowest activities for this enzyme were observed in the dietary groups with n-6-to-n-3 fatty acid ratios of 8 and 40 respectively. However, the EMS of rats fed diets with a n-6-to-n-3 fatty acid ratio of 40 alone had significantly higher Ca2+,Mg2+-ATPase compared to those of other two groups. Significant increases were observed in absolute amounts of cholesterol, phospholipids and molar ratio of cholesterol to phospholipids in the EMS of rats fed a diet with a very high 18:2 n-6-to-18:3 n-3 fatty acid ratio (120) as compared to those from the dietary group with 18:2 n-6-to-18:3 n-3 fatty acid ratio (40), which had the lowest levels of cholesterol, phospholipids and cholesterol-to-phospholipid molar ratio. On the other hand, the EMS from rats fed a diet with a very low n-6-to-n-3 fatty acid ratio (8) had significantly lower cholesterol and higher proportions of stearic (18:0), oleic (18:1 n-9), eicosapentaenoic (20:5 n-3), and docosahexaenoic acids, and a higher ratio of docosahexaenoic (22:6 n-3) acid-to-a-linoleic (18:3 n-3) acid compared to the EMS from a very high n-6-to-n-3 fatty acid ratio of 120. Although these changes in EM fatty acid profiles were expected of the respective dietary regimens, the observed changes in the activities of membrane-bound enzymes could have resulted from their interaction with membrane cholesterol, phospholipids and fatty acyl chains.

Insulin-like effect of (-)epicatechin on erythrocyte membrane acetylcholinesterase activity in type 2 diabetes mellitus.

1. Changes in the activity of acetylcholinesterase (AChE) have been reported in diabetes mellitus that have been linked to certain brain defects. The erythrocyte membrane AChE is reported to be similar to AChE present in the brain. 2. Epicatechin, a member of a group of polyphenolic compounds collectively known as "catechins" that are present in tea and belong to the flavonoid family, has been reported to possess insulin-like activity. 3. In the present study, the in vitro effect of (-)epicatechin and/or insulin was tested on erythrocyte membrane AChE in normal and type 2 diabetic patients. The aim of the study was to test the efficacy of (-)epicatechin to mimic insulin in its effect on erythrocyte membrane AChE. 4. Acetylcholinesterase activity was significantly lower in type 2 diabetic patients than in normal controls and in vitro insulin treatment restored this activity to normal levels. Epicatechin (1 mmol/L) also caused an elevation in AChE activity in diabetic erythrocytes, an effect that was similar to the effect of insulin. 5. Epicatechin has a pronounced insulin-like effect on erythrocyte membrane-bound AChE in type 2 diabetic patients; however, the mechanism of action of epicatechin remains speculative.

Two Distinct Proteins Are Associated with Tetrameric Acetylcholinesterase on the Cell Surface

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.

Changes of acetylcholinesterase activity in brain areas and liver of sucrose- and ethanol-fed rats.

The effects of chronic ethanol or sucrose administration to rats on acetylcholinesterase from brain and liver were investigated. Membrane-bound and soluble acetylcholinesterase activities were determined in fractions prepared by centrifugation. The thermal stability and the effects of temperature and different types of alcohols on acetylcholinesterase activity were also studied. Membrane-bound acetylcholinesterase activity increased (p < 0.01) in the liver after chronic ethanol administration, whereas no differences among groups in the encephalic areas, except in the brain stem soluble form, were found. Membrane-bound acetylcholinesterase from the ethanol- and sucrose-treated groups was more stable at the different temperatures assayed between 10 and 50 degrees C than that corresponding to the control group. Non-linear Arrhenius plots were obtained with preparations of membrane-bound acetylcholinesterase from rat liver, with discontinuities at 30 degrees C (control or sucrose groups) or 34-35 degrees C (alcohol group). Assays made with membrane-bound or soluble enzyme from brain showed linear Arrhenius plots in all groups studied. The inhibitory effects of increasing concentrations of ethanol, n-propanol and n-butanol on acetylcholinesterase preparations from forebrain, cerebellum, brain stem and liver of the three experimental groups (control, sucrose-fed and ethanol-fed) were very similar. However, n-butanol displayed a biphasic action on particulate or soluble preparations of rat forebrain. n-butanol inhibited (competitive inhibition) at higher concentrations (250-500 mM), while at lower concentrations (10-25 mM), the alcohol inhibited at low substrate concentrations but activated at high substrate concentration. These results suggest that the liver is more affected by ethanol than the brain. Moreover, the lipid composition of membranes is probably modified by ethanol or sucrose ingestion and this would affect membrane fluidity and consequently the behaviour of acetylcholinesterase.

Enhanced Expression of Acetylcholinesterase Activity in Bovine Satellite Cells Treated with Insulin-like Growth Factor I

The activity of membrane-bound acetylcholinesterase (AChE, EC 3. 1. 1. 7) in bovine muscle cells was enzyme-histochemically detailed in the M. longissimus thoracis during myogenesis in vivo and in vitro. AChE activity was detected in muscle satellite cells of regenerating muscles after an injection of the myotoxic agent, bupivacaine. In the myogenic cultures of satellite cells derived from regenerating muscles, AChE activity was found in mononucleated cells although they were a very low density, while all myotubes expressed AChE activity. The mononucleated cells positive for AChE activity (AChE+mononucleated cells) increased in number with an addition of insulin-like growth factor-I (IGF-I), but not with an addition of acidic fibroblast growth factor (aFGF) to the cultures. aFGF stimulated cell proliferation and IGF-I significantly induced the mononucleated cells positive for desmin, a muscle specific intermediate-filament protein (desmin+cells). The number of AChE+mononucleated cells and desmin+cells similarly changed in the myogenic cultures with IGF-I. The results support that AChE activity is expressed in postmitotic myoblasts which have been triggered to commit to the differentiation.

Human erythrocyte acetylcholinesterase inhibition by cis-diamminediaquaplatinum (II): a novel kinetic approach

The present work addresses the analyses of some novel kinetic parameters ( k t, K v, t 50, K ir, t c, m c, IC 50, IC 99 and K i) of human erythrocyte membrane-bound acetylcholinesterase (AChE, EC 3.1.1.7) inhibition by cis-diamminediaquaplatinum II (PDC). PDC is under a clinical trial for use as an antineoplastic drug. The authors recently reported that PDC and cisplatin have the ability to inhibit AChE activity in vitro. Therefore this study was designed to determine the estimation of time constant ( k t), velocity constant ( K v), 50% inhibition time ( t 50), inhibition rate constant ( K ir), transition concentration ( t c), meeting concentration ( m c), 50% inhibition (IC 50), 99% inhibition (IC 99) and inhibition constant ( K i) by novel methods. The details are described in the text.