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PubMed1999

Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens: a new member of the cholinesterase family resistant to diisopropyl fluorophosphate.

Acetylcholinesterase cDNA was cloned by screening a library from Loligo opalescens optic lobes; cDNA sequence analysis revealed an open reading frame coding for a protein of 610 amino acids that showed 20-41% amino acid identity with the acetylcholinesterases studied so far. The characteristic structure of cholinesterase (the choline binding site, the catalytic triad, and six cysteines that form three intrachain disulfide bonds) was conserved in the protein. The heterologous expression of acetylcholinesterase in COS cells gave a recovery of acetylcholinesterase activity 20-fold higher than in controls. The enzyme, partially purified by affinity chromatography, showed molecular and kinetic features indistinguishable from those of acetylcholinesterase expressed in vivo, which displays a high catalytic efficiency. Both enzymes are true acetylcholinesterase corresponding to phosphatidylinositol-anchored G2a dimers of class I, with a marked substrate specificity for acetylthiocholine. The deduced amino acid sequence may explain some particular kinetic characteristics of Loligo acetylcholinesterase, because the presence of a polar amino acid residue (S313) instead of a nonpolar one [F(288) in Torpedo] in the acyl pocket of the active site could justify the high substrate specificity of the enzyme, the absence of hydrolysis with butyrylthiocholine, and the poor inhibition by the organophosphate diisopropyl fluorophosphate.

Acetylcholinesterase↗

PubMed1990

Sympathectomy alters acetylcholinesterase expression in adult rat heart.

STUDY OBJECTIVE: The aim of the study was to determine the significance of adrenergic nerve associated acetylcholinesterase for the pool of total acetylcholinesterase molecules. DESIGN: Acetylcholinesterase was analysed after destruction of adrenergic nerves by 6-hydroxydopamine or bilateral stellate sympathectomy. Effectiveness of treatment was verified by determining noradrenaline concentrations in right ventricle. Acetylcholinesterase activity was assayed in homogenates of atria and portions of left ventricular free wall. SUBJECTS: Adult male Sprague-Dawley rats were used, weight 225-260 g, n = 5 per experimental group. MAIN RESULTS: Sympathectomy caused a small decrease in acetylcholinesterase activity, due to a decrease in the activity of the tetrameric globular form of the enzyme. Choline acetylcholinesterase activity was not altered by sympathectomy, which is an indication that cholinergic nerves were not affected. CONCLUSIONS: The contribution of adrenergic neurones to the cardiac pool of acetylcholinesterase is measurable and consists primarily of the tetrameric globular form of the enzyme.

Acetylcholinesterase↗

PubMed1981

A second class of acetylcholinesterase-deficient mutants of the nematode Caenorhabditis elegans.

In JOHNSON et al. (1981), the Caenorhabditis elegans mutant strain PR1000, homozygous for the ace-1 mutation p1000, is shown to be deficient in the class A subset of acetylcholinesterases, which comprises approximately one-half of the total C. elegans acetylcholinesterase activity. Beginning with this strain, we have isolated 487 new behavioral and morphological mutant strains. Two of these, independently derived, lack approximately 98% of the wild-type acetylcholinesterase activity and share the same specific uncoordinated phenotype; both move forward in a slow and uncoordinated manner, and when mechanically stimulated to induce reversal, both hypercontract and become temporarily paralyzed. In addition to the ace-1 mutation, both strains also harbor recessive mutations in the same newly identified gene, ace-2, which maps to chromosome I and is therefore not linked to ace-1. Gene dosage experiments suggest that ace-2 is a structural gene for the remaining class B acetylcholinesterases, which are not affected by ace-1.--The uncoordinated phenotype of the newly isolated, doubly mutant strains depends on both the ace-1 and ace-2 mutations; homozygosity for either mutation alone produces normally coordinated animals. This result implies functional overlap of the acetylcholinesterases controlled by ace-1 and ace-2, perhaps at common synapses. Consistent with this, light microscopic histochemical staining of permeabilized whole mounts indicates some areas of possible spatial overlap of these acetylcholinesterases (nerve ring, longitudinal nerve cords). In addition, there is at least one area where only ace-2-controlled acetylcholinesterase activity appears (pharyngeo-intestinal valve).

Acetylcholinesterase↗

PubMed2000

Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II.

Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.

Acetylcholinesterase↗

PubMed1985

Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica.

The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non-reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity.

Acetylcholinesterase↗

PubMed1981

Solubilization of collagen-tailed acetylcholinesterase from chick retina: effect of different extraction procedures.

We have extracted acetylcholinesterase from young chick retinas by homogenization in different solutions combining high salt concentration, ionic and nonionic detergents, and EDTA, looking for an optimum procedure for the solubilization of collagen-tailed, asymmetric structural forms of the enzyme. High salt and EDTA seem to be the only necessary requirements for the solubilization of acetylcholinesterase as the A12 form (20S), and the presence of detergent in the homogenization medium does not significantly improve the yield of tailed enzyme. Extraction in the absence of detergent has the potential advantage of a threefold enrichment of tailed enzyme, because only about one-third of the total retinal acetylcholinesterase activity is solubilized. Divalent cations, especially Ca2+, seem to be involved in the attachment of the tailed enzyme to the retinal membranes, at the tail level. High salt-EDTA-extracted 20S acetylcholinesterase (without detergent) aggregates in the presence of exogenous Ca2+ and becomes "insoluble." However, the aggregated 20S acetylcholinesterase can be completely recovered and brought back into solution by further addition of EDTA. Besides, the aggregation can be prevented by the inclusion of Triton X-100 in the homogenization buffer or by adding the detergent concurrently with Ca2+. It is postulated that the acetylcholinesterase collagenous tail is coated by acidic lipid molecules hydrophobically bound to the tail protein so that Ca2+ ionic bridges would actually link these lipid molecules (and consequently the tail) to the membrane matrix. Removal of the lipid coat (e.g., by Triton X-100) produces tailed acetylcholinesterase molecules that no longer aggregate in the presence of Ca2+ and are fully accessible to collagenase digestion.

Acetylcholinesterase↗

PubMed2003

Brain acetylcholinesterase activity in mild cognitive impairment and early Alzheimer's disease.

OBJECTIVE: Brain acetylcholinesterase activity was determined in healthy controls and in patients with mild cognitive impairment and early Alzheimer's disease. METHODS: A specific acetylcholinesterase tracer, [methyl-(11)C]N-methyl-piperidyl-4-acetate ([(11)C]MP4A), and a three dimensional PET system with magnetic resonance coregistration were used for imaging. RESULTS: There was a significant difference in the acetylcholinesterase activity in the hippocampus between the groups (p = 0.03), the mean (SD) acetylcholinesterase activity (k(3) values, min(-1)) being 0.114 (0.036) in controls, 0.098 (0.023) in mild cognitive impairment, and 0.085 (0.022) in Alzheimer's disease. The mini-mental state examination score showed no significant relation with acetylcholinesterase activity in any brain area in the combined mild cognitive impairment/Alzheimer group. CONCLUSIONS: Hippocampal acetylcholinesterase activity is only slightly reduced in mild cognitive impairment and early Alzheimer's disease and so the value of in vivo acetylcholinesterase measurements in detecting the early Alzheimer process is limited.

Acetates↗

PubMed1991

Inhibition of acetylcholinesterase activity in human brain tissue and erythrocytes by galanthamine, physostigmine and tacrine.

Galanthamine, physostigmine and 9-amino-1,2,3,4-tetrahydroacridine (tacrine) were evaluated as inhibitors of human acetylcholinesterase activity from samples of postmortem human brain, fresh brain cortex biopsies and human erythrocytes. Acetylcholinesterase activity was most effectively inhibited in all tissues by physostigmine, followed by tacrine and galanthamine. The respective inhibitor concentrations exerting a half maximal effect (IC50) on acetylcholinesterase in postmortem human brain frontal cortex were 14 nmol/l, 1.0 mumol/l and 3.2 mumol/l versus 15 nmol/l, 1.1 mumol/l and 2.8 mumol/l in the hippocampus region. In addition, the inhibition of acetylcholinesterase by galanthamine was similar in postmortem brain and brain cortical biopsies from patients submitted to brain-tumour removal, indicating that postmortem changes up to 28 h after death probably did not influence the measurement of acetylcholinesterase inhibition. While physostigmine and tacrine acted equally on acetylcholinesterase from different sources, galanthamine was 10-fold less potent in inhibiting the enzyme activity from human brain that from human erythrocytes. Comparison with issues from mice revealed that galanthamine was selectively more potent in suppressing acetylcholinesterase in human erythrocytes. The results are discussed in the light of pharmacokinetic data, and conclusions are drawn for further clinical studies.

Acetylcholinesterase↗

PubMed1990

Substance P, acetylcholinesterase, and beta-endorphin levels in the plasma and pericardial fluid of patients with and without angina pectoris.

We measured substance P-like immunoreactivity (SPLI), beta-endorphin-like immunoreactivity (BELI), acetylcholinesterase activity, and total protein content in pericardial fluid and plasma of patients with angina pectoris and patients with no angina pectoris. SPLI and BELI levels, acetylcholinesterase activity, and total protein content were determined by radioimmunoassay, a colorimetric method, and by the method of Lowry et al. (J Biol Chem 1951; 193:265-75), respectively. In the pericardial fluid, patients with angina had SPLI, BELI, acetylcholinesterase, and total protein values of 1.69 +/- 0.23 fmol/mg protein, 0.16 +/- 0.13 fmol/mg protein, 0.06 +/- 0.02 units, and 25.7 +/- 3.2 mg/ml, respectively. Patients with no angina had SPLI, BELI, acetylcholinesterase, and total protein values of 0.93 +/- 0.17 fmol/mg protein, 0.19 +/- 0.10 fmol/mg protein, 0.16 +/- 0.02 units, and 44.6 +/- 5.3 mg/ml, respectively. SPLI levels were significantly higher (p less than 0.03), and acetylcholinesterase (less than 0.002) and total protein content (less than 0.004) were significantly lower in the pericardial fluid of patients with angina when compared with those of patients with no angina. BELI levels were not significantly different between the two groups. In the plasma, no significant differences were found in SPLI, BELI, acetylcholinesterase, and total protein values between the two groups of patients. Patients with angina had SPLI, BELI, acetylcholinesterase, and total protein values of 0.47 +/- 0.26 fmol/mg protein, 0.06 +/- 0.06 fmol/mg protein, 0.29 +/- 0.15 units, and 68.2 +/- 8.7 mg/ml, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

PubMed1989

False-positive acetylcholinesterase with early amniocentesis.

Among 93 acetylcholinesterase determinations performed on amniotic fluid samples from pregnancies at 11-14 weeks' gestation, five unexplained false-positive results were observed. In four of the five cases, the ratio of acetylcholinesterase to pseudocholinesterase was compatible with that observed in association with open neural tube defects in later gestation. In contrast, no false-positive results were noted among 951 acetylcholinesterase determinations performed on samples from women at 15-20 weeks' gestation. Repeat amniocentesis was performed several weeks after the first procedure in four of the five cases of early amniocentesis and false-positive results; in each case, the acetylcholinesterase was negative on the second sample. All four pregnancies had a normal outcome. In the remaining case, trisomy 21 was diagnosed in the fetus and the pregnancy was terminated. Positive acetylcholinesterase results should be interpreted cautiously in samples from early amniocentesis, especially when the amniotic fluid alpha-fetoprotein level is not markedly elevated. The acetylcholinesterase-to-pseudocholinesterase ratio is not useful in identifying fetal neural tube defects before 15 weeks' gestation. Repeat amniocentesis may help in determining the significance of a positive acetylcholinesterase result from early amniocentesis when no fetal defect is identified by ultrasonography.

Acetylcholinesterase↗

PubMed1984

Structural requirements and species specificity of the inhibition by beta-endorphin of heavy acetylcholinesterase from vertebrate skeletal muscle.

The molecular forms of acetylcholinesterase in extracts of gastrocnemius muscle from four vertebrate species and in electric eel (Electrophorus) electric organ were separated and identified by low-salt precipitation and velocity sedimentation. The activity of the heavy insoluble (A12) form of human muscle acetylcholinesterase was inhibited by synthetic human beta-endorphin (500 mM). The homologous form in rat muscle extracts was poorly inhibited by human beta-endorphin at the same concentration, but was more effectively inhibited by camel beta-endorphin. The activities of heavy forms of pseudocholinesterase, present in small amounts in both species, were not reduced by beta-endorphin. Selective inhibition of homologous heavy forms of acetylcholinesterase activity by camel and human beta-endorphin was also seen in skeletal muscle extracts from frog and pigeon, but with decreased effectiveness. No inhibition was detectable in the heavy acetylcholinesterase form from extracts of electric organ tissue of the electric eel. The inhibition of heavy acetylcholinesterase activity in human muscle by human beta-endorphin was dependent on the presence of its NH2-terminal pentapeptide sequence. Maximal inhibitory potency depended on the presence of the entire amino acid sequence, since potency was considerably reduced in synthetic peptide analogues lacking either middle or COOH-terminal segments of beta-endorphin. The relative potency of beta-endorphin from various species as inhibitors of rat heavy acetylcholinesterase activity was also investigated. beta-Endorphin sequences most closely resembling that of the rat peptide (camel, equine) were most potent, whereas those with sequence differences of more than one amino-acid were less potent (turkey, human) or had no inhibitory activity (ostrich). The selective inhibition of heavy acetylcholinesterase by beta-endorphin thus exhibits species specificity, even among mammals, in which homologues of this molecular form of the enzyme are otherwise indistinguishable.

Acetylcholinesterase↗

PubMed1982

Prenatal diagnosis of open neural tube defects using the amniotic fluid acetylcholinesterase assay.

The most reliable tool at present for the prenatal detection of open neural tube defects is the amniotic fluid alpha-fetoprotein assay. Nevertheless its nonspecificity and false-positive results produced by fetal blood admixture and other less well-defined causes have spurred the search for a more specific test. Increased amniotic fluid acetylcholinesterase activity has been reported in association with open neural tube defects. The authors have assayed acetylcholinesterase in second-trimester amniotic fluid samples with previously determined alpha-fetoprotein values from 200 pregnancies with 96.5% of the outcomes known. These pregnancies were selected because of known fetal abnormalities or difficulties in prenatal diagnosis. High alpha-fetoprotein and acetylcholinesterase activity was detected in all 66 cases of open neural tube defects. The authors were able to reclassify correctly 89% of normal pregnancies with spuriously high alpha-fetoprotein by demonstrating no acetylcholinesterase band after slab gel in electrophoresis. Nine cases with a normal outcome but high alpha-fetoprotein values and acetylcholinesterase activity constitute the remaining false-positive results. Notwithstanding the false-positive results, which may have been due to undetected fetal blood admixture, the acetylcholinesterase gel electrophoresis assay is clearly a valuable adjunctive test to the alpha-fetoprotein assay for the prenatal detection of neural tube defects. Although no specific for open neural tube defects, acetylcholinesterase analysis by gel electrophoresis of amniotic fluid is significantly less influenced by fetal blood than is alpha-fetoprotein and, furthermore, may prove as reliable a diagnostic test for open neural tube defect.

Acetylcholinesterase↗

PubMed1983

Production and characterization of separate monoclonal antibodies to human acetylcholinesterase and butyrylcholinesterase.

Butyrylcholinesterase purified from human plasma and acetylcholinesterase purified from human red blood cells were used to immunize separate groups of BALB/c mice. A solid-phase immunoadsorbance assay was developed to screen and characterize antibodies specific for the cholinesterases. Immunized spleen cells were fused with a non-immunoglobulin-secreting myeloma cell line (FO). After two subcultures at limiting dilution, several clones secreting antibodies to acetylcholinesterase or butyrylcholinesterase were obtained. Selected clones were expanded as ascites tumors in immunosuppressed BALB/c mice. All tested immunoglobulins consisted of kappa light chains and either G1 or G2b heavy chains. Two-dimensional gel electrophoresis confirmed the monoclonal nature of each isolated antibody. None of the antibodies to acetylcholinesterase cross-reacted with butyrylcholinesterase, and vice versa. All tested antibodies exhibited high avidity for human enzyme, independent of the tissue source (apparent dissociation constants: 1-3 nM for acetylcholinesterase antibodies; 2-13 nM for butyrylcholinesterase antibodies). Treatment of enzymes with monoclonal antibodies increased the sedimentation coefficients (from 6.5 S to 12 S for acetylcholinesterase, from 11 S to 18 S or 20 S for butyrylcholinesterase). All of the monoclonal antibodies displayed marked species specificity. Several antibodies reacted only with human enzyme; others reacted with enzyme from nonhuman primates as well. A few of the butyrylcholinesterase antibodies cross-reacted weakly with enzyme from dog, cat, and horse, but none reacted with the enzyme from rat, guinea pig, and chicken. One acetylcholinesterase antibody cross-reacted with acetylcholinesterase of rabbit and guinea pig. The avidity, species selectivity, and other properties of these antibody reagents will be useful in future studies on the regulation and disposition of cholinesterases.

Acetylcholinesterase↗

PubMed1984

[Inhibition of acetylcholinesterase using fluorescence-labeled inhibitors].

The inhibition of acetylcholinesterase with fluorophores type II and III linked with the OH-selective fluorophosphonoyl groups is kinetically investigated by comparing the changes in activity and fluorescence. The hydrolysis of the fluorescent phosphonoylfluorides 1 and 4 to 7 in aqueous buffer solutions does not interfere with the inhibition kinetics. The inhibition constants of the investigated compounds are unexpectedly high (10(6) to 10(8) S-1M-1). They increase with increasing spacer length, but arrive at an optimal value with four methylene groups in the inhibitor 6. The fluorescence is quenched by the interaction of 6 and acetylcholinesterase. This fact can be used for the determination of acetylcholinesterase by fluorescence titration (Fig. 9). Fluorescence once more increases slowly during the aging process, leading to the degradation products 9, 11 and 12. In acetylcholinesterase inhibited by 1, a sensitized fluorescence is observed, produced by tryptophane intrinsically linked to the esterase. In the presence of quaternary ammonium salts like acetylcholine chloride or gallamine triethochloride 15, the decrease of fluorescence is lower. Acetylcholinesterase inhibited in this way is reactivated quantitatively by toxogonine. No reactivation is possible with acetylcholinesterase inhibited in the absence of the above mentioned quaternary ammonium salts. As a result of the investigation using fluorescent inhibitors the conclusion can be drawn that not only the active site of acetylcholinesterase is blocked by phosphonoylation. The conformation too seems to be influenced by interactions of the inhibitors with the hydrophobic areas of the enzyme.

Acetylcholine↗

PubMed2001

Differential inhibition of [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding to muscarinic receptors in rat brain membranes with acetylcholinesterase inhibitors.

The potential interaction of acetylcholinesterase inhibitors with cholinergic receptors may play a significant role in the therapeutic and/or side-effects associated with this class of compound. In the present study, the capacity of acetylcholinesterase inhibitors to interact with muscarinic receptors was assessed by their ability to displace both [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding in rat brain membranes. The [3H]-quinuclinidyl benzilate/[3H]-oxotremorine-M affinity ratios permitted predictions to be made of either the antagonist or agonist properties of the different compounds. A series of compounds, representative of the principal classes of acetylcholinesterase inhibitors, displaced [3H]-oxotremorine-M binding with high-to-moderate potency (ambenonium>neostigmine=pyridostigmine=tacrine>physostigmine> edrophonium=galanthamine>desoxypeganine) whereas only ambenonium and tacrine displaced [3H]-quinuclinidyl benzilate binding. Inhibitors such as desoxypeganine, parathion and gramine demonstrated negligible inhibition of the binding of both radioligands. Scatchard plots constructed from the inhibition of [3H]-oxotremorine-M binding in the absence and presence of different inhibitors showed an unaltered Bmax and a reduced affinity constant, indicative of potential competitive or allosteric mechanisms. The capacity of acetylcholinesterase inhibitors, with the exception of tacrine and ambenonium, to displace bound [3H]-oxotremorine-M in preference to [3H]quinuclinidyl benzilate predicts that the former compounds could act as potential agonists at muscarinic receptors. Moreover, the rank order for potency in inhibiting acetylcholinesterase (ambenonium>neostigmine=physostigmine =tacrine>pyridostigmine=edrophonium=galanthamine >desoxypeganine>parathion>gramine) indicated that the most effective inhibitors of acetylcholinesterase also displaced [3H]-oxotremorine-M to the greatest extent. The capacity of these inhibitors to displace [3H]-oxotremorine-M binding preclude their utilisation for the prevention of acetylcholine catabolism in rat brain membranes, the latter being required to estimate the binding of acetylcholine to [3H]-oxotremorine-M-labelled muscarinic receptors. However, fasciculin-2, a potent peptide inhibitor of acetylcholinesterase (IC50 24 nM), did prevent catabolism of acetylcholine in rat brain membranes with an atypical inhibition isotherm of [3H]-oxotremorine-M binding, thus permitting an estimation of the "global affinity" of acetylcholine (Ki 85 nM) for [3H]-oxotremorine-M-labelled muscarinic receptors in rat brain.

Animals↗

PubMed1983

Ultrastructural observations of the cholinergic neuron in the rat striatum as identified by acetylcholinesterase pharmacohistochemistry.

In order to examine the ultrastructural features of the cholinergic neuron in the striatum (caudatoputamen) of the rat, cytochemistry for acetylcholinesterase was conducted 2-12 h after intramuscular injection of the irreversible acetylcholinesterase inhibitor diisopropylphosphorofluoridate. Light microscopic examination of Epon sections reacted from acetylcholinesterase showed that only large-sized cells in the striatum (25-35 microns in the long axis) were stained intensely. In the case of longer survival periods (10-12 h), some lightly stained cells (medium-sized) were seen dispersed amongst the large acetylcholinesterase-rich cells. Electron microscopic observations were made on ultrathin sections of selected large acetylcholinesterase-rich neurons that were first studied by light microscopy. The nucleus of these cells has an eccentric position and possesses several indentations of the nuclear envelope. The cytoplasm contains abundant organelles, many exhibiting features unique to this cell type. Many stacks of granular endoplasmic reticulum, arranged in a parallel manner and forming typical Nissl bodies, were observed in the periphery of the perikarya, and many distinct golgi complexes were seen in the perinuclear zone. At all post-diisopropylphosphorofluoridate survival times, heavy deposits of acetylcholinesterase reaction product were found within the perikarya of this cell type, for the most part within the cisternae of the granular endoplasmic reticulum. At the longer post-diisopropylphosphorofluoridate survival times, reaction product within the cytoplasm was very dense and appeared to have reached a maximum level. At these times reaction product also appeared in the secondary and tertiary dendritic branches of the large-sized neurons. Of the other cell types in the striatum, two types of medium-sized cells displayed a light deposit of reaction product in their perikarya, but this was observed only at longer recovery times (8-12 h). The majority of cells in the striatum lacked reaction particles. Throughout the early post-diisopropylphosphorofluoridate period, the recovery of enzyme activity in the neuropil was moderate compared to that seen within cell bodies. These findings indicate that the large-sized neuron is the only striatal structure that shows rapid regeneration of acetylcholinesterase activity during the early recovery phase after diisopropylphosphorofluoridate administration. Previous studies have indicated that this type of neuron represents the cholinergic interneuron of the striatum. (ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗

PubMed2004

Pharmacology of selective acetylcholinesterase inhibitors: implications for use in Alzheimer's disease.

Cholinesterase inhibitors vary in their selectivity for acetylcholinesterase versus butyrylcholinesterase. We examined several cholinesterase inhibitors and assessed the relative role of acetylcholinesterase versus butyrylcholinesterase inhibition in central and peripheral responses to these medications. Donepezil and icopezil are highly selective for acetylcholinesterase, whereas tacrine and heptylphysostigmine demonstrated greater potency for butyrylcholinesterase over acetylcholinesterase. All four compounds increased acetylcholine levels in mouse brains. Dose-response curves for tremor (central effect) and salivation (peripheral effect) showed that donepezil and icopezil possess a more favourable therapeutic index than the nonselective inhibitors, tacrine and heptylphysostigmine. Co-administration of the selective butyrylcholinesterase inhibitor tetraisopropylpyrophosphoramide (iso-OMPA) potentiated peripheral, but not central, effects of the selective acetylcholinesterase inhibitor icopezil. The improved therapeutic index observed in mice with icopezil is due to a high degree of selectivity for acetylcholinesterase versus butyrylcholinesterase, suggesting that high selectivity for acetylcholinesterase may contribute to the clinically favourable tolerability profile of agents such as donepezil in Alzheimer's disease patients.

Acetylcholine↗

PubMed1993

Inhibition patterns of brain acetylcholinesterase and hepatic and plasma aliesterases following exposures to three phosphorothionate insecticides and their oxons in rats.

Rats were administered high sublethal intraperitoneal dosages of the phosphorothionate insecticides parathion, methyl parathion, and chlorpyrifos, and their oxons. Acetylcholinesterase activities in cerebral cortex and medulla oblongata and aliesterase activities in liver and plasma were monitored at 2 hr and 1, 2, and 4 days after exposure. The maximal inhibition of brain acetylcholinesterase activity was not immediate with parathion and chlorpyrifos, reflecting the time required for bioactivation of the phosphorothionates as well as the effectiveness of the aliesterases to inactivate much of the hepatically generated oxons. In contrast, brain acetylcholinesterase activities were more quickly inhibited following administration of paraoxon and chlorpyrifos-oxon, which do not require bioactivation. Brain acetylcholinesterase was also rapidly inhibited following administration of methyl parathion and methyl paraoxon, reflecting the low sensitivity of the aliesterases to methyl paraoxon. Aliesterases were inhibited to a greater extent than acetylcholinesterase at each sampling time with parathion and chlorpyrifos and their oxons, whereas the reverse was true with methyl parathion and methyl paraoxon. All of the above patterns correlate with the in vitro sensitivities of acetylcholinesterase and aliesterases to the oxons. The very prolonged inhibition of esterase activities following chlorpyrifos treatment probably results from its substantially greater lipophilicity compared to the other compounds, which would allow it to be stored and released for gradual bioactivation. The data reported indicate that the disposition and effects of different phosphorothionate insecticides will be influenced by the sensitivities of target and nontarget esterases for their oxons and by their lipophilicity, and that predictions of in vivo responses can be made from in vitro data.

Acetylcholinesterase↗