Ultrastructural distribution of AChE in Catenula leptocephala (Nuttycombe, 1956).

Acetylcholinesterase activity (AChE, E.C. 3.1.1.7) was examined in different tissues of Catenula leptocephala (Nuttycombe, 1956). Eserine and iso-OMPA were used to distinguish AChE from non-specific cholinesterases (ChE, E.C. 3.1.1.8). The enzyme was located mainly in the brain neuropil, the peripheral nervous system, neuromuscular junctions, on the membrane of muscle cells and of cells with rhabdites. The distribution of the enzyme suggests that cholinergic transmission occurs in Catenula leptocephala, while simultaneously the presence of AChE on the membranes of muscle cells points to the receipt of cholinergic stimulation. The role of AChE in differentiation and maturation of cells with rhabdites is also discussed in this paper.     

A CHOLINERGIC COMPONENT IN THE INNERVATION OF THE LONGITUDINAL SMOOTH MUSCLE OF THE GUINEA PIG VAS DEFERENS : The Fine Structural Localization of Acetylcholinesterase

Acetylcholinesterase (AChE) has been detected on the plasma membrane of about 25% of the axons in the longitudinal smooth muscle tissue of guinea pig vas deferens. These axons are presumably cholinergic. No enzyme was detected in the remaining 75% of axons. These axons are presumably adrenergic. The plasma membrane of the Schwann cells associated with the cholinergic axons also stained for AChE. Some axon bundles contained only cholinergic or adrenergic axons while others contained both types of axon. When a cholinergic axon approached within 1100 A of a smooth muscle cell, there was a patch of AChE activity on the muscle membrane adjacent to the axon. It is suggested that these approaches are the points of effective transmission from cholinergic axons to smooth muscle cells. Butyrylcholinesterase activity was detected on the plasma membranes of all axons and smooth muscle cells in this tissue.     

Trypanosoma evansi: immune response and acetylcholinesterase activity in lymphocytes from infected rats

The existence of cholinergic receptors in the immune system cells is well documented. This study aimed to evaluate the acetylcholinesterase activity (AChE) in lymphocytes from rats infected with Trypanosoma evansi in acute and chronic phase disease. Twenty animals were infected with 10⁶ trypomastigotes forms each and 10 were used as negative controls. The two groups of inoculated rats were formed according to the degree of parasitemia and the period post-infection (PI). Group A: rats with 4 days PI and between 24 and 45 parasites/field (1000×); group B: rats with 30 days PI and parasitemia with jagged peaks between 0 and 1 parasites/field; group C: not-infected animals. At 4 days PI (acute phase) and 30 days PI (chronic phase) the rats were anesthetized to collect blood for hemogram and separation of lymphocytes. After separation, the AChE activity was measured in lymphocytes. It was observed that the number of lymphocytes increased significantly in group A compared to group C. The activity of AChE in lymphocytes significantly increased in acute phase and decreased in chronic phase in the infected rats when compared to not-infected (P < 0.05). Statistical analysis showed a positive correlation between the number of lymphocytes and AChE activity in lymphocytes in 4 days PI (r²: 0.59). Therefore, the infection by T. evansi influences AChE activity in lymphocytes of rats indicating changes in the responses of cholinergic system in acute phase, possibly due to immune functions performed by these enzymes.     

Cholinergic neurons in the lamina ganglionaris of the blowfly Calliphora erythrocephala

Summary The distribution of putative cholinergic neurons in the lamina of the blowfly Calliphora erythrocephala was studied by immunocytochemical and histochemical methods. Three different antibodies directed against the AChsynthesizing enzyme, choline acetyltransferase (ChAT), revealed a cholinergic population of fibres running parallel to the laminar cartridges, which have branch-like structures at the distal lamina border. Cell bodies in the chiasma next to the lamina border were also labelled by the anti-ChAT antibodies. Monopolar cell bodies in the nuclear layer were faintly labelled. The distribution of the acetylcholine hydrolyzing enzyme, acetylcholine esterase (AChE), was revealed by histochemical staining and was similar to the ChAT immunocytochemistry. The arrangement of ChAT positive fibres in transverse and longitudinal sections and the distribution of AChE stained fibres indicate that the amacrine cells of the lamina are cholinergic cells.We dedicate this work to Prof. F. Zettler who passed away in fall 1988: K.-H. Datum, I. Rambold     

Acetylcholinesterase in central vocal control nuclei of the zebra finch (<Emphasis Type="Italic">Taeniopygia guttata</Emphasis>)

The distribution of acetylcholinesterase (AChE) in the central vocal control nuclei of the zebra finch was studied using enzyme histochemistry. AChE fibres and cells are intensely labelled in the forebrain nucleus area X, strongly labelled in high vocal centre (HVC) perikarya, and moderately to lightly labelled in the somata and neuropil of vocal control nuclei robust nucleus of arcopallium (RA), medial magnocellular nucleus of the anterior nidopallium (MMAN) and lateral magnocellular nucleus of the anterior nidopallium (LMAN). The identified sites of cholinergic and/or cholinoceptive neurons are similar to the cholinergic presence in vocal control regions of other songbirds such as the song sparrow, starling and another genus of the zebra finch (Poephila guttata), and to a certain extent in parallel vocal control regions in vocalizing birds such as the budgerigar. AChE presence in the vocal control system suggests innervation by either afferent projecting cholinergic systems and/or local circuit cholinergic neurons. Co-occurrence with choline acetyltransferase (ChAT) indicates efferent cholinergic projections. The cholinergic presence in parts of the zebra finch vocal control system, such as the area X, that is also intricately wired with parts of the basal ganglia, the descending fibre tracts and brain stem nuclei could underlie this circuitry’s involvement in sensory processing and motor control of song.     

Pharmacophore-based design and discovery of (−)-meptazinol carbamates as dual modulators of cholinesterase and amyloidogenesis

Multifunctional carbamate-type acetylcholinesterase (AChE) inhibitors with anti-amyloidogenic properties like phenserine are potential therapeutic agents for Alzheimer’s disease (AD). We reported here the design of new carbamates using pharmacophore model strategy to modulate both cholinesterase and amyloidogenesis. A five-feature pharmacophore model was generated based on 25 carbamate-type training set compounds. (?)-Meptazinol carbamates that superimposed well upon the model were designed and synthesized, which exhibited nanomolar AChE inhibitory potency and good anti-amyloidogenic properties in in vitro test. The phenylcarbamate 43 was highly potent (IC₅₀ 31.6?nM) and slightly selective for AChE, and showed low acute toxicity. In enzyme kinetics assay, 43 exhibited uncompetitive inhibition and reacted by pseudo-irreversible mechanism. 43 also showed amyloid-β (Aβ) lowering effects (51.9% decrease of Aβ₄₂) superior to phenserine (31% decrease of total Aβ) in SH-SY5Y-APP₆₉₅ cells at 50?µM. The dual actions of 43 on cholinergic and amyloidogenic pathways indicated potential uses as symptomatic and disease-modifying agents.     

Epitope Mapping of Form-Specific and Nonspecific Antibodies to Acetylcholinesterase

We have mapped the epitopes to which two monoclonal antibodies against acetylcholinesterase (AChE) from Torpedo californica are directed. One antibody, 2C9, has equivalent affinity for both the 5.6S (amphiphilic) and 11S (hydrophilic) enzyme forms; the other, 4E7, recognizes only the amphiphilic form and has been shown previously to require an N-linked oligosaccharide residue on the protein. Isolation of cyanogen bromide peptides from the amphiphilic form and assay by a competition ELISA for 2C9 and by a direct binding ELISA for 4E7 identified the same peptide, residues 44–82, as containing epitopes against both antibodies. The epitope for 4E7 includes the oligosaccharide conjugated to Asp⁵⁹, an N-linked glycosylation site not present in mouse AChE. A 20-amino-acid synthetic peptide, RFRRPEPKKPWSQVWNASTY, representing residues 44–63, was synthesized and found to inhibit completely 2C9 binding to 5.6S enzyme at molar concentrations comparable to those of the cyanogen bromide peptide. It was unreactive with 4E7. Fractionation of the synthetic peptide further localized the 2C9 epitope. Peptides RFRRPEPKKPW and KPWSGVWNASTY both reacted but less so than the entire synthetic peptide at equivalent molar concentrations, whereas the peptide RPEPKKPWSGVWNASTY was as effective as the larger synthetic peptide. The crystal structure of AChE shows the peptide to be on the surface of the molecule as part of a convex hairpin loop starting before the first α-helix.     

Localization of adenylate cyclase activity in the tissues of an intact planarian Dugesia lugubris (O. Schmidt)

Summary Adenylate cyclase was localized in tissues of an intact planarian Dugesia lugubris (O. Schmidt) by ultracytochemical methods. The enzyme was found in epithelium, muscles, secretory cells (mucous), and rhabdites forming cells and neoblasts. Adenylate cyclase occurred on the external side of cell membranes in cisterns of the endoplasmic reticulum, nuclear envelope and mitochondria. The problems of ultracytochemical localization of AC are discussed.     

Cellular expression patterns of acetylcholinesterase activity during grasshopper development

We examined the expression of acetylcholinesterase (AChE) in the nervous system and epidermal body structures during embryonic and larval development of two grasshopper species: Locusta migratoria and Schistocerca americana. Histochemical labelling was blocked by the enzyme inhibitors eserine and BW284c51, but not by iso-OMPA, showing that the staining reflected true AChE activity. The majority of staining was localized on the cell surface but granular intracellular staining was also visible in many cell bodies. In both species, the cellular expression of AChE followed a similar but complex spatiotemporal staining pattern. Initially, mainly epidermal tissue structures were stained in the various body appendages (stages 25%–30%). Labelling subsequently appeared in outgrowing neurons of the central nervous system (CNS) and in the nerves innervating the limbs and dorsal body wall (stages 30%–40%). The latter staining originated in motoneurons of the ventral nerve cord. In a third phase (after 45%), the somata of certain identified mechanosensory neurons started to express AChE activity, presumably reflecting cholinergic differentiation. Staining was also found in repo-positive glial cells of the CNS, longitudinal glia of connectives, glia of the stomatogastric nervous system and glial cells ensheathing peripheral nerves. Glial cells remained AChE-positive during larval to adult development, whereas motoneurons lost their AChE expression. The expression pattern in non-neuronal cells and glutamatergic motoneurons and the developmental appearance of AChE prior to synaptogenesis in the CNS suggest non-cholinergic functions of AChE during grasshopper embryogenesis. Financial support was provided by the Deutsche Forschungsgemeinschaft (Bi 262/7-1 and 262/11-1)     

Evidence for a cerebral cholinergic system and suggested pharmacological patterns of neural organization in the prostomium of the polychaete Nereis virens (SARS)

The histological visualization of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) on frozen sections of prostomia of Nereis virens indicate a concentration of cholinergic activity in the anterior brain. Components are probably sensory epithelial cells with cholinergic axons entering the brain in cephalic nerves and efferent cholinergic axons to prostomial muscle leaving the brain in the same nerves. There are also subepidermal cholinergic cells that may be second order motor neurons serving epidermal mucous cells. The smaller, second lobe of the corpora pedunculata and its associated vertical fibre tract are CAT⁴ and appear continuous, on each side of the cerebral ganglion, with a dorsal and a ventral longitudinal bundle of AChE⁺ fibers. This system tapers to nothing at the level of the posterior eyes. There is a small AChE⁺ component to each optic nerve and AChE is present in the nuchal epithelium. These observations are discussed in relation to earlier studies on aminergic and neurosecretory activity in the same ganglion.     

Acetylcholinesterases.

Acetylcholinesterase (E.C.3.1.1.7) is a widely distributed enzyme, particularly in excitable tissues such as muscle, nerve, and electric organs but also found in erythrocytes and snake venoms. The function of the enzyme at postsynaptic sites in excitable tissues is considered to be termination of synaptic transmission via the hydrolytic inactivation of acetylcholine. The functional role of the enzyme at extrajunctional sites of excitable tissues, in nerve endings and in the erythrocyte has not been established. In the past five or six years substantial progress has been made in terms of our understanding of acetylcholinesterases (AChE) particularly with regard to their molecular characterization, their subunit structure and their immunological properties. These advances have been due in part to successful purification of enzymes from various tissues by the application of affinity chromatography techniques. In addition, some progress has been made regarding physiological aspects of the development and regulation of AChE in excitable tissues. This review will focus on these aspects of AChE by reference to work utilizing the enzyme from the following sources: electric tissue of the eel, $\text{Electrophorus electricus}$, or electric fish, $\text{Torpedo}$, species; mammalian and avian skeletal muscle; neural tissues, particularly mammalian brain; and the mammalian erythrocyte. For more comprehensive reviews of AChE readers are referred to the following references (1, 2).     

Cholinesterasen mit Nebenjobs

Moonlighting cholinesterases in non-synaptic cholinergic mechanisms The early phylogenetic and ontogenetic appearance of acetylcholine (ACh) and its cholinergic protein components render their possible functionalities, apart from purely neuronal ones, most likely. The capacities of cholinesterases (ChEs) to form large protein complexes opened wide functional fields for them. Already existent in stem cells, ChEs in cooperation with components of the cell matrix (ECM) promote cell differentiation, whereby their enzymatic activity is (at least partially) dispensable. This is independently supported by effects of inactive AChE protein exerted in non-neuronal cells, as well as the discovery of cholinesterase-like adhesion molecules (CLAMs). Therefore, much evidence supports the conclusion that the original functionalities of cholinesterases, and, more generally of cholinergic systems, are to be sought in cell-cell-communication. Here, these views were exemplified by some in vitro and in vivo model studies. In the vertebrate retina early differentiating amacrine cells co-regulate network formation. Similarly potent are cholinergic mechanisms during skeletogenesis. ACh accelerates bone formation, and ChEs not only regulate its concentration, but exert additional structural functions. As much convincing, a study on tadpoles documented that gut formation in Xenopus laevis depends strictly on the AChE protein, but not on its enzymatic activity. A full elucidation of ChE functionalities is essential, since a multitude of anticholinesterases (ChE inhibitors) are widely applied in public life (agriculture, health, security). It is timely that cholinergic research focuses on elucidation of non-synaptic ChEs, and on analyzing non-neuronal cholinergic systems (NNCS) in general.     

Neurotrophic control of 16S acetylcholinesterase from mammalian skeletal muscle in organ culture

The effects of rat obturator nerve extracts on total and 16S acetylcholinesterase (AChE) activity were studied in endplate regions of denervated anterior gracilis muscles maintained in organ culture for 48 hr. The decrease of total AChE activity in cultured muscles was similar to that observed in denervated muscles in vivo. This decrease in activity was partly prevented by addition of either 100 or 200 μl nerve extract (2.7 mg/ml protein) to the nutrient medium. Nerve extract treatment also decreased the release of AChE activity from the muscle into the bathing medium. Conversely, rat serum (20 μl; 90 mg/ml protein) had no effect on total AChE activity in muscle endplates, nor on release of the enzyme by the muscle. The 16S form of AChE was confined to motor endplate muscle regions and its activity was drastically decreased by denervation in both organ culture and in vivo preparations in a comparable manner. Nerve-extract supplemented cultures contained a significantly (p ? 0.001) larger amount of endplate 16S AChE activity (140–145%) than the corresponding controls (100-). Our results suggest that some nerve soluble substance, other than serum contaminants or 16S AChE itself, affects the maintenance of 16S AChE at the neuromuscular junction.     

A comparison of the Xenopus laevis oocyte acetylcholinesterase with the muscle and brain enzyme suggests variations at the post-translational level.

1. Xenopus laevis oocytes express endogenously two components of the cholinergic system: the muscarinic receptors and the acetylcholinesterase (AChE). 2. A biochemical characterization of this enzyme was carried out. 3. The results established that the activity found in the oocytes correspond to 'true' AChE with a molecular weight of 65,000 Da and a sedimentation coefficient of 3-4 S. 4. The enzyme aggregates in the absence of detergent suggesting that it possess an hydrophobic character; despite that, it is not sensitive to PIPLC. 5. A comparison with the Xenopus brain and muscle AChE shows different post-translational modifications and catalytic properties with the oocyte AChE.     

Acetylcholinesterase in the ocellus of Apis mellifica

The ultrastructural localization of the enzyme acetylcholinesterase (AChE) in the ocellus of the honey bee (Apis mellifica) was studied by electron microscopy. High AChE activity was found both in the receptor-cell axons and in the surrounding glial cells. Second order neurones exhibited a remarkably lower anzyme activity. AChE was also detected in the intercellular spaces between the receptor-cell axons and the second order neurones. These results provide additional support to the assumed cholinergic nature of the photoreceptor cells in the insect ocellus.     

Targeting acetylcholinesterase: identification of chemical leads by high throughput screening, structure determination and molecular modeling

Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e.g. in Alzheimer''s disease), but may also act as dangerous toxins (e.g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685•mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators.     

Overexpressed Monomeric Human Acetylcholinesterase Induces Subtle Ultrastructural Modifications in Developing Neuromuscular Junctions of Xenopus laevis Embryos

Abstract: Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle-specific proteins, among them the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DMA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over-expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell-associated rHAChE accumulated in myotomes of 2-and 3-day-old embryos within the same sub-cellular compartments as native Xenopus AChE. NMJs from 3-day-old DNA-injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.     

Carboxylic esterases in developing myoneural junctions of rat striated muscle

Summary The distribution of acetylcholinesterase (AChE; E.C. 3.1.1.7), other cholinesterases (ns.ChE; E.C. 3.1.1.8) and eserine-resistant carboxylic esterases (ns. E; E.C. 3.1.1.1) was studied in the developing myoneural junction of the rat tibialis anterior muscle from the 16th intrauterine day onwards. Acetyl-and butyrylthiocholine were used as substrates for AChE and ns.ChE, and -naphthyl acetate for ns. E.Acetylcholinesterase was first visible in 18-day rat embryos, ns.ChE in 21-day embryos and ns. E in 1-day-old postnatal rats and thereafter. Both AChE and ns.ChE activities were localized at the level of the plasma membrane in the middle of the muscle fibres. In the early stages this area of activity had the appearance of a plate-like structure, which deepened to form a depression in the surface of the muscle fibre by the 2nd to the 4th postnatal day. About 5 days later subneural lamellaes appeared in this structure, and ramification and segmentation took place, their extent increasing concomitantly with the increase of enzyme activity. The adult pattern was attained by the age of one month. Precise localization of ns. E was not possible in the immature stages, mainly owing to the granularity of the reaction end-product. After the age of about 10 days, however, the distribution of the reaction end-product suggested a mainly presynaptic location. Other cholinesterases and ns. E, but not AChE, were detected in the neurilemma cells along nerve fibres. This neurilemmal enzyme activity gradually diminished after birth and was lost at about the age of 3 weeks.These observations demonstrate that the formation of the junction between the nerve and the muscle fibre takes place just before the first appearance of AChE activity in a sharply delineated area of the plasma membrane. The structural changes made apparent by the distribution of the reaction end-products are assumed to be linked to the spatial rearrangement of the synaptic membranes, seen in earlier electron microscopic studies.     

Temporospatial localization of acetylcholinesterase activity in the dental epithelium during mouse tooth development

Acetylcholinesterase (AChE), a principal modulator of cholinergic neurotransmission, also has been demonstrated to be involved in the morphogenetic processes of neuronal and non-neuronal tissues. This study shows that AChE exhibits temporospatial activity in the dental epithelium of the developing mouse tooth. To identify the AChE activity in the mouse tooth during development, we performed enzyme histochemistry on the mouse embryos from embryonic day 13 (E13) to E18 and on the incisors and molars of the neonatal mouse at 10 days after birth (P10). In the developing molars of mouse embryos, AChE activity was not found in the dental epithelium at E13 (bud stage). AChE activity first appeared in the developing cervical loops of the enamel organ at E14 (cap stage), but was not found in the enamel knot. At E18 (bell stage), AChE activity was localized in the inner enamel epithelium except the cervical-loop area. In the incisors and molars of neonatal mice (P10), AChE activity was localized in the inner enamel epithelium of the cervical-loop and enamel-free area. Overall, AChE activity was localized in the differentiating dental epithelium while the activity of butyrylcholinesterse, another cholinesterase, was located primarily in the cells of the dental follicle. The results suggest that AChE may play a role in the histo- and cytodifferentiation of dental epithelium during tooth development.