Molecular Forms Of Acetylcholinesterase Research Articles

Biological activity identification of newly synthesized plumbagin‐lipoic acid conjugate: In vitro and in silico analyses of interaction with acetylcholinesterase and butyrylcholinesterase

AbstractBackgroundIdentification of novel compounds with inhibitory potential against cholinesterases (ChEs) such as acetylcholinesterase (AChE) and butrylcholinesterase (BChE) is considered important strategy to overcome Alzheimer’s disease (AD). The ratio of major globular tetrameric form of AChE (G4) to the minor lower species (G1; monomers and G2; dimers) is decreased in the AD brain. Also, in recent time, the involvement of BChE in progression of AD is recognized. Hence, it is imperative to understand the inhibitory potential of any new compound against BChE and various globular forms of AChE to identify as lead molecule to further explored for AD treatment. Keeping this view, plumbagin (PLU) and lipoic acid (LA) conjugate was synthesized and characterized for its inhibition against AChE and BChE. Further in silico analysis was carried out to get an insight into the interaction of PLU‐LA with the AChE and BChE.MethodNewly synthesized PLU‐LA conjugate by esterification reaction was characterized by nuclear magnetic resonance spectroscopy (NMR) and mass spectroscopy (MS). The synthesized PLU‐LA was studied for the inhibition against BChE and different molecular forms of AChE. Kinetic analysis was carried out to determine the type of inhibition and inhibitor constant. In silico analysis such as molecular docking and molecular dynamics simulation was carried out using AutoDock and Desmond, respectively.ResultPLU‐LA synthesized was confirmed by NMR and MS analysis. PLU‐LA inhibited the different molecular forms (G2 and G4) of AChE at micromolar concentration. Kinetic analysis revealed the type of inhibition with low Ki value. Molecular docking analysis revealed the orientation of PLU‐LA at the active site of human AChE and BChE forming interaction with catalytic site amino acid. Molecular dynamics simulation confirmed the stable binding of PLU‐LA with ChEs.ConclusionPLU‐LA was synthesized and it’s inhibition against ChEs (AChE and BChE) is understood. In silico analysis highlighted the interaction of PLU‐LA within the active site gorge of the ChEs.

Identifying amine fragments as potential inhibitors of acetylcholinesterase and butyrylcholinesterase through in silico analysis and validation through in vitro enzyme inhibition analysis

AbstractBackgroundThe cholinesterases [ChEs; acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] which are involved in cholinergic neurotransmission are target for drug development against cognitive dysfunction in Alzheimer’s disease. The search for novel ChEs inhibitors with improved biological profiles continues to be of great interest. Currently know inhibitors of ChEs such as galatamine, tacrine, donepezil and rivastigmine are amine‐based compound. Keeping this view, in the present study virtual screening was carried out to identify the potential amine fragment that interacted with the ChEs in in silico. The selected molecules were analyzed by molecular docking and molecular dynamics simulation. The best interacted molecule in in silico was further characterized for its inhibition against AChE and BChE in in vitro.MethodHigh Throughput Virtual Screening (HTVS) of various amine fragments was carried out in Schrödinger suite. Identified top ten molecules were subjected to molecular docking analysis using Induced Fit Docking (IFD) module. Molecular dynamics (MD) simulation of top three scored compounds were carried out to understand the stability of the interaction with ChEs. In silico quantum crystallography studies was done to understand the involvement of functional group in the interaction with ChEs. In vitro inhibition and kinetic analysis of these compounds against ChEs was performed to determine the inhibitory constant and type of inhibition.ResultAmong the amine compounds screened, three compounds 2‐aminoquinoline (2AQ), 2‐aminobenzimidazole (2AB) and 2‐amino‐1‐methylbenzimidazole (2AMB) was identified to interact with ChEs with lowest docking score. Further MD simulation confirmed the stable binding of these compounds in the active site of the ChEs. Topological and electrostatic properties of intermolecular interactions between these compounds with ChEs showed that amine group played important role in interacting with protein structure. These amine compounds inhibited the BChE and different molecular forms of AChE at micromolar concentration. Kinetic analysis revealed the mixed‐type of inhibition.ConclusionAmine fragments screened for interaction with ChEs in in silico. Three compounds (2AQ, 2AB and 2AMB) showed better interaction in molecular docking and molecular dynamics analysis. These compounds showed inhibition against ChEs in in vitro.

Isolation of bioactive components from Corallocarpus epigaeus tuber and inhibitory potential against various molecular forms of acetylcholinesterase

AbstractBackgroundIn human, acetylcholinesterase (AChE) is involved in cholinergic neurotransmission. AChE exhibits as different globular form such as G4 (tetramer), G2 (dimer) and G1 (monomer). For the most part of the adult brain G4 forms is present. Nevertheless, under dementia such as Alzheimer’s disease (AD) conditions, the ratio between G1 and G4 form is increased which is involved in disease progression. Inhibition of AChE is considered important therapeutic intervention to overcome the AD. Corallocarpus epigaeus comes under the family of Cucurbitacea has known for the biological properties such as anti‐snake venom, analgesic, anthelminthic properties and antidiabetic activity. Identifying natural compounds with AChE inhibition is considered important for AD treatment. In the present study, bioactive fraction extracted from tuber of C. epigaeus was investigated for its inhibition against different molecular forms of AChE in in vitro.MethodDried tuber powder of C. epigaeus was extracted using various solvents sequentially by increasing polarity in Soxhlet apparatus. Methanol extract was further purified by silica column chromatography. The partially purified fraction was studied for the inhibition against different molecular forms of AChE. Kinetic analysis was carried out to determine the type of inhibition and inhibitor constant. The active compounds in partially purified fraction were determined using gas chromatography mass spectrum analysis (GCMS).ResultPartially purified fraction of methanolic extract of C. epigaeus tuber inhibited all the molecular forms (G1, G2 and G4) of AChE at microgram per milliliter concentration. Kinetic analysis revealed the mixed type non‐competitive mode of inhibition with low Ki value. GCMS analysis 4H‐pyran‐4‐one, 2,3‐dihydro‐3,5‐dihydroxy‐6‐methyl and alpha‐linolenic acid as major constituents in the extract.ConclusionThe bioactive components in tuber of C. epigaeus inhibited AChE in in vitro. This is the first report on the inhibitory effect of C. epigaeus tuber extract against AChE. Further in vivo studies are necessary to understand the anti‐dementia effect of C. epigaeus.

Biochemical adaptation in brain Acetylcholinesterase during acclimation to sub-lethal temperatures in the eurythermal fish Tilapia mossambica

Tilapia mossambica is a eurythermal tropical fish. We studied the effect of temperature on the kinetics of brain Acetylcholinesterase (AChE) during adaptation to sublethal temperatures by acclimating the fish to 37 °C, and controls to 25 °C. Electrophoresis showed the presence of two AChE bands that did not change in position or intensity with acclimation period or temperature. The apparent Km was 0.23 ± 0.01 mM ATChI and remained relatively constant over the in vitro assay temperature range 10 °C to 40 °C. Biochemical characterization suggested that the enzyme is a ‘eurytolerant protein’ in its stability of kinetic and thermal properties over a wide temperature range. Thermal stability and arrhenius plots suggested that the AChE was made up of two forms that differed in their thermal properties.The two molecular forms of acetylcholinesterase were purified from the brain of T. mossambica. Molecular weight studies revealed that the two forms were size isomers: a monomer of 59 KDa and a tetramer of 244 KDa. They differed in their Kms, thermal stabilities and energies of activation. We suggest that biochemical adaptation to temperature in the brain acetylcholinerase system of the fish Tilapia mossambica is based on the aggregation-dissociation of these size isomers.

In vitro and in silico analysis of novel astaxanthin-s-allyl cysteine as an inhibitor of butyrylcholinesterase and various globular forms of acetylcholinesterases

In Alzheimer's disease (AD) and diabetes-associated cognitive decline, the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity is increased. AChE exists as different globular molecular forms: tetramer (G4), dimer (G2) and monomer (G1). In adult brain, G4 form is abundant however in AD, the ratio of lower molecular forms (G1) to G4 form increased. Hence, the present study delineated the inhibition of novel astaxanthin-s-allyl cysteine (AST-SAC) against BChE and various molecular forms of AChE. Cobra venom, human erythrocyte and Electrophorus electricus was used as source of G1, G2 and G4 form of AChE. AST-SAC showed inhibition against G1 (IC50 = 0.72 μM, competitive, Ki = 0.66 μM), G2 (IC50 = 0.65 μM, mixed, Ki = 0.50 μM) and G4 (IC50 = 0.67 μM, competitive, Ki = 0.67 μM) form of AChE. AST-SAC inhibited human brain AChE (IC50 = 0.84 μM, competitive, Ki = 0.53 μM) and human serum BChE (IC50 = 0.80 μM, competitive, Ki = 0.58 μM). In silico analysis revealed the interaction of AST-SAC with the amino acids present in peripheral anionic and catalytic site of human AChE and BChE. Molecular dynamics simulation confirmed the stable interaction of AST-SAC in the active site gorge of AChE and BChE.

Reduced Expression of P2Y2 Receptor and Acetylcholinesterase at Neuromuscular Junction of P2Y1 Receptor Knock-out Mice.

ATP is co-stored and co-released with acetylcholine (ACh) at the pre-synaptic vesicles in vertebrate neuromuscular junction (nmj). Several lines of studies demonstrated that binding of ATP to its corresponding P2Y1 and P2Y2 receptors in the muscle regulated post-synaptic gene expressions. To further support the notion that P2Y receptors are playing indispensable role in formation of post-synaptic specifications at the nmj, the knock-out mice of P2Y1 receptor (P2Y1R (-/-)) were employed here for analyses. In P2Y1R (-/-) mice, the expression of P2Y2 receptor in muscle was reduced by over 50 %, as compared to P2Y1R (+/+) mice. In parallel, the expression of acetylcholinesterase (AChE) in muscle was markedly decreased. In the analysis of the expression of anchoring subunits of AChE in P2Y1R (-/-) mice, the proline-rich membrane anchor (PRiMA) subunit was reduced by 60 %; while the collagen tail (ColQ) subunit was reduced by 50 %. AChE molecular forms in the muscle were not changed, except the amount of enzyme was reduced. Immuno-staining of P2Y1R (-/-) mice nmj, both AChE and AChR were still co-localized at the nmj, and the staining was diminished. Taken together our data demonstrated that P2Y1 receptor regulated the nmj gene expression.

Induction of soluble AChE expression via alternative splicing by chemical stress in Drosophila melanogaster

Various molecular forms of acetylcholinesterase (AChE) have been characterized in insects. Post-translational modification is known to be a major mechanism for the molecular diversity of insect AChE. However, multiple forms of Drosophila melanogaster AChE (DmAChE) were recently suggested to be generated via alternative splicing (Kim and Lee, 2013). To confirm alternative splicing as the mechanism for generating the soluble form of DmAChE, we generated a transgenic fly strain carrying the cDNA of DmAChE gene (Dm_ace) that predominantly expressed a single transcript variant encoding the membrane-anchored dimer. 3′ RACE (rapid amplification of cDNA ends) and western blotting were performed to compare Dm_ace transcript variants and DmAChE forms between wild-type and transgenic strains. Various Dm_ace transcripts and DmAChE molecular forms were observed in wild-type flies, whereas the transgenic fly predominantly expressed Dm_ace transcript variant encoding the membrane-anchored dimer. This supports alternative splicing as the major determinant in the generation of multiple forms of DmAChE. In addition, treatment with DDVP as a chemical stress induced the expression of the Dm_ace splice variant without the glycosylphosphatidylinositol anchor site in a dose-dependent manner and, accordingly, the soluble form of DmAChE in wild-type flies. In contrast, little soluble DmAChE was expressed in the transgenic fly upon exposure to DDVP. DDVP bioassays revealed that transgenic flies, which were unable to express a sufficient amount of soluble monomeric DmAChE, were more sensitive to DDVP compared to wild-type flies, suggesting that the soluble monomer may exert non-neuronal functions, such as chemical defense against xenobiotics.

Interaction of metal chelators with different molecular forms of acetylcholinesterase and its significance in Alzheimer's disease treatment

The peripheral anionic site (PAS) of acetylcholinesterase (AChE) is involved in amyloid beta (Aβ) peptides aggregation of Alzheimer's disease (AD). AChE exhibits an aryl acylamidase (AAA) activity along with the well known esterase activity. Numerous studies have reported the beneficiary effect of metal chelators in AD treatment. Hence, a comparative study on the effect of metal chelators on both the esterase and AAA activity of AChE globular (G4 and G1) molecular forms was performed. The inhibitory effect of 1,10-phenanthroline was high towards AChE esterase activity. The corresponding IC50 values for esterase activity of G4 and G1-form was 190 µM and 770 µM and for AAA activity it was 270 µM and 2.74 mM, respectively. Kinetic studies on both forms of AChE show that 1,10-phenanthroline inhibits esterase in competitive and AAA activity in non-competitive manner. Protection studies further revealed that the nature of 1,10-phenanthroline inhibition on AChE is through its direct binding to protein rather than its metal chelation property. Molecular docking studies shows orientation of 1,10-phenathroline in the PAS through hydrophobic interactions with the PAS residues (Trp286, Tyr124 and Tyr341) and hydrogen bonding with Phe295. Further molecular dynamics simulation of "hAChE-1,10-phenanthroline" complex revealed that both hydrogen and hydrophobic interaction contribute equally for 1,10-phenanthroline binding to hAChE. Such an interaction of 1,10-phenanthroline on PAS may hinder "AChE-Aβ peptide" complex formation. Hence, 1,10-phenanthroline can act as a lead molecule for developing drug(s) against AD ailment with dual functions namely, anti-cholinesterase and anti-amyloid aggregation potency in a single chemical entity.

Molecular Assembly and Biosynthesis of Acetylcholinesterase in Brain and Muscle: the Roles of t-peptide, FHB Domain, and N-linked Glycosylation

Acetylcholinesterase (AChE) is responsible for the hydrolysis of the neurotransmitter, acetylcholine, in the nervous system. The functional localization and oligomerization of AChE T variant are depending primarily on the association of their anchoring partners, either collagen tail (ColQ) or proline-rich membrane anchor (PRiMA). Complexes with ColQ represent the asymmetric forms (A12) in muscle, while complexes with PRiMA represent tetrameric globular forms (G4) mainly found in brain and muscle. Apart from these traditional molecular forms, a ColQ-linked asymmetric form and a PRiMA-linked globular form of hybrid cholinesterases (ChEs), having both AChE and BChE catalytic subunits, were revealed in chicken brain and muscle. The similarity of various molecular forms of AChE and BChE raises interesting question regarding to their possible relationship in enzyme assembly and localization. The focus of this review is to provide current findings about the biosynthesis of different forms of ChEs together with their anchoring proteins.

Organophosphate-resistant forms of acetylcholinesterases in two scallops—the Antarctic Adamussium colbecki and the Mediterranean Pecten jacobaeus

We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G 2, G 4) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G 2); a further amphiphilic-low-salt-soluble G 2 acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G 4 and an amphiphilic G 2 acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G 2) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G 2 forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.

Junctional and extrajunctional acetylcholinesterase in skeletal muscle fibers

The asymmetric A 12 acetylcholinesterase (AChE) molecular form, consisting of three tetrameric catalytic oligomers and three non-catalytic subunits of collagen Q (ColQ), is the functional AChE form in the neuromuscular junction. Its extremely high concentration and sharp localization in the junction is mostly due to the binding of this AChE form to perlecan in the synaptic basal lamina. In the rat neuromuscular junctions, about two-thirds of AChE molecules appear to be bound by ionic interactions involving calcium and the rest is probably bound covalently. In immature rat muscles, the A 12 AChE forms are expressed also extrajunctionally. During the early post-natal period, this expression is completely suppressed in rat fast muscles, whereas it extends into adulthood in the slow soleus muscles because of the differences in the extrajunctional expression of ColQ between fast and slow muscles. The level of the A 12 molecular forms of AChE in the extrajunctional muscle regions is regulated by the motor nerve, probably via the pattern of muscle fibre activations triggered by the nerve. The pattern of muscle activations also regulates the extrajunctional expression of the catalytic subunits of AChE: phasic, infrequent, high frequency activations enhance expression, whereas prolonged tonic low-frequency activations tend to decrease it. Calcineurin signalling pathway seems to be involved.

Localization, specific activity, and molecular forms of acetylcholinesterase in developmental stages of the cestode Mesocestoides corti

The nervous system of flatworms is quite simple although there is increasing evidence indicating that it is chemically complex. Studies of the nervous system in these animals have only been performed in the larval stage or in the adult worms, which are easy to obtain in nature, while the description of the nervous system in developing stages of these organisms is missing. Mesocestoides corti is a parasitic platyhelminth whose larvae can be induced in vitro to develop to adult, sexually mature worms, opening the possibility of studying the nervous system of a flatworm in different stages of development. Here, we describe the presence, activity, location, and molecular forms of acetylcholinesterase (AChE) in different stages of development of M. corti, from the larvae to adult forms of this endoparasite, obtained in in vitro cultures after induction of the larval stage with trypsin. Our results point to AChE as a molecular marker of the nervous system in platyhelminthes. The change in molecular forms of this enzyme and the increase in its activity during development from larvae to adult worm may reflect the presence of a more complex nervous system, necessary to adjust and coordinate the movement of a much bigger structure. A relationship between the development of the reproductive apparatus in segmented and adult worms with a more complex nervous system in these stages is also apparent. Finally, our study opens the possibility of applying anti-AChE as more effective therapeutic strategies against cestode parasites.

Molecular forms of acetylcholinesterase in the rat extensor digitorum longus and soleus muscles regenerating from notexin-induced necrosis

The activity of acetylcholinesterase molecular forms were examined after separation on sucrose gradients during notexin-induced necrosis and the following regeneration in rat extensor digitorum longus (EDL) and soleus (SOL) muscles. All forms dropped rapidly in both muscles in the first few days after single notexin injection. After a delay small globular forms (G1+G2) started to regenerate from day 7 and larger forms (G4 and A12) from day 10 in EDL. The A8 form which cannot be detected in normal EDL was present between day 7 and day 28. In SOL the recovery of AChE forms begun already on day 3. The small globular forms displayed a more rapid increase between day 3 and day 7 then the other forms. In SOL we observed a temporary overshooting peak at day 7 in the activity of all molecular forms. Both muscles recovered their normal AChE pattern by that time when muscle fibres regained their normal diameter (day 28). Most of the events of regeneration of AChE forms resembled those of normal myogenesis.

Acetylcholinesterase activity is affected by stress conditions in Paracentrotus lividus coelomocytes

The presence and function of acetylcholinesterase (AChE) in blood cells and plasma is well known. The enzyme activity is associated to red blood cell membranes, as well as to the perinuclear region of leucocytes, suggesting a role in the function of the latter. Recently coelomocytes, acknowledged progenitors of immune cells in vertebrate systems, have been shown to respond to stress conditions, both in laboratory experiments and field studies. Bearing in mind the similarity between coelomocytes and vertebrate blood cells, we investigated the presence of AChE activity in coelomocytes of the sea urchin Paracentrotus lividus characterized by specific inhibitors, in relation to cold stress induced in the laboratory. Exposure to stress was followed by electrophoretic analysis of AChE activity and Western blot analysis of AChE-like immunoreactivity. Moreover, quantitative analysis of the different molecular forms of AChE was carried out with the aid of a spectrophotometric method. The effects of stress on AChE activity were time dependent. In fact, the enzyme activity was detected by the catalytic activity in cleaving the substrate acetyl thiocholine iodide, which appears to be formed by different isoforms of the enzyme, compared to that of Electrophorus, which was used as a control. Western blot analyses showed the presence of different immunoreactive molecules, with molecular mass ranging from 265 kDa (corresponding to Electrophorus molecules) to about 180 kDa. According to quantitative analysis, the isoforms that were differentiated corresponded to an AChE with the capacity to cleave acetyl-β-methyl thiocholine iodide (the so-called true AChE, E.C. 3.1.1.7, corresponding to the 265 kDa band) and to a ChE with the capacity to cleave propionyl thiocholine iodide as well. The latter is an ancestral form of AChE, mainly present in invertebrates. Each of these forms was affected differently by varying time exposures.

Alternative acetylcholinesterase molecular forms exhibit similar ability to induce neurite outgrowth.

Several groups have reported that acetylcholinesterase (AChE), through a mechanism not involving its catalytic activity, may have a role in fiber elongation. These observations were performed on experimental systems in which acetylcholine synthesis was active. Because neurite outgrowth can be modulated by neurotransmitters, we used the N18TG2 neuroblastoma line, which is defective for neurotransmitter production, to evaluate whether AChE may modulate neurite sprouting in nonenzymatic ways. To avoid the possibility that differences between transfected and mock-transfected clones may be due to the selection procedure, N18TG2 cells were previously subcloned, and the FB5 subclone was used for transfections. We performed transfections of FB5 cells with three distinct constructs encoding for the glycosylphosphoinositol-anchored AChE form, the tetrameric AChE form, and a soluble monomeric AChE form truncated in its C-terminus. A morphometric analysis of retinoic acid-differentiated clones was also undertaken. The results revealed that higher AChE expression following transfection brings about a greater ability of the clones to grow fibers with respect to nontransfected or mock-transfected cells irrespective of the used construct. Having observed no differences between the morphology of the transfected clones, we tested the possibility that the culture substrate can affect the capability of the clones to extend fibers. Also in this case we revealed no differences between the clones cultured on uncoated or collagen-pretreated dishes. These data indicate that alternative AChE molecular forms that differ in their C-teminal region exhibit similar ability to induce fiber outgrowth and suggest that the protein region responsible for this role is located in the invariant portion of the AChE molecule.

Effects of huperzine A on acetylcholinesterase isoforms in vitro: comparison with tacrine, donepezil, rivastigmine and physostigmine

Five inhibitors of acetylcholinesterase, huperzine A, donepezil, tacrine, rivastigmine and physostigmine, were compared with regard to their effects on different molecular forms of acetylcholinesterase in cerebral cortex, hippocampus, and striatum from the rat brain. In general, huperzine A preferentially inhibited tetrameric acetylcholinesterase (G4 form), while tacrine and rivastigmine preferentially inhibited monomeric acetylcholinesterase (G1 form). Donepezil showed pronounced selectivity for G1 acetylcholinesterase in striatum and hippocampus, but not in cortex. Physostigmine showed no form-selectivity in any brain region. In cortex, the most potent inhibitors of G4 acetylcholinesterase were huperzine A ( K i 7×10 −9 M) and donepezil ( K i 4×10 −9 M). The potent inhibitors of cortical G1 acetylcholinesterase were donepezil ( K i 3.5×10 −9 M) and tacrine ( K i 2.3×10 −8 M). In hippocampus, huperzine A and physostigmine were the most potent inhibitors of G4 acetylcholinesterase, while donepezil and tacrine were most potent against G1 acetylcholinesterase. In striatum, huperzine A and donepezil were the most potent against G4 acetylcholinesterase, while again donepezil was the most potent against G1. Although the inhibition constants ( K i) of these acetylcholinesterase inhibitors differed significantly from region to region, the nature of the inhibition did not vary. These results suggest that the use of acetylcholinesterase inhibitors in treatment of Alzheimer's disease must consider both form-specific and region-specific characteristics of acetylcholinesterase inhibition.

Different expressions of organophosphate‐resistant acetylcholinesterases in the bivalve mollusk Scapharca inaequivalvis living in three different habitats

The benthic mollusk Scapharca inaequivalvis was collected in spring 1999 from three areas of the northern Adriatic Sea. From the mollusk, molecular forms of acetylcholinesterase (AChE), consisting of two prevailing spontaneously soluble (SS) forms present in the blood, were obtained. These forms are a globular tetramer (SSG4), a dimer (SSG2) of catalytic subunits, and a minor amphiphilic globular dimer (low-salt Triton [LST] G2) phosphatidylinositol tailed. All SS and LST forms, partially purified by affinity chromatography, are AChEs with a marked substrate specificity for acetylthiocholine and poor hydrolysis with butyrylthiocholine. They are poorly inhibited by carbamate eserine and show a different pattern of inhibition by organophosphate diisopropylfluorophosphate (DFP), with totally resistant SS forms from clams collected from the two stations nearest the Po River. Acetylcholinesterase SS and LST forms are expressed at highest, lowest, and middle levels in clams collected from the northern station, closer to the Po delta, and from the two more southern ones, respectively. The possibility that the expression pattern of AChE forms is due to effects of single or mixed classes of chemical pollutants is discussed.

Effects of chronic metrifonate treatment on cholinergic enzymes and the blood–brain barrier

After an acute (4 h) treatment with an irreversible cholinesterase inhibitor organophosphate, metrifonate (100 mg/kg i.p.), the activities of both acetyl- and butyrylcholinesterase were inhibited (66.0–70.7% of the control level) in the rat brain cortex and hippocampus. There were no significant changes in the acetyl- and butyrylcholinesterase activities in the olfactory bulb, or in the choline acetyltransferase activity in all three brain areas. After chronic (2 or 5 week) metrifonate treatment (100 mg/kg daily i.p.), the activities of both cholinesterases were substantially inhibited in the rat brain cortex and hippocampus (15.8–31.8% of the control levels), but there was no inhibition of the choline acetyltransferase activity. Moreover, chronic metrifonate treatment did not have any effect on the distribution of the acetylcholinesterase molecular forms. In vitro, metrifonate proved to be a more potent inhibitor of butyryl- than of acetylcholinesterase in both the cortex and the hippocampus. In the hippocampus, the butyrylcholinesterase activity was twice as sensitive to metrifonate inhibition as that in the cortex (IC 50 values 0.22 and 0.46 μM, respectively). The effects of chronic (5 week) metrifonate treatment on the blood–brain barrier of the adult rat were examined. The damage to the blood–brain barrier was judged by the extravasation of Evans’ blue dye in three brain regions: the cerebral cortex, the hippocampus, and the striatum. No extravasation of Evans’ blue dye was found in the brain by fluorometric quantitation. These data indicate that chronic metrifonate treatment may increase the extracellular acetylcholine level via cholinesterase inhibition, but it does not have any effects on the blood–brain barrier. Therefore, it appears reasonable to hypothesize that cholinesterase activities do not play a role in the blood–brain barrier permeability.

Acetyl- and butyrylcholinesterase molecular forms in normal and streptozotocin-diabetic rat retinal pigment epithelium

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinal pigment epithelium (RPE). Tissues were sequentially extracted with saline (S 1) and saline-detergent buffers (S 2). About a 50% decrease in AChE molecular forms was observed in the diabetic RPE compared to the controls. Approximately 70% of the BChE activity in normal RPE was brought into solution and evenly distributed in S 1 and S 2. Analysis of the fractions from RPE revealed the presence of G A 1, G A 4 and a small proportion of G H 4 BChE forms in S 1; whereas G A 4 and G A 1 molecules predominate in S 2. A 40% decrease in the activity of G A 4 in S 2 was observed in the diabetic RPE. Our results show that diabetes caused a remarkable decrease in the activity of cholinesterases molecular forms in the RPE. This might be related to the alterations observed in diabetic retinopathy.

Streptozotocin-induced diabetes alters the oligomerization pattern of acetylcholinesterase in rat skeletal muscle.

Diabetes mellitus has a serious effect on most of the properties of skeletal muscles. Changes in neuromuscular transmission are also involved in propagating the disease. In our experiments, acetylcholinesterase was extracted from the fast extensor digitorum longus and slow soleus muscles of control, non-treated 6-week-diabetic and insulin-treated diabetic rats. The extracts were applied to velocity sedimentation and acetylcholinesterase activity was determined. We observed considerable differences in the distribution of individual acetylcholinesterase molecular forms in diabetic fast muscles. This included a 59% decline in G4 content together with a fivefold increase in A8 and a 53 % increase in A12 activity resulting in a shift of acetylcholinesterase profile characteristically towards slow muscles. These alterations were partly reversed by insulin treatment. In slow muscles diabetes caused an increase in G4 activity without affecting the sedimentation profile. Decline in G4 content in fast muscles could contribute to enhanced desensitization of acetylcholine receptors in diabetes.