Total Acetylcholinesterase Research Articles

Characterization of acetylcholinesterase from human heart auricles: Evidence for the presence of a G-form sensitive to phosphatidylinositol-specific phospholipase c

1. 1. Acetylcholinesterase (AChE) is an important enzyme of the cholinergic system in mammals. 2. 2. We report here the subcellular association of the AChE molecular forms in the normal human heart auricle. 3. 3. Both globular (G) and asymmetric (A) forms were identified using velocity sedimentation and sequential extraction procedures. 4. 4. G forms corresponds to 84% and A forms account for 16% of the total AChE activity. 5. 5. Of G forms 64% of AChE activity correspond to the G 1 monomer and of the A forms the class I-A account for 80% of AChE activity. 6. 6. In addition, treatment of the cardiac membranes with the enzyme phosphatidylinositol-specific phospholipase c (PIPLC) results in the solubilization of AChE activity. 7. 7. This means that a G 2 AChE dimer with a glycolipid anchoring domain is present in the human heart.

Acetylcholinesterase activity in regions of mouse brain following acute and chronic treatment with a benzodiazepine inverse agonist

1. Chronic administration of the benzodiazepine inverse agonist FG 7142 has previously been shown to induce seizure activity in mice. In the present study we have investigated the effects of acute and chronic treatment with FG 7142 in mice on the levels of acetylcholinesterase activity in cortex, hippocampus, midbrain and striatum. We have also investigated the effects of acute and chronic stress in the form of handling (vehicle-injection) on acetylcholinesterase levels. 2. A single dose of FG 7142 produced a marked elevation of total acetylcholinesterase activities in the hippocampus and midbrain when compared with vehicle-injected control levels, but the levels were not different from those in unhandled animals. 3. Acute stress, in the form of vehicle-injection produced decreases in cortical and hippocampal soluble acetylcholinesterase activity but FG 7142 had no effect upon these stress-induced changes. 4. Total cortical and hippocampal acetylcholinesterase activities were increased by 56% and 16% respectively in the chronic FG 7142-treated mice that exhibited seizure activity (compared with vehicle-injected controls). 5. Soluble acetylcholinesterase activity in the midbrain was decreased to 82% of control levels only in animals that had undergone FG 7142-induced kindling. Smaller or no changes in acetylcholinesterase activity in the midbrain were observed in chronically FG 7142-treated animals that exhibited no seizure activity. 6. Mice that did not demonstrate seizure activity in response to chronic FG 7142 treatment showed alterations in the soluble acetylcholinesterase activities of the hippocampus and midbrain. 7. It is concluded that chronic treatment with the benzodiazepine inverse agonist FG 7142 produces alterations in the acetylcholinesterase activities of various brain regions, in a manner related to the kindling that can be produced by this treatment. 8. Chronic mild stress, in the form of repeated handling (vehicle injection), induced changes in brain activity with decreases in total activity occurring in the cortex and hippocampus, and an increase in soluble acetylcholinesterase activity occurring in the midbrain. 9. All these stress-induced changes appeared to be prevented by administration of FG 7142 at the time of the stress. It would appear therefore that FG 7142 can prevent the effects of chronic stress on brain acetylcholinesterase activity.

Changes in membrane-bound and soluble molecular forms of acetylcholinesterase in mouse hippocampus after cholinergic denervation

In order to observe acetylcholinesterase (AChE) and its distribution into soluble and membrane-bound molecular forms in cholinergically denervated hippocampus, AChE was analyzed in the hippocampus of adult mice with and without bilateral fornix lesions made by stereotactically positioned knife cuts. Homogenates from lesioned mice contained an average of 3% of choline acetyltransferase-specific activity, 12% of AChE-specific activity and 98% of protein compared to homogenates from controls. After lesioning, the relative proportion of membrane-bound G 4 decreased from 77% to 56% of total AChE while soluble G 4, membrane-bound G 1–G 2, and soluble G 1–G 2 each increased in relative abundance.

Differential effects of denervation on acetylcholinesterase activity in parasympathetic and sympathetic ganglia of the frog, Rana pipiens

The transsynaptic regulation of acetylcholinesterase (AChE) was studied by recording the changes in enzymatic activity following denervation in two types of autonomic ganglia in the frog, Rana pipiens. Opposite effects on AChE were found in the parasympathetic cardiac ganglion and in the sympathetic lumbar ganglion; denervation produced a significant increase in AChE activity in cardiac ganglia but a significant decrease in lumbar ganglia. The relative effects of denervation on intracellular and total AChE were examined by selectively inhibiting extracellular AChE with echothiophate, a poorly lipid-soluble cholinesterase inhibitor. Denervation resulted in a significant increase in intracellular AChE in cholinergic cardiac ganglia but had no effect on intracellular AChE activity in adrenergic lumbar ganglia. Histochemical studies revealed little change in extracellular AChE staining upon denervation in the cardiac ganglion, whereas in the lumbar ganglia there was a loss of AChE-specific reaction product. These results raise the possibility that the transsynaptic control of AChE activity by innervation in the frog is influenced by the transmitter synthetic properties of the postsynaptic ganglion cells.

Sympathectomy alters acetylcholinesterase expression in adult rat heart

The aim of the study was to determine the significance of adrenergic nerve associated acetylcholinesterase for the pool of total acetylcholinesterase molecules. 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. Adult male Sprague-Dawley rats were used, weight 225-260 g, n = 5 per experimental group. 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. The contribution of adrenergic neurones to the cardiac pool of acetylcholinesterase is measurable and consists primarily of the tetrameric globular form of the enzyme.

Interaction of an organophosphate with a peripheral site on acetylcholinesterase

O-Ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (MPT) is an active site directed inhibitor of acetylcholinesterase (AChE). Inhibition of the Electrophorus electricus (G4) enzyme follows classical second-order kinetics. However, inhibition of total mouse skeletal muscle AChE and inhibition of the individual molecular forms from muscle, including the monomeric species, do not proceed as simple irreversible bimolecular reactions. Similarly, complex inhibition kinetics are observed for the purified enzyme from Torpedo californica. AChE can be cross-linked with glutaraldehyde into a semisolid matrix. Under these conditions the abnormal concentration dependence for MPT inhibition is accentuated, and a range of MPT concentrations can be found where inhibition of polymerized AChE is far less than that observed at lower concentrations. Inhibition in certain concentration ranges is partially reversible after removal of all unbound ligand. Thus, there are two different modes of organophosphorus inhibition by MPT: the classical irreversible phosphorylation of the active site and a reversible interaction at a site peripheral to the active center. Propidium, a well-studied peripheral site ligand, can prevent the later interaction. Hence, the second site of MPT interaction with AChE may overlap or be linked to the peripheral anionic site of AChE characterized by the binding of propidium and other peripheral site inhibitors.

Cardiac Acetylcholinesterase Molecular Forms and Choline Acetyltransferase in the Dahl (Salt-Sensitive) Hypertensive Strain of Rats: A Regional Study

A regional study of acetylcholinesterase (AChE) molecular forms and choline acetyltransferase (ChAT) in the hearts of Dahl-salt sensitive (DS) and salt resistant (DR) rats was performed in animals administered either 8% or 0.35% dietary NaCl. Atria isolated from DS rats, regardless of dietary NaCl intake, had lower activities of all of the AChE molecular forms and ChAT when compared to their dietary-matched DR controls. In the ventricles, the activities of AChE molecular forms and ChAT were lower in DS rats compared to dietary-matched DR rats only when 8% NaCl diets were administered. The percent contribution of each of the molecular forms to the total AChE pool was not affected by animal strain or diet.

Molecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells

Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G2a form. In Triton X-100 extracts of the subline K562-243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G2a AChE into a hydrophilic dimer (G2h), indicating that the G2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562-243 cells with PtdIns-PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562-48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562-48 AChE resembles the G2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503-508]. In both K562 sublines, sialidase decreased the migration of the G2a form but not of the G1a form of AChE. G1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. The sialidase-resistant G1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented.

Carrageenans solubilize asymmetric acetylcholinesterase from nicotinic cholinergic synapses

1. Acetylcholinesterase (AChE) catalyzes the hydrolysis of acetylcholine at cholinergic synapses in both vertebrate and invertebrates organisms. 2. The asymmetric synaptic AChE is attached to the extracellular matrix (ECM) of the neuromuscular junction through heparin sulphate proteoglycans (HSPGs). 3. It has been shown previously that heparin-like glycosaminoglycans (GAGs) can solubilize this enzyme from the cholinergic synapses. 4. The present paper describes the solubilization of asymmetric AChE by different marine macroalgal polysaccharides, called carrageenans. 5. Important differences were found among all the carrageenans tested; they released 15–50% of the total AChE activity normally solubilized by heparin. 6. Carrageenans extracted from tetrasporic stages of Iridaea ciliata and I. membranacea were always better extracting agents than those from the cystocarpic stages of these algae, suggesting that lambda-like carrageenans are involved. 7. This hypothesis was confirmed by extracting AChE with purified carrageenans.

Evidence for the presence of nematode-derived acetylcholinesterase in sheep infected with Trichostrongylus colubriformis

The distribution of acetylcholinesterase isoenzymes in the ovine intestinal nematode Trichostrongylus colubriformis was compared with that in chronically infected and worm-free lambs. Total acetylcholinesterase activity in homogenates of adult T colubriformis was resolved into five isoenzyme peaks following gel electrophoresis and specific esterase staining. Two isoenzymes with electrophoretic mobilities similar to those present in adult worm homogenates were detected in mucosal homogenates and plasma extracts from all of six sheep chronically infected with T colubriformis, but not in similar preparations from two uninfected animals.

Changes in the expression of acetylcholinesterase molecular forms during rat heart development

The development of the molecular forms of acetylcholinesterase was studied in rat heart during the perinatal period. In this study, the activity of acetylcholinesterase increased both per unit wet weight and per unit protein from post-conception day 14 to day 42. Additionally, the activity of atrial acetylcholinesterase per unit wet weight increased more rapidly after birth than that of ventricular acetylcholinesterase. The percent contribution of the various molecular forms to the total acetylcholinesterase pool in heart changed dramatically from the fetal to the neonatal period. This switch primarily consisted of a decrease in the ratio of the asymmetric to globular forms. Thus, the specific activity of globular forms increased while that of the asymmetric forms remained relatively stable. When the atria and ventricles were examined separately at 19 days post-conception, the percent contribution of the individual molecular forms in the two cardiac areas was different. The atria contained a pool of acetylcholinesterase forms similar to postnatal heart while the ventricles contained a pool of acetylcholinesterase forms with a lower globular to asymmetric ratio. Finally, this study showed that greater than half of the acetylcholinesterase pool was inhibited by incubating hearts from fetal rats with echothiopate iodide, suggesting that a large portion of acetylcholinesterase catalytic sites are externalized in fetal heart.

Serum cholinesterase activity in infantile and juvenile spinal muscular atrophy.

Serum acetylcholinesterase (AChE) and pseudocholinesterase (ChE) activity in infantile and juvenile spinal muscular atrophy (SMA) was determined. The total AChE activity was either normal or decreased in the childhood SMA (Type 1), the other SMA groups and disease controls (ALS, X-linked SMA). In the majority of SMA Type 1 cases (6/7 tested) an absence of the asymmetric A12 form was found. This was accompanied by changes in the other asymmetric and globular forms. The latter was, however, not specific for SMA Type 1 cases. The ChE activity was increased in the majority of SMA cases as well as disease controls. The asymmetric A12 ChE form was increased in all SMA Type 3 cases, the values of this form in SMA Type 1 was variable. A change in the ChE globular forms in SMA Type 1 and SMA Type 2 was a frequent finding. It is suggested that the absence of the asymmetric A12 AChE form in SMA Type 1 arises because of muscle cell immaturity and undeveloped muscle-nerve interactions. The reason of ChE changes is obscure.

Axonal transport in mdx mouse sciatic nerve

Anterograde and retrograde flows of acetylcholinesterase (AChE) in sciatic nerves of adult mdx mice were compared with those of normal mice. Specific molecular forms of AChE were resolved by high-performance liquid chromatography such that slow anterograde (G 1 + G 2), fast anterograde and fast retrograde (G 4 and A 12) flows could be simultaneously studied. Although we found no difference in the total AChE activity and the molecular forms in non-ligated nerves between mdx and the normal mice, ligated nerves showed significant differences. The total AChE activity accumulated at the proximal segment of ligated nerve was higher in mdx mice than in normal mice after 24 h ligation. The G 1 + G 2 molecular forms were accumulated more in the proximal segment of mdx than the normal. A 12, on the other hand, was more abundant in both segments of mdx mice than the normal. No statistically significant difference in the accumulated amount of G 4 molecular form was present between mdx and the normal mice at either proximal or distal segment. These results indicated that axonal flow in sciatic nerve likely plays a role in muscle regeneration, and that the transport machinery in dystrophin-deficient mdx neuron is probably normal.

Regional distribution of the molecular forms of acetylcholinesterase in adult rat heart.

Acetylcholinesterase (AChE), the enzyme that degrades acetylcholine, exists as a multiple molecular forms that differ in their quaternary structure and mode of attachment to the cell surface. The distribution of the individual molecular forms of AChE in various cardiac regions with distinct anatomical characteristics was investigated. The results confirmed those of others by showing that the total pool of cardiac AChE had a nonuniform distribution in heart that paralleled the distribution of choline acetyltransferase. The rank order of this distribution was right atrial appendage greater than interatrial septum greater than left atrial appendage = right ventricle = interventricular septum greater than left ventricle. Velocity sedimentation in sucrose gradients of extracts from selected cardiac areas showed that four molecular forms were present in all areas but that the proportions of these forms differed as a function of area. The right and left ventricular walls, the apical portion of the interventricular septum, and the left atrial appendage contained G1 and G4 (globular) AChE in near-equal proportions, but in the basal portion of interventricular septum, the contribution of G4 AChE was greater than that of G1 AChE. The right atrial appendage and the interatrial septum had the largest amount of activity attributable to G4 AChE and the lowest amount attributable to G1 AChE. In all cardiac regions, A12 (asymmetric) AChE comprised 8-10% of the total AChE pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Vesiculation induced by amphiphiles in erythrocytes

The ability of shape-transforming cationic, anionic, zwitterionic, and nonionic amphiphiles to induce vesiculation in human erythrocytes was studied. At concentrations where they exhibit maximum protection against hypotonic haemolysis (CAHmax) echinocytogenic amphiphiles induced a rapid release of exovesicles. Following 5 min of incubation, the vesicle release (acetylcholinesterase release) amounted from 4% (sodium alkyl sulphates) to 13% (zwittergents) of the total acetylcholinesterase activity of the erythrocytes. At concentrations corresponding to CAH50 the vesicle release was less than 15% of that released at CAHmax. The size and the appearance of the vesicles varied with the type of amphiphile. Stomatocytogenic amphiphiles which do not pass the erythrocytes through echinocytic stages, did not induce release of exovesicles. Electron and fluorescence microscopic observations of erythrocytes treated with stomatocytogenic amphiphiles strongly indicated that an endovesiculation had occurred. Amphiphiles which pass the erythrocytes through echinocytic stages before stomatocytic shapes are attained, induced a release of both exo- and endovesicles.

Acetylcholine receptor density and acetylcholinesterase activity in skeletal muscle of rats following thermal injury.

Thermal injury causes systemic changes that result in altered sensitivity to many drugs including nondepolarizing muscle relaxants. In an effort to identify the mechanism(s) responsible for the resistance to nondepolarizing muscle relaxants, the density of acetylcholine receptors (AChR) and the activity of acetylcholinesterase (AChE) were determined in rats following a 30% total body surface thermal injury at a time when resistance to atracurium is maximum. AChR density in gastrocnemius and diaphragm was unchanged by thermal injury. Furthermore, the ratio of junctional to extrajunctional AChR in diaphragm was unaltered. Total AChE activity was unchanged in thermally injured rats compared with that in sham-injured animals. Separation of the molecular forms of AChE by sucrose gradient centrifugation also showed no changes in the relative proportions of these species. The mechanism of resistance to nondepolarizing muscle relaxants does not appear to be explained by changes in AChR number or changes in the activity of AChE.

Reduced Axonal Transport of the G4 Molecular Form of Acetylcholinesterase in the Rat Sciatic Nerve During Aging

Aging in the sciatic nerve of the rat is characterized by various alterations, mainly cytoskeletal impairment, the presence of residual bodies and glycogen deposits, and axonal dystrophies. These alterations could form a mechanical blockade in the axoplasm and disturb the axoplasmic transports. However, morphometric studies on the fiber distribution indicate that the increase of the axoplasmic compartment during aging could obviate this mechanical blockade. Analysis of the axoplasmic transport, using acetylcholinesterase (AChE) molecular forms as markers, demonstrates a reduction in the total AChE flow rate, which is entirely accounted for by a significant bidirectional 40-60% decrease in the rapid axonal transport of the G4 molecular form. However, the slow axoplasmic flow of G1 + G2 forms, as well as the rapid transport of the A12 form of AChE, remain unchanged. Our results support the hypothesis that the alterations observed in aged nerves might be related either to the impairment in the rapid transport of specific factor(s) or to modified exchanges between rapidly transported and stationary material along the nerves, rather than to a general defect in the axonal transport mechanisms themselves.

Interaction of 9-amino-1,2,3,4-tetrahydroaminoacridine (THA) with human cortical nicotinic and muscarinic receptor binding in vitro

Tetrahydroaminoacridine (THA) has recently been reported to be more useful in the treatment of Alzheimer's disease than physostigmine. A comparison of the effects of these two anticholinesterase agents on in vitro enzyme and receptor activities of human cerebral cortex (obtained at autopsy) revealed similarities in their interactions with acetylcholinesterase (AChE) but striking differences in their ability to displace both nicotinic and muscarinic radioligands from membrane preparations. IC 50 values (the concentration required to reduce enzyme activity by 50%) for the inhibition of total tissue AChE were 7.9 × 10 −7 M and 4.5 × 10 −8 M for THA and physostigmine, respectively, and similar values were also obtained for individual molecular forms of AChE (monomer G 1, dimer G 2 and tetramer G 4) separated by sucrose density gradient centrifugation. In contrast, IC 50 values for [ 3H]nicotine displacement (a measure of nicotinic cholinergic receptor binding) differed 1000-fold for THA (2 × 10 −5 M) and physostigmine (2 × 10 −2 M) and 100-fold for [ 3 H]N- methylscopolamine displacement (a measure of muscarinic cholinergic receptor binding). Differences were also noted in the inhibition of carbachol stimulated polyphosphoinositide (PI) hydrolysis (a measure of muscarinic receptor induced second messenger activity) in isolated rat cortical miniprisms. It is suggested that variations in clinical efficacy of THA and physostigmine may be related less to their anticholinesterase properties and more to their interactions with other activities such as cholinergic receptors.

Effects of glycyl-L-glutamine in vitro on the molecular forms of acetylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.

Normal and preganglionically denervated cat superior cervical ganglia were sectioned and cultured for 24 or 48 hr, with or without preliminary inactivation of acetylcholinesterase, and in the presence or absence of 10(-5) M glycyl-L-glutamine. They were then homogenized, and the molecular forms of acetylcholinesterase were analyzed by sucrose gradient sedimentation. We observed an increased proportion of the globular monomeric G1 form, and to a lesser extent of the dimeric G2 and tetrameric membranous G4 forms, of acetylcholinesterase in the glycyl-L-glutamine-treated compared with the control cultures. There was only a small increase in the total acetylcholinesterase activity and no significant variation in the activity of the metabolic enzyme lactate dehydrogenase. It therefore seems likely that glycyl-L-glutamine, or the endogenous neurotrophic factor, maintains acetylcholinesterase in the preganglionically denervated ganglia in vivo by specifically increasing the biosynthesis of the monomeric G1 form, but not that of other proteins; these trophic factors do not seem to promote the polymerization of G1 into the more complex G2 and G4 forms.

Regulation of neurotransmitter metabolic enzymes and tyrosine hydroxylase mRNA level by nerve growth factor in cultured sympathetic neurones.

The survival of new-born rat sympathetic neurones in culture was increased in a dose-dependent manner by 7S nerve growth factor (NGF). NGF also increased, in a parallel manner, the specific activities of tyrosine hydroxylase (TOH) and choline acetyltransferase (CAT). Total acetylcholinesterase (AcChE) activity increased with NGF concentration, although less distinctly than TOH and CAT. However, NGF caused a large induction of the asymmetric A12 form of AcChE, and to a lesser extent of the globular G1 and G2 forms, whereas the globular G4 form was little affected. This suggests that NGF differentially regulates the synthesis and/or assembly of the various AcChE molecular forms. The levels of TOH mRNA in neurone cultures grown with increasing NGF concentrations were measured by Northern blot analysis with a rat cDNA probe. To correct for variations in the total mass of RNA per neurone, the filters were rehybridized with an 18S rRNA probe. The level of TOH mRNA, measured by the ratio (TOH:18S) of the hybridization signals increased 3.4-fold between 92 and 740 ng ml-1 7S NGF. Increases of TOH specific activity of the same order of magnitude were observed in sister cultures. The deficit in the level of mature TOH mRNA at low NGF concentration was not accompanied by a compensatory accumulation in unprocessed TOH transcripts. As TOH induction is insensitive to RNA polymerase inhibitors, we suggest that NGF regulates the maturation of TOH pre-mRNAs, and that the unprocessed transcripts are rapidly degraded. The long-term regulation of TOH by NGF may thus constitute a case of process-versus-discard control, as defined by J.E. Darnell.