Specific Activity Of Acetylcholinesterase Research Articles

Regulation of Expression of Cholinergic Neuronal Phenotypic Markers in Neuroblastoma LA‐N‐2

Cholinergic neurons in PNS and CNS are identified by the presence of choline acetyltransferase and the accumulation of choline by a high-affinity, sodium-coupled choline transporter to be used for acetylcholine synthesis. It appears that expression of choline acetyltransferase can be altered by several physiological conditions, including hormones and trophic factors, but little is known about control of expression of the sodium-coupled choline carrier or whether these two phenotypic markers are regulated similarly. In the present study, the cholinergic human neuroblastoma LA-N-2 was used to investigate regulation of expression of choline acetyltransferase and choline uptake activity associated with differentiation and neurite extension. Cells grown in serum-containing basal medium maintained a relatively undifferentiated morphology, expressed low levels of choline acetyltransferase activity, and accumulated choline by a sodium-dependent process followed by conversion to acetylcholine. Transfer of cells to an enriched, serum-free defined medium resulted in morphological and neurochemical differentiation, with an enhancement of cholinergic phenotype. Hemicholinium-sensitive choline uptake activity was increased about sixfold over a 4-day period, with no change in choline acetyltransferase or acetylcholinesterase specific activity. Acetylcholine synthesis was increased in parallel with the changes in choline accumulation; choline metabolism in the differentiated cells differed significantly from that observed in the undifferentiated cells, with proportionally less converted to phosphorylcholine and proportionally more remaining as unmetabolized choline and converted to acetylcholine. The enhanced choline accumulation appeared to be mediated by an increased number of choline carriers, demonstrated by increased binding of the affinity ligand [3H]-choline mustard to the transporter and by an increased Vmax for the uptake process. The increased expression of the transport function appeared to be under transcriptional control, as the enhancement of uptake was blocked by the RNA polymerase II inhibitor alpha-amanitin as well as by the protein synthesis inhibitor cycloheximide. These results show that expression of sodium-coupled choline carriers and choline acetyltransferase may be regulated separately in the differentiating neuroblastoma LA-N-2 and that neurotransmitter synthesis is controlled by provision of precursor rather than at the level of the biosynthetic enzyme.

Parallel development of acetylcholinesterase in vivo and in primary neuron surface culture

The development of acetylcholinesterase (AChE) activity in primary surface cultures of mouse cortical neurons and in mouse brain was examined. The specific activity of AChE in culture increased over 600% during a 3 week period and closely paralleled the development of AChE observed in vivo. The results obtained in this study show that a developmental increase in AChE can be obtained in primary surface neuron cultures, and that the high degree of cellular organization previously deemed necessary for this development in vitro is not as important as previously thought.

Regeneration of acetylcholinesterase in clonal neuroblastoma-glioma hybrid NG108-15 cells after soman inhibition: effect of glycyl-l-glutamine

Acetylcholinesterase (AChE) in the clonal NG108-15 cell line has been previously characterized. This cell line represents an in vitro system to study AChE regulation and effects of chemical compounds that may alter AChE activity. Recently, glycyl-L-glutamine (GLG) was demonstrated to function as a neurotrophic factor for maintenance of AChE content in cat denervated superior cervical ganglion cells. In the present study, regeneration of AChE activity in cultures of undifferentiated NG108-15 cells after soman inhibition was investigated in the presence and absence of GLG. Cells were treated with soman (5.5 x 10(-6) M) for 15 min and then washed to remove excess soman. Culture medium containing either GLG (10(-6), 10(-5), or 10(-4) M) or glycyl-L-glutamic acid (10(-6) M) was added to cultures after soman treatment and remained in the medium until cell harvest. Cells were physically detached at various times after soman treatment and specific AChE activity was determined. After soman, AChE activity dramatically decreased to less than 1% of untreated cellular activity at 1 hr. AChE activity gradually increased after 5 hr, while untreated cell AChE activity was regained 20 hr after soman. The t1/2 for AChE regeneration was approximately 10 hr. GLG did not increase the rate of AChE regeneration after soman inhibition. These results indicate that GLG is not a directly acting neurotrophic factor for AChE synthesis in NG108-15 cells after chemical AChE inactivation.

Cholinergic differentiation of clonal rat pheochromocytoma cells (PC12) induced by retinoic acid: increase of choline acetyltransferase activity and decrease of tyrosine hydroxylase activity

The effects of retinoic acid (RA), a naturally occurring metabolite of vitamin A, on the growth, morphology and neurochemical differentiation of the PC12 clone of rat pheochromocytoma cells were investigated. RA added to the medium inhibited the growth of PC12 cells in a dose-dependent manner up to 10 μM without affecting their morphology. In PC12 cells cultured in the presence of 10 μM RA for 8 days, the specific activity of choline acetyltransferase (ChAT) was increased2-fold, while the specific activity of tyrosine hydroxylase (TH) was decreased 0.5-fold compared with cells cultured in the absence of RA. Specific activities of acetylcholinesterase (AChE), glutamate decar☐ylase (GAD) and lactate dehydrogenase (LDH) were not affected by RA. Both the increase of ChAT and the decrease of TH induced by RA exhibited similar time and dose dependencies. RA inhibited the increase of TH activity induced by nerve growth factor (NGF), an adrenergic neuronotrophic factor on PC12 cells. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of PC12 cells independent of a morphological differentiation.

Recovery of benthiocarb-inhibited AChE in fish brain: an in vitro study

The effects in vivo and in vitro of benthiocarb, an organocarbamate, on acetylcholinesterase (AChE) activity and its recovery in vitro in the brain of the freshwater fish Sarotherodon mossambicus have been studied. Findings revealed that the specific activity of AChE was inhibited both in vivo and in vitro in a concentration-dependent manner. Of the different modulators tested, dexamethasone (0.72 mg), prednisolone (0.74 mg), testosterone (0.9 mg), and ADP (9.5 mg) were found to neutralize the inhibitory effect (IC 50 = 0.30 μ M) of benthiocarb on AChE.

Acetylcholinesterase in the development of chick dorsal root ganglia

Acetylcholinesterase is expressed in chick dorsal root ganglia neurons very early in development. Since the physiological role of the enzyme in these cells is still obscure, it appeared of interest to investigate its modifications in the course of development.The specific activity of acetylcholinesterase in chick dorsal root ganglia increases, during in ovo development, from day E5 to day E13; after day E13 there is a decrease. Conversely, when acetylcholinesterase activity was expressed on a per ganglion basis, a continuous increase in the level of the enzyme until day E20 was observed.Acetylcholinesterase is a polymorphic enzyme and its molecular forms have different cellular localizations. Two globular forms, a tetramer (G4) and a dimer (G2), are present in the ganglia, as in chick brain. G4 is the major form at day E5, where it represents about 85% of the activity. This form shows a progressive decrease since day E8, and at day E20 exhibits activity levels similar to those of G2.It is known that acetylcholinesterase-producing cells are also able to release the enzyme in the extracellular space. We determined the release of acetylcholinesterase by cultured dorsal root ganglia neurons at various developmental stages: acetylcholinesterase release is significantly increased at day E20, as compared to younger stages, and 90% of the enzyme released is G4.

Effect of aging and acetyl- l-carnitine on energetic and cholinergic metabolism in rat brain regions

The effect of aging and subchronic treatment with acetyl- l-carnitine (50 mg/kg per day) was studied on mitochondrial bioenergetics and cholinergic metabolism in non-synaptic mitochondria and synaptosomes isolated from cerebral cortex, hippocampus and striatum of rats aged 4, 11 and 18 months. Respiratory activity and cytochrome oxidase specific activity were unaffected by aging in non-synaptic mitochondria. In synaptosomes, pyruvate dehydrogenase, choline acetyltransferase and acetylcholinesterase specific activity remained unchanged, but the high-affinity choline uptake decreased in cerebral cortex and striatum of 18-month-old rats. Acetyl- l-carnitine treatment increased the high-affinity choline uptake in cerebral cortex of 18-month-old rats. The treatment caused also an increase in cytochrome oxidase activity in all the three cerebral regions and in choline uptake in the hippocampus, parameters that were not directly affected by aging processes.

Intra and juxtavagal paraganglia: a topographical, histochemical, and ultrastructural study in the human.

The topographical, ultrastructural, and histochemical features of 23 human vagal paraganglia were analyzed. Nineteen of the 23 paraganglia were found in previously unreported sites; 18 of the 19 were in the cervical part of the nerve, between the carotid bifurcation and the superior thoraco-cervical inlet, and one paraganglion was located in the retrothyroidal part of the left inferior laryngeal nerve. The results of ultrastructural studies (2 cases), the histochemical and formaldehyde-induced-fluorescence studies (3 cases), and specific acetylcholinesterase activity (one case) demonstrate that these structures fulfill many of the modern criteria for paraganglionic tissue. In addition to paraganglia, single, isolated neurons or true micro-ganglia were always found along the trunk and branches of the vagus nerve when multiple sections were examined.

Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS.

Myelinated axons were isolated by flotation from bovine pons, middle cerebellar peduncle, cervical spinal cord and three regions of the subcortical white matter. The myelinated axons were osmotically and mechanically shocked, followed by fractionation on a linear 15% sucrose to 45% sucrose density gradient. Axolemma-enriched fractions (AEF) found in the 28% to 32% sucrose region of the gradient from brainstem and cord white matter had high acetylcholinesterase (AChE) while little or nil AChE activity was found in corresponding AEF derived from the subcortical white matter. Morphologically, the subcortical white matter from all regions contained a heterogeneous population of well-myelinated to thinly myelinated axons, while brainstem and cord regions contained a more homogeneous population of well-myelinated axons. Histochemical analysis of AChE localized this enzyme to axonal elements. The AEF derived from any white matter source had similar polypeptide compositions. AEF derived from subcortical white matter contained two-fold more myelin basic protein and a three-fold greater content of 2' 3' cyclic nucleotide 3' phosphodiesterase (CNP) compared with AEF derived from well myelinated white matter. We conclude that the purity of the AEF is related to the degree of myelination of the white matter from which the AEF is derived. Homogeneously well myelinated white matter (pons, cerebellar peduncle, cervical spinal cord) yields the highest purity AEF, as judged by the low CNP and myelin basic protein content and highest enrichment in AChE specific activity.

Neonatal enucleations reduce specific activity of acetylcholinesterase but not choline acetyltransferase in developing rat visual cortex

Infant rats were subjected to unilateral or bilateral enucleation or sham lesions on the day of birth. On postnatal day 13 or 14, tissue blocks from cortex were assayed for acetylcholinesterase (AChE) and choline acetyltransferase (ChAT). Bilateral enucleation resulted in a decrease in specific activity of AChE in occipital cortex, as compared with control animals. Unilateral enucleation resulted in a decrease of AChE activity in the hemisphere contralateral to the enucleated orbit. However, neither type of enucleation resulted in significant changes of ChAT specific activity. These data suggest that reductions in AChE activity resulting from neonatal enucleations are not due to a direct involvement of cholinergic afferents to visual cortex.

Polymorphisme de l'acétylcholinestérase et de la myosine au cours du développement des muscles rapide et lent dénervés chez le lapin nouveau-né

The rabbit Semimembranosus proprius (SMp) and Semimembranosus accessorius (SMa) muscles represent good models for studying the transformations of muscle properties during postnatal differentiation. In the adult, these muscles are homogeneous in slow twitch (SMp) and fast twitch (SMa) fibers, respectively. However, they are heterogeneous at birth and express their adult characteristics from two months onwards. During this period we studied the influence of motor innervation on the development of their properties, particularly at the level of acetylcholinesterase (AChE) molecular forms and myosin slow (LCs) and fast (LCf) light chains. The postnatal alteration of SMa and SMp muscles was characterized by the disappearance of the neonatal heterogeneity and the acquisition of the homogeneous fast or slow fiber type pattern. The fibers of these muscles denervated at birth were altered differently: dramatic atrophy of fast twitch fibers whatever the muscles studied, preservation of SMp slow twitch fiber characteristics and fatty degeneration of SMa. At birth, both muscles presented a similar pattern of myosin fast and slow LC. In control muscles, the alteration of fiber populations to homogeneous types led to the disappearance of supernumerary chains from 15 days onwards. In the slow muscle, neonatal denervation prevented LCf disappearance. In the fast muscle, denervation influenced essentially the installation of LCf which was delayed by 15 days. At birth, the polymorphism of AChE was similar in SMp and SMa muscles. One month after denervation, the specific activity of AChE was twice that of the control. Its polymorphism was not much disturbed, while in the adult denervation induced a large increase in AChE specific activity (x 10) and particularly a great alteration in its polymorphism according to the fast or slow muscle fiber types.

VIP-sensitive adenylate cyclase, guanylate cyclase, muscarinic receptors, choline acetyltransferase and acetylcholinesterase, in brain tissue afflicted by alzheimer's disease/senile dementia of the Alzheimer type

Biochemical parameters were determined in autopsy material from several brain regions of thirteen patients with Alzheimer's disease/senile dementia of Alzheimer type (AD/SDAT) (mean age 75 years) and from brains of ten age-matched controls (mean age 76 years). Choline acetyltransferase specific activity was significantly lower in the nucleus caudatus, putamen, left thalamus, hippocampus and the cortex from gyrus hippocampus and the temporal lobe in AD/SDAT, acetylcholinesterase specific activity was significantly lower in the hippocampus, parietal and left frontal lobe in AD/SDAT samples than in corresponding samples from aged-matched controls. A compensatory increase of muscarinic receptors was found in the nucleus caudatus and left frontal lobe samples in AD/SDAT. Guanylate cyclase activity was not significantly altered in AD/SDAT in the brain regions examined. The basal, non-stimulated activity of adenylate cyclase was significantly (p less than 0.05) elevated in hippocampus samples from AD/SDAT patients and the enzyme activity stimulated by the vasoactive intestinal polypeptide VIP (2 microM) or forskolin (10 microM) was also elevated in AD/SDAT although not significantly.

Chronic bombesin treatment increased the [ 3H]spiperone binding, glutamate decar☐ylase and choline acetyltransferase activity in the rat brain

The effects of chronic bombesin (BBS) on [ 3H]spiperone (SPD) binding activity, choline acetyltransferase (ChAT), acetylcholinesterase (AChE) and glutamate decar☐ylase (GAD) were investigated in the rat brain corpus striatum (CS). The chronic i.p. administration of BBS to rats increased: (1) the specific [ 3H]SPD binding to the striatal P m (plasma membrane) 16%, P < 0.03and34%, P < 0.008 at 5 μg/kg and 10 μg/kg respectively), (2) the specific GAD activity in the CS by 52% (5 μg/kg, n.s.) and 46% (10 μg/kg, P <0.05) respectively, (3) the specific ChAT activity in the CS by 54% (10 μg/kg, P < 0.002) and (4) the specific AChE activity by 23% (10 μg/kg, P < 0.02) after 14 days. It increased only: (1) the specific [ 3H]SPD binding by 29% ( P < 0.001, at 10μg/kg) and (2) the specific GAD activity by 23% ( P < 0.015, 10 μg/kg), after 7 days. Neither ChAT nor AChE activity was affected after 7 days treatment of BBS at 10 μg/kg. In vitro study showed that BBS at 0.2μM did not affect any of the neurochemical parameters examined in the CS. Thus, the changes in brain chemistry caused by chronic BBS were not due to direct effects of BBS but may be mediated through its metabolites or CCK release. Data indicate that the central effects of peripherally administered BBS are dependent on both the duration and the dosage of the drug treatment and that the dopaminergic and GABAergic systems seem to be more vulnerable to chronic BBS than the cholinergic system in the rat brain CS. Results suggest that BBS may play a role in regulating the dopaminergic, GABAergic and cholinergic systems in the brain.

Hypertrophy of pheochromocytoma cells treated with nerve growth factor and activators of adenylate cyclase.

PC12 pheochromocytoma cells treated with nerve growth factor (NGF) in combination with high concentrations of the activators of adenylate cyclase, forskolin or cholera toxin, become more neuron-like in size than cells treated with NGF or with activators of adenylate cyclase alone. Cells treated simultaneously with NGF plus forskolin or cholera toxin paradoxically show less process outgrowth than cells treated with NGF alone. Addition of forskolin or cholera toxin to cells pretreated with NGF, however, produces enlarged cells with intact processes that are indistinguishable from cultured neurons. One possible implication of these findings is that NGF might act in concert with agents that increase intracellular cyclic AMP to cause neuronal maturation during embryogenesis, and that the proper sequence of exposure to these signals is necessary for normal development. Specific activity of acetylcholinesterase is increased by NGF but is unaffected or slightly decreased by forskolin, suggesting that individual aspects of the developing neuronal phenotype are subject to different types of control.

Acetylcholinesterase is regulated by action potential generation and not by muscle contractile activity per se in mouse muscle in vitro

Dysgenic ( mdg/mdg) skeletal muscle of the mouse, grown in primary cell culture, fires action potentials in the absence of contractions, thus allowing analysis of the role of electrical activity (without contraction) on the specific activity and molecular forms of acetylcholinesterase (AChE). Specific activity of AChE was assessed by the spectrophometric method of Ellman (Biochem. Pharmacol., 7 (1961) 88–95) and found to increase by 2–5 times in the active myotubes (contraction and action potentials in normal and action potentials, alone in dysgenic muscle) compared to quiescent muscle. Sucrose density sedimentation analysis of muscle homogenates revealed an increase, by 2–3 times, in the proportion of the asymmetric (16S) molecular form of AChE in active muscle of both genotypes. Thus, electrical membrane activity, and not contraction per se, is directly involved in the regulation of levels of specific activity of and accumulation of the asymmetric (16S) form of AChE in muscle cells in culture.

Autonomic innervation of interstitial cells in the nasal respiratory mucosa.

We analyzed the microscopic innervations of the pars respiratoria of the nasal mucosa in humans, cats, and rabbits. To this end, the techniques of Jabonero, Champy-Maillet, and Koelle-Friedenwald were employed to detect specific acetylcholinesterase activity. The supremum colli ganglion was also removed from cats in order to observe any tissue changes produced. Using our histochemical techniques, we were able to demonstrate for the first time that Cajal's interstitial cells in the nasal mucosa are acetylcholinesterase-positive. These cells also appear to be totally integrated into the structure of the terminal vegetative neural formations. Additionally, the fibers surrounding these cells were found to show early degeneration after experimental cervical sympathectomies had been performed.

Acetylcholinesterase activity in the developing olfactory bulb: a biochemical study on normal maturation and the influence of peripheral and central connections.

In the newborn rat olfactory bulb (OB), the specific activity of acetylcholinesterase (AChE) is about 20% of the adult value. During postnatal development, the specific activity remains unchanged until day 10; a growth spurt of 5X is observed between days 10 and 25, when adult values (90 nmoles ACh/min/mg protein; 6 nmol/min/mg wet wt.) are reached. However, total activity shows continuous increase slowly at first and rapidly between days 10 to 30, reaching a plateau by day 60. Between birth to day 60 total activity increases 65X. To determine the influence of peripheral and central connections on the development of AChE activity in the OB, rats were subjected to unilateral olfactory denervation and/or transection of olfactory peduncle, carried out either neonatally or at day 30; the bulbs were assayed a month later (days 30 and 60 respectively). It was found that in both neonatal and 30-day-old rats, denervation caused a 15% decrease in total activity, while transection led to more than 60% reduction. In the older rats, the reduction due to transection represented the degenerative loss of activity, but in the neonatally transected bulbs the growth of some cholinergic elements continued although very slowly. Between birth to day 30, total AChE activity increased only 12X in completely isolated OB, 25X in transected OB and 40X in denervated OB, compared to 45-50X in control OB. In the transected and isolated bulbs specific activity of AChE was also reduced significantly (25-50% depending on age and operation). These results suggest that while centrifugal fibers are the main source of cholinergic activity in the mature as well as the developing OB, the olfactory nerve and some intrabulbar sources such as cholinergic cells or cholinoceptive membranes also contribute to AChE activity in the OB. These intrinsic sources of AChE activity can persist and even show some growth in the developing olfactory bulb in the absence of the centrifugal fibers and/or the olfactory afferents.

Inhibition of neurotoxic esterase and acetylcholinesterase by organophosphorus compounds in selected ectothermic vertebrates

The sensitivity of brain acetylcholinesterase and neurotoxic esterase to inhibition by several organophosphorus compounds was studied in selected ectothermic vertebrates. These enzymes are associated with organophosphorus compound acute and delayed toxicity, respectively. In addition, the susceptibility of several of these species to delayed neurotoxicity induced by organophosphorus compounds was studied. Larvae of the gray treefrog, Southern leopard frog, and narrow-mouthed toad were exposed dermally to tri- o-tolyl phosphate or phenyl saliginen cyclic phosphate (PSCP); no symptoms of delayed neurotoxicity were observed in any of these animals up to 2 weeks after metamorphosis. No symptoms of delayed neurotoxicity were seen in juvenile bullfrogs exposed to multiple ip doses of PSCP. The specific activity of neurotoxic esterase was highest in the larval bullfrog, with juvenile channel catfish and adult mosquitofish demonstrating intermediate levels. The larval Southern leopard frog, adult Northern leopard frog, juvenile green treefrog, and adult marine toad exhibited extremely low activities. The specific activity of acetylcholinesterase was highest in the juvenile channel catfish. Neurotoxic esterase in the larval bullfrog was more sensitive to organophosphate inhibition than that in either fish. PSCP was a more potent neurotoxic esterase inhibitor than leptophos-oxon. The juvenile channel catfish had the acetylcholinesterase most sensitive to organophosphate inhibition. Under the conditions tested, no evidence of in vivo sensitivity to the organophosphate-induced delayed neurotoxicity phenomenon was observed.

A rapid percoll gradient procedure for the preparation of acetylcholine receptor-rich vesicles from Torpedo marmorata electric organ

A rapid method for the isolation of acetylcholine receptor-rich membranes from Torpedo marmorata electric organ, using a Percoll density gradient, is presented. The preparation of purified membranes appeared on electron microscope examination as a homogeneous population of sealed vesicles, covered with the characteristic rosettes identified as acetylcholine receptor clusters. Biochemical characterization revealed an α-bungarotoxin specific binding activity of 1.6–2.1 nmol/mg of protein, low acetylcholinesterase specific activity, a protein:lipid ratio (w/w) of 2.1 with high cholesterol content. Polyacrylamide gel electrophoresis under denaturing conditions showed the polypeptide bands characteristic of the receptor (α, β, γ and δ), together with 43 kDa and ∼100 kDa proteins (already described as ν and ϵ). The method is simple and rapid, and maintains constant osmotic conditions throughout. It thus represents an improvement over previous methods, and will be useful for routine preparation and specially for studying post-synaptic membrane components interactions.

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10 −7 and 10 −6 M with maximum AChE values detected at 10 −6–10 −5 M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.