Acetylcholinesterase In Skeletal Muscle Research Articles

Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits

The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.

Evaluation of male-mediated reproductive toxic effects of methamidophos in the mouse

The reproductive toxicity of the insecticide methamidophos was studied in male mice. Adult male mice were treated by gavage with methamidophos at doses of 0, 1, 2 and 3 mg kg(-1) day(-1) for 4 weeks before mating with untreated females. Brain and skeletal muscle acetylcholinesterase activity was inhibited in the middle- and high-treated groups. Methamidophos treatment was associated with a decreased number of live foetuses and an increased number of dead and resorption foetuses at 2 and 3 mg kg(-1) day(-1) treated groups. The per cent morphologically normal spermatozoa were affected in the 2 and 3 mg kg(-1) day(-1) dose groups; however, sperm motility and count were decreased in the same treated groups compared to the control. Histological examination of brain, muscles, testes and epididymis revealed histological abnormalities in a dose-dependent manner. The current study demonstrated adverse effects of male methamidophos exposure on pregnancy outcome with effects on sperm parameters at 2 and 3 mg kg(-1) day(-1) .

Chlorpyrifos induced reproductive toxicity in male mice

The reproductive toxicity of the insecticide chloropyrifos was studied in male mice. Adult male mice were treated by gavage with chloropyrifos at doses of 0, 5, 15, and 25 mg/kg-d for 4 weeks before mating with untreated females. Signs of cholinergic effects were observed in the 15 and 25 mg/kg-d treated groups. Brain and skeletal muscle acetylcholinesterase activity was inhibited in the same groups. Chloropyrifos treatment was associated with a decreased number of live fetuses, and an increased number of dead fetuses at 25 mg/kg-d. The number of early resorption was decreased in both 15 and 25 mg/kg-d treated groups. The percent morphologically normal spermatozoa were affected in 15 and 25 mg/kg-d dose groups; however, sperm motility and count were decreased in the same treated groups compared to the control. Histologic examination of brain revealed histological abnormalities in the middle and high groups. Dose related histologic changes, including degeneration of muscle fibers, were observed in the muscles of male mice treated with all the doses of chloropyrifos. The current study demonstrated adverse effects of male chloropyrifos exposure on pregnancy outcome with effects on sperm parameters at 15 and 25 mg/kg-d.

Dissociation of Transcription, Translation, and Assembly of Collagen-tailed Acetylcholinesterase in Skeletal Muscle

The synaptic form of acetylcholinesterase (AChE) in skeletal muscle ColQ-AChE derives from two separate genes encoding the catalytic and the non-catalytic collagenic tail (ColQ) subunits, respectively. ColQ-AChE expression is regulated by muscle activity; however, how this regulation takes place in skeletal muscle remains poorly understood. In this study, we overexpressed or knocked down ColQ expression in skeletal muscle and found that the level of this non-catalytic component by itself was sufficient to change the levels of total AChE activity by promoting assembly of higher order oligomeric forms including the collagen-tailed forms. These results initially suggested that ColQ could be limiting in the assembly of synaptic ColQ-AChE during development and differentiation. We then determined the levels of ColQ protein and ColQ mRNA during primary quail muscle cell development and differentiation in culture (QMCs) and as a function of muscle activity. Surprisingly, we found dissociation between transcription and translation of the non-catalytic subunit from its assembly into ColQ-AChE. Furthermore, we found that the vast majority of the steady state ColQ molecules in mature quail muscle cultures are not assembled into ColQ-AChE, suggesting that they are either rapidly degraded or have alternative function(s).

Assessment of reproductive toxicity of orally administered technical dimethoate in male mice

The effect of organophosphate insecticide dimethoate at three dosage levels (7, 15, and 28 mg/kg/day) on male reproduction in mice was studied. Dimethoate was given orally by gavage to male mice for 20 days before mating with untreated females. Signs of cholinergic effects were observed in the 15 and 28 mg/kg/day treated groups. Brain and skeletal muscle acetylcholinesterase activities were inhibited in both the middle and high dose groups. Dimethoate was associated with a decreased number of implantations and live fetuses, and an increased number of dead and early resorptions at 28 mg/kg/day treated group. The percent morphologically normal spermatozoa were unaffected in any of dose groups. However, sperm production and percent motile sperm were decreased in the 15 and 28 mg/kg/day treated groups compared to the control. Histological changes in testis were observed in the middle and high treated groups. The current study demonstrated the adverse effects of dimethoate on the reproductive performance of male mice and pregnancy outcomes following mating with untreated female mice at dose levels of 15 and 28 mg/kg/day. The No Observed Effect Level (NOEL) in the present study for reproductive performance was 7 mg/kg/day.

Reproductive toxicology of acephate in male mice

The reproductive toxicity of the insecticide acephate was studied in male mice. Adult male mice were treated by gavage with acephate at doses of 0, 7, 14, and 28 mg/kg/day for 4 weeks before mating with untreated females. Signs of cholinergic effects were observed in the 28 mg/kg/day group. Brain and skeletal muscle acetylcholinesterase activity was inhibited only in this group. Acephate treatment was associated with a decreased number of implantations and live fetuses, and an increased number of early resorptions at 28 mg/kg/day. The percent morphologically normal spermatozoa was unaffected in all dose groups; however, sperm motility and count were decreased in the 14 and 28 mg/kg/day groups compared to the control. Histologic examination of brain did not reveal any abnormalities. Dose related histologic changes, including degeneration of muscle fibers, were observed in the muscles of male mice treated with any of the doses of acephate. The current study demonstrated adverse effects of male acephate exposure on pregnancy outcome with effects on sperm parameters at 14 and 28 mg/kg/day.

Modification of skeletal muscle AChE expression by a novel method of stimulus application to the peripheral nerve

We have developed a new method for chronic application of electrical stimuli to the rat peripheral nerve in vivo. This method has the following advantages: (1) the amplitude, duration and pattern of stimulation can be adjusted before and during the course of experiment, (2) the set-up allows the animal to move freely during the experiment, and (3) the set-up is constructed from inexpensive, of-the-shelf components that can be reused several times. The new method was used to study the influence of the pattern of muscle activation on acetylcholinesterase (AChE) regulation in extensor digitorum longus (EDL) and tibialis anterior (TA), fast skeletal muscles. Northern blot analysis of the chronically stimulated, fast EDL and TA revealed a rapid decrease of AChE mRNA level to a level typical for a slow, skeletal muscle.

Ciliary neurotrophic factor: regulation of acetylcholinesterase in skeletal muscle and distribution of messenger RNA encoding its receptor in synaptic versus extrasynaptic compartments.

Several recent studies have shown that the ciliary neurotrophic factor exerts myotrophic effects in addition to its well-characterized neurotrophic actions on various neuronal populations. Since expression of acetylcholinesterase in skeletal muscle has been shown to be regulated by putative yet unknown nerve-derived trophic factors, we tested the hypothesis that the ciliary neurotrophic factor is a neurotrophic agent capable of influencing expression of acetylcholinesterase in adult rat skeletal muscle in vivo. To this end, we first determined the impact of daily ciliary neurotrophic factor administration on expression of acetylcholinesterase in both intact and denervated rat soleus muscles. The results of our experiments indicate that although chronic administration of ciliary neurotrophic factor partially counteracted the atrophic response of soleus muscles to surgical denervation, thus confirming its myotrophic effects, it failed to either increase acetylcholinesterase expression in intact muscles or prevent the decrease normally occurring in seven-day denervated muscles. In fact, acetylcholinesterase messenger RNA and enzyme levels were further reduced by ciliary neurotrophic factor treatment in denervated muscles without significant modifications in the pattern of acetylcholinesterase molecular forms. Conversely, transcript levels of the epsilon subunit of the acetylcholine receptor in intact and denervated soleus muscles treated with the ciliary neurotrophic factor were similar to those observed in their respective counterparts from vehicle-treated animals. In addition, we also determined whether transcripts encoding the receptor for the ciliary neurotrophic factor selectively accumulate in junctional domains of rat skeletal muscle fibres. In contrast to the preferential localization of transcripts encoding acetylcholinesterase and the epsilon subunit of the acetylcholine receptor within the postsynaptic sarcoplasm, messenger RNAs for the ciliary neurotrophic factor receptor appeared homogeneously distributed between junctional and extra-junctional compartments of both diaphragm and extensor digitorum longus muscle fibres, with no compelling evidence for a selective accumulation within the postsynaptic sarcoplasm. These data show that the ciliary neurotrophic factor exerts an inhibitory influence on expression of acetylcholinesterase in muscle fibres. Furthermore, the lack of an effect on expression of the epsilon acetylcholine receptor transcripts indicates that treatment with ciliary neurotrophic factor does not lead to general adaptations in the expression of all synaptic proteins. Given the distribution of transcripts encoding the ciliary neurotrophic factor receptor along multinucleated muscle fibres, we propose a model whereby the ciliary neurotrophic factor, or a related unknown molecule that also utilizes the receptor for the ciliary neurotrophic factor, contributes to the maintenance of low levels of enzyme activity in extrajunctional regions of muscle fibres by acting as a repressor of acetylcholinesterase expression that functions directly or indirectly via a pretranslational regulatory mechanism. Accordingly, these results further highlight the complexity of the regulatory mechanisms presiding over acetylcholinesterase expression in vivo.

Trophic regulation of acetylcholinesterase isoenzymes in adult mammalian skeletal muscles

This work addresses the physiological regulation of skeletal muscle acetylcholinesterase (AChE) isoforms by examining endplate-enriched samples from adult rat gracilis muscles 48 h after: low-intensity treadmill exercise; obturator nerve transection; nerve impulse conduction blockade by tetrodotoxin; acetylcholine (ACh) receptor (AChR) inactivation by alpha-bungarotoxin; and, addition of obturator nerve extracts to muscles in organ culture. Results document the important role(s) of functional AChRs and ACh-AChR interactions in the differential control of individual AChE isoenzymes. A theoretical model based on these and other findings considers that: AChR activation by spontaneously released ACh is the only neural factor required for the maintenance of G1 + G2 AChE; the amount of A12 AChE is determined by the combined effects of ACh and another neurogenic substance; although mechanisms intrinsic to myofibers control normal levels of G4 AChE, enhanced production of this isoform is initiated through increasing the frequency of ACh-AChR interactions.

Increased skeletal muscle acetylcholinesterase activity in porcine malignant hyperthermia.

The content and distribution of acetylcholinesterase (AChE) activity in the skeletal muscle disorder malignant hyperthermia (MH) was examined. The AChE activity of sarcolemma membranes isolated from MH-susceptible (MHS) swine was increased twofold when compared with normal sarcolemma. The total AChE activity of muscle extracts was also doubled in MHS tissue; however, the relative distribution between low-salt extractable (globular forms) and high-salt extractable (asymmetric forms) AChE activities were similar in MHS and normal muscle. Our results suggest that, for as yet unexplained reasons, both the sarcolemmal and total AChE activity of skeletal muscle is increased in porcine MH.

Skeletal muscle acetylcholinesterase molecular forms in amyotrophic lateral sclerosis.

Acetylcholinesterase (AChE) molecular forms in muscle biopsies from control and amyotrophic lateral sclerosis (ALS) patients were extracted under low (G: globular forms) and high (A: asymmetric forms) ionic strength conditions and were evaluated by velocity sedimentation analysis. Total AChE activity in endplate-containing ALS muscle sections was reduced by an average of 65% of control muscle levels. This decrement resulted from an almost complete disappearance of 9.5S (G4) and 8.0S (A4) AChE and significant decreases in the 3.8S (G1), 12.1S (A8), and 15.8S (A12) forms (66%, 9%, and 25% of control, respectively). In most of the ALS biopsies examined, ultrastructural-cytochemical analysis revealed large reductions in AChE reaction product of both synaptic infoldings (extracellular) and sarcoplasmic reticulum (intracellular) of the muscles' motor endplate regions. These data are compatible with the view that alterations observed in AChE forms from ALS muscles are related to disturbances in the normal "trophic" interactions between nerve and muscle.

Two classes of collagen-tailed, asymmetric molecular forms of acetylcholinesterase in skeletal muscle: differential effects of denervation

Skeletal muscles of different vertebrate species contain, as it is the case in other cholinergic tissues, two classes of collagen-tailed, asymmetric forms (A-forms) of acetylcholinesterase (AChE). Class I A-forms are readily brought into solution in the presence of high salt, while class II A-forms do additionally require a chelating agent, such as EDTA, for solubilization. All A-forms aggregate at low ionic strength but only class II A-forms are reaggregated by excess Ca(++), even in the presence of 1M NaCl. This Ca(++)-mediated aggregability of class II A-forms is slowly lost upon exposure to detergents such as Triton X-100. Although these two classes of AChE tailed forms seem to be present in endplate and non-endplate areas, and in both the extra- and intracellular compartments, class II A-forms are predominantly extracellular and endplate-specific, at least in the rat diaphragm. On the other hand, well-characterized fast- and slow-twitch muscles show no preference for either class of asymmetric AChE species. Upon denervation, class I A-forms are degraded faster and disappear earlier than their class II counterparts, which are still easily detectable 17 days after nerve section. Class I and class II AChE molecular species exist in similar relative proportions in many vertebrate muscles. Thus, collagen-tailed forms may be altogether more abundant, in skeletal muscle, than it was hitherto realized. It is expected that this further example of AChE polymorphism will contribute to a better understanding of cholinergic transmission in skeletal muscle and, more specially, of nerve-muscle interactions.

Content and release of acetylcholinesterase in skeletal muscle of rats with experimental autoimmune myasthenia gravis

The effects of experimental autoimmune myasthenia gravis (EAMG) on acetylcholinesterase (AChE) were investigated in diaphragms of adult female Lewis rats. Both total AChE activity per muscle and release of enzyme activity during a 3-h incubation in vitro were measured. Two groups of myasthenic animals were used. Acute EAMG was induced by intravenous injection 48 h earlier with a syngeneic monoclonal autoantibody against the nicotinic acetylcholine receptor (AChR) of rat skeletal muscle; age- and weight-matched controls received a monoclonal anti-AChR antibody nonreactive with mammalian muscle. Chronic EAMG was induced by immunization 4 weeks earlier with AChR purified from Torpedo electroplax; controls received only adjuvants. When preparations from rats with acute or chronic EAMG were compared with the appropriate controls, no statistically significant differences in content or release of AChE activity were detected. Neither was there any change in the relative amounts of the various molecular forms of AChE in samples from animals with chronic EAMG. We conclude that the structural and functional changes arising in EAMG are highly specific for the acetylcholine receptor and associated elements of the neuromuscular junction, but have little impact on the biology of AChE.

Abnormal distribution of skeletal muscle acetylcholinesterase molecular forms in dystrophic mice

Acetylcholinesterase (AChE) activities and molecular forms, as separated by density gradient centrifugation, were studied in dystrophic and clinically normal mouse muscle. Dystrophic hemidiaphragms exhibited normal AChE activity, but there was little or no 10 S enzyme, a form that constitutes 27% of control tissue AChE. The 10 S-AChE abnormality was similarly present in dystrophic extensor digitorum longus (EDL) muscle, but this muscle exhibited significantly reduced AChE activity. The EDL muscles also had reduced 16 S-AChE but normal 4 S enzyme activity. Chronic denervation of EDL muscles resulted in proportionally similar reductions of weight, total AChE, and 16 S enzyme in dystrophic and control muscles. We conclude that murine dystrophy involves some alterations that resemble denervation, but that there are major qualitative and quantitative differences in AChE that cannot be explained by a denervation-like effect.

Partial purification and characterization of a nerve trophic factor regulating muscle acetylcholinesterase activity

A protein influencing acetylcholinesterase activity of newt triceps muscle in organ culture has been identified and partially purified from rat brain homogenates by gel filtration, ion-exchange chromatography, and isoelectric focusing. Muscles treated with fractions containing the active factor had higher acetylcholinesterase activity than paired untreated muscles after 1 week in organ culture. Analysis of the molecular forms of acetylcholinesterase by velocity sedimentation showed that the factor had the greatest effect on the 16.5 S acetylcholinesterase, increasing it more than 100% of untreated controls. With isoelectric focusing, activity was present in a fraction focusing at pH 8.3. On SDS-polyacrylamide gel electrophoresis, a protein with a molecular weight of 34,000 was present in active fractions and was the major component of the isoelectric focusing pH 8.3 fraction. These findings indicate that a small basic protein, which we have termed neurotrophin, mediates the neuronal trophic effect on skeletal muscle acetylcholinesterase.

Muscle cholinesterase: Effect of phencyclidine and ketamine on rat and human muscle cholinesterase activity

Phencyclidine (PCP) and ketamine were found to weakly inhibit rat skeletal muscle acetylcholinesterase (AChE) activity. PCP was a slightly more effective inhibitor of AChE than ketamine in vitro. PCP inhibited AChE activity in a noncompetitive manner which suggests the interaction of PCP at the secondary anionic site(s) of AChE whereas ketamine inhibited in a competitive manner, suggesting the interaction at the active site(s) of the enzyme. Both drugs also inhibited human muscle AChE activity in vitro. In vivo, both drugs stimulated rat skeletal muscle AChE activity whereas no effect was observed on rat brain AChE activity. When combined with restraints for 30 min following PCP and ketamine, the stimulation of AChE activity was greater than that produced by drug alone or restraint alone.

Parallel regulation of acetylcholinesterase and pseudocholinesterase in normal, denervated and dystrophic chicken skeletal muscle.

IN vertebrate skeletal muscle acetylcholinesterase (AChE, EC 3.1.1.7) is usually considered to be involved in the termination of impulse transmission by hydrolysis of acetylcholine1. AChE is often accompanied by a second enzyme2, pseudocholinesterase (ψ/ChE, EC 3.1.1.8), which differs from AChE in substrate specificity and in susceptibility to anticholinesterases3,4. No role comparable with that of AChE in synaptic transmission is known for ψChE, despite its abundance at some sites and its occurrence (as shown histochemically) with ψChE in various muscle endplates1,5. It has been suggested that ψChE is a precursor of AChE in the superior cervical ganglion6,7. Moreover, in ganglia and skeletal muscle of rats, forms of 0ChE have been demonstrated8 homologous with forms of ψChE observed previously9–11. Elevated levels of AChE have been demonstrated in aqueous extracts of fast-twitch muscles of dystrophic as compared with normal chickens12,13; ψChE levels increased similarly13, and also in such extracts of denervated muscles of normal chickens14. We show here that the total amounts and forms of AChE change in parallel with those of ψChE during development of skeletal muscles of both normal and dystrophic chickens, as well as on denervation of normal muscle. Our data strongly indicate that in these tissues the two enzymes are controlled by a common regulatory mechanism.

Intrinsic forms of acetylcholinesterase in skeletal muscle

Intrinsic forms of acetylcholinesterase in skeletal muscle

Role of axoplasmic transport in neurotrophic regulation of muscle end plate acetylcholinesterase.

THE mechanism underlying neural control of skeletal muscle acetylcholinesterase (AChE) is not fully understood1. The experimental evidence points to acetylcholine (ACh)2 and nerve-evoked muscle activity3 as the “trophic” mediators, but neither is sufficient to account for the entire effect of innervation on muscle4. It remains to be seen whether factors such as cell-to-cell contact, and/or some regulatory substance(s) released by motor nerves also contribute to the regulation of AChE activity. In turn, the regulatory substance(s) might be conveyed by axoplasmic transport, for blockage of this process causes denervation-like changes without disturbing neurotransmitter release or the consequent muscle contractile activity5. We report here a reversible change in the activity of endplate AChE that can be ascribed to a temporary interruption of axoplasmic transport.

Acetylcholinesterase activity in developing skeletal muscle cells in vitro

Acetylcholinesterase activity has been demonstrated biochemically and cytochemically in developing chick embryo skeletal muscle cells growing in culture. The enzyme shows the same pattern of drug sensitivity as that of adult skeletal muscle acetylcholinesterase and in present in cultured myogenic cells before the time of cell fusion, the formation of myotubes, and the subsequent increase in rate of myosin synthesis. Myogenic cell fusion is accompanied, however, by a large increase in activity of acetylcholinesterase. The enzyme activity is restricted in these cultures to myogenic cells. Neighboring fibroblasts show no cytochemical responses when challenged with techniques showing intense activity in myoblasts and myotubes. In addition, evidence is presented which strongly suggests that acetylcholinesterase activity in dividing myogenic cells is not constant over the cell cycle.