Regulation of the Expression of Acetylcholinesterase by Muscular Activity in Avian Primary Cultures

Primary cultures of avian muscle cells express both globular and asymmetric molecular forms of acetylcholinesterase (AChE) when grown in a simple defined culture medium. Under these conditions, we analyzed the role of various agents interfering with muscular activity: tetrodotoxin (TTX) and veratridine, as well as a depolarizing concentration of KCl. These treatments caused the complete cessation of contractions in mature myotubes. We observed no influence on cellular AChE activity. The paralyzing treatments induced different effects on AChE secretion: TTX increased the secretion by approximately 25%, whereas KCl and veratridine reduced it by approximately 30%. The proportions of secreted molecular forms (mostly hydrophilic G4 and G2) were not modified significantly. TTX did not affect the pattern of molecular forms of cellular AChE (in particular, the proportion of A forms was not changed). Depolarization by veratridine or KCl induced an increase in the proportion of A forms in mature myotubes by a factor of 2-3. Similar results were obtained with quail myotubes cultured under the same conditions. This study shows that, in avian muscle cultures, the ionic balance across myotube membranes, rather than muscular activity per se, can regulate the level of A forms and the rate of AChE secretion. These results do not exclude the possible involvement of other factors, such as Ca2+ and/or peptidic factors. In addition, taking together our results and data from the literature. we conclude that the expression of AChE molecular forms depends both on the species and on the culture conditions used.     

Presynaptic or postsynaptic origin of acetylcholinesterase at neuromuscular junctions? An immunological study in heterologous nerve-muscle cultures

Numerous studies have shown that the acetylcholine receptor (AChR) is inserted in the plasma membrane of the muscle fiber, and that it is focalized at the site of neuromuscular junctions, as an effect of neural influence. In contrast, acetylcholinesterase (AChE) may be presynaptic or anchored in the basal lamina, as well as postsynaptic at neuromuscular junctions. We investigated the origin of the junctional enzyme, particularly the collagen-tailed asymmetric A12 forms, by studying the AChE contents of heterologous rat and chicken neuromuscular cocultures by immunohistochemical and biochemical methods. We found that the overall content of AChE, in the neuromuscular cocultures, including the A12 form, was essentially identical to the sum of the contents of separate myotube and motoneuron cultures. The sedimentation coefficients of the rat and chicken asymmetric forms are sufficiently different to clearly differentiate these enzymes in sucrose gradients: 16 S for rat, 20 S for chicken A12 AChE. Sedimentation analyses of AChE in cocultures thus showed that the A12 form was of muscular origin. In the case of aneural cultures of myotubes, histochemical staining of AChE activity or immunohistochemical staining with specific antibodies showed only very scarce, faint concentrations of enzyme. Some patches of acetylcholine receptor (AChR) were, however, visible in these cultures. Neuromuscular contacts are readily established in cocultures of myotubes with embryonic motoneurons from spinal cords. In the presence of motoneurons, the myotubes presented a larger number of AChR patches. The most remarkable feature of neuromuscular cocultures was the presence of numerous intense AChE patches which always coincided with AChR clusters. By specifically staining nerve terminals with tetanus toxin, we could show an excellent correlation between neuromuscular contacts and the presence of AChE-AChR patches. We found that the AChE patches in heterologous cocultures could be stained exclusively by the anti-myotube AChE antiserum. The focalized enzyme is therefore exclusively, or very predominantly, provided by the myotube.     

Muscular differentiation of chicken myotubes in a simple defined synthetic culture medium and in serum supplemented media: Expression of the molecular forms of acetylcholinesterase

We attempted to cultivate muscle cells from chick embryos in a serum-free, defined medium similar to that proposed by Bottenstein and Sato (1979) for the growth and differentiation of a murine neuronal cell-line. (1) We found that muscle cells from the legs of 11-day old chick embryos can be cultivated in a medium containing the different components indicated by Bottenstein and Sato, with 2 g/l bovine serum albumin, without serum or chick embryo extract. Myoblasts attached to the gelatin-coated dishes without any addition of attachment factors. They differentiated into myotubes in a similar manner as in classical serum supplemented media. (2) The level of cellular AchE activity was comparable in cultures grown in the presence of fetal calf serum (FCS), of horse serum (HS) and in the defined medium. The percentage of A₁₂ form was however higher in the defined medium (25–30%) than in FCS supplemented medium (about 5–6%). In HS supplemented medium the A₁₂ form was not detectable, partly because horse serum contains immunoglobulins which bind chicken AChE. The addition of defined medium components to FCS medium cultures did not lead to an increase of A₁₂. In contrast, the addition of a small amount (1%) of fetal calf serum to DM cultures reduced the level of A₁₂ in a drastic manner. FCS components therefore seem to repress the biosynthesis of A₁₂ AChE, or increase its degradation. (3) We estimated intracellular and extracellular compartments of AChE. The ratio of endocellular to ectocellular AChE decreased with the age of the cultures. The G₁ form was intracellular at all stages analyzed, but the other molecular forms were located in both cellular compartment, in different proportion: A₁₂ and G₄ seemed to be located preferentially in the external compartment, whereas G₂ was preferentially intracellular. (4) Muscle cultures grown in the defined medium and in the presence of serum secreted globular forms of AChE in a similar manner.     

Globular and asymmetric acetylcholinesterase in the synaptic basal lamina of skeletal muscle

The aim of this study was to characterize the molecular forms of acetylcholinesterase (AChE) associated with the synaptic basal lamina at the neuromuscular junction. The observations were made on the neuromuscular junctions of cutaneous pectoris muscles of frog, Rana pipiens, which are similar to junctions of most other vertebrates including mammals, but are especially convenient for experimentation. By measuring relative AChE activity in junctional and extrajunctional regions of muscles after selective inactivation of extracellular AChE with echothiophate, or of intracellular AChE with DFP and 2-PAM, we found that > 66% of the total AChE activity in the muscle was junction- specific, and that > 50% of the junction-specific AChE was on the cell surface. More than 80% of the cell surface AChE was solubilized in high ionic strength detergent-free buffer, indicating that most, if not all, was a component of the synaptic basal lamina. Sedimentation analysis of that fraction indicated that while asymmetric forms (A12, A8) were abundant, globular forms sedimenting at 4-6 S (G1 and G2), composed > 50% of the AChE. It was also found that when muscles were damaged in various ways that caused degeneration of axons and muscle fibers but left intact the basal lamina sheaths, the small globular forms persisted at the synaptic site for weeks after phagocytosis of cellular components; under certain damage conditions, the proportion of globular to asymmetric forms in the vacated basal lamina sheaths was as in normal junctions. While the asymmetric forms required high ionic strength for solubilization, the extracellular globular AChE could be extracted from the junctional regions of normal and damaged muscles by isotonic buffer. Some of the globular AChE appeared to be amphiphilic when examined in detergents, suggesting that it may form hydrophobic interactions, but most was non-amphiphilic consistent with the possibility that it forms weak electrostatic interactions. We conclude that the major form of AChE in frog synaptic basal lamina is globular and that its mode of association with the basal lamina differs from that of the asymmetric forms.     

Evidence for the Neurotrophic Regulation of Collagen-Tailed Acetylcholinesterase in Immature Skeletal Muscle by β-Endorphin

Abstract: Acetylcholinesterase (AChE) was extracted in a high-saline medium from gastrocnemius muscles of rat embryos and young rats aged 14 days'gestation to 40 days post partum. The molecular forms of the enzyme were separated by low-salt precipitation, followed by velocity sedimentation. During gestation, all molecular forms increased in activity, particularly the 16 S (A₁₂) form. During the first 2 weeks of life, there was a large increase in the activity of soluble AChE (G forms), whilst the activity of insoluble AChE (A forms) was reduced. Denervation of the muscle reversed the change in the relative proportions of the molecular forms. The embryonic pattern of activities of AChE forms persisted in cultures of myotubes obtained at 20 days'gestation and maintained in the absence of spinal cord. When myotubes were maintained in medium previously conditioned by developing spinal cord explants, 16 S AChE declined while the soluble (4 and 6 S) forms increased in activity in a manner resembling that seen in early postnatal muscles in vivo . β-Endorphin (β-EP) immunoreactivity was detected in the spinal cord-conditioned medium and was identified by HPLC and ion-exchange chromatography as β-EP-(l–31) plus its shortened and N -acetylated forms. Cultivation of myotubes in the presence of synthetic camel β-EP resulted in a reversible change in the pattern of AChE forms which was similar to that seen with spinal cord-conditioned medium. These studies provide evidence for the neuroregulation of AChE A and G forms in immature skeletal muscle. A major candidate for this role is β-EP, produced and released by developing spinal cord.     

Difference in the expression of asymmetric acetylcholinesterase molecular forms during myogenesis in early avian dermomyotomes and limb buds in ovo and in vitro

The A12 (asymmetric) form of acetylcholinesterase (AChE) is generally considered to be synthesized in leg muscle tissues by myotubes under neural influence, but not by myoblasts. We have examined the expression of the different molecular forms of AChE in explants of developing limb buds and dermomyotomes (the myogenic part of the somites) obtained from 3-day-old chick and quail embryos, either directly after removal or during in vitro culture. We describe a muscular differentiation of both territories in vitro, leading to the formation of myotubes which are morphologically similar to the class of early muscle cells described by Bonner and Hauschka (1974). In vivo the A12 form is present in quail dermomyotomes which are almost entirely composed of mononucleated poorly differentiated cells; in contrast, it is absent from similar cells in chick dermomyotomes and from limb buds in both species. This shows that in the case of quail embryos the appearance of the A12 form precedes the fusion of myoblasts into myotubes. In both species, dermomyotome explants express asymmetric and globular forms of the enzyme during muscular differentiation in vitro, whereas limb buds synthesize only globular forms. After surgical removal of neural tube and/or neural crest at 2 days in ovo, the biosynthesis of the A forms in quail dermomyotomes is not suppressed and is consequently not dependent upon prior connection of the dermomyotomes to central neurons or upon the presence of autonomic precursors. Since limb bud muscle cells derive from somites our results raise questions concerning the differentiation of migrating somitic cells in this territory where a neural influence appears necessary to induce the biosynthesis of asymmetric AChE forms.     

Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes

A factor in extracts of the electric organ of Torpedo californica causes the formation of clusters of acetylcholine receptors (AChRs) and aggregates of acetylcholinesterase (AChE) on myotubes in culture. In vivo, AChRs and AChE accumulate at the same locations on myofibers, as components of the postsynaptic apparatus at neuromuscular junctions. The aim of this study was to compare the distribution of AChRs, AChE, and butyrylcholinesterase (BuChE), a third component of the postsynaptic apparatus, on control and extract-treated myotubes. Electric organ extracts induced the formation of patches that contained high concentrations of all three molecules. The extract-induced aggregation of AChRs, AChE, and BuChE occurred in defined medium, and these components accumulated in patches simultaneously. Three lines of evidence indicate that a single factor in the extracts induced the aggregation of all three components: the dose dependence for the formation of patches of AChRs was the same as that for patches of AChE and BuChE; the AChE- and BuChE-aggregating activities co-purified with the AChR-aggregating activity; and all three aggregating activities were immunoprecipitated at the same titer by a monoclonal antibody against the AChR-aggregating factor. We have shown previously that this monoclonal antibody binds to molecules concentrated in the synaptic cleft at neuromuscular junctions. Taken together, these results suggest that during development and regeneration of myofibers in vivo, the accumulation at synaptic sites of at least three components of the postsynaptic apparatus, AChRs, AChE, and BuChE, are all triggered by the same molecule, a molecule similar if not identical to the electric organ aggregating factor.     

Two-Site Immunoradiometric Assay of Chicken Acetylcholinesterase: Active and Inactive Molecular Forms in Brain and Muscle

Abstract: Several monoclonal antibodies were raised against chicken acetylcholinesterase (AChE; EC 3.1.1.7). Some of these antibodies react with quail AChE but not with AChEs from nonavian vertebrates or invertebrates and not with butyrylcholinesterase. They may be classified in several mutually compatible groups, i.e., that can bind simultaneously to the monomeric form of AChE. Most antibodies recognize a peptidic domain that does not exist in mammalian AChE and that may be digested by trypsin without loss of activity or dissociation of quaternary structure. The only exception is the antibody C-131, which is conformation dependent and preferentially recognizes active AChE. We have set up two-site immunoradiometric assays, using an immobilized capture antibody, C-6 or C-131, and a radiolabeled antibody, ¹²⁵I-C-54. The C-6/C-54 assay quantifies the totality of inactive and active AChE subunits: It detects 10^?3 Ellman unit (～40 pg of protein) and yields a linear response up to at least 25 10^?3 Ellman units. An analysis of gradient fractions, using C-6/C-54 and C-131/C-54 assays as well as activity determination, shows that the A₁₂ and G₄ forms are exclusively composed of active subunits, whereas inactive molecules cosediment with the active G₂ and G₁ forms. Both active and inactive G₂ and G₁ forms are amphiphilic, as indicated by the influence of detergents on their sedimentation coefficients and Stokes radii. In brain, the proportion of inactive forms decreases from 40% at embryonic day 11 (E11) to 20% at birth [day 1 (D1)]. In muscle, we observed no inactive AChE at E11 and a small proportion of inactive G₁ at D1. The proportion of inactive forms was much higher in cultured myotubes, obtained from E11 myoblasts. These results show that the proportion of inactive AChE depends on the tissue and varies during development. Thus, the cells seem to control actively the acquisition of AChE activity, as well as the formation of the various oligomeric forms.     

Acetylcholinesterase from the Skeletal Muscle of the Lamprey Petromyzon marinus Exists in Globular and Asymmetric Forms

To obtain information about the evolution of acetylcholinesterase (AChE), we undertook a study of the enzyme from the skeletal muscle of the lamprey Petromyzon marinus, a primitive vertebrate. We found that the cholinesterase activity of lamprey muscle is due to AChE, not pseudocholinesterase; the enzyme was inhibited by 1,5-bis(4-allyldimethylammonium phenyl) pentane-3-one (BW284C51), but not by tetramonoisopropyl pyrophosphortetramide (iso-OMPA) or ethopropazine. Also, the enzyme had a high affinity for acetylthiocholine and was inhibited by high concentrations of substrate. A large fraction of the AChE was found to be glycoprotein, since it was precipitated by concanavalin A-agarose. Optimal extraction of AChE was obtained in a high-salt detergent-containing buffer; fractional amounts of enzyme were extracted in buffers lacking salt and/or detergent. These data suggest that globular and asymmetric forms of AChE are present. On sucrose gradients, enzyme that was extracted in high-salt detergent-containing buffer sedimented as a broad peak of activity corresponding to G4; additionally, there was usually a peak corresponding to A12. Sequential extraction of AChE in conjunction with velocity sedimentation resolved minor forms of AChE and revealed that the G1, G2, G4, A4, A8, and A12 forms of AChE could be obtained from the muscle. The identity of the forms was confirmed through high-salt precipitation and collagenase digestion. The asymmetric forms of AChE were precipitated in low ionic strength buffer, and their sedimentation coefficients were shifted to higher values by collagenase digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholinesterase molecular forms in the fast or slow muscles of the chicken and the pigeon

Molecular forms of acetylcholinesterase (AChE) were examined in various skeletal muscles of the chicken and the pigeon. In chicken pectoralis m., AChE was found to be restricted to endplate containing segments, and no asymmetric form could be detected in aneural samples. In the chicken muscles studied, a relation has been established between globular (G1,G2,G4) forms or asymmetric (A8,A12) forms, and muscle fibre types. Asymmetric forms are preponderant in fast-twitch muscles, whereas in slow tonic muscles 80% of the AChE activity is due to globular forms. However, comparison with pigeon muscles shows that AChE chicken muscle patterns may not be generalized.     

H and T subunits of acetylcholinesterase from Torpedo, expressed in COS cells, generate all types of globular forms

We analyzed the production of Torpedo marmorata acetylcholinesterase (AChE) in transfected COS cells. We report that the presence of an aspartic acid at position 397, homologous to that observed in other cholinesterases and related enzymes (Krejci, E., N. Duval, A. Chatonnet, P. Vincens, and J. Massoulié. 1991. Proc. Natl. Acad. Sci. USA. 88:6647-6651), is necessary for catalytic activity. The presence of an asparagine in the previously reported cDNA sequence (Sikorav, J.L., E. Krejci, and J. Massoulié. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1865-1873) was most likely due to a cloning error (codon AAC instead of GAC). We expressed the T and H subunits of Torpedo AChE, which differ in their COOH-terminal region and correspond respectively to the collagen-tailed asymmetric forms and to glycophosphatidylinositol-anchored dimers of Torpedo electric organs, as well as a truncated T subunit (T delta), lacking most of the COOH-terminal peptide. The transfected cells synthesized similar amounts of AChE immunoreactive protein at 37 degrees and 27 degrees C. However AChE activity was only produced at 27 degrees C and, even at this temperature, only a small proportion of the protein was active. We analyzed the molecular forms of active AChE produced at 27 degrees C. The H polypeptides generated glycophosphatidylinositol-anchored dimers, resembling the corresponding natural AChE form. The cells also released non-amphiphilic dimers G2na. The T polypeptides generated a series of active forms which are not produced in Torpedo electric organs: G1a, G2a, G4a, and G4na cellular forms and G2a and G4na secreted forms. The amphiphilic forms appeared to correspond to type II forms (Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:776-785; Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:786-794), which are abundant in the nervous tissue and muscles of higher vertebrates (Bon, S., T. L. Rosenberry, and J. Massoulié. 1991. Cell. Mol. Neurobiol. 11:157-172). The H and T catalytic subunits are thus sufficient to account for all types of known AChE forms. The truncated T delta subunit yielded only non-amphiphilic monomers, demonstrating the importance of the T COOH-terminal peptide in the formation of oligomers, and in the hydrophobic character of type II forms.     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.     

Acetylcholinesterase in chick embryo latissimus dorsii muscles: effects of curarization and electrical stimulation

The accumulation of acetylcholinesterase (AChE), the changes in AChE-specific activity and in AChE molecular form distribution were studied in slow-tonic anterior latissimus dorsi (ALD) and in fast-twitch posterior latissimus dorsi (PLD) muscles of the chick embryo. From stage 36 (day 11) to stage 42 (day 17) of Hamburger and Hamilton, the AChE-specific activity decreased, while the relative proportion of asymmetric A 12 and A 8 forms increased. Repetitive injection of curare resulted at stage 42 (day 17) in a decrease in AChE-specific activity, in the accumulation of the synaptic AChE and in the expression of AChE asymmetric forms. Electrical stimulation at a relatively high frequency (40 Hz) of curarized ALD and PLD muscles resulted in a normal increase in AChE asymmetric forms, whereas a lower frequency (5 Hz) resulted in a dominance of globular forms. Both patterns of stimulation partly prevented the loss in synaptic AChE accumulations. These results suggest that in chick embryo muscles, muscle activity and its rhythms are involved in the normal evolution of AChE.     

Stabilization of collagen-tailed acetycholinesterase in muscle cells through extracellular anhorage by transglutaminase-catalysed cross-linking

A component of collagen-tailed acetylcholinesterase (asymmetric; A-AChE) in muscle forms a metabolically-stable pool which can be released from the cell surface only by collagenase, suggesting that part of the enzyme is covalently bound by its tail (COL.Q) subunits. We have investigated whether this insoluble pool forms through covalent cross-linking of A-AChE to extracellular matrix glycoproteins by tissue transglutaminase (Tg; type 2 transglutaminase). Tg catalyzed the incorporation of the polyamine substrate³[H]-putrescine into the collagen tail of affinity-purified avian A12-AChE. Complexes between A12-AChE and cellular fibronectin were also formed in vitro by Tg. In quail myotubes, retinoic acid, which stimulates the formation of (-glutamyl)lysine isodipeptide bonds by Tg in myotubes, increased the proportion of extraction-resistant (er) A-AChE. Following irreversible inactivation of AChE by diisopropylfluorophosphate, entry of newly-synthesized A-AChE into the extraction-resistant pool was inhibited by a competitive Tg inactivator RS48373-007. The quantity of exogenously-added A12 AChE incorporated into the extraction-resistant pool in living myotubes was increased by Tg in the presence of calcium. The inhibition of cross-bridge formation in fibrillar collagen by -aminopropionitrile, and pre-exposure of myotubes to a monoclonal antibody to fibronectin, resulted in a reduction in the size of the erA-AChE pool present on the cell-surface. The evidence supports the hypothesis that a component of insoluble collagen-tailed AChE, once subject to clustering influences mediated via reversible docking to proteoglycans and their receptors, is anchored at the cell surface through covalent cross-linking by Tg. The high stability of the (-glutamyl)lysine isopeptide bond is likely to contribute to the observed low turnover of the erA-AChE fraction.     

Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in culture

The pattern of acetylcholinesterase (AChE) molecular forms, obtained by sucrose gradient sedimentation, was studied at different in vitro developmental stages of myogenic cells isolated from adult mouse skeletal muscle. Only the globular forms were present in rapidly dividing satellite cells during the first days in culture. After myotube formation, a pattern similar to that described in mammalian fast-twitch skeletal muscle was observed. This pattern did not change during the following period in culture (up to 1 month) nor could it be modified by co-culturing with spinal cord motoneurons or by addition of brain-derived extracts. The internal-external localization of AChE molecular forms has been determined by the use of echothiophate iodide, a membrane-impermeant irreversible inhibitor of AChE. Echothiophate-treated cultures showed about 40% of both asymmetric and globular forms localized on the sarcolemma, with their active sites oriented outward. Analysis of culture medium from untreated cultures revealed the presence of both asymmetric and globular forms. When the same analysis was repeated on cultures of myoblasts derived from 16-day-old mouse embryos, the pattern of AChE forms was different. The myotubes derived from these cells exhibit a very small proportion of asymmetric form, which was not released into the medium. This pattern was not further modified during the following days of culture, nor by co-cultures with spinal cord motoneurons or by incubations with brain-derived extracts. Thus, the myotubes derived from myoblasts express in culture a clear phenotypic difference when compared to the corresponding myotubes from satellite cells, supporting the view that these two myogenic cells are endowed with different developmental programs.     

Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide

To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.     

Retrograde Effect of Muscle on Forms of Acetylcholinesterase in Peripheral Nerves

In the peripheral nerves of birds and mammals, acetylcholinesterase (AChE) exists in four main molecular forms (G1, G2, G4, and A12). The two heaviest forms (G4 and A12) are carried by rapid axoplasmic transport, whereas the two lightest forms (G1 and G2) are probably much more slowly transported. Here we report that nerves innervating fast-twitch (F nerves) and slow-twitch (S nerves) muscles of the rabbit differ both in their AChE molecular form patterns and in their anterograde and retrograde axonal transport parameters. Since we had previously shown a selective regulation of this enzyme in fast and slow parts of rabbit semimembranosus muscle, we wondered whether the differences observed in the nerve could be affected by the twitch properties of muscle. The results reported here show that in F nerves that reinnervate slow-twitch muscles, both the AChE molecular form patterns and axonal transport parameters turn into those of the S nerve. These data suggest the existence of a retrograde specific effect exerted by the muscles on their respective motoneurons.     

The Calcitonin Gene-Related Peptide-Induced Acetylcholinesterase Synthesis in Cultured Chick Myotubes Is Mediated by Cyclic AMP

Abstract: In vertebrate neuromuscular junctions, post-synaptic specialization includes aggregation of acetylcholine receptors (AChRs) and acetylcholinesterase (AChE). The motor nerve provides soluble factors and electrical activity to achieve this striking localization of AChRs/AChE. Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, is able to stimulate the expression of AChR in cultured myotubes. Similar to AChR regulation, synthesis of AChE in cultured chick myotubes is also stimulated by CGRP. Application of CGRP onto cultured myotubes stimulated the accumulation of intracellular cyclic AMP (cAMP) as well as the expression of AChE mRNA and protein. However, the enzymatic activity of AChE remained unchanged. In cultured myotubes, various drugs affecting the intracellular level of cAMP, such as N ⁶, O ^2'-dibutyryladenosine 3',5'-cyclic monophosphate, cholera toxin, and forskolin, could mimic the effect of CGRP in stimulating the expression of AChE. When myotubes were transfected with cDNA encoding constitutively active mutant Gα_s, the intracellular cAMP synthesis was increased. The increase in cAMP level was in parallel with an increase in the expression of AChE, whereas transfection of active mutant Gα_i cDNA decreased the cAMP level as well as the AChE expression. In addition, expression of collagen-tailed AChE was up-regulated by the cAMP pathway. These findings indicated that CGRP-induced AChE regulation is mediated by the cAMP pathway and represented the first evidence to suggest that the regulation of mRNA synthesis of AChR and AChE can be mediated by the same neuron-derived factor.