Recovery of acetylcholinesterase and of its multiple molecular forms in motor end-plate-free and motor end-plate-rich regions of mouse striated muscle, after irreversible inactivation by an organophosphorus compound (methyl-phosphorothiolate derivative)

Most of mouse diaphragm muscle acetylcholinesterase (AChE) is irreversibly inhibited after a single intraperitoneal injection of a methyl-phosphorothiolate derivative (MPT), an organophosphorus compound which phosphorylates the active site. The muscle recovers its AChE (de novo synthesis) and we studied the time course of reappearance of AChE and its multiple active molecular forms. After inhibition, there is an initial (3 to 15 hr) rapid recovery of total AChE (which evolves from 20-28% to 50-60% of the control values), followed by a slow phase of AChE return. After 3 days, the recovery is still incomplete (reaching 70-80% of control values). Among the main molecular forms present in diaphragm muscle (16 S, 10 S and 4 S, accompanied by minor components), the 16 S and 10 S forms are the most sensitive to MPT treatment. During the rapid initial phase of AChE recovery, the absolute rate of recovery of the 4 S form is faster than for the other forms with a correspondingly much higher relative proportion to total AChE. These observations are consistent with the hypothesized precursor role of the 4 S form. The 16 S form, which is found concentrated in the motor end-plate (MEP)-rich regions and in low amounts in MEP-free regions, is similarly partially recovered in both regions, suggesting that there is 16 S biosynthesis not only in the MEP-rich regions but also in the MEP-free regions.     

Effects of Acute and Chronic Denervation on Release of Acetylcholinesterase and Its Molecular Forms in Rat Diaphragms

Abstract: Hemidiaphragms were removed from rats at various times after intrathoracic transection of the left phrenic nerve and were incubated in organ baths containing 1.5 ml of oxygenated, buffered physiologic saline solution, with added glucose and bovine serum albumin. After incubation, the acetylcholinesterase (AChE; EC 3.1.1.7) activities of the bath fluid and of the muscle were determined. Innervated left hemidiaphragms were found to release 107 units of AChE over a 3-h period, corresponding to 1.9% of their total AChE activity. Denervation led to a rapid loss of AChE from the muscle coincident with a transient increase in the outpouring of enzyme activity into the bath fluid. Thus, 1 day after nerve transection the left hemidiaphragm contained only 68% of the control amount of AChE activity, but released 140% as much as control. After 3 or 4 days of denervation, the AChE activity of the diaphragm stabilized at 35% of the control value. Release also fell below control by this time, but not as far. One week after denervation the release, 69 units per 3 hr, corresponded to 3.3% of the reduced content of AChE activity in the muscle, indicating that denervation caused an increase in the proportion of AChE released. Sucrose density gradient ultracentrifugation showed that 10S AChE accounted for more than 80% of the released enzyme activity at all times. The results did not rule out the possibility, however, that the released enzyme originally stemmed from 4S or 16S AChE in the diaphragm.     

Junctional form of acetylcholinesterase restored at nerve-free endplates.

Rat soleus muscles were ectopically innervated by implanting a foreign nerve in an endplate-free region of muscle and, 2–3 weeks later, cutting the original nerve. The junctional, 16 S form of acetylcholinesterase (AChE) and focal staining for AChE disappeared from the old endplate region within a few days after denervation. In muscles with an ectopic nerve, but not in paired control muscles, 16 S AChE and focal staining were restored in the old endplate region 1–2 weeks after denervation even though nerve fibers could not be detected in that region. These results suggest that the nerve exerts a local effect, specifying the site at which junctional AChE appears, and a nonlocal effect, perhaps mediated by muscle activity, regulating the amount of junctional AChE.     

Neurotrophic control of 16S acetylcholinesterase from mammalian skeletal muscle in organ culture

The effects of rat obturator nerve extracts on total and 16S acetylcholinesterase (AChE) activity were studied in endplate regions of denervated anterior gracilis muscles maintained in organ culture for 48 hr. The decrease of total AChE activity in cultured muscles was similar to that observed in denervated muscles in vivo. This decrease in activity was partly prevented by addition of either 100 or 200 μl nerve extract (2.7 mg/ml protein) to the nutrient medium. Nerve extract treatment also decreased the release of AChE activity from the muscle into the bathing medium. Conversely, rat serum (20 μl; 90 mg/ml protein) had no effect on total AChE activity in muscle endplates, nor on release of the enzyme by the muscle. The 16S form of AChE was confined to motor endplate muscle regions and its activity was drastically decreased by denervation in both organ culture and in vivo preparations in a comparable manner. Nerve-extract supplemented cultures contained a significantly (p ? 0.001) larger amount of endplate 16S AChE activity (140–145%) than the corresponding controls (100-). Our results suggest that some nerve soluble substance, other than serum contaminants or 16S AChE itself, affects the maintenance of 16S AChE at the neuromuscular junction.     

Turnover of the Molecular Forms of Acetylcholinesterase in the Rat Diaphragm

Abstract: The turnover of acetylcholinesterase (AChE) and its molecular forms was measured by following the loss of enzyme activity in the right hemidiaphragms of Sprague-Dawley rats treated with cycloheximide, 20 mg/kg, every 4 h. This treatment inhibited 96% of the incorporation of [³H]leucine into muscle protein. After 8 h of treatment, the total AChE activity of the diaphragm decreased by 17% ( P < 0.01). Assuming first-order exponential kinetics, a half-life of 30 h and an hourly turnover of 180 units were calculated. The measured accumulation of AChE activity at a ligature on the phrenic nerve indicated that axonal transport contributed trivially to this turnover. Sucrose density gradient experiments showed that the cycloheximide-induced loss of AChE activity was restricted to the 4S enzyme, which had an apparent half-life of 6.2 h.     

Structural differences of pale and strongly stained motor end-plates of the rat diaphragm.

According to the staining intensities for AChE, the motor end-plates of the rat diaphragm can be classified into strong (S) and pale (P) types. About 34% of the total end-plates of the rat diaphragm are of S type and 50% of P type. The P end-plates differ from S end-plates in two aspects. First, the secondary subneural clefts of the S end-plates are well developed. They are numerous, long, closely packed and often branched. On the other hand, the secondary subneural clefts of the P end-plates are short, sparse and usually unbranched. Secondly, there seems to be a variation in AChE activity in the P end-plates. Focal negative AChE areas are found in the subneural apparatus of some P end-plates. It is concluded that the less well developed secondary subneural clefts and focal areas of negative AChE activity contribute to the paler staining of the P end-plates.     

Characterization of 125I-Lysergic Acid Diethylamide Binding to Serotonin Receptors in Rat Frontal Cortex

Acetylcholinesterase (AChE) is found both in motor end-plate (MEP)-free and MEP-rich regions of rat or mouse muscle. We studied the developmental aspects of the localization of asymmetric 16S AChE in both regions of the sternocleidomastoid muscle, which has a well-defined zone of motor innervation. In the rat, the proportion of 16S AChE to total AChE increases in the MEP-rich region, and becomes significantly higher than in the MEP-free regions between the first and the second weeks after birth. In the mouse, at birth, the MEP-rich region already has a higher relative content in 16S AChE than the MEP-free regions. Total 16S AChE amounts increase during postnatal development, not only in the MEP-rich region but also in the MEP-free regions. Thus, 16S AChE is not eliminated from MEP-free regions during muscle maturation and growth. Two distinct pools of 16S AChE are distinguished in the muscles, both of which increase during postnatal development: junctional and background 16S AChE.     

Extrasynaptic accumulations of acetylcholinesterase in the rat sternocleidomastoid muscle after neonatal denervation. Light and electron microscopic localization and molecular forms

Denervated neonatal rat sternocleidomastoid muscle has decreased levels of total AChE when compared to control muscle. Denervated versus control values of total muscle AChE present a three-phase curve in function of time after denervation. There is a rapid initial fall 0-3 days after denervation, an increase during about 2 weeks, then again a decrease in total AChE. Thus, there is a transitory net accumulation of AChE after the initial fall of activity in denervated developing muscle. Extrasynaptic areas of high AChE activity develop between 1 and 2 weeks after denervation and remain visible up to 1 month after denervation before vanishing. An electron microscope study shows that these accumulations are internal to the muscle fiber, close to a limited number of muscle nuclei and associated to the sarcoplasmic reticulum and nuclear envelope, but not to the T-tubule system. As found in adult rat muscle, the initial fall in AChE affects first the 16 S AChE form, and soon after, the 4 S and 10 S AChE forms. A main difference with adult muscle is the sudden increase and predominance over other forms of 10 S AChE 2 weeks after denervation at birth. Later, the decrease in AChE affects 16 S and 4 S AChE before 10 S AChE. The regions rich in extrasynaptic sites of AChE accumulation possess a very high proportion of 10 S AChE. Thus, the mechanisms of biosynthesis, intracellular transport and/or secretion of AChE may be very different in young, developing muscle compared to adult muscle.     

Disease expression in +-/+- ----mdg/mdg mouse chimeras: evidence for an extramuscular component in the pathogenesis of both dysgenic abnormal diaphragm innervation and skeletal muscle 16 S acetylcholinesterase deficiency

Homozygous mdg/mdg mice die at birth and express a syndrome of abnormalities, the most striking of which is a gross failure of skeletal muscle development. Recently, additional abnormalities in the development of nerve-muscle relationships have been recognized; in particular, on muscle fibers within the diaphragm, motor end plates are inappropriately dispersed and, in all muscles, there is a paucity of the 16 S form of acetylcholinesterase (AChE). These abnormalities could result entirely as secondary consequences of the primary muscle defect or from expression of the mdg defect in additional cell types, e.g., motor neurons. To determine if the muscle genotype alone is responsible for these defects in dysgenic mice, chimeras composed of both dysgenic and normal cells have been investigated. Different glucosephosphate isomerase variants existed in the mdg/mdg and normal cells comprising these chimeras and the mutant, normal, or mosaic genotypes of chimera diaphragm and skeletal muscle was estimated by measuring the relative proportions of each isozyme. In two chimeras, the diaphragm innervation pattern was revealed by AChE cytochemistry and in both, discrete regions of abnormally dispersed and normally restricted motor end-plate zones were observed. No correlation between these patterns of innervation and the assessed genotype of the muscle fibers existing in each area was observed. The relative 16 S AChE content in the limbs of four chimeras was found to range from 2.5 to 42.0%. Here also, no correlation between 16 S AChE content and the muscle genotype was observed. The results of these investigations are not consistent with a model of mdg/mdg pathogenesis in which only the skeletal muscle is primarily affected; an extramuscular deficiency responsible for at least part of the full mdg/mdg syndrome is therefore suggested.     

Attachment of acetylcholinesterase to structures of the motor endplate.

The kinetics of AChE solubilization from intact motor endplates of mouse diaphragm, by collagenase, papain and hyaluronidase, was studied in parallel with the ultrastructural localization of AChE in treated neuromuscular junctions. Hyaluronidase did not solubilize more AChE from isolated motor endplate regions than Ringer's solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached. Less than 35% of junctional AChE is left after collagenase, and less than 20% after papain treatment, as estimated by the percentage of AChE activity left in the isolated endplate region of the diaphragm after protease treatment. Cytochemically, both proteases had a similar effect on postsynaptic AChE. Residual AChE activity was distributed randomly, adhering to the sarcolemma of junctional clefts. Presynaptic AChE localized in the gap between axon terminal and Schwann cell appears to be resistant to collagenase but not to papain treatment. The mode of AChE attachment or the composition of the intercellular material in this gap may differ from that of the primary and secondary clefts.     

Attachment of acetylcholinesterase to structures of the motor endplate

Summary The kinetics of AChE solubilization from intact motor endplates of mouse diaphragm, by collagenase, papain and hyaluronidase, was studied in parallel with the ultrastructural localization of AChE in treated neuromuscular junctions. Hyaluronidase did not solubilize more AChE from isolated motor endplate regions than Ringer's solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached. Less than 35% of junctional AChE is left after collagenase, and less than 20% after papain treatment, as estimated by the percentage of AChE activity left in the isolated endplate region of the diaphragm after protease treatment. Cytochemically, both proteases had a similar effect on postsynaptic AChE. Residual AChE activity was distributed randomly, adhering to the sarcolemma of junctional clefts. Presynaptic AChE localized in the gap between axon terminal and Schwann cell appears to be resistant to collagenase but not to papain treatment. The mode of AChE attachment or the composition of the intercellular material in this gap may differ from that of the primary and secondary clefts.     

Stimulated Release of Phosphatidylinositol Phosphodiesterase in an Isolated Phrenic Nerve-Diaphragm Preparation

Electrical stimulation of the phrenic nerve in an isolated nerve-diaphragm preparation resulted in the release of phosphatidylinositol phosphodiesterase into the organ bath. The released enzyme was Ca2+-dependent and exhibited two pH optima. The enzyme was released in response to nerve stimulation even in the presence of d-tubocurarine in concentrations that block neuromuscular transmission, and was not therefore released from the muscle as a consequence of its contractile activity. Phosphatidylinositol phosphodiesterase activity was determined in the soluble cytosol fractions prepared from different regions of skeletal muscles and from normal peripheral nerves and nerves that were degenerating after transection. The specific activity of the enzyme in the cytosol from the endplate-rich region of the diaphragm was significantly greater than that in cytosol from either the endplate-free region of the diaphragm or from the phrenic nerve. In degenerating nerve the activity of the enzyme was greater in the distal stump than in the proximal stump at 36 h after nerve section. Possible roles for released phosphatidylinositol phosphodiesterase at the neuromuscular junction are discussed.     

Comparison of changes in AChE activity in the brain of the laboratory rat after soman and tabun intoxication

The aim of this study was to compare changes in activity of acetylcholinesterase (AChE) in the brain and motor endplates of rat after administration of soman and tabun. We took brain and diaphragm from laboratory rats administered a median lethal dose (LD(50)) of soman or tabun. Enzyme activity of AChE was studied in selected structures of brain and in motor endplates in the diaphragm. Histochemical detection of AChE by Karnovski and Roots with simultaneous histochemical detection of alkaline phosphatase in case of brain sections was used. The highest activity of AChE in the control group was found in the striatum, amygdaloid nuclei, substantia nigra, superior colliculi, and motor nuclei of cranial nerves V, X a XII. LD(50) of both nerve agents dramatically decreased the activity of AChE in the structures studied--both brain and diaphragm. After intoxication by either agent, activity in above mentioned nuclei was characterized as low or focally moderate. Very low activity was seen in some structures (CA3 field of hippocampus, some nuclei of the tegmentum and cerebellar cortex). We found minimal differences in the histochemical picture of soman or tabun intoxication, apart from the striatum and the superior colliculi which showed stronger inhibition by tabun.     

Difference in the ability of neonatal and adult denervated muscle to accumulate acetylcholinesterase at the old sites of innervation

In adult rat sternocleidomastoid muscle, AChE is concentrated in the region rich in motor end-plates (MEP). All major AChE forms, "16 S," "10 S," and "4 S," are accumulated at high levels, and not only "16 S" AChE. After denervation, muscle AChE decreases; 2 weeks after denervation, low levels (20-40% of control) are reached for all forms. During the following weeks, a slow but steady increase in "10 S" and "16 S" AChE occurs in the denervated muscle. At this stage, all forms are again observed to be highly concentrated in the region containing the old sites of innervation. Thus, in adult rat muscle the structures able to accumulate "16 S," "10 S," and "4 S" AChE in the MEP-rich regions remain several months after denervation. In normal young rat sternocleidomastoid muscle at birth, all AChE forms are already accumulated in the MEP-rich region. After denervation at birth, the denervated muscle loses its ability to keep a high concentration of "4 S," "10 S," and "16 S" AChE in the old MEP-rich region. All AChE forms are still present 1 month after denervation, but they are decreased and diffusedly distributed over the whole length of the muscle. In particular, "16 S" AChE is detected in the same proportion (10-15%) all along the denervated muscle. Thus, the diffuse distribution of AChE, and especially "16 S" AChE, after neonatal denervation, contrasts with the maintained accumulation observed in adult denervated muscle. It seems that denervation of young muscle results in a specific loss of the muscle ability to concentrate high levels of all AChE forms at the old sites of innervation.     

Rapid changes in levels of individual molecular forms of acetylcholinesterase after denervation of mouse sternocleidomastoid muscle

Experimental denervation of adult mouse sternocleidomastoid muscle results in a decrease in total AChE. The most rapid change essentially affects the tailed, asymmetric 16 S AChE, since one day after nerve section, “16S” AChE is already significantly decreased to about 70% of its control value. We found that both background and junctional “16S” AChE are affected by this rapid decrease. Later, a sharp fall in “10S” and “4S” AChE occurs about seven days after denervation when muscle atrophy develops with loss of weight and proteins. A gaussian analysis of the sedimentation profiles of AChE extracted from denervated muscle shows that there is not only an early rapid decrease in 16 S AChE but also a decrease in the monomeric 3.3S AChE. This result suggests that there is a very rapid turn-over of two molecular forms of AChE, the supposedly monomeric precursor and the complex asymmetric 16S AChE.     

Developmental regulation of 16S acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line

We have studied the appearance, distribution and regulation of acetylcholinesterase (AChE) and acetylcholine receptors (AChRs) in a mouse skeletal muscle cell line (C2), that was originally isolated and described by Yaffe & Saxel [54]. In culture, cells from this line form spontaneously contracting myotubes, with overshooting action potentials that are TTX-sensitive. After fusion of myoblasts into myotubes, there was a dramatic increase in the amount of both AChE and AChR. Three forms of AChE, distinguished by their sedimentation on sucrose gradients, were synthesized: 4-6S, 10S, and 16S. The 4-6S and 10S forms appeared 1 day after the cells began to fuse, whereas the 16S form appeared only 2 days after fusion began. Maximal levels of the 16S AChE form (25-30% of the total) were obtained by reducing the concentration of horse serum in the fusion medium. Prevention of myoblast fusion by reducing the calcium levels in the medium decreased the total AChE by 70%, and only the 4-6S form was synthesized. Blocking spontaneous contractile activity of the myotubes by tetrodotoxin (TTX) led to a 50% reduction in all three esterase forms. Thus, the 16S, or endplate form of AChE is not specifically regulated by electrical or contractile activity in the C2 cell line. After fusion the number of AChRs increased rapidly for 3-4 days and then stabilized. Receptor clusters, ranging from 10-30 micron in length, appeared 1 day after myoblast fusion began. When cells were grown in medium containing reduced Ca2+, the total number of AChRs was decreased by 20-50%. Reduction of Ca2+ after myotubes and AChR clusters had formed resulted in dispersal of AChR clusters. Inhibition of muscle contractions with TTX did not affect the number of AChRs or their distribution.     

Bidirectional axonal transport of 16S acetylcholinesterase in rat sciatic nerve

Axonal transport of the 16S Molecular form of acetylcholinesterase (16S-AChE) in doubly ligated rat sciatic nerves was studied by means of velocity sedimentation analysis on sucrose gradients. This form of AChE was selectively confined to motor, and not to sensory, fibers in the sciatic nerve, where it represented 3--4% of total AChE. Its activity increased linearly with time (4--20 hr) in nerve segments (7 mm) proximal to the central ligature (4.5 mU/24hr) and distal to the peripheral ligature (2.0 mU/24 hr). From the linear rates of accumulation of 16S-AChE, we conclude that the enzyme is conveyed by anterograde and retrograde axonal transport at velocities close to those previously defined for the movement of total AChE (410 mm/day, anterograde; 220 mm/day, retrograde). The transport of AChE molecular forms, other than the 16S form, could not be resolved presumably due to their presence in blood as well as at extraaxonal sites. The present findings are consistent with the view that in rat sciatic nerve most, if not all, of the small portion of total AChE (approximately 3%) which is transported may be accounted for by 16S-AChE.     

Recovery of Acetylcholinesterase in the Diaphragm, Brain, and Plasma of the Rat After Irreversible Inhibition by Soman: A Study of Cytochemical Localization and Molecular Forms of the Enzyme in the Motor End Plate

Abstract Recovery of AChE activity in the motor end plate region and end plate free region of the rat diaphragm was studied after irreversible inhibition by soman. Recovery was slow during the first 2 days and only 4 S and 10 S molecular forms of AChE were present in the end plate region. However, cytochemical evidence indicates that synaptic AChE has already started to accumulate and that the synthesis of AChE in muscle and Schwann cell might even be enhanced. Tubular structures, observed underneath the motor end plate, may serve to transport the enzyme from its sites of synthesis in the sarcoplasmic reticulum. Asymmetric molecular forms of AChE in the end plate region appeared later during recovery and, one week after poisoning, their activity was only about 50% of normal value. The limited ability of newly synthesized AChE to attach to the subcellular structures and, therefore, to be retained in the muscle, may explain the phase of slow recovery. In accordance with this view, AChE activity in brain recovered in a similar way as in muscle, whereas soluble plasma cholinesterases recovered faster, apparently without a slow initial phase.     

SUBCELLULAR ANALYSIS OF THE MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN RAT SKELETAL MUSCLE

Acetylcholinesterase (AChE, EC 3.1.1.7) activity of rat gastrocnemius muscle homogenized in 1 M-NaCl and 0.5% Triton X-100 was separated by velocity sedimentation in sucrose gradients into three molecular forms with sedimentation coefficients of about 4S, 10S and 16S. The distribution of homogenate AChE activity among the three peaks was 53, 34 and 13% respectively. The different molecular forms were found to be heterogeneously distributed in subcellular fractions prepared from sucrose homogenates of muscle, as follows: Subfractions of the crude sarcolemmal fraction were prepared by discontinuous sucrose gradient centrifugation. AChE was recovered in the greatest yield and with the highest specific activity in a light density subfraction (0.6/0.8 M-sucrose interface). The AChE activity in this light density subfraction was mainly (81-88%) the 10S form of the enzyme. The velocity sedimentation profiles of the AChE activity in the more dense subfractions were markedly different in that 16S AChE was a major component.     

Atypical Distribution of Asymmetric Acetylcholinesterase in Mutant PC12 Pheochromocytoma Cells Lacking a Cell Surface Heparan Sulfate Proteoglycan

We studied the distribution of the molecular forms of acetylcholinesterase (AChE) in a stable variant (F3) of the rat pheochromocytoma cell line, PC12, that lacks a heparan sulfate proteoglycan on the cell surface. After treatment with nerve growth factor F3 cells synthesize less 4S enzyme, and more 10S and 16S enzyme than normal PC12 cells. This distribution is similar to that seen in normal cells after incubation with beta-D-xylosides, molecules that interfere with proteoglycan assembly. Using collagenase treatment and membrane-permeable and -impermeable inhibitors of AChE, we determined the cellular location of the AChE forms. Although in normal cells greater than 90% of the 16S AChE is on the cell surface, approximately 60% is present in an internal pool in the variant. Following irreversible inhibition of all forms of AChE in the variant, the newly synthesized 16S AChE appears in the internal pool after a 1-h lag, but is not detected on the cell surface until after 2.5 h. Our results thus show that 16S AChE is assembled internally within neuronal cells and that alterations in the synthesis and distribution of proteoglycans affect the total amount and cellular localization of the 16S AChE form.