Demonstration of acetylcholinesterase molecular forms in a continuous tubular lysosomal system of rat pancreatic acinar cells.

By applying the highly sensitive cytochemical Gautron's technique, we were able to reveal AChE activity in rat pancreatic acinar cells, particularly at the level of a complex membrane-bound network formed by tubules with varicosities located around the nuclei and close to the basolateral membrane. The Golgi apparatus was devoid of cytochemical reaction beside the trans-Golgi network cisternae, which showed a positive reaction. The RER of some acinar cells also presented a signal, demonstrating their capability of synthesizing AChE. Immunogold using a specific anti-AChE antibody yielded similar results. Double-labeling experiments corroborated the presence of enzyme cytochemical and immunocytochemical signals in the same lysosomal tubular network. Biochemical sedimentation assays confirmed the presence of AChE in acinar cells, which exists as two globular molecular forms, G(1) and G(4). These results were obtained with pancreatic tissue in situ as well as with isolated acinar cells maintained in culture and devoid of neural elements. The existence of a continuous tubular lysosomal network containing AChE is in agreement with previous reports on acinar and other cell types, and supports a more general hypothesis on dynamic continuities among cell structures. Whether AChE is being secreted by the acinar cells or internalized through this endo-lysosomal system was not defined. However, the capability of the acinar cells to synthesize AChE and to channel it through a tubular system is a good indication that the cells can modulate their cholinergic stimulation for optimal secretion of digestive enzymes.

Fatigability of rat hindlimb muscles after acute irreversible acetylcholinesterase inhibition.

The purpose of this study was to investigate the functional impact of acute irreversible inhibition of acetylcholinesterase (AChE) on the fatigability of medial gastrocnemius and plantaris muscles of Sprague-Dawley rats. After treatment with methanesulfonyl fluoride (a lipid-soluble anticholinesterase), which reduced their AChE activity by >90%, these muscles were subjected to an in situ indirect stimulation protocol, including a series of isolated twitch and tetanic contractions preceding a 3-min fatigue regimen (100-ms trains at 75 Hz applied every 1.5 s). During the first minute of the fatigue regimen, the effects of AChE inhibition were already near maximal, including marked reductions in peak tension and the force-time integral (area), as well as a decrement of compound muscle action potential amplitudes within a stimulus train. Neuromuscular transmission failure was the major contributor of the force decreases in the AChE-inhibited muscles. However, despite this neuromuscular transmission failure, muscles of which all AChE molecular forms were nearly completely inhibited were still able to function, although abnormally, during 3 min of intermittent high-frequency nerve stimulation.

Regulation of gene expression by trans-synaptic activity: a role for the transcription factor NF-kappa B.

Earlier studies in the sympathetic ganglion have led to the proposal that adaptation of transcription to trans-synaptic activity is controlled by a signal transduction pathway featuring a transcription factor which translocates to the nucleus upon its release from the post-synaptic membrane by after-hyperpolarization. In light of recent progress, it is proposed here that NF-kappa B constitutes the postulated transcription factor.

Fast and slow skeletal muscles express a common basic profile of acetylcholinesterase molecular forms.

Recent evidence suggests that the high content of acetylcholinesterase (AChE) globular form G4, characteristic of fast muscles, is controlled by phasic high-frequency activity performed by these muscles. This indicates that inactive, though still innervated, fast muscles should be devoid of their characteristic G4 pool. Accordingly, in the absence of phasic activity, both fast and slow muscles should exhibit a common basic profile of AChE molecular forms of the slow type. We first tested this hypothesis by examining the AChE content in cultures of myotubes obtained from the fusion of satellite cells originating from fast and slow muscles. These two cell populations produced AChE molecular-form profiles of the slow type characterized by modest levels of G4 together with an increased proportion of the asymmetric forms A8 relative to A12. Second, we determined the impact of muscle paralysis on the specific content of AChE molecular forms of adult rat fast and slow muscles. Complete paralysis of hindlimb muscles was achieved by chronic superfusion of tetrodotoxin (TTX) onto the sciatic nerve. Ten days after TTX inactivation, the distributions of AChE molecular forms of both fast extensor digitorum longus (EDL) and plantaris muscles were transformed into ones resembling the slow soleus, the latter showing no significant modifications in its AChE profile. Finally, we investigated the impact of nerve-mediated phasic high-frequency stimulation of TTX-inactivated fast and slow muscles on the content of AChE molecular forms. This stimulation produced a profile of AChE molecular forms similar to that observed in control EDL muscles, indicating that phasic activation counteracted the TTX-induced transformation in the distribution of AChE molecular forms in fast EDL muscles. Together, these results are consistent with the proposal that adult fast muscles constitutively express a basic profile of AChE molecular forms of the type displayed by slow muscles, onto which varying levels of G4 are added according to the amount of phasic activity performed by the muscles.

Diffuse transmission by acetylcholine in the CNS.

Recent immunoelectron microscopic studies have revealed a low frequency of synaptic membrane differentiations on ACh (ChAT-immunostained) axon terminals (boutons or varicosities) in adult rat cerebral cortex, hippocampus and neostriatum, suggesting that, besides synaptic transmission, diffuse transmission by ACh prevails in many regions of the CNS. Cytological analysis of the immediate micro-environment of these ACh terminals, as well as currently available immunocytochemical data on the cellular and subcellular distribution of ACh receptors, is congruent with this view. At least in brain regions densely innervated by ACh neurons, a further aspect of the diffuse transmission paradigm is envisaged: the existence of an ambient level of ACh in the extracellular space, to which all tissue elements would be permanently exposed. Recent experimental data on the various molecular forms of AChE and their presumptive role at the neuromuscular junction support this hypothesis. As in the peripheral nervous system, degradation of ACh by the prevalent G4 form of AChE in the CNS would primarily serve to keep the extrasynaptic, ambient level of ACh within physiological limits, rather than totally eliminate ACh from synaptic clefts. Long-lasting and widespread electrophysiological effects imputable to ACh in the CNS might be explained in this manner. The notions of diffuse transmission and of an ambient level of ACh in the CNS could also be of clinical relevance, in accounting for the production and nature of certain cholinergic deficits and the efficacy of substitution therapies.

Acetylcholinesterase adaptation to voluntary wheel running is proportional to the volume of activity in fast, but not slow, rat hindlimb muscles.

Chronic enhancement of neuromuscular activity by forced exercise training programmes results in selective adaptation of the G4 acetylcholinesterase (AChE) molecular form in hindlimb fast muscles of the rat, with only minor and non-selective AChE changes in the soleus. In order to shed further light on the physiological significance of this G4 adaptation to training, we turned to a voluntary exercise model. The impact of 5 days and 4 weeks of voluntary wheel cage running on AChE molecular forms was examined in four hindlimb fast muscles and the slow-twitch soleus from two rat strains. Inbred Fisher and Sprague-Dawley rats, placed in live-in wheel cages, exercised spontaneously for distances which progressively increased up to an average of approximately 3 and 18 km/day, respectively, by the end of week 4. Fast muscles responded to this voluntary activity by massive G4 increases (up to 420%) with almost no changes in A12, so that by week 4 the tetramer became the main AChE component of these muscles. The additional G4 was composed primarily of amphiphilic molecules, suggesting a membrane-bound state. The G4 content of fast muscles was highly correlated with the distance covered by the rats during the 5 days before they were killed (r = 0.850-0.879, P < 0.001 in three muscles). The soleus muscle, in turn, responded to wheel cage activity by a marked selective reduction of its asymmetric forms--up to 45% for A12. This A12 decline, already maximal by day 5 of wheel cage running, showed no relationship with the distance covered.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle acetylcholinesterase adapts to compensatory overload by a general increase in its molecular forms.

We have investigated the impact of compensatory overload on the content of acetylcholinesterase (AChe) molecular forms in the rat fast-twitch medial gastrocnemius (MG). Overload was induced by way of a bilateral tenotomy of the MG's functional synergists coupled to a daily walking training program (15 m/min, 30% incline, up to 60 min per session, 12-18 wks). This latter condition ensured that the MG were used on a regular basis. In comparison to control values, overloaded MG showed 25 and 19% increases (P less than 0.05) in muscle wet weight and protein concentration, respectively. The content in AChe (activity per muscle) was also increased in these MG (28%, P less than 0.05). Sedimentation analyses revealed a general elevation in the content of AChe molecular forms, with A8, G2, and G1 displaying significant changes (35-42%, P less than 0.05). In a second group of rats, daily running training (27 m/min, 30% incline, using the same timetable) was supplemented to the compensatory overload. In this group, the additional running training led to a greater hypertrophic response as attested to by increases (P less than 0.05) in the MG wet weight (41%) and protein concentration (35%) in comparison to controls. However, total AChe content of these muscles was increased to an extent similar to that observed in the MG subjected only to compensatory overload (24%, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Swimming training increases the G4 acetylcholinesterase content of both fast ankle extensors and flexors.

The effect of endurance swimming training on AChE molecular forms was examined in 2 groups of functionally antagonist rat muscles, including ankle extensors and flexors. This exercise regimen, which entails predominant dynamic activity (i.e., involving extensive shortening) of both groups of muscles, resulted in marked selective G4 increases in all fast muscles. The G4 elevation exhibited by the ankle flexors was in sharp contrast to the G4 reduction reported in these same muscles following running training, during which their action is predominantly tonic. The results strengthen the conclusion that predominantly dynamic activity increases the G4 content of mature innervated fast muscles.

Regulation by exercise of the pool of G4 acetylcholinesterase characterizing fast muscles: opposite effect of running training in antagonist muscles.

Fast muscles of rodents characteristically differ from their slow-twitch counterparts by exhibiting high levels of G4, i.e., the tetrameric acetylcholinesterase (AChE) molecular form. Converging evidence suggests that this additional G4 pool is specifically regulated by the type of activity actually performed by the muscle. This hypothesis was tested by studying the effect of a chronic increase in neuromuscular activity on the AChE content and distribution of molecular forms of functionally antagonist rat hindlimb muscles. They included the fast ankle extensors gastrocnemius (GAST) and plantaris (PL), the fast ankle flexors tibialis anterior (TA) and extensor digitorum longus (EDL), as well as the slow-twitch soleus (SOL). Neuromuscular activity was enhanced by subjecting the rats to a 12-week training program consisting of repeated sessions of prolonged endurance running on a rodent treadmill. This exercise regimen preferentially affected the G4 pool characterizing fast muscles which exhibited marked and opposite changes according to the functional role of the muscles. The amount of G4 was increased by more than 50% in the ankle extensors GAST and PL, which play a dynamic role, and reduced by about 40% in the ankle flexors TA and EDL, which exhibit a predominant tonic activity during running. The asymmetric forms A12 and A8 were slightly elevated in the fast muscles. In the case of the slow-twitch SOL, running training resulted in a small, nonspecific decrease in AChE content which affected most of the molecular forms. These data indicate that the size of the G4 pool characteristic of fast muscles depends on the type, dynamic or tonic, of activity actually performed. The present results support the conclusion that this G4 pool fulfills a specific and essential function, distinct from that of A12.

Localization of the pool of G4 acetylcholinesterase characterizing fast muscles and its alteration in murine muscular dystrophy.

The distributions of acetylcholinesterase and its molecular forms within muscles of normal and dystrophic 129/ReJ mice were established by a concomitant cytochemical and biochemical study performed on 1-mm serial sections of three predominantly fast muscles, i.e., anterior tibialis, extensor digitorum longus, and sternomastoid, as well as the slow-twitch soleus. This comparative study showed the following main findings. 1) In every muscle of both normal and dystrophic mice a) the three asymmetric forms were confined to the motor zone where they systematically codistributed with the endplates, and b) all globular forms, including G4, were concentrated at the motor zone from which they extended over the entire muscle length along a concentration gradient. 2) In the normal muscles, the perijunctional sarcoplasmic cytochemical reaction exhibited by individual fibers was grouped into a well-defined cojunctional acetylcholinesterase compartment in which the endplates were embedded. The overall intensity of the cojunctional cytochemical reaction was either high or low according to whether the muscle was predominantly fast or slow. 3) This cojunctional acetylcholinesterase compartment varied in close parallelism with G4 and thus appeared as the cytochemical correlate of the G4 molecules concentrated around the endplates. In particular, as the shape of the motor zone progressively increased in complexity along with the intricacy of the muscle fiber organization, from sternomastoid to extensor digitorum longus to anterior tibialis, so did both the relative volume occupied by the cojunctional acetylcholinesterase compartment and the proportion of G4. 4) The motor zone of the normal fast-twitch muscles characteristically differed from that of the soleus by the presence of a G4-rich environment around the endplates, which was cooperatively provided by the surrounding fibers. 5) In dystrophic muscles, this cojunctional G4-rich compartment was lost: the cojunctional cytochemical compartment was no longer discernable, while G4 was reduced to a minimal low level similar to that characteristic of the normal soleus.

Role of hyperpolarization generated by the Na+ - K+ pump in the trans-synaptic induction of RNA synthesis in sympathetic neurons.

1. The mechanism which enables activation of the nicotinic receptors to modify the synthesis of RNA was investigated in the incubated superior cervical ganglion of the rat. RNA labelling with [5-3H] uridine was used in order to screen the effects of varying the ionic environment and altering Na+ channels on the sequence of the three changes in ganglionic RNA synthesis induced by stimulation, i.e. a) an initial decrease, b) an increase during the late stages of stimulation, c) another increase taking place after the end of stimulation. These successive variations were obtained by either repetitive excitation of the preganglionic nerve, or application of ACh or carbachol to the ganglion. 2. The three changes in RNA synthesis mediated by ACh or carbachol were prevented when the KCl concentration of the medium was increased up to 37 mM or when NaCl of the medium was replaced with Tris or sucrose. This confirmed previous indications that the sequence of activity-induced changes is initiated by the transmembrane ionic fluxes mediated by nicotinic activation and not by depolarization per se. 3. Application of aconitine to resting ganglia for 1 h decreased the RNA synthesis to the same extent as a 1 h preganglionic or ACh stimulation. This effect was prevented by a concomitant application of tetrodotoxin (TTX) which also restored the ability of carbachol to modify RNA synthesis. This suggested that the initial decrease in RNA synthesis is caused by the increase in [Na+]i which seems to interfere directly with the transcription process. 4. The increase of RNA synthesis occurring during the late stages of synaptic activation was selectively inhibited by replacing Cl- of the medium with SO4-. On the other hand, the post-stimulation increase was selectively inhibited when the generation of after-hyperpolarization resulting from the electrogenic extrusion of Na+ was prevented by substituting LiCl for NaCl. This indicated that increases in RNA synthesis during and after the stimulation are triggered by different ionic events. 5. An induction of RNA synthesis was obtained, without previous activation of the nicotinic receptors, by incubating the ganglia at rest in conditions which entail the generation of an hyperpolarization resulting from the activation of the Na(+)-K- pump, i.e. low external KCl as well as application of TTX following an aconitine treatment. However, in these cases, the increase in RNA synthesis was delayed by about 2 h as compared to that observed after the end of nicotinic activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous labelling of basal lamina components and acetylcholinesterase at the neuromuscular junction.

A double labelling technique has been developed which permits the concomitant localization of basal lamina constituents together with acetylcholinesterase in mouse skeletal muscles. First, using the protein A-gold technique, type IV collagen and laminin were revealed on basal laminae ensheathing skeletal muscle fibres. The immunolabelling for both proteins was higher in synaptic than extrasynaptic regions. At synaptic sites the anti-type IV collagen immunolabelling exhibited an asymmetry; it was more intense on the portion of basal lamina closest to the postsynaptic membrane, whereas the anti-laminin immunolabelling was more uniformly distributed. It was also observed that the laminin immunoreactivity associated with Schwann and perineural cells was higher than that of skeletal muscle fibres. Secondly, the two basal lamina antigens were revealed simultaneously with another synaptic protein, acetylcholinesterase, using a refined cytochemical technique prior to the immunolabelling. The cytochemical reaction, which facilitates the location of endplates, did not alter the immunolabelling pattern. This double labelling procedure permits ready comparison of the distributions of type IV collagen and laminin with that of acetylcholinesterase, and may prove to be a useful approach in studies on synaptic components in developing and diseased muscle.

Decreased G4 (10S) acetylcholinesterase content in motor nerves to fast muscles of dystrophic 129/ReJ mice: lack of a specific compartment of nerve acetylcholinesterase?

Acetylcholinesterase (AChE; EC 3.1.1.7) activity and the distribution of its molecular forms were studied in the nervous system of normal and dystrophic 129/ReJ mice, including the sciatic-tibial nerve trunk and motor nerves to slow- and fast-twitch muscles. In normal mice, motor nerves to the slow-twitch soleus exhibited a low AChE activity together with a low level of G4 (10S form) as compared with nerves of the predominantly fast-twitch plantaris and extensor digitorum longus. In contrast, in dystrophic mice, the AChE activity as well as the G4 content of nerves to the fast-twitch muscles were low, displaying an AChE content similar to that of the nerve of the soleus muscle. In the sciatic-tibial nerve trunk, the AChE activity decreased along the nerve in an exponential mode, at rates that were similar in both conditions. However, in dystrophic mice, the AChE activity was reduced throughout the nerve length by a constant value of approximately 180 nmol/h/mg protein. Further analyses indicated that AChE in this nerve trunk was distributed among two compartments, a decaying and a constant one. The decay involved exclusively the globular forms. The activity of A12 (16S form) remained constant along the nerve and was similar in both normal and dystrophic mice. In addition, according to the equation describing the decay of AChE, the reduction in enzymatic activity observed in the dystrophic mice affected mainly G4 in the constant compartment. Brain, spinal cord, sympathetic ganglia, and serum, which were also examined, showed no remarkable differences between the two conditions in their G4 content. The AChE abnormalities that we found in nervous tissues of 129/ReJ dystrophic mice were confined to the motor system.

Asymmetric and globular forms of AChE in slow and fast muscles of 129/ReJ normal and dystrophic mice.

Acetylcholinesterase activities and molecular forms were studied in normal and dystrophic 129/ReJ mice, focusing on four predominantly fast-twitch muscles and the slow-twitch soleus. The asymmetric and globular forms were analyzed separately so that the effect of dystrophy on each form could be determined. This comparative study showed the following. (1) In the normal condition, each muscle exhibited a distinct distribution of the molecular forms. (2) The diversity among the fast muscles resulted mainly from variations in the proportions of the three globular forms; in contrast, these muscles showed a constant and precise A12/A8/A4 ratio. (3) The slow-twitch soleus clearly differed from the other muscles in its low acetylcholinesterase activity and distinct distribution of the molecular forms, characterized by a low level of G4 and a peculiar ratio among its asymmetric forms, resulting from a relative increase of the A8 and A4 forms. (4) In dystrophic mice, the diversity of the acetylcholinesterase distribution was lost; all the fast muscles displayed profiles exhibiting the characteristics typical of the soleus. The fast-twitch extensor digitorum longus, sternomastoid, and plantaris converged towards an identical set of acetylcholinesterase molecules. (5) In contrast, the acetylcholinesterase activity and molecular forms of the soleus were only slightly affected by the disease. These results reveal that the dystrophy modifies both categories of molecular forms of acetylcholinesterase in a very precise manner. Such complex changes, which are highly reproducible in a variety of different muscles, are unlikely to result from nonspecific reactions secondary to the disease.

Acetylcholinesterase content in both motor nerve and muscle is correlated with twitch properties.

The activity and molecular forms of acetylcholinesterase (AChE) were studied in the rat soleus muscle and its nerve, as compared to their fast-twitch counterparts. The soleus muscle and its nerve exhibited both significantly lower AChE activity and less of the G4 (10S) molecular form. In addition, the soleus muscle displayed a specific increase in the A8 (13S) and A4 (8.8S) asymmetric forms, not seen in any of the fast-twitch muscles examined. These results indicate that the AChE content of a muscle and its nerve are linked and depend on the twitch properties, and that the slow-twitch muscle is characterized by a specific set of AChE molecular forms.

Correlation between the acetylcholinesterase content in motor nerves and their muscles.

A comparative study of the activity and distribution of the molecular forms of acetylcholinesterase (AChE) in slow and fast-twitch systems was performed in normal and dystrophic 129/ReJ mice and in rats. As compared to its fast-twitch counterparts, the soleus muscle of the normal mouse as well as of the rat showed a distinct content in AChE characterized by a slow activity, a low proportion in G4 and a relative increase in the A8 form. In the dystrophic condition, the fast muscles exhibited an AChE content typical of the soleus muscle, in particular low G4 and a relative increase in A8. In contrast, the soleus muscle was only slightly affected by the disease. The soleus nerve in the normal mouse and rat showed a significant reduction in both total AChE activity and G4 content as compared to nerves of fast-twitch muscles. In the dystrophic mouse, the AChE content of the nerves to fast muscles was similar to that of the soleus nerve in the normal condition. These results reveal a strict correlation between the AChE content in both nerve and muscle, and the twitch properties. The results obtained in dystrophic mice suggest that neural AChE is the determinant factor in this relationship. A mechanism by which neural AChE may regulate muscle AChE is proposed and discussed in the light of recent advances on the neuromuscular junction.

"Nonspecific" cholinesterase and acetylcholinesterase in rat tissues: molecular forms, structural and catalytic properties, and significance of the two enzyme systems.

"Nonspecific" cholinesterase (acylcholine acylhydrolase; EC 3.1.1.8) from various rat tissues has been found to exist in several stable molecular forms that appear as exact counterparts of molecular forms of acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7). The sedimentation pattern of cholinesterase was similar to that of acetylcholinesterase with a small but significant shift between the sedimentation coefficients of the corresponding forms. Extraction yields in different media also demonstrated a close parallelism between the two enzyme systems. Other properties, such as thermal stability and catalytic characteristics, indicated both differences and similarities. In spite of the structural resemblance implied by their physicochemical properties, cholinesterase did not crossreact with antibodies against acetylcholinesterase. The nature of the relationships revealed by these studies and their bearing on the physiological significance of cholinesterases are discussed.