Muscle activity pattern regulates postnatal development of acetylcholinesterase molecular forms in normal mice and mice with motor endplate disease

We have studied the relative contributions of muscle activity and nerve-supplied materials to the regulation of AChE molecular forms during postnatal development of muscles in normal mice and in mice with motor endplate disease (med mice). Onset of this hereditary disease causes a progressive failure of evoked release of ACh from the motor neuron, which prevents contraction in muscles such as biceps and soleus. In these innervated but inactive muscles, one can examine the consequences of inactivity on the distribution of AChE forms. In normal mouse biceps the distribution of AChE forms, as shown by sucrose-gradient analysis, change substantially after birth; the most dramatic alteration is an increase in G4 AChE from 15 to 45% of total AChE during the third postnatal week. AChE profiles in normal or med biceps are indistinguishable until 10-12 d after birth, but the changes in distribution of AChE forms does not occur in med biceps nor in normal biceps denervated 2 weeks after birth. In contrast, the distributions of AChE forms in a predominantly slow muscle, the soleus, are similar in med and normal mice both early (10 d) and late (20 d) in the course of the disease, and the distributions are affected little by denervation. The profiles of AChE forms seen in normal soleus at all times studied resembled those seen in newborn biceps or biceps inactivated by denervation or the med disease. We conclude that neither innervation, age-dependent changes intrinsic to muscle, nor muscle activity is sufficient to induce the changes we seen in AChE forms in biceps. These results support the hypothesis that neonatal, inactive, or tonically active muscles produce an intrinsic pattern of AChE molecular forms, and that a phasic pattern of activity induces a postnatal redistribution of the AChE molecular forms expressed by the muscle.     

Influence of innervation on molecular forms of acetylcholinesterase in regenerating fast and slow skeletal muscles

Nerve-intact muscle regenerates were prepared by ischemic-toxic injury of slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles of the rat. Rapid innervation of regenerating myotubes modified intrinsic patterns of AChE molecular forms, revealed by velocity sedimentation in linear sucrose gradients. Regarding their onset, the effects of innervation can be classified as early and late. The earliest changes in the SOL regenerates appeared a few days after innervation by their motoneurons: the activity of the 13 S AChE form (A 8) increased significantly in comparison to non-innervated regenerates. The pattern of AChE molecular forms became similar to that in the normal SOL muscle during the 2nd week after injury. In contrast, no major differences were observed between 8 day-old innervated and non-innervated EDL regenerates. Their patterns of AChE molecular forms resembled that in the normal EDL. However, the predominance of the 10 S AChE form (G 4) characteristic for the 2-week old non-innervated regenerates was prevented by innervation. Early effect of innervation observed in the SOL regenerates but not in the EDL may be due to intrinsically different response of the regenerating SOL myotubes to innervation. Rather high extrajunctional activity of the asymmetric 16 S (A 12) molecular form of AChE in early regenerates was reduced to adult level in about 3 weeks in the SOL, and nearly completely suppressed in 5 weeks after innervation in the EDL regenerates. This reduction is assumed to be a late effect of innervation, as well as a decrease of the activity of the 4 S AChE form (G 1) in the SOL regenerates. A suppressive mechanism is activated in the extra-junctional regions of the innervated muscle regenerates during their maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific impulse patterns regulate acetylcholinesterase activity in skeletal muscles of rats and rabbits

In rats, acetylcholinesterase (AChE) activity in the fast muscles is several times higher than in the slow soleus muscle. The hypothesis that specific neural impulse patterns in fast or slow muscles are responsible for different AChE activities was tested by altering the neural activation pattern in the fast extensor digitorum longus (EDL) muscle by chronic low-frequency stimulation of its nerve. In addition, the soleus muscle was examined after hind limb immobilization, which changed its neural activation pattern from tonic to phasic. Myosin heavy-chain (MHC) isoforms were analyzed by gel electrophoresis. Activity of the molecular forms of AChE was determined by velocity sedimentation. Low-frequency stimulation of the rat EDL for 35 days shifted the profile of MHC II isoforms toward a slower MHCIIa isoform. Activity of the globular G₁ and G₄ molecular forms of AChE decreased by a factor of 4 and 10, respectively, and became comparable with those in the soleus muscle. After hind limb immobilization, the fast MHCIId isoform, which is not normally present, appeared in the soleus muscle. Activity of the globular G₁ form of AChE increased approximately three times and approached the levels in the fast EDL muscle. In the rabbit, on the contrary to the rat, activity of the globular forms of AChE in a fast muscle increased after low-frequency stimulation. The results demonstrate that specific neural activation patterns regulate AChE activity in muscles. Great differences, however, exist among different mammalian species in regard to muscle AChE regulation. J. Neurosci. Res. 47:49–57, 1997. © 1997 Wiley-Liss, Inc.     

Changes in the cholinergic system of rat sciatic nerve and skeletal muscle following suspension-induced disuse

Muscle disuse-induced changes in the cholinergic system of sciatic nerve, slow-twitch soleus (SOL), and fast-twitch extensor digitorum longus (EDL) muscles were studied in rats. Rats with hind limbs suspended for 2 to 3 weeks showed marked elevation in the activity of choline acetyltransferase in sciatic nerve (38%), in the SOL (108%), and in the EDL (67%). Acetylcholinesterase (AChE) activity in the SOL increased 163% without changing the molecular forms pattern of 4S, 10S, 12S, and 16S. No significant (P greater than 0.05) changes in the activity and molecular forms pattern of AChE were seen in the EDL or in AChE activity of sciatic nerve. Nicotinic receptor binding of [3H]acetylcholine was increased in both muscles. When measured after 3 weeks of hind limb suspension the normal distribution of type I fibers in the SOL (87%) was reduced (to 58%) and a corresponding increase in types IIa and IIb fibers occurred. In the EDL no significant change in fiber proportion was observed. Muscle activity, such as loadbearing, appeared to have a greater controlling influence on the characteristics of the slow-twitch SOL muscle than on the fast-twitch EDL muscle.     

Expression of myosin heavy chain isoforms in stimulated fast and slow rat muscles

The expression of 4 myosin heavy chain (MHC) isoforms was analyzed in the rat soleus (SOL) and extensor digitorum longus (EDL) muscles after denervation and chronic electric stimulation. The stimulation frequencies used were 20 and 150 Hz and the amount of stimulation was either large (20 Hz), intermediate (150 Hz), or small (150 Hz). These patterns resemble some features of normal motor unit activity in SOL and EDL of freely moving rats (Hennig and L?mo, 1985). The relative expression of each MHC isoform depended strongly on the stimulation pattern. Furthermore, for any particular stimulation pattern, fibers in SOL and EDL expressed different MHCs. Coexistence of different MHC types in the same fiber was frequently observed in stimulated muscles. 20-Hz stimulation preserved normal expression of type 1-MHC in SOL but failed to induce type 1-MHC in type 2 fibers of the EDL, where type 2A- and 2X-MHC expression dominated and type 2B-MHC expression was completely suppressed. 150-Hz low-amount stimulation preserved nearly normal 2B-MHC expression in many type 2 fibers of the EDL but failed to induce type 2B-MHC expression in the SOL, where 2X-MHC became predominant. 150-Hz high-amount stimulation differed from 150-Hz small amount stimulation by suppressing almost all type 2B-MHC expression in EDL and by inducing considerable type 2A-MHC expression in the SOL. Scattered fibers in EDL that were probably the original type 1 fibers responded differently from both type 2 fibers in the EDL and from type 1 fibers in the SOL to stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholinesterase molecular forms in C57BL/6J dystrophic mice

Acetylcholinesterase (AChE) was extracted from normal and dystrophic C57BL/6J mouse hindlimb muscles and its molecular forms fractionated by sucrose density gradient ultracentrifugation. In the soleus muscles from 6- to 7-week-old mice an increase in the 3 Svedberg unit (S) and a decrease in the 16S AChE molecular forms was observed in dystrophic animals compared to controls. At 12-13 weeks of age, no major significant differences in the relative proportions of AChE molecular forms were noted. In the extensor digitorum longus (EDL) muscles of 6- to 7-week-old dystrophic mice a significant decrease in the proportion of the 10S AChE molecular form and an increase in the 3S and 5S forms was observed. At 12-13 weeks, the dystrophic EDL muscles again displayed a decrease in the 10S form; however, the increase in the 3S and 5S AChE forms, while still apparent, was not significant. These results provide evidence for a biochemical abnormality in the distribution of specific AChE molecular forms, and a differential expression of this abnormality in the soleus and EDL muscles.     

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.     

Continuous low amplitude direct current stimulation of the crushed peripheral nerve accelerates the early recovery of choline acetyltransferase but not of acetylcholinesterase activity in fast and slow muscles

We investigated if continuous 1 μA direct current stimulation of the injured nerve, with the cathode electrode at the distal end of the nerve crush injury (cathode stimulation), accelerated the recovery of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity in transiently denervated extensor digitorum longus (EDL) and soleus (SOL) rat muscles. ChAT is a specific marker of cholinergic nerve terminals and may reflect axon ingrowth, and AChE reflects the re-establishment of neuromuscular junctions and recovery of muscle activity. Compared to sham operated animals, the cathode (CA) stimulated rats had a statistically significant larger ChAT activity in the EDL and SOL muscles on days 12 and 14 after nerve crush (P < 0.01, n = 6). The difference in ChAT activity between the groups decreased thereafter. Regarding recovery of muscle AChE, CA stimulation of the crushed sciatic nerve did not detectably accelerate the normalization of activity and pattern of AChE molecular forms in the EDL and SOL muscles. This means that the early rise in ChAT muscle activity in CA stimulated rats was not followed by an accelerated normalization of the neuromuscular transmission in the same group. It is more likely that the higher ChAT activity observed after cathode stimulation indicates a higher ChAT content in regenerating motor nerve endings, rather than a greater number of motor axons entering the muscles. It seems possible that cathode stimulation increased ChAT axonal transport, causing the early increase of ChAT content in the nerve endings. This raises the possibility that the axon transport and subsequent secretion of a trophic factor(s) from the nerve to the reinnervated muscle are enhanced as well, thus shortening the overall time of muscle force recovery in the absence of an appreciable acceleration of recovery of the neuromuscular transmission.     

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.     

Reinnervation of a denervated slow muscle triggers high extrajunctional expression of the asymmetric molecular forms of acetyicholinesterase

Expression of acetylcholine receptor and of the asymmetric molecular forms of acetylcholinesterase (AChE) in the extrajunctional regions of rat muscles is suppressed during early postnatal development. In mature muscles, the extrajunctional synthesis of acetylcholine receptor, but not of the asymmetric molecular forms of AChE, becomes reactivated after denervation. The hypothesis that a denervated muscle needs reinnervation in order to revert transiently to an immature state characterized by high extrajunctional production of the asymmetric AChE forms, was examined in rat muscles recovering after nerve crush. Molecular forms of AChE were analysed by velocity sedimentation. Activity of the asymmetric A12 AChE form in the extrajunctional regions of the slow soleus (SOL) muscle increased during the first week after reinnervation to about 9 times its control level, remained high for about one week, and declined towards normal thereafter. If the nerve was crushed close to the muscle and reinnervation occured very rapidly, the extrajunctional increase of the A₁₂ AChE form still occured but was less pronounced than after late reinnervation. In contrast, a transient paralysis of the SOL muscle due to acetylcholine receptor blockade by α-bungarotoxin, followed by spontaneous recovery of muscle activity after 3–5 days, did not revert AChE regulation into an immature state. Disuse of the SOL muscle caused by leg immobilization, which is known to change the tonic pattern of neural stimulation of the SOL muscle into a phasic one, did not prevent the reversion of AChE regulation during reinnervation. This indicates that neural stimulation pattern is not crucial for this reversion. In contrast to slow SOL, the fast extensor digitorum longus muscle did not revert to an immature state in respect to AChE regulation after reinnervation. This muscle type-specific response may be due to intrinsic differences between the myogenic cells of slow and fast muscle fibres. © 1995 Wiley-Liss, Inc.     

Satellite cells in slow and fast rat muscles differ in respect to acetylcholinesterase regulation mechanisms they convey to their descendant myofibers during regeneration

The hypothesis of satellite cell diversity in slow and fast mammalian muscles was tested by examining acetylcholinesterase (AChE) regulation in muscles regenerating (1) under conditions of muscle disuse (tenotomy, leg immobilization) in which the pattern of neural stimulation is changed, and (2) after cross-transplantation when the regenerating muscle develops under a foreign neural stimulation pattern. Soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were allowed to regenerate after ischemic-toxic injury either in their own sites or had been cross-transplanted to the site of the other muscle. Molecular forms of AChE in regenerating muscles were analyzed by velocity sedimentation in linear sucrose gradients. Neither tenotomy nor limb immobilization significantly affected the characteristic pattern of AChE molecular forms in regenerating SOL muscles, suggesting that the neural stimulation pattern is probably not decisive for its induction. During an early phase of regeneration, the general pattern of AChE molecular forms in the cross-transplanted regenerating muscle was predominantly determined by the type of its muscle of origin, and much less by the innervating nerve which exerted only a modest modifying effect. However, alkali-resistant myofibrillar ATPase activity on which the separation of muscle fibers into type I and type II is based, was determined predominantly by the motor nerve innervating the regenerating muscle. Mature regenerated EDL muscles (13 weeks after injury) which had been innervated by the SOL nerve became virtually indistinguishable from the SOL muscles in regard to their pattern of AChE molecular forms. However, AChE patterns of mature regenerated SOL muscles that had been innervated by the EDL nerve still displayed some features of the SOL pattern. In regard to AChE regulation, muscle satellite cells from slow or fast rat muscles convey to their descendant myotubes the information shifting their initial development in the direction of either slow or fast muscle, respectively. The satellite cells in fast or slow muscles are, therefore, intrinsically different. Intrinsic information is expressed mostly during an early phase of regeneration whereas later on the regulatory influence of the motor nerve more or less predominates. © 1994 Wiley-Liss, Inc.     

The effect of continuous pyridostigmine administration on functional (A12) acetylcholinesterase activity in guinea-pig muscles

Pyridostigmine which causes a reversible inhibition of acetylcholinesterase (AChE), was administered continuously for 6 days to guinea-pigs, via a subcutaneously implanted osmotic pump. This produced 40-50% inhibition of red cell acetylcholinesterase (AChE). Controls were animals treated with saline via pumps, and untreated animals. The activities of the functional A12 molecular form of AChE were compared in diaphragm, extensor digitorum longus (EDL) and soleus muscles in the three animal groups at 6 days. The pumps were removed at 6 days and the A12 AChE activities were determined at various times thereafter As the enzyme separation procedure was lengthy, drug-induced inhibition was no longer present when the enzyme activity was measured. At 6 days, the activity was significantly higher in EDL (over 50% higher) and soleus (over two-fold higher) in pyridostigmine-treated animals than saline-treated animals. In the diaphragm, the activities in pyridostigmine and saline-treated animals were similar but both were significantly (over two-fold) higher than in untreated animals. At 1 day after pump removal (day 7) the activity had declined in all three muscles of the pyridostigmine-treated animals and in the diaphragm of saline-treated animals. Thereafter, in the diaphragm (but not the EDL or soleus) in pyridostigmine-treated animals, there were marked variations in the enzyme activity up to day 20. In saline-treated animals there was a marked transient increase in activity at day 13 in all muscles. The results indicate that the homeostatic control offunctional AChE had been affected in both the pyridostigmine and saline treatment groups.     

Effect of denervation on sarcolemmal proteins and glycoproteins of fast and slow mammalian skeletal muscle

We compared the protein and glycoprotein composition of a sarcolemmal membrane fraction isolated from normal and denervated rat extensor digitorum longus (EDL) and soleus muscles. Membranes from EDL and soleus muscles showed significantly different protein compositions. A relatively small number of glycoproteins, which were all minor proteins, accounted for the majority of concanavalin A (ConA) and Ricinus communis agglutinin (RCA120) binding. These glycoproteins appear to be common to EDL and soleus but bound different relative amounts of lectin in the two muscles. A large proportion of the ConA binding sites in EDL, but not soleus, were cryptic (not accessible by ConA unless the membrane structure was disrupted). Denervation had a differential effect on sarcolemma from the two muscles with EDL exhibiting large changes and soleus changing little if at all. Several major proteins changed their relative concentrations after denervation and the relative amount of RCA120 bound to the major glycoproteins also changed. The major ConA-binding glycoproteins did not change in either membrane but denervation resulted in the exposure of most of the cryptic ConA-binding sites in EDL membranes. Endogenous sialyl- and galactosyl-transferase activities in the membrane fractions significantly increased in EDL, but did not change in soleus, suggesting that the turnover of the glycoproteins is increased in EDL after denervation.     

The effect of low-frequency electrical stimulation on the denervated extensor digitorum longus muscle of the rabbit

Both extensores digitorum longi (EDL) muscles of rabbits were denervated by crushing the common peroneal nerves. The EDL muscle on one side was directly stimulated at 10-12 Hz via implanted electrodes. This treatment reduced the changes of twitch/tetanus ratios produced by denervation and prevented the slowing of contraction and relaxation that follows denervation. It is concluded that the stimulation reduced the duration of the active state of denervated muscles.
These effects of stimulation were reduced after 5 weeks, probably because by that time the slowing effect of low-frequency activity on the fast muscles became apparent.     

A distinct difference between slow and fast muscle in acetylcholinesterase recovery after reinnervation in the rat

The changes in acetylcholinesterase (AChE) and choline acetyltransferase (CAT) activity in nerve proximal and distal to the crush site as well as in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle were studied during denervation and reinnervation in rat. Within 24 h after nerve crush, conduction in the distal nerve and neuromuscular transmission was lost. In the distal nerve segment, AChE and CAT activity showed no initial increase and was reduced to 25% 14 days after crush. During the reinnervation period, AChE and CAT activity recovered to 50% (AChE) and 80% (CAT) of control values and CAT activity in the EDL and SOL muscles followed closely the changes in distal nerve. In muscle, AChE activity was reduced to 15% by 2 weeks postoperatively. Enzyme activity in EDL recovered to 50% of control activity in 5 weeks after crush. In the SOL, end-plate and non-end-plate regions' AChE activity recovered at a faster rate, resulting in a temporary increase in AChE activity to more than control values during the third and fourth week. By the end of the fifth week, AChE activity had returned to control activity. Turnover values for AChE based on the reinnervation data showed a half-life value for AChE in proximal nerve of 32 days, in distal nerve 42 days, in EDL 23 days, and for SOL 5.1 days. The half-life for AChE in both muscles was significantly shorter than that of the nerve, indicating that the nerve did not supply AChE to the muscles. Half-lives for CAT calculated on the basis of the reinnervation data were 11.6 days for proximal nerve, 18.4 days for distal nerve, and 30 days for SOL and EDL muscles. It is concluded that the ability to synthesize AChE in end-plate and non-end-plate regions of muscle is an endogenously programmed event in the development of both fast and slow muscles. The nerve initiates and maintains the synthesis and can modify the rate of synthesis in individual muscle fibers. The mechanism by which the nerve stimulates and maintains AChE synthesis in muscle may be related to the release of trophic factors muscle activity or to a combination of these and other factors still to be investigated.     

Carnitine and acyltransferase in experimental neurogenic atrophies: changes with treatment

Summary Carnitine level and carnitine palmityl transferase (CPT) activity were investigated in muscles of patients with infantile and juvenile spinal muscular atrophy and polyneuropathies. A significant decrease of both carnitine and CPT was found in the infantile spinal muscular atrophy, but not in the other neurogenic muscle atrophies. These findings were compared with the experimental effect of denervation and reinnervation upon the lipid metabolism in soleus and extensor digitorum longus (EDL) of adult and newborn rats. Twenty-one days after denervation free and total carnitine decreased significantly in both EDL (P<0.001) and soleus (P<0.05) of adult animals. CPT activity was significantly decreased in the soleus 50 days after denervation (P<0.005). Long-term reinnervation restored the level of carnitine fraction and CPT activity. l-carnitine treatment for 21 days restored the level of free carnitine to normal in the soleus of denervated adult animals. Denervation in newborn rats influenced carnitine concentration in soleus and EDL to a lesser extent; the treatment with l-carnitine raised short-chain acylcarnitines in denervated muscles, while reinnervation restored carnitine level within 50 days.Presented as a preliminary report at the Fifth International Congress on Neuromuscular Diseases, Marseille, France, September 1982     

Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles

The mitotic activity in muscles of growing rats and the effect of denervation were studied by means of continuous infusion of 5-bromo-2-deoxyuridine (BRDU). Denervated muscles after 10 weeks contained 20 to 60% fewer muscle nuclei than normal; BRDU labeled about 25% of the nuclei of normal soleus and extensor digitorum longus (EDL) and of denervated EDL muscles but only 5% in the denervated soleus muscle. Labeled nuclei persisted in denervated but not in normal muscles. After the main growth period, the turnover of myonuclei was at most 1 to 2% per week. The behavior of connective tissue nuclei was similar to that in muscle fibers. Infusion of BRDU had no effect on contractile properties. It is suggested that the exceptionally rapid atrophy of the denervated rat soleus associated with loss of satellite cells was due to loss of myonuclei and differentiation and fusion of satellite cells. The cause may possibly be that the phase of postdenervation fibrillation is shorter than in other muscles.     

Trophic control of cholinesterase activity in a testosterone-dependent muscle of the rat. II. Effects of testosterone administration

The effects of testosterone on the weight, protein content, and acetylcholinesterase (AChE) activity were investigated in the hormone-dependent levator ani and nondependent extensor digitorum longus and soleus muscles from normal or castrated male rats. In either group some muscles were also chronically denervated. Testosterone propionate treatment (3 mg/week for 2 weeks, s.c.) of normal rats increased the weight and protein content of the levator ani, respectively, by 19% and 63%; the muscle AChE was not affected. Protein content, but not the weight of the normal extensor digitorum longus and soleus was also increased after testosterone; AChE was reduced by 20% in the extensor digitorum longus and unaltered in the soleus. In castrated rats, testosterone reversed the levator ani atrophy and slowed down the decay of AChE, but it did not restore the normal enzyme activity. Testosterone did not prevent the atrophy and AChE decrease induced by denervation of either muscle. The weight and protein content of the denervated levator ani from castrated rats were increased by testosterone to the values found in denervated muscles from normal rats; AChE in the same muscles was not increased. The results confirm that separate mechanisms regulate protein synthesis and AChE in the rat levator ani. AChE is mainly regulated by neural factors which in turn appear to be influenced by circulating androgens. Similar hormonal influence on the muscle AChE was not detected in the extensor digitorum longus and soleus muscles.