Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity

Denervation of adult skeletal muscle results in increased sensitivity to acetylcholine in extrajunctional regions of the muscle fiber. This increase in acetylcholine sensitivity is accompanied by a large increase in the level of mRNAs coding for the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. To determine whether muscle activity is sufficient to regulate expression of extrajunctional acetylcholine receptor mRNA levels, denervated muscles were stimulated with extracellular electrodes. Direct stimulation of denervated muscle suppresses both the increase in extrajunctional acetylcholine sensitivity and the expression of mRNA encoding the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. These results show that muscle activity regulates the level of extrajunctional acetylcholine receptors by regulating the expression of their mRNAs.     

Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.     

Acetylcholine Receptors : Revised Estimates of Extrajunctional Receptor Density in Denervated Rat Diaphragm

The number of extrajunctional acetylcholine receptors (¹²⁵I-labeled α-bungarotoxin binding sites) per unit length of muscle fiber and the average fiber circumference were determined for rat diaphragm muscle fibers denervated 0, 2, 4, 7, 10, and 14 days. From these data receptor densities (sites per square micrometer of surface) were calculated. Values thus obtained were considerably lower than those estimated previously by autoradiography. Receptor density increased from < 6 sites/µm² in innervated muscle to 635 ± 29 sites/µm² 14 days after denervation. The form of the relationship between receptor density and acetylcholine sensitivity and the time-course of change in receptor density after denervation are as previously reported.     

Effects of innervation on the distribution of acetylcholine receptors in regenerating skeletal muscles of adult chickens

In order to determine the roles of nerves in the formation of clusters of acetylcholine receptors (AChRs) during synaptogenesis, we examined the distribution of AChRs in denervated, nerve-transplanted (neurotized) muscles and in regenerated skeletal muscles of adult chickens by fluorescence microscopy using curaremimetic toxins. In the denervated muscles, many extrajunctional clusters developed at the periphery of some of the muscle nuclei of a single muscle fiber and continued to be present for up to 3 months. The AChR accumulations originally present at the neuromuscular junctions disappeared within 3 weeks. In the neurotized muscles, line-shaped AChR clusters developed at 4 days after transection of the original nerve, but no change in the distribution of AChRs had occurred even at 2 months after implantation of the foreign nerve. The line-shaped AChR clusters were found to be newly formed junctional clusters as they were associated with nerve terminals of similar shape and size. Some of both the line-shaped and extrajunctional clusters were formed at least partly by the redistribution of preexisting AChRs. Finally, based on the above observations, the regenerating muscle fibers in normal muscles and in denervated muscles were examined: The extrajunctional clusters appeared in both kinds of muscles at 2 weeks after injury. Afterward, during the innervation process, the line-shaped AChR clusters developed while the extrajunctional clusters disappeared in the innervated muscles. In contrast with this, in the absence of innervation, only the extrajunctional clusters continued to be present for up to 3 months. These results demonstrate clearly that the nerve not only induces the formation of junctional clusters at the contact site, but also prevents the formation of clusters at the extrajunctional region during synaptogenesis.     

Nonacceptance of innervation by innervated neonatal rat muscle

Denervated adult muscle accepts innervation and has high levels of extrajunctional acetylcholine (ACh) receptor, compared to innervated adult muscle. If the high receptor density or any externally oriented part of the receptor molecule permitted or triggered functional synaptogenesis, then innervated neonatal muscle, with its known high extrajunctional sensitivity, should also accept extra synapses from implanted motor nerves. This prediction was tested by implanting the common peroneal nerve into innervated lateral gastrocnemius muscle in 25 neonatal rats and studying the innervation achieved 1–8 weeks later. With one exception, zero or negligible twitch tensions were obtained when the implanted nerve was stimulated. Intracellular recording in two cases showed no evidence of subthresholdevoked potentials in surface muscle fibers. In contrast, when the original motor nerve was cut at the time of common peroneal nerve implantation, reinnervation occurred as soon as 4 days later, and substantial indirect twitches (most observed qualitatively) were invariably found 6–7 days after operation. Four to eight weeks after nerve implantation into denervated muscle, substantial twitch tensions were obtained upon stimulation of the implanted nerve. α-Bungarotoxin binding to extrajunctional ACh receptors per unit surface area was similar in innervated neonatal and denervated adult muscle. Therefore, nonacceptance of additional functional innervation in neonatal muscle implies that a high average density of extrajunctional ACh receptor is not sufficient to permit or trigger functional neuromuscular junction formation.     

Development of the neuromuscular junction in the chick embryo: The number,distribution, and stability of acetylcholine receptors

The number, distribution, and stability of skeletal muscle acetylcholine receptors during development of the neuromuscular junction in the chick embryo were studied. The distribution and turnover of receptors labeled with ¹²⁵I-labeled α-bungarotoxin were determined by quantitative autoradiography on individual teased muscle fibers. Each posterior latissimus dorsi muscle fiber, which in the adult is singly innervated, has a high density of acetylcholine receptors at a single spot from embryonic Day 10 through hatching. The spots stain more intensely than elsewhere for acetylcholinesterase and are assumed to be end plates. The receptors at these spots are presumed to be junctional receptors. The junctional receptor density remains constant at 10⁴/μm² from embryonic Day 14 through adult life, although the area of the junction increases 40-fold. In contrast, the extrajunctional receptor density drops precipitously from 250/μm² on Day 14 to only 10/μm² on Day 19. This decrease in extrajunctional receptor density can be prevented by chronic paralysis with curare. The rate of autoradiographic grain loss from junctional and extrajunctional regions after a pulse injection of ¹²⁵I-labeled α-bungarotoxin indicates that both classes of embryonic receptors turn over at the same rate ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 30}\text{hr}$).     

Single channel properties of newly synthesized acetylcholine receptors following denervation of mammalian skeletal muscle

We have examined the single channel properties of newly synthesized acetylcholine (ACh) receptors in denervated adult mouse muscle. Patch-clamp recordings were made on freshly isolated fibers from flexor digitorum brevis (fdb) muscles that had been denervated in vivo for periods up to 3 wk. Muscles were treated with alpha-bungarotoxin (alpha-BTX), immediately before denervation, in order to block pre-existing receptors. Denervated fibers exhibited two types of ACh receptor channels, which differed in terms of single channel conductance (45 and 70 pS) and mean channel open time (approximately 7 and 2.5 ms, respectively). In contrast to innervated muscle, where only 3% of the total openings were contributed by the low-conductance channel type, greater than 80% of the openings in the nonsynaptic membrane of denervated muscle were of this type. Importantly, a similar increase in the proportion of low-conductance channels was observed for recordings from synaptic membrane after denervation. These data argue against the proposal that, in denervated muscle, the low-conductance channels undergo continued conversion to the high-conductance type focally at the site of former synaptic contact. Rather, our findings provide additional support for the idea that the functional properties of ACh receptors are governed uniformly by the state of innervation of the fiber and not by proximity to the site of synaptic contact.     

Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure

A scanning electron microscope (SEM) autoradiographic technique was calibrated and used to determine the site density of acetylcholine receptors within 250 micron of the neuromuscular junction in innervated as well as 3- and 10-d denervated sternomastoid muscle of the mouse. In all these groups sharp gradients of receptor site density are seen around the endplates in the first 2-7 micron, continuing less sharply to between 25 and 50 micron. Beyond 50 micron (to 250 micron) a spatial density gradient is present 3 d after denervation, but none exist by 10 d. These results suggest that the postdenervation steady-state extrajunctional receptor site density is reached sooner near the junction than away from the junction. The usefulness of SEM autoradiography to study the expression and distribution of membrane molecules at high resolution is demonstrated.     

Acetylcholine receptors and nerve terminal distribution at the neuromuscular junction of long-term regenerated muscle fibers

Mdx mice are deficient in dystrophin and show muscle fiber regeneration. Changes in the distribution of acetylcholine receptors have been reported at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration. In this study, we examined whether the distribution of receptors was still altered in long-term, regenerated muscle fibers from C57Bl/10 mice. The left sternomastoid muscle of adult mice was injected with 60 μl of lidocaine hydrochloride to induce muscle degeneration-regeneration. In some mice, the sternomastoid muscle was denervated at the time of lidocaine injection. After 90 and 150 days, the nicotinic acetylcholine receptors were labeled with rhodamine-α-bungarotoxin for confocal microscopy. At both intervals studied, the receptors were distributed in spots. In denervated-regenerated fibers, the receptors were distributed as regular branches similar to denervated muscles without lidocaine treatment. These findings suggested that nerve-dependent mechanisms were involved in the changes in receptor distribution seen in regenerated muscle fibers after lidocaine treatment, and that a similar phenomenon could explain the changes in receptor distribution seen in dystrophic muscle fibers.     

Effects of α-Bungarotoxin and Reversible Cholinergic Ligands on Normal and Denervated Mammalian Skeletal Muscle

α-Bungarotoxin (BuTX; 5 μg/ml) completely blocked the endplate potential and extrajunctional acetylcholine (ACh) sensitivity of surface fibers in normal and chronically denervated mammalian muscles, respectively, in about 35 min. A 0.72 ± 0.033 mV amplitude endplate potential returned in normal muscle fibers after 6.5 hr. of washout of α-BuTX, and an ACh sensitivity of 41.02 ± 3.95 mV/nC was recorded in denervated muscle after 6.5 hr of wash (control being 1215 ± 197 mV/nC). A two-step reaction of BuTX with binding sites which may allosterically interact is postulated.

Several pharmacologic differences were noted between the ACh receptors at the normal endplate and those appearing extrajunctionally following denervation. In normal innervated muscles exposed to BuTX in the presence of 20 μM carbamylcholine or decamethonium, washout of both drugs restored twitch to control levels within 2 hr. Endplate potentials large enough to initiate action potentials were also recorded in most surface fibers. In contrast, these agents, in much higher concentrations (50 μM), were almost ineffective in preventing BuTX blockade of ACh sensitivity in denervated muscle. Hexamethonium (10 and 50 mM) depressed neuromuscular transmission and blocked the action of BuTX in normal muscle in a dose-dependent fashion. On the extrajunctional receptors, hexamethonium (50 mM) was ineffective in protecting against BuTX. We may conclude that at the normal endplate region there are two distinct populations of ACh receptors, both of which react with cholinergic ligands and BuTX, but that a small population (representing ± 1% of the total) reacts with BuTX reversibly. Our findings further suggest a clear distinction between ACh receptors located at the normal endplate region and those of the extrajunctional region of the chronically denervated mammalian muscle.     

Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses

To elucidate the nature of signals that control the level and spatial distribution of mRNAs encoding acetylcholine receptor (AChR), alpha-, beta-, gamma-, delta- and epsilon-subunits in muscle fibers chronic paralysis was induced in rat leg muscles either by surgical denervation or by different neurotoxins that cause disuse of the muscle or selectively block neuromuscular transmission pre- or postsynaptically and cause an increase of AChRs in muscle membrane. After paralysis, the levels and the spatial distributions of the different subunit-specific mRNAs change discoordinately and seem to follow one of three different patterns depending on the subunit mRNA examined. The level of epsilon-subunit mRNA and its accumulation at the end-plate are largely independent on the presence of the nerve or electrical muscle activity. In contrast, the gamma-subunit mRNA level is tightly coupled to innervation. It is undetectable or low in innervated normally active muscle and in innervated but disused muscle, whereas it is abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked. The alpha-, beta-, and delta-subunit mRNA levels show a different pattern. Highest amounts are always found at end-plate nuclei irrespective of whether the muscle is innervated, denervated, active, or inactive, whereas in extrasynaptic regions they are tightly controlled by innervation partially through electrical muscle activity. The changes in the levels and distribution of gamma- and epsilon-subunit-specific mRNAs in toxin-paralyzed muscle correlate well with the spatial appearance of functional fetal and adult AChR channel subtypes along the muscle fiber. The results suggest that the focal accumulation at the synaptic region of mRNAs encoding the alpha-, beta-, delta-, and epsilon-subunits, which constitute the adult type end-plate channel, is largely determined by at least two different neural factors that act on AChR subunit gene expression of subsynaptic nuclei.     

Acetylcholine Receptors : Distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity

Using ¹²⁵iodine-labeled α-bungarotoxin (α-BGT-¹²⁵I) and quantitative radioautography, we have studied the time-course of the change in acetylcholine (ACh) receptor distribution and density occurring in rat diaphragm after denervation. In innervated fibers, ACh receptors are localized at the neuromuscular junction and the extrajunctional receptor density is less than five receptors per square micrometer. The extrajunctional receptor density begins to increase between 2 and 3 days after denervation and increases approximately linearly to 1695 receptors/µm² at 14 days, subsequently decreasing to 529 receptors/µm² at 45 days. We have isolated plasma membranes from rat leg muscles at various times after denervation and find that the change in concentration of ACh receptors in the membranes measured by α-BGT-¹²⁵I binding and scintillation counting follows a time-course similar to the change in ACh receptor density measured radioautographically. Furthermore, we have correlated extrajunctional ACh receptor density measured by radioautography with extrajunctional ACh sensitivity measured by iontophoretic application of ACh and intracellular recording and find that the log of ACh receptor density is related to 0.53 times the log of ACh sensitivity. These results are discussed in terms of the electrophysiological experiments on the ACh receptor and the recent, more biochemical approaches to the study of ACh receptor control and function.     

Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies.

Acetylcholine receptor located at the neuromuscular synapse of normal innervated adult muscle fibers is extremely stable metabolically. We have studied the kinetics of receptor degradation in both normal innervated and denervated rat diaphragms in organ culture. These studies show that degradation of receptor-bound 125I-alpha-bungarotoxin is a valid measure of junctional receptor degradation. Degradation of junctional receptor is similar or identical to degradation of extrajunctional receptor in many ways: 1) both require energy, 2) both are inhibited by specific lysosomal protease inhibitors, 3) both are inhibited by treatment with colchicine, and 4) both are stimulated by treatment with anti-acetylcholine receptor antibodies. The one important distinction between degradation of junctional and extrajunctional receptor is a 10-fold difference in rate constant for the process.     

Developmental regulation of interstitial cell density in bullfrog skeletal muscle

Denervation of skeletal muscle results in striking connective tissue remodelling in junctional areas of muscle. Since extracellular matrix molecules mediate axonal growth and synaptic differentiation, it is likely that the interstitial cells and matrix molecules that accumulate near synaptic sites after denervation influence the regrowth and regeneration of synaptic connections. The experiments presented here addressed the question of whether the junctional connective tissue in developing bullfrog skeletal muscle was also specialized in its cellular and molecular composition. Denervation responses of muscle, such as extrajunctional sensitivity to acetylcholine, often reproduce the characteristics of developing muscle during synaptogenesis. In developing muscle, the distribution of interstitial cells was nonuniform during the period of muscle fibre birth and synaptogenesis. Interstitial cells were concentrated near synaptic sites as in denervated adult muscle. Unlike denervated adult muscle, there were no junctional accumulations of fibronectin or tenascin, matrix molecules produced by interstitial cells, in developing muscles. These results demonstrate that the junctional connective tissue in developing muscle is identified by a high density of interstitial cells that may play a role in the identification and formation of synaptic sites. Further, the junctional matrix environment of developing muscle is distinct from the matrix remodelling that occurs in response to denervation, suggesting that the matrix production by interstitial cells during development is regulated differently from that after denervation of the neuromuscular junction.     

Neural Control of Muscle Androgen Receptors

The number of cytosolic androgen receptors in rat skeletal muscle increases following denervation and disuse. This increase was postulated to represent altered intracellular distribution and consequent diminished sensitivity of skeletal muscle to androgens. To test this hypothesis, we measured total (homogenate) androgen receptor levels after denervation. Total (homogenate) androgen receptor binding did not change in response to denervation of leg muscles from adult male rats. An increase in cytosolic receptor number with no increase in total (homogenate) receptor levels supports the hypothesis of altered intracellular distribution of androgen receptors in denervated muscle. Cytosolic androgen receptor binding in muscle from male rats increased by 40% after denervation, whereas in females the increase was 17%. These increases could not be altered by endocrine manipulations of males or females.     

Histological evidences of reparative and regenerative effects of beta-adrenoceptor agonists, clenbuterol and isoproterenol, in denervated rat skeletal muscle

The aim of this study was to determine the contribution of beta-adrenoceptor activation in the reconstruction of the structural and functional organization of denervated skeletal muscle. beta-agonists, clenbuterol (1.2 mg/kg body weight) and isoproterenol (2 mg/kg body weight), administration (daily oral administration; maximum 7 days) to normal innervated rats as well as denervated animals caused muscle hypertrophy. An increase in mean fiber diameter confirmed this stimulated growth both in normal innervated and denervated rat gastrocnemius muscle. Examination of muscle nuclei from treated but normal innervated rat gastrocnemius exhibited features like large size, active nucleoplasm and an increase in their number per fiber cross section and per mm mean fiber length indicating towards an elevated biosynthetic activity in tissue in the presence of beta adrenoceptor agonists. Administration of drugs to normal innervated animals resulted in an emergence of central muscle nuclei. The hyperactive and enlarged muscle nuclei ultimately organized themselves into unusually elongated nuclear streaks. beta agonist treatment to denervated rats resulted in amelioration of atrophic state of tissue characterized by hypertrophy of muscle fibers thus lending to a restoration of structural organization of tissue. Bizarre shapes of nuclei in denervated muscle tend to recover to that characteristic to normal innervated muscle in presence of clenbuterol and isoproterenol hydrochloride. All observations were confirmed by administering butoxamine, a beta-adrenoceptor antagonist along with beta-agonists. The results suggests that both clenbuterol and isoproterenol hydrochloride are capable of mimicking normal innervation functions in skeletal muscle and thus play important role in the structural and functional reorganization of tissue. Amelioration of denervation atrophy in rat gastrocnemius in the presence of beta-agonists supports this.