Nicotinic acetylcholine receptors in vertebrate muscle: properties, distribution and neural control.
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Aging↗
Biomedical subjects
M M Salpeter
Publications and source records attributed to M M Salpeter.
Recovery of acetylcholinesterase at intact neuromuscular junctions after in vivo inactivation with di-isopropylfluorophosphate.
Recovery of acetylcholinesterase (AChE) at endplates of mouse sternomastoid muscle was studied after inactivation with di-isopropylfluorophosphate in vivo. A short incubation with alpha-bungarotoxin was used to prevent muscle necrosis which usually occurs after esterase inactivation. Under these conditions there was no delay in AChE recovery, unlike what we had previously seen in necrotic muscle. However, even in non-necrotic muscle, the overall recovery of AChE at the postjunctional membrane was very slow, with a half-life of about 20 days. The fact that, at adult neuromuscular junctions, both esterases and receptors turn over about 10 times slower than they do in embryonic muscle suggests a similar regulation of these two molecules by the nerve.
Acetylcholinesterase↗
Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.
In previous papers we studied the rising phase of a miniature endplate current (MEPC) to derive diffusion and forward rate constants controlling acetylcholine (AcCho) in the intact neuromuscular junction. The present study derives similar values (but with smaller error ranges) for these constants by including experimental results from the falling phase of the MEPC. We find diffusion to be 4 X 10(-6) cm2 s-1, slightly slower than free diffusion, forward binding to be 3.3 X 10(7) M-1 s-1, and the distance from an average release site to the nearest exit from the cleft to be 1.6 micron. We also estimate the back reaction rates. From our values we can accurately describe the shape of MEPCs under different conditions of receptor and esterase concentration. Since we suggest that unbinding is slower than isomerization, we further predict that there should be several short "closing flickers" during the total open time for an AcCho-ligated receptor channel.
Acetylcholine↗
Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction.
The distribution and turnover rate of acetylcholine receptors labeled with 125I-alpha-bungarotoxin were examined in innervated mouse sternomastoid muscle by electron microscope autoradiography using the "mask" analysis procedure. We compared the total population of receptors with receptors newly inserted at the junction 2 d after inactivation with nonradioactive alpha-bungarotoxin, both at the top (thickened) region of the postjunctional folds (pjm) and the nonthickened bottom folds. We found that the receptor site density was approximately 10 times greater on the thickened pjm than on the nonthickened bottom folds for both total and newly inserted receptors. This ratio does not change significantly during a 6-d period after labeling the new receptors. Furthermore, calculated values for turnover time of receptors show that both total and newly inserted receptors at both regions of the junctional folds have half-lives for degradation within the range given in the literature for slow junctional receptors. These data exclude a simple migration model whereby receptors are preferentially inserted in the nonthickened region of the junctional folds and then migrate into the thickened membrane at a rate equal to the turnover rate of the receptors.
Animals↗
Soluble compound electron microscope (EM) autoradiography: a resolution source to test redistribution of soluble tritiated compounds during processing.
The development of a resolution source that can be labeled with either a soluble or insoluble tritiated compound, and of a method for applying a dry, uniform monolayer of emulsion is reported. Influences due to redistribution of the soluble isotope during emulsion coating were measured by comparing the grain density distributions around the resolution source for soluble tritiated proline (3H-PRO) with that obtained for cross-linked tritiated bovine serum albumin (3H-BSA). The grain density distributions resulting from a standard method of emulsion application (partly gelled/loop method) are compared to that obtained from a dry stripping film. It was found that only the dry stripping film gave a grain distribution which was statistically not different for the soluble and insoluble specimens.
Autoradiography↗
Absence of [125I] alpha-bungarotoxin binding to motor nerve terminals of frog, lizard and mouse muscle.
The existence of nicotinic acetylcholine receptors (AChRs) on the motor nerve terminals of vertebrates has long been controversial. We have re-examined this issue by electron microscope autoradiography with [125I] alpha-bungarotoxin, following separation of nerve terminals from muscle fibers by collagenase and protease treatment. We found no label over nerve terminal membranes other than that due to background, and we calculate upper limits of less than 0.1% of the postsynaptic AChR density for nerve terminals in frogs, lizards, and mice. We conclude that there are essentially no presynaptic acetylcholine receptors that bind alpha-bungarotoxin at vertebrate neuromuscular junctions.
Animals↗
Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions.
The distribution of acetylcholine receptors (AChRs) in neuromuscular junctions of embryonic and newborn mice (strain 129/ReJ) was examined on the fine structural level using [125I]alpha-bungarotoxin labeling and quantitative electron microscope autoradiography. Changes in junctional receptor site density were related to changes in the structure of the postjunctional membrane, in particular the differentiation of morphologically distinct regions of thickened membrane and the formation of junctional folds. The following sequence of development is described: (1) At the earliest age examined, embryonic day 16 of gestation (i.e., at approximately the time when junctional receptor accumulations are first detected), subneural receptor aggregates have poorly defined boundaries and seem to extend beyond the region of direct axonal or Schwann cell contact. At that time the subneural AChR site density is somewhat variable but averages approximately 2000 to 3000 sites/micron 2, and the subneural muscle membrane shows discontinuous membrane specialization (membrane thickening plus a cytoplasmic amorphous layer). There seems to be no preferential labeling of contact regions rich in such specialization compared with those where membrane thickening was less obvious or absent. (2) By birth, junctional AChRs are strongly correlated with morphologically specialized membrane, at a constant density of approximately 9000/micron 2 (comparable to that in adult animals of this strain of mouse). During the first postnatal week, a large fraction of the primary cleft is covered by Schwann cell rather than axon terminal. Receptor-rich dense membrane and incipient junctional folds are found under axon terminals and, to a lesser extent, under Schwann cell extensions, but they are not seen beyond the edge of the "junctional complex." Coated vesicles are a prominent feature of the subjunctional sarcoplasm in neonatal junctions, and their number declines sharply during the first week. (3) Junctional folds differentiate mainly after the first postnatal week. Development of folds is associated with a 1.5- to 1.8-fold increase in length of specialized thickened membrane and a parallel increase in number of receptor sites per endplate. Since this is also a period of rapid overall endplate growth (Steinbach, J.H. (1981) Dev. Biol. 84: 267-276) the total number of endplate receptors increases greater than 30-fold during the maturation period following the first week while the concentration of receptors/micron 2 of thickened membrane remains constant.
Animals↗
125I-alpha-bungarotoxin. Effects of radiodecomposition on specific activity.
Radiodecomposition of 125I-alpha-bungarotoxin has been followed by a variety of assays, including an ion-exchange assay which reflects the ability of toxin to bind to receptor and a competition assay that is sensitive to changes in specific activity. The rate of degradation of labeled toxin can be much faster than the decay rate of 125I. The radiodecomposition rate increases with increasing specific activity but is dramatically reduced by the addition of bovine serum albumin. Our results indicate that radiodecomposition affects labeled and unlabeled molecules equally and is due mostly to secondary radiodecomposition. As expected from this mechanism, we have found that radiodecomposition does not affect the specific activity of 125I-labeled alpha-bungarotoxin. Our results from iodinated preparations of moderate specific activity (40-500 Ci/mmol), containing a mixture of unlabeled and monolabeled toxin molecules, are also applicable to preparations of pure monoiodo-alpha-bungarotoxin of similar specific activity.
Animals↗
Calcium-mediated myopathy at neuromuscular junctions of normal and dystrophic muscle.
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Animals↗
Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions.
We studied the effect of rat brain extract on rat muscle cells in vitro by light and electron microscope (EM) autoradiography after labeling acetylcholine receptors (AChR's) with 125I-alpha-bungarotoxin. We found that: (a) In the absence of brain extract, peak site densities within AChR clusters usually do not exceed 4,000 sites/micrometer2. (b) Within hours after exposure to brain extract, AChR's redistribute to form clusters in which the peak site densities are greater than 10,000 sites/micrometer2. Receptor concentration within extract-induced clusters is thus within a factor of 2 of that at the neuromuscular junction (nmj). (c) In the absence of extract, the AChR's and AChR clusters are predominantly on the bottom surface of the myotubes (facing the tissue culture dish). After extract treatment, they are predominantly at the top surface. (d) Plasma membrane in regions of high-density AChR clusters is enriched in membrane with enhanced electron density and surface basal lamina whether or not cells are treated with extract. Extract causes an increase in both these specializations on the top surface of the myotubes. (e) Brain extract does not produce an overall increase in AChR site density or a marked change in degradation rate of receptors in either clustered or nonclustered regions. By producing AChR clusters with junctional site densities and enhanced surface specialization, and by causing an overall shift in AChR's distribution, brain extract mimics early events reported at developing neuromuscular junctions.
Animals↗
Denervated endplates have a dual population of junctional acetylcholine receptors.
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Animals↗
Kinetic parameters for acetylcholine interaction in intact neuromuscular junction.
The dependency of miniature endplate current (mepc) rise time upon mepc amplitude and acetylcholine receptor site density was measured in lizard intercostal muscles and used to fit the rate constants in a simple kinetic scheme. The kinetic scheme included diffusion, two-step sequential binding of acetylcholine to receptor, and opening of the ion channel. Numerical simulation of the observed mepc behavior yielded the following kinetic constants; (i) diffusion constant, 4 X 10(-6) cm2 sec-1; (ii) forward binding rates, 4.7 X 10(7) M-1 sec-1; (iii) channel relaxation rate, 25 msec-1. The value above for the forward binding rates assumed both rates to be equal. If they are different, the slower of the two is in the range of 2-5 X 10(7) M-1 sec-1. A radial profile of bound receptor indicated that activation of the receptor was very local, occurring essentially within a radius of about 0.3 micrometers from the point of acetylcholine release.
Acetylcholine↗
High resolution analysis of the secretory pathway in mammotrophs of the rat anterior pituitary.
The secretory process in pituitary mammotrophs was analyzed by quantitative electron microscope autoradiography. Dispersed pituitary cells from estrogen-treated female rats were subjected to pulse-labeling with [3H]leucine (5 min) followed by a chase incubation of up to 4 h. Autoradiograms were prepared using fine-grained emulsion (Kodak 129-01), and analyzed using a three-step "mask analysis' procedure: (a) the distribution of autoradiographic grains is determined as in a simple grain density analysis; (b) masks (transparent overlays) are used to generate expected grains from assumed sources; and (c) a computer program compares these two distributions and varies the expected distribution to match the observed distribution, thereby identifying the radioactive sources in the tissue. The overall route of intracellular transport of prolactin from rough endoplasmic reticulum (ER) leads to Golgi complex leads to immature secretory granules leads to mature secretory granules was as established in previous studies. However, by use of the high resolution emulsion and method of analysis, the precision with which label could be localized within individual source compartments was much greater and the time resolution was much sharper than achieved previously using Ilford L4 emulsion and simple grain density analysis. The main new findings were as follows: (a) the ER was essentially drained of radioactivity by 30 min, the Golgi complex by 1 h, and the immature secretory granules by 2h postpulse. This indicates that the secretory product (prolactin) is rapidly and efficiently transported out of these compartments. (b) approximately 30% of the total radioactivity remains located in the ground cytoplasm over the entire postpulse period examined (up to 4 h), and by 30 min postpulse the grain density in the ground cytoplasm exceeded that of the ER. This indicates the ability to resolve ER-associated label (presumably associated mainly with secretory products) from the cytoplasmic label (presumably associated with nonsecretory proteins). (c) the specific activity of immature secretory granules was much greater than previously appreciated; at 1 h postpulse it was greater than 200 times that of the adjacent Golgi complex cisternae. This large dynamic range in observed grain density demonstrates the ability to effectively correct for radiation spread and thus to detect with great accuracy high concentration of label even from very small structures (20-100 nm) which constitute a small percentage (less than 1%) of the total cell area.
Animals↗
Neuronal control of acetylcholine receptor turnover rate at a vertebrate neuromuscular junction.
The turnover rate of acetylcholine receptors at neuromuscular junctions in mice increases progressively after denervation and, after 15 days, reaches a half-time of 30 z 5 hours. Denervation thus causes the clustered junctional acetylcholine receptors to assume the rapid turnover characteristic of extrajunctional receptors before innervation.
Animals↗
Denervation increases turnover rate of junctional acetylcholine receptors.
The turnover rates of junctional acetylcholine receptors were measured in innervated and denervated mouse sternomastoid neuromuscular junctions by 125I-labeled alpha-bungarotoxin binding. First, we determined that the density of labeled toxin initially bound to the neuromuscular junction was essentially unchanged up to 16 days after denervation. Innervated muscles and muscles that had been denervated 8 days previously were then saturated with labeled toxin, and the specific label at the endplate regions was compared by gamma counting 7 days later. At that time, the residual junctional label seen in innervated muscle was 3.2 times greater than in denervated muscle. Electron microscope autoradiography further showed that, after saturation with unlabeled toxin, new binding sites appeared rapidly at the specialized receptive region of the postsynaptic membrane with an apparent half-time of turnover of 2-3 days. At innervated junctions, the half-time of turnover was about 10 days. These data show that the mechanisms that control receptor turnover rates are different from those that control high-density receptor clustering. The slow turnover rate of junctional receptors appears to be more directly dependent on the presence of the nerve than is the clustering of junctional receptors.
Acetylcholine↗
Acetylcholine receptor site density affects the rising phase of miniature endplate currents.
The relationship between acetylcholine receptor (AcChoR) site density (sigma) and the rising phase of the miniature endplate current was determined in esterase-inactivated lizard intercostal neuromuscular junctions. The currents were recorded by using a voltage clamp. The receptor site density was determined by electron microscope autoradiography after labeling with 125I-labeled alpha-bungarotoxin in normal endplates and in those partially inactivated with nonradioactive alpha-bungarotoxin. We found that as sigma is decreased the rise time in increased and the amplitude is decreased. These results are compatible with a previously stated "saturating disk" model, which suggests that a quantum of acetylcholine (AcCho) acts on a small postsynaptic area at saturating concentration. We conclude that in the normal neuromuscular junction the most likely number of AcCho molecules needed to open an ion channel is 2, and that the 20--80% rise time of < 100 musec is influenced both by the sigma-dependent factors such as diffusion and binding of AcCho to AcChoR and by the sigma-independent time delays such as the conformation change time to open the ion channels. From our data we calculate the lower limits to the forward rate constant of AcCho binding to AcChoR greater than or equal to 3 X 10(7) M-1 sec-1 and the diffusion constant for AcCho in the cleft greater than or equal to 4 X 10(-6) cm2 sec-1.
Acetylcholine↗
Sensitivity in electron microscope autoradiography for calcium-45.
The sensitivity of Ilford L4 emulsion to 45Ca was determined for electron microscope autoradiographic conditions. Sensitivity values were obtained for monolayers and double layers of emulsion in combination with various developing procedures. The dependence of sensitivity upon radiation dose was determined. All results are compared to previously calibrated isotopes.
Autoradiography↗
Endplates after esterase inactivation in vivo: correlation between esterase concentration, functional response and fine structure.
Mouse sternomastoid muscles were incubated with diisopropylfluorophosphate (DFP) in vivo, and the time course of recovery was studied using histochemistry, EM autoradiography and physiology. We found that: (1) the ability of the muscle to sustain tetanus in response to nerve stimulation is eliminated when the esterases at the neuromuscular junctions are saturated with DFP. This ability is regained partially when less than 10% of the DFP-binding sites have recovered. (2) There is a positive correlation between the frequency of stimulation at which the tetanic response can be maintained and the extent of acetylcholinesterase (AChE) recovery. (3) Tetanic responses at fusion frequency (about 100 Hz) appear indistinguishable from controls with only about 25% of normal AChE. (4) Butyrylcholinesterase (BuChE) possibly of Schwann cell origin recovers more rapidly than does AChE. (5) The muscle shows fine structural changes involving Z band dissolution and the breakdown of sarcoplasmic reticulum within hours after esterase inactivation. (6) This myopathy reaches a peak at three days after esterase inactivation and is almost fully recovered by two weeks. (7) It can be eliminated if, at the time of esterase inactivation, the nerve is cut or the acetylcholine receptors at the endplate are inactivated by alpha-bungarotoxin. We suggest that the myopathy, seen after DFP, is mediated by Ca2+ fluxes due to prolonged action of acetylcholine (ACh) in the absence of esterases.
Acetylcholinesterase↗