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Post-natal disappearance of transient calcium channels in mouse skeletal muscle: effects of denervation and culture.

1. The whole-cell voltage clamp technique was used to record Ba2+ currents in voltage-sensitive Ca2+ channels in mouse flexor digitorum brevis muscles developing in situ from day 1 to 30 after birth. Effects of denervation and tissue culture on the Ca2+ channel currents were also studied. 2. The muscle fibres in newborn mice showed two distinct types of Ca2+ channel currents, a low-threshold transient current and a high-threshold sustained current. 3. The specific amplitude of the transient current was 2.7 +/- 1.7 (S.D.) A/F in response to -30 mV test pulses in medium containing 30 mM-Ba2+ on day 1 after birth. The transient current decreased progressively in the post-natal days and became undetectable by day 17. In contrast, the specific amplitude of the sustained current in response to +20 mV test pulses increased 4-fold from 6.9 A/F on day 1 to 27.7 A/F on day 30. 4. The disappearance of the transient current could not be accounted for by either shifts in voltage dependence of activation and inactivation or changes in activation and inactivation times of the two types of current during development. 5. Denervating muscle fibres on day 8 after birth did not prevent the disappearance of the transient current. Denervating them on day 17 did not allow reappearance of the transient current. However, the increase of the sustained current was suppressed by the denervation either on day 8 or day 17. 6. In muscle fibres isolated on day 8 after birth and cultured thereafter, the transient current did not disappear until day 19 in culture (27 days after birth), while the sustained current was maintained at the level on day 8. 7. In muscle fibres isolated on day 17, when the transient current had become undetectable, and cultured thereafter, the transient current did not reappear until day 15 in culture (32 days after birth), while the sustained current was maintained at a level similar to that on day 17. 8. We conclude that innervation has little influence on the developmental disappearance of the transient Ca2+ channel current in mouse muscle fibres, and suggest that some influencing factors from surroundings other than the nerve may be required for the disappearance of the functional transient channels.

Action Potentials↗

Electrical activity suppresses nicotinic acetylcholine receptor gamma subunit promoter activity.

Nicotinic acetylcholine receptors are pentameric, transmembrane, ligand-gated ion channels critical for neuromuscular signal transmission. Prior to innervation, the genes encoding these receptors are expressed in nuclei throughout the muscle fiber. Muscle innervation leads to a dramatic decrease in expression of these genes in extrasynaptic nuclei. This reduction in gene expression can be reversed by muscle denervation. The effects of denervation on receptor gene expression can be blocked by electrical stimulation of muscle using extracellular electrodes. The molecular mechanisms by which muscle electrical activity leads to altered patterns of nicotinic acetylcholine receptor gene expression are not well understood. Using an in vitro electrical stimulation paradigm to induce muscle activity, we have been able to mimic the effect of innervation on extrasynaptic acetylcholine receptor gene expression. We have found that a 93-bp region of 5'-flanking DNA, spanning nucleotides -150 to -57 relative to the transcription start site of the gamma subunit gene, is required for the suppression of gene expression in response to muscle activity. Sequences downstream of this region are transcriptionally active but are not responsive to muscle activity. However, these downstream sequences become responsive to muscle activity when placed under the control of the gamma subunit muscle-specific enhancer.

Animals↗

Botulinum toxin A for the treatment of lateral periorbital rhytids.

The lateral periorbital area represents one of the earliest and most bothersome facial stigmata of aging. Although traditional techniques have had limited success in treating the static cutaneous component to rhytid formation in this region, they have been largely unsuccessful in addressing the dynamic hyperkinetic lines that are created by the activity of the lateral orbicularis oculi muscle. Botox A injection represents a safe and reproducible technique to selectively denervate muscle activity in this troublesome area. This minimally invasive technique has led to significant improvement of periorbital lines, either when used as a single agent or in combination with other methods (Fig. 3). As more experience is gained with this toxin, as well as other Botox serotypes, continued progress is likely to occur in this area, as well as in other facial regions.

Botulinum Toxins, Type A↗

Subunit structure and peptide mapping of junctional and extrajunctional acetylcholine receptors from rat muscle.

We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated.

Acetylcholine↗

Interaction of myasthenic serum globulin with the acetylcholine receptor.

A serum factor from patients with myasthenia gravis which inhibited the binding of 125I-labeled alpha-bungarotoxin to acetylcholine receptor extracted with Triton X-100 from rat muscle has been studied in detail. The inhibitory activity was localized to the IgG fraction based upon the fractionations by sodium sulfate precipitation and DEAE chromatography as well as reaction with anti-IgG globulin. The myasthenic globulin inhibited toxin binding to receptors extracted from degenerated muscle but did not inhibit toxin binding to normal junctional receptors. At saturation levels of myasthenic globulin, the number of denervated acetylcholine receptors available for toxin binding was reduced approx. 50 percent. The myastehnic globulin was found to bind to denervated acetylcholine receptors but not to normal acetylcholine receptors by a radioimmunoassay technique in which myasthenic globulin incubated with 125I-labeled alpha bungarotoxin-receptor complexes was precipitated by anti-IgG serum. The globulin binding was saturable over the same range as inhibition of toxin binding. The data suggest that the myasthenic IgC binds to a site on the receptor complex juxtaposed to the acetylcholine receptor site. The myasthenic globulin appears to be a useful probe for investigation differences between acetylcholine receptors extracted from normal and denervated muscle and for investigating the pathogenesis of myasthenia gravis.

Acetylcholine↗

Recruitment of gastrocnemius muscles during the swing phase of stepping following partial denervation of knee flexor muscles in the cat.

In walking cats, the biarticular medial and lateral gastrocnemius (MG-LG) muscles act to produce extension and flexion torques at the ankle and knee, respectively, and they usually display only one burst of activity beginning just before ground contact and ending near the end of the stance phase. Currently, the MG-LG muscles are considered to function primarily to control extension movements around the ankle joint during the stance phase. However, their flexion action at the knee means that they have the capacity to regulate rotations at the knee, but this role has not yet been clearly defined. Following partial denervation of the other muscles that normally act to flex the knee during swing, we observed that the MG-LG muscles, but not the Soleus muscle (a pure ankle extensor), often generated strong bursts of activity during early swing. These bursts were enhanced following mechanical stimulation of the paw, and they were especially prominent when the leg trailed over an object. They were absent when the leg led over an object. During treadmill walking the swing-related bursts in MG and LG had little influence on ankle flexion at the beginning of swing, but they were associated with slowing of ankle flexion when the leg trailed over an object. We hypothesized that the recruitment of these bursts functions to partially compensate for the reduction in knee torque resulting from the denervation of other knee flexors. Consistent with this hypothesis was our finding that the magnitude of the swing-related activity in the MG-LG muscles was linearly correlated to the extent of the knee flexion and to the peak angular velocity of knee flexion, and that the timing of the bursts was similar to that in the denervated muscles prior to denervation. Our findings suggest that an excitatory pathway exists from the flexor half-center of the central pattern-generating network to MG-LG motoneurons, and that this pathway is strongly regulated by central and/or peripheral signals.

Animals↗

Denervation exposes cryptic concanavalin A binding sites in skeletal muscle membranes.

Denervation produces significant changes in glycoproteins on the surfaces of skeletal muscles which can be detected as an increase in membrane-bound carbohydrate and an increase in specific binding of labelled lectins. The increased binding of concanavalin A to denervated membranes is detectable only in intact membranes and is abolished by treatments which disrupt or perturb membrane structure whereas the increased binding of Ricinus communis agglutinin is detectable under both conditions. We suggest that, in addition to chemical modification of existing carbohydrate chains in glycoproteins and the synthesis of new glycoproteins, denervation results in an altered geometric arrangement of existing membrane glycoproteins.

Animals↗

Glutamine transport and metabolism in denervated rat skeletal muscle.

Rat skeletal muscle glutamine fell by 40% from 4.18 to 2.5 mumols/g wet weight (P less than 0.01) after 4 days of denervation. Over the same period net glutamine efflux from denervated hindlimbs [i.e., arteriovenous (a-v) concentration differences x blood flow] increased 3.5-fold (from -6.72 +/- 1.73 to -26 +/- 4.81 nmol.min-1.g-1, P less than 0.001). Gastrocnemius glutamine synthetase activity fell 48% after denervation (from 475 +/- 81 to 248 +/- 39 nmol.min-1.g-1, P less than 0.001), but glutaminase activity was not significantly altered (17 nmol.min-1.g-1). The maximal activity (Vmax) of the unidirectional Na(+)-dependent glutamine transporter (system Nm) was depressed by 45% from 1,020 +/- 104 to 571 +/- 9 nmol.min-1.g-1 (P less than 0.01), but the concentration at which transport was half maximal (Km) was not significantly altered (control 8.1 +/- 0.6 mM; denervated 6.52 +/- 0.12). Hindlimb denervation resulted in an increase of intramuscular Na+ by 17% and a fall of K+ by 12%, and the resting membrane potential in isolated muscles decreased from -75 +/- 10 to -59.5 +/- 5.5 mV. Membrane potential of perfused denervated muscle, isolated after acute addition of the Na+ channel blocker tetrodotoxin (TTX, 3 microM), repolarized to -66.4 +/- 3.2 mV. In perfused denervated preparations TTX caused an acute recovery of Vmax of unidirectional glutamine transport to 848 +/- 75 nmol.min-1.g-1; Km was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Membrane Ca2+ interactions and contraction in denervated rat soleus muscle.

Under voltage clamp conditions contractile responses and ionic currents of single fibres isolated from rat soleus, denervated for more than 20 days, were recorded in Na-free TEA containing solutions. The relationship between membrane potential and contraction has been analysed under various conditions. The addition of trivalent cations (La3+, Gd3+) resulted in a dose dependent reduction of the contractile response and similar effects were produced by polymyxin B (0.05-0.5 mM). By contrast in the presence of phospholipase D (1-5 U/ml) contractions were significantly increased for all values of depolarization. The time course of the change of tension amplitude after the application of Ca-free medium, was dependent on the amplitude, the duration and the frequency of the depolarization. Upon depolarization glycerol-treated fibres generated contractile responses which were similar to those recorded in normal muscle and were also dependent on [Ca]o. It is proposed that in denervated soleus muscle the negatively charged phospholipids at the outside of the membrane were involved in the depolarization-contraction coupling by means of their Ca binding properties. The quantity of Ca binding sites would be dependent on [Ca]o and membrane potential and their binding properties modified during and/or following variation in membrane potential.

Animals↗

Selective resection and denervation of cervical muscles in the treatment of spasmodic torticollis: results in 60 cases.

The author presents a series of 60 patients with torticollis who were treated successfully by operation. He describes the clinical types of torticollis: rotational torticollis, retrocollis, antecollis, lateroflexional torticollis, and combined types. The muscular mechanism of torticollis is detailed, and the operative technique of resecting or denervating only the principal or motive muscles is explained and illustrated for each type of torticollis. The results in this series were very good: 83.3% recovered completely or were improved markedly, and there were no complications.

Adolescent↗

Skeletal muscle protein metabolism under denervation atrophy in dog, Canis domesticus.

Denervated dog gastrocnemius muscle has shown a progressive decrease in total protein content, alanine aminotransferase (AIAT), aspartate aminotransferase (AAT) and glutamate dehydrogenase (GDH) activity levels and elevation in free amino acid, ammonia, urea, glutamine contents and AMP deaminase activity levels during post-neurectemic days. The possible implications of these findings are discussed in relation to denervation atrophy.

Alanine Transaminase↗

Influence of phospholipase C on some electrical properties of the skeletal muscle membrane.

1. A study was made of the effects of phospholipase C (PhC) on the resting membrane potential, input resistance, action potential and acetylcholine sensitivity of innervated and chronically denervated single muscle fibres of the rat.2. In doses higher than 1.5 mug/ml. PhC significantly increased the ionic permeability of the muscle membrane (indicated by a fall in input resistance) and reduced the resting membrane potential of innervated fibres. The ;fast' or ;white' extensor digitorum longus muscle was the most sensitive and the ;slow' or ;red' soleus the least sensitive muscle. A similar difference was observed among ;fast' and ;slow' muscles of the chicken, the posterior latissimus dorsi being far more sensitive than the ;slow' anterior latissimus dorsi muscle. Chronically denervated muscles were more resistant to these actions of PhC than innervated ones but even after denervation the ;fast' muscles remained more sensitive than the ;slow' muscles.3. The action-potential generating mechanism in the ;fast' and ;slow' muscle was completely and irreversibly blocked by 1.5 mug/ml. of PhC within 1 hr. Furthermore the innervated and chronically denervated muscles were equally sensitive to this effect of PhC. The enzyme caused a gradual increase in the threshold for excitation, reduction in the rate of rise and the amplitude of the action potential. The input resistance and the resting membrane potential were not reduced by this dose of PhC.4. Acetylcholine sensitivity of chronically denervated muscles was not affected by PhC in doses that abolished the electrical excitability of the membrane. When PhC reduced the input resistance and the resting membrane potential a decrease was also observed in the response to applied acetylcholine.5. The results suggest that PhC by acting at the polar heads of membrane phospholipids interferes with the ionic carrier mechanism which generates the action potential. The differences in sensitivity between ;fast' and ;slow' muscles and between innervated and chronically denervated ones are tentatively explained on the basis of heterologous membrane phospholipids and/or variations in their stereochemical arrangement.

Acetylcholine↗

Ultrastructural and histochemical study of the neuromuscular junctions in the denervated intrinsic laryngeal muscle of the cat.

Morphological changes and acetylcholinesterase (AchE) activity in the neuromuscular junctions (NMJ) of the normal and denervated posterior cricoarytenoid muscle (PCA muscle) of the cat were studied. Two days after denervation, the nerve terminals at the NMJ had almost disappeared. Six weeks after denervation, intensity of AchE activity at the former junctional site (FJS) was unchanged histochemically. At this stage, primary synaptic clefts were distorted and the Schwann's cells covered the FJS. Fourteen weeks after denervation, AchE activity at the FJS had decreased in contrast to that of the non-affected side. Calcitonin gene-related peptide (CGRP) was also investigated at the NMJ of the normal PCA muscles immunocytochemically. The present study shows that CGRP coexists with Ach in the nerve terminals of the PCA muscles and may be involved in the regulation of the contractile function of the intrinsic laryngeal muscle.

Acetylcholinesterase↗