Effects of nerve activity on the postsynaptic membrane of skeletal muscle.

The effect of nerve activity on the sensitivity of the neuromuscular junction to acetylcholine was studied. After 1 h of nerve stimulation the flexor hallucis longus muscles of cats become more sensitive to the blocking effect of suxamethonium than before stimulation. Similar results were found using rat soleus muscles. The increased sensitivity to suxamethonium was still observed in muscles from rats which had been curarized during the period of stimulation. Intracellular recordings from end plates of rat soleus muscles revealed an initial transient increase in miniature end-plate potential (MEPP) frequency after stimulation and an increase in the amplitude of MEPPs which persisted for as long as 4 h. Moreover the depolarising responses to acetylcholine and suxamethonium added to the bath were always greater in stimulated muscles. It was therefore concluded that activity of the motor nerve increases the sensitivity of the end plates to transmitter.

Electrophysiology of the neuromuscular junction of the laminin-2 (merosin) deficient C57 BL/6J dy2J/dy2J dystrophic mouse.

The C57 BL/6J dy2J/dy2J dystrophic mouse expresses an abnormal truncated form of the alpha2 subunit of the protein laminin-2 (or merosin), which is unable to form a stable link between the extracellular matrix and the dystrophin-associated proteins, resulting in muscular dystrophy. Morphological abnormalities of the peripheral nervous system and neuromuscular junction have also been reported. The electrophysiological properties of the neuromuscular junctions of diaphragm, extensor digitorum longus (EDL), and soleus from C57 BL/6J dy2J/dy2J mice and controls are described. No evidence for the presence of denervated fibres were found. Mean MEPP amplitudes were significantly increased in EDL and soleus but reduced in the diaphragm from affected mice. Mean MEPP frequencies were raised in all the dy2J/dy2J muscles studied. dy2J/dy2J muscles were paralysed by low concentrations of mu-conotoxin suggesting that embryonic (tetrodotoxin and mu-conotoxin resistant) sodium channels are not widespread on dy2J/dy2J muscle as has previously been reported. EPP latencies were significantly prolonged in the diaphragm and EDL but not soleus from dy2J/dy2J mice. Quantal contents were higher in all dy2J/dy2J muscles. In the dy2J/dy2J diaphragm failures in neurotransmission occurred and a faster rate of rundown of EPPs were apparent. Some changes appear from a direct effect of dystrophy, whilst increased MEPP frequency and quantal content, and failures in neurotransmission indicate neuronal abnormalities.

Effects of denervation and immobilisation during development upon [3H]ouabain binding by slow- and fast-twitch muscle of the rat.

The effect of innervation and of muscle inactivity upon the normal production of Na+-K+-ATPase sites, assayed by [3H]ouabain binding, in muscle surface membranes has been determined for the rat. In both slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles a large increase was found to occur in [3H]ouabain binding per unit weight of muscle over the first 3 weeks of life. Interruptions of development, brought about by fixation of muscles at different lengths at 5 days of age, had no significant effect upon [3H]ouabain binding by EDL. In contrast, fixation led to a decrease in binding in SOL. When fixed in a shortened position profound morphological changes occurred, although these were not apparent when SOL was fixed in a stretched position. Denervation of SOL at 5 days of age significantly reduced the age related increase in the density of [3H]ouabain binding, whilst denervation of EDL had little effect. It was concluded that normal development of SOL is dependent upon innervation and possibly the resulting muscle activity, whereas development of EDL was relatively independent of innervation.

Potassium channel activity in sarcolemmal vesicles formed from skeletal muscle fibres of normal and dystrophic mice.

Sarcolemmal vesicles were produced from adult mouse extensor digitorum longus muscle (EDL) by treating swollen muscle fibres with collagenase. Vesicles formed from dystrophic (C57BL/6J dy/dy) and phenotypically normal animals were patch clamped and the single channel activity was recorded. Three types of K+ channel were observed in excised patches taken from normal and dystrophic muscle. A large conductance (300 pS) Ca2(+)-dependent K+ channel (KCa) was the most frequently observed of the K+ channels in both types of muscle preparation. In a number of patches taken from dystrophic muscle the open probability-voltage relationship for the KCa channel was markedly different from that in normal muscle, suggesting a possible reduction in Ca2+ sensitivity. An ATP-sensitive K+ channel (90 pS) was common to both normal and dystrophic muscle vesicles and was present in a large number of patches. An inwardly rectifying K+ channel (40 pS) was also observed in both types of sarcolemmal vesicles. The properties of all three K+ channels types were broadly consistent with other observations of skeletal muscle K+ channels, though all had higher conductances than had previously been noted in other species.

Sarcolemmal K+ channel activity in developing rat skeletal muscle membranes.

A method has been adapted to produce membrane vesicles suitable for routine membrane patch clamping from neonate rat skeletal muscle. Single K+ channel activity was recorded from cell-free inside-out patches. Most Ca2(+)-activated voltage sensitive channels had large conductances of up to 300 pS, as determined from their current/voltage relationship, and an open probability (Po) approaching unity at positive membrane potentials. A lower conductance K+ channel, probably responsible for inward rectification, had a lower conductance of about 100 pS. Outward rectifying K+ channels were also observed with the lowest conductance, about 40 pS. 0.1 mM ATP when applied to the inner membrane surface reduced or blocked activity, drastically reducing Po without altering single channel conductance. Such an effect has been reported in other preparations but was different in the neonate preparation in that it blocked channels with conductances as high as 300 pS. The simple preparation described, which we have also used successfully on mature rat and mouse skeletal muscle, has potential in the analysis of channel activities in various conditions and pathologies without the need for tissue culture to produce suitable membrane preparations.

[3H]Ouabain binding in normal and dystrophic mouse skeletal muscles and the effect of age.

The numbers of Na+-K+ ATPase sites in skeletal muscles of normal and dystrophic mice between 3 and 17 months of age have been estimated using [3H]ouabain binding assays. In normal mice, at all ages, slow twitch muscle, soleus (SOL), bound significantly more [3H]ouabain than fast-twitch muscle, extensor digitorum longus (EDL). [3H]Ouabain binding did not alter in either SOL or EDL from normal mice over the age range studied. The numbers of Na+-K+ ATPase sites did alter in muscles taken from dystrophic mice (C57BL/6J dy2J/dy2J). In EDL there was an increase and in SOL a decrease in [3H]ouabain binding. This may be related to a change in muscle fibre metabolism from glycolytic to oxidative or to an altered activity pattern. Increasing age resulted in a progressive reduction in [3H]ouabain binding of both SOL and EDL from dystrophic mice. Part of this reduction may be only apparent and due to an increase in connective tissue composition of dystrophic muscles. A limited study of muscles from neonate dystrophic mice indicated that abnormal [3H]ouabain binding was not present in EDL before two weeks of age.

Effect of the beta 2-adrenergic agonist clenbuterol on the growth of fast- and slow-twitch skeletal muscle of the dystrophic (C57BL6J dy2J/dy2J) mouse.

Clenbuterol (4mg/kg in diet for 21 days) had no statistically significant effect on whole body growth. It did cause a significant increase (18.2%) in wet weight of the fast twitch muscle extensor digitorum longus (EDL) and a corresponding 14.9% increase in total muscle protein. In transverse sections through dystrophic muscle fibre sizes were more variable than in normal muscle. Clenbuterol treatment resulted in a reduction in the proportion of small diameter fibres, and therefore an increase in mean fibre diameter, in dystrophic EDL. Clenbuterol had no significant effect upon the slow twitch muscle soleus.

Effect of denervation and ouabain on the response of the resting membrane potential of rat skeletal muscle to potassium.

The effects of denervation and ouabain on the relationship between resting membrane potential (EM) and extracellular potassium concentration were determined for rat soleus (SOL) and extensor digitorum longus (EDL) muscles. At concentrations above 20 mM for both SOL AND EDL fibres the relationship could be described adequately by the Nernst equation. Denervation resulted in reduction of EM and a decrease in the slope of the relationship between EM and potassium concentration. 10(-3) M ouabain produced the same effects as denervation. The results are discussed in relation to the alterations in membrane permeability previously shown to occur as a consequence of denervation. It is concluded that the low EM of denervated muscle is the result of an increase in membrane permeability to sodium.

Rundown and reactivation of ATP-sensitive potassium channels (KATP) in mouse skeletal muscle.

Dissociated single fibers from the mouse flexor digitorum brevis (FDB) muscle were used in patch clamp experiments to investigate the mechanisms of activation and inactivation of KATP in mammalian skeletal muscle. Spontaneous rundown of channel activity, in many excised patches, occurred gradually over a period of 10-20 min. Application of 1.0 mM free-Ca2+ to the cytoplasmic side of the patch caused irreversible inactivation of KATP within 15 sec. Ca(2+)-induced rundown was not prevented by the presence of 1.0 microM okadaic acid or 2.0 mg ml-1 of an inhibitor of calcium-activated neutral proteases, a result consistent with the conclusion that phosphatases or calcium-activated neutral proteases were not involved in the rundown process. Application of 1.0 mM Mg.ATP to Ca(2+)-inactivated KATP caused inhibition of residual activity but little or no reactivation of the channels upon washout of ATP, even in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase (10 U ml-1). Mg.ATP also failed to reactivate KATP, even after only partial spontaneous rundown, despite the presence of channels that could be activated by the potassium channel opener BRL 38227. Nucleotide diphosphates (500 microM; CDP, UDP, GDP and IDP) caused immediate and reversible opening of Ca(2+)-inactivated KATP. Reactivation of KATP by ADP (100 microM) increased further upon removal of the nucleotide. In contrast to KATP from cardiac and pancreatic cells, there was no evidence for phosphorylation of KATP from the surface sarcolemma of dissociated single fibers from mouse skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of denervation during development upon membrane potential and intracellular potassium and sodium activities of skeletal muscle of the rat.

Potassium and sodium ion-selective microelectrodes were used in vitro to investigate the effect of denervation of fast-twitch skeletal muscle (extensor digitorum longus) at 5 days of age upon subsequent development of the resting membrane potential. Normally, during the first 3 weeks of life the balance of intracellular potassium and sodium changed to elevate potassium and to lower sodium. These changes were reflected in the development of a more hyperpolarized resting membrane. Periods of denervation delayed, or prevented, the changes in ion activities and membrane hyperpolarization from occurring. The results are compared with those found after denervation of adult skeletal muscle.

Membrane potential and intracellular potassium and sodium activities after denervation of soleus muscles of neonatal rats.

Potassium and sodium ion-selective microelectrodes were used in vitro to investigate the depolarization of slow-twitch skeletal muscle fibers (soleus) following denervation at 5 to 6 days of age. The normal increase in intracellular potassium ion activity and decrease in sodium ion activity which occurs during the first 3 weeks of life were delayed or prevented by denervation. The results are compared with those found after similar periods of denervation of neonatal rat fast-twitch muscle.

Effect of clenbuterol on skeletal muscle atrophy in mice induced by the glucocorticoid dexamethasone.

1. The ability of clenbuterol to antagonize the catabolic effect of the glucocorticoid dexamethasone on the skeletal muscles, soleus, gastrocnemius and extensor digitorum longus was studied in mice. 2. Daily injections of 5 mg dexamethasone/kg body weight over 10 days caused a significant (20%) loss of muscle weight and protein content in fast twitch but not in slow twitch muscles. 3. Inclusion of clenbuterol (4 mg/kg) in the diet for the period of dexamethasone treatment partly prevented glucocorticoid-induced muscle atrophy, and increasing the concentration of clenbuterol to 8 mg/kg diet totally prevented glucocorticoid-induced protein loss in all muscles.

Some factors affecting spontaneous transmitter release in dystrophic mice.

Neuromuscular junctions of slow- and fast-twitch skeletal muscles from dystrophic (dy2J/dy2J) and control mice of the C57BL/6J strain were used to investigate the effect of muscular dystrophy on nerve-terminal regulation of their intracellular concentration of free calcium ions. The frequency of spontaneous miniature endplate potentials (MEPPs) was taken as an indicator of the intraterminal free calcium ion concentration. Dicoumarol, 2,4-dinitrophenol, ruthenium red, and the calcium ionophore A-23187 all potentiated the MEPP frequency in dystrophic muscles at concentrations which had negligible effects on normal muscles. Dystrophic muscle preparations were also more sensitive to an increased extracellular calcium concentration. Usually, these manipulations had more effect on the nerve terminals of dystrophic slow muscle than on those of dystrophic fast muscle. We conclude that muscular dystrophy alters the nerve terminal's ability to regulate the concentration of intracellular free calcium ions.

Effects of denervation on resting membrane and action potentials of neonate rat fast and slow twitch muscles.

During the first 20 days postpartum of the rat, a period of rapid muscle growth, resting potentials and maximum rate of rise and overshoot of action potentials recorded from fast and slow twitch muscle fibres all increased, although did not reach adult values. The action potential of fast twitch muscle fibres had a greater maximum rate of rise and overshoot than that of slow twitch muscle fibres of the same age. Innervation had a marked influence upon maintenance of the electrical properties of muscle fibres in the neonate since denervation reduced the value of the resting potential and maximum rate of rise and overshoot of the action potential. Neverthless, some differences between action potentials recorded from the two fibre types remained 4 days after denervation.

Intracellular activity of sodium in normal and dystrophic skeletal muscle from C57BL/6J mice.

Potassium and sodium ion-selective microelectrodes were used in vitro to investigate the depolarization of skeletal muscle fibers associated with muscular dystrophy. In dystrophy there was a large increase of intracellular Na activity and an associated decrease in K activity in fibers of extensor digitorum longus muscles. Despite this, the recorded membrane potential was very close to the calculated potassium equilibrium potential (Ek) in dystrophic fibers. In contrast, in normal muscle fibers, Em was significantly depolarized with respect to Ek. The data suggest that in dystrophic fibers there is an increase in the relative membrane permeability to potassium over sodium.

A comparison of the mechanical properties of oesophageal striated muscle with skeletal muscles of the guinea pig.

An in vitro comparative study has been made of the contractile properties of guinea pig oesophageal striated muscle with fast twitch (extensor digitorum longus) and slow twitch (soleus) muscles. Histochemical analysis of sections showed oesophageal fibres to react in a manner typical of fast twitch muscles. Isometric measurements made at 23 and 37 degrees C indicated that oesophageal striated muscle contracts at speeds similar to, but still significantly faster than soleus. Isotonic measurements of oesophageal preparations made at 23 degrees C gave an intrinsic speed of shortening and an a/Po value derived from Hill's equation, comparable with that obtained for soleus. The reason for the contradiction between the results from histochemistry, and the results from isometric and isotonic contractions are not apparent, but may be due to differences in fibre arrangement between oesophagus and the two skeletal muscles.

Effect of clenbuterol on normal and denervated muscle growth and contractility.

The reported anabolic action of some beta 2 agonists may have clinical applications in certain muscle wasting states. Administration of clenbuterol (2 mg/kg diet for 14 days) to rats resulted in a limited degree of hypertrophy of normal muscles; the effect was more pronounced on fast-twitch muscles than on slow-twitch muscles. The anabolic effect was greatest in denervated muscles, where it was significantly more effective on the slow-twitch type. Clenbuterol significantly improved the contractile properties of denervated slow-twitch muscle, reverting them toward normal, but had little effect on contractile properties of denervated fast-twitch muscle. Such differential effects of clenbuterol must be taken into consideration in the evaluation of any future human intervention study.

Changes in membrane potential and potassium and sodium activities during postnatal development of mouse skeletal muscle.

Dual-channel potassium-selective and single-channel sodium-selective microelectrodes were used to investigate the cause of changes in resting membrane potential of muscle fibers of the mouse during early development. The resting membrane of extensor digitorum longus fibers hyperpolarized during the postnatal period from -41.8 mV at 4 days of age to -76.4 mV at 27 days. During this period intracellular potassium activity increased by 42.1% from 82.5 mM at 8 days to 117.2 mM at 29 days. Intracellular sodium activity was high at 8 days, 23.7 mM, but decreased rapidly to adult values by 27 days when it was 9.98 mM, a 57.9% reduction in sodium activity. The time course of the change in resting membrane potential was different from that of the potassium equilibrium potential calculated from the data. If only potassium and sodium ions were to make significant contributions to the potential, then it was calculated that the permeability ratio PNa:PK would have to change from a value of 0.0659 at 8 days to 0.0227 at 27 days. The results indicated that other factors might be involved in generating the membrane potential inasmuch as, although both intracellular potassium and sodium activities did not change significantly after 27 to 30 days, the membrane potential had not attained adult values at that time. The possibility that increases in muscle activity during the postnatal period might initiate the changes in membrane polarization and intracellular ion activities is discussed together with possible complications in interpretation due to great variations in fiber diameters.

Low quantal content of the endplate potential reduces safety factor for neuromuscular transmission in the diaphragm of the newborn rat.

We have used phrenic nerve-hemidiaphragm preparations from rats aged 11-28 days to determine the electrophysiological basis of the three-fold increase in sensitivity of the neonatal neuromuscular junction to non-depolarizing neuromuscular blocking drugs. The mean resting membrane potential (RMP) of intact muscle fibres at 19-21 degrees C increased from -67.1 (SEM 0.6) mV at 11 days to -75.8 (0.06) mV at 28 days. Over the same age range the mean size of miniature endplate potentials (MEPP) decreased from 1.7 (0.1) mV to 1.4 (0.03) mV, and the threshold depolarization to produce a muscle action potential increased from 6.8 (0.4) mV to 9.5 (0.6) mV. The quantal content of the evoked endplate potential (EPP) calculated from EPP and MEPP measured in cut muscle fibres was only 6.6 (0.9) at 11 days, but this increased to 21.1 (2.3) at 21 days. As a result of the low quantal content of the EPP, the safety factor for neuromuscular transmission was only 1.7 at 11 days, compared with 2.8 at 20 days. A safety factor of 1.7 in the youngest rats indicates that block of only about 40% of postjunctional acetylcholine receptors would result in failure of neuromuscular transmission. Hence, a low quantal content of EPP, leading to a low safety factor for neuromuscular transmission, probably underlies the increased sensitivity to non-depolarizing neuromuscular blocking drugs of young rats and, by inference, human neonates.

Age-dependent variation in response to tubocurarine in the isolated rat diaphragm.

The EC50 of tubocurarine was determined in phrenic nerve-hemidiaphragm preparations obtained from 35 Sprague-Dawley rats aged 0-46 days. We measured also the ratio of the fourth to the first twitch in the train-of-four (T4:T1) when the first twitch of the train was depressed to 50% of control. The preparation was not unduly sensitive to tubocurarine at 0 days and there was little evidence of T4:T1 fade. However, by age 11 days the preparation exhibited fade and a three-fold sensitivity to tubocurarine similar to that in the human neonate. We conclude that the phrenic nerve-hemidiaphragm preparation from 11-day-old rats should be a suitable model in which to investigate the biochemical and electrophysiological basis of the sensitivity seen in humans.