Effect of length and caffeine on isometric tetanus relaxation of frog sartorius muscles.

In an isometric tetanus of frog sartorius muscle the total relaxation time increased linearly with change in length from 0.7 to 1.4 times rest length. Maximal rate of relaxation, measured from the time derivative (dp/dt) of tension decay, decreased with both decrease and increase from rest length in correlation with the generated tetanus tension. Stretching the muscle did not significantly affect the times to maximal rate, positive and negative inflexion points but greatly increased the time to total relaxation from the negative inflexion point. Caffeine at 2 mM, acting on muscles at rest length, also slowed the relaxation and decreased the maximal rate of tension decay. However, caffeine increased the times to maximal rate, positive and negative inflexion points without significantly affecting time to total relaxation from the negative inflexion point. These results suggest that caffeine slows an earlier step in relaxation, while stretch slows a later step. It is proposed that muscle relaxation is a two step process: an initial step that is regulated by the rate of Ca2+ uptake by sarcoplasmic reticulum, and a later step that is mostly controlled by the speed of dissociation of remaining cross-bridges.

Multiple effects of alpha-toxins on the nicotinic acetylcholine receptor.

Very low concentrations (5 nM) of alpha-toxin from the venom of Naja naja atra produced a characteristic fade in muscle compound action potential and tetanus induced by repetitive nerve stimulation which was identical to the effects of curare. High concentrations of alpha-toxin and all concentrations of alpha-bungarotoxin reduced the response but produced very little fade in comparison to curare. These results suggest that alpha-toxins have more than one effect at the neuromuscular junction.

Neuromuscular transmission in neonatal mice injected with serum globulin of myasthenia gravis patients.

Neuromuscular transmission was studied in neonatal mice following injection with serum globulin of patients with myasthenia gravis (MG). Compared to controls, these mice showed significant reduction in successive muscle action potentials evoked by repetitive nerve stimulation, amplitude of miniature endplate potentials, and postjunctional sensitivity to acetylcholine. There was no change in evoked isometric tension, quantal content of endplate potentials, or input resistance of the endplate membrane. These results confirm earlier reports of neuromuscular block in animals following injection of globulin of myasthenic patients, and demonstrate that decrease in amplitude of evoked potentials and of miniature endplate potentials is due to reduction in sensitivity to acetylcholine rather than in input resistance of the postsynaptic membrane. These findings are compatible with a postsynaptic defect in MG caused by a humorally mediated autoimmune mechanism.

Peripheral mechanisms of fatigue in muscles of normal and dystrophic mice.

We evaluated the contribution of different processes to fatigue of normal and dystrophic mouse muscles using an in vitro electromyography chamber. Fatigue was induced by repetitive nerve stimulation at 30 Hz for 0.5 s, every 2.5 s until tension decreased by about 50%. We monitored the compound nerve action potential (AP), compound muscle AP, and isometric tension responses to nerve stimulation, and compound muscle AP and tension responses to direct muscle stimulation. In normal mice, about 50% reduction in nerve-evoked tension occurred by 2.4 min in extensor digitorum longus (EDL), 4.8 min in diaphragm, and 9 min in soleus. Analysis of the responses revealed that the fatigue was caused by failure of more than one process in all muscles, and failure of nerve conduction did not contribute to fatigue in any muscle. Failure of neuromuscular transmission, muscle membrane excitation, and excitation-contraction (E-C) coupling and contractility accounted for 55, 45, and 0%, respectively, of the fatigue in EDL, for 21, 74, and 5% of the fatigue in diaphragm, and for 2, 54, and 44% of the fatigue in soleus. In dystrophic mice, while about 50% reduction in nerve-evoked tension occurred by 8.1 min in EDL and 5.6 min in diaphragm, only 29% reduction in tension occurred by 80 min in soleus. Failure of neuromuscular transmission, muscle membrane excitation, E-C coupling and contractility accounted for 22, 63 and 15% of the fatigue in EDL, for 21, 79, and 0% of the fatigue in diaphragm, and for 15, 59, and 26% of the fatigue in soleus. The proportion of slow-twitch oxidative fibers was more than normal in dystrophic EDL, but the same as normal in dystrophic diaphragm and soleus. The slower onset of fatigue was attributable to lesser failure of neuromuscular transmission in dystrophic EDL, and to lesser failure of E-C coupling and contractility in dystrophic soleus.

Desensitization to acetylcholine at motor end plates in normal humans, patients with myasthenia gravis, and experimental models of myasthenia gravis.

Desensitization to ionophoretically applied ACh at the motor end plates of normal humans and patients with MG was studied. The pattern of desensitization at the normal human motor end plate was completely consistent with that observed in muscles of various animals. At motor end plates of MG patients there was greater desensitization and slower recovery from desensitization than in normals. A similar pattern of desensitization was observed at normal end plates when they were exposed to serum globulins of MG patients, and at end plates of muscles from mice than were repeatedly injected with serum globulins of MG patients. Despite the reported reduction in the number of ACh receptors, our calculations show that there is a large pool of spare receptors forming a sizeable margin of safety at the end plates of MG patients. The number of receptors may not show additional progressive reduction in number during repetitive motor activity. Our results indicate that desensitization could play a significant role in the development of neuromuscular block following repetitive motor activity in MG patients. The mechanism by which desensitization is augmented in MG patients in not yet clear.

Curare can reverse the failure in muscle contraction caused by an AChE inhibitor.

Diisopropyl fluorophosphate, which is an organophosphate inhibitor of acetylcholinesterase, caused an irreversible block of neuromuscular transmission in the rat diaphragm preparation. This block could be reversed by the addition of a competitive antagonist of acetylcholine such as D-tubocurarine.

Effect of fiber types, fascicle size and halothane on caffeine contractures in rat muscles.

Caffeine contractures were recorded from thin bundles and whole extensor digitorum longus (EDL) and soleus muscles of rat, and correlated with preparation size and fiber types. Thin bundles were more sensitive to caffeine and halothane than whole muscles, and bundles of 100% type I fibers were more sensitive than bundles of 100% type II fibers. Magnitude of contracture had significant correlation with maximal tetanic tension, total number of fibers, thickness of the preparation, and proportion of type I fibers. These results suggest that fascicle size and fiber types significantly affect results of in vitro contracture test for susceptibility to malignant hyperthermia.

Effect of Cleistanthus collinus leaf extract on neuromuscular function of the isolated mouse phrenic nerve-diaphragm.

Cleistanthus collinus is a toxic plant whose leaves have been used for homicidal or suicidal purposes. Since the toxic effects include muscle cramps and weakness, the effect of the leaf extract on the electrical and mechanical responses to nerve and muscle stimulation was studied in the isolated phrenic nerve-diaphragm preparation of the mouse. Following a 1 hr exposure to 0.015% leaf extract, the response of the compound nerve action potential to supramaximal nerve stimulation was reduced by 38%. The compound muscle action potential was reduced by 97%, and isometric tension by 99%. In response to direct muscle stimulation the compound muscle action potential and isometric tension were reduced by 38%. There was only an 11% reduction in resting membrane potential, but a 51% reduction in the amplitude of miniature endplate potentials. Endplate potentials could be evoked by nerve stimulation without prior treatment of the muscle with curare or a high concentration of magnesium. These studies indicate that the leaf extract markedly inhibits muscle contraction by reducing excitability of the nerve and muscle membranes, and by blocking neuromuscular transmission, without affecting excitation-contraction coupling or contractility of the muscle fibers.

Effect of cocaine on responses of mouse phrenic nerve-diaphragm preparation.

Effects of 5 to 40 microM cocaine on the compound action potential (AP) and tension responses of the mouse phrenic nerve-diaphragm preparation were monitored following nerve and muscle stimulation at 37 degrees C. Cocaine caused concentration dependent reduction in amplitude of the nerve AP, muscle AP, and tension response to a single nerve stimulus, and greater reduction in amplitude of these responses to repetitive nerve stimuli at 100 Hz for 0.5 sec. Cocaine caused similar reduction in the muscle AP and tension responses to direct muscle stimulation in the presence or absence of curare, and markedly reduced the overshoot, total potential, and maximum rate of rise and fall of intracellularly recorded muscle AP, without affecting the resting potential, or the contracture responses evoked by caffeine. These results indicate that cocaine reduces skeletal muscle function by reducing the excitability of muscle and nerve membranes, without significantly affecting neuromuscular transmission, excitation-contraction coupling or contractility.

Twitch potentiation of skeletal muscle by physostigmine at different pH.

This report extends earlier research, done with external media at pH 7.2, to new studies at pH 6.4 and 8.4 as well as 7.2, to determine the roles of the protonated and neutral forms of physostigmine (a weak base with pKalpha = 8.2) in causing twitch potentiation of the frog sartorius muscle. Physostigmine, especially at relatively high pH (8.4) and concentration (1.5 mM), considerably blocks excitation. However, the results show in general that physostigmine potentiation increased peak contraction time and thereby indicate that potentiation is occurring in terms of prolongation of the active state. At pH 6.4 and 8.4, 1 mM physostigmine causes no change in the mechanical threshold of K depolarization contractures of toe muscles, as found previously at pH 7.2. Physostigmine increasingly prolongs the action potential as pH rises, i.e., in positive correlation with the twitch potentiation, thus indicating that this electrical change is the prime determinant of the potentiation.

Imaging caffeine-induced Ca2+ transients in individual fast-twitch and slow-twitch rat skeletal muscle fibers.

Fast-twitch and slow-twitch rat skeletal muscles produce dissimilar contractures with caffeine. We used digital imaging microscopy to monitor Ca2+ (with fluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study this difference. Changes in Ca2+ in individual fibers were markedly different from average responses of a population. All fibers showed discrete, nonpropagated, local Ca2+ transients occurring randomly in spots about one sarcomere apart. Caffeine increased local Ca2+ transients and sarcomere motion initially at 4 mM in soleus and 8 mM in extensor digitorum longus (EDL; approximately 23 degrees C). Ca2+ release subsequently adapted or inactivated; this was surmounted by higher doses. Motion also adapted but was not surmounted. Prolonged exposure to caffeine evidently suppressed myofilament interaction in both types of fiber. In EDL fibers, 16 mM caffeine moderately increased local Ca2+ transients. In soleus fibers, 16 mM caffeine greatly increased Ca2+ release and produced propagated waves of Ca2+ (approximately 1.5-2.5 microns/s). Ca2+ waves in slow-twitch fibers reflect the caffeine-sensitive mechanism of Ca2(+)-induced Ca2+ release. Fast-twitch fibers possibly lack this mechanism, which could account for their lower sensitivity to caffeine.

Physostigmine-induced contractures in frog skeletal muscle.

Physostigmine in 15 mM concentration at pH 8.4 produces reversible contractures of up to 0.3 Po tension output in frog's whole toe muscle or in 7-10 fiber bundles of these muscles, At pH 7.2, the 15 mM physostigmine contracture output is only about 0.10 Po. The 15 mM, pH 8.4 contractures are essentially unaffected by lack of external Ca2+, complete depolarization of the fibers, detubulation by glycerol treatment, and 0 degrees C ambient temperature. These results and other evidence indicate that physostigmine produces contracture by directly releasing activator Ca2+ from the sarcoplasmic reticulum (SR). Pretreatment of muscles with 4 mM procaine reduces physostigmine's capacity to produce contracture, evidently by means of a competitive inhibition at SR sites. The above results indicate similarities between physostagmine and caffeine contractures. But the physostigmine action differs in that it is reversible, and, especially, it lacks the ability, strongly characteristic of caffeine, to sensitize a muscle to produce a rapid cooling contracture. The internal action of physostigmine requires that it be permeant, and, since it is a weak base (pKa = 8.2), this property is provided by its uncharged base. But, once internal, where the pH = 6.8, most of the drug will be protonated and it may act on the SR in this form, in contrast with caffeine which, since its pKa is about 1.0, acts on the SR as uncharged base.

Detubulation effects on the action of zinc on frog skeletal muscle action potential.

Detubulation of the untreated fiber decreases the time constant of the action potential's foot (tauf) and increases the maximal rate of rise of the spike (Vmax). Zinc at all concentrations, and regardless of whether the fiber is intact or detubulated, increases tauf and decreases Vmax, and thus seems to decrease Na activation of the fiber. Detubulation was used principally to elucidate the localization and mechanism of the Zn2+-induced retardation of the falling phase of the frog sartorius fiber action potential, which evidently results from a general depression of delayed rectification. At 50 to 1000 mum, Zn2+ not only prolongs repolarization of intact fibers (measured by increase in t0.5, the half-time of the spike's fall), but also produces a marked hump early in this phase. Detubulation of zinc-free fibers reduces t0.5 to about 80% of its intact value, and under Zn2+ treatment t0.5 is increased but only to about 80% of that produced in the inus, detubulation decreases t0.5 in Zn2+-treated fibers not only to about 80% of that produced in the intact fiber, and the falling-phase hump is completely obliterated. Thus, detubulation decreases t0.5 in Zn2+-treated fibers not only by generally eliminating T-tubular participation in action potential generation, but also by subtracting a Zn2+-induced retardation of tubular delayed rectification. Tubular delayed rectification must therefore be an intrinsic feature of normal excitation. These results are further analyzed by means of (i) Stanfield's findings about retardation of delayed rectification by Zn2+ and (ii) Adrian-Peachey's theory of T-tubule participation in action potential generation, which suggests that the Zn2+-evoked repolarization hump signals onset of Zn2+-altered active participation of T-tubules in determining the spike's shape.

Failure of neuromuscular transmission and contractility during muscle fatigue.

In previous studies of muscle fatigue, tension was monitored from whole muscle, while action potentials were recorded from a few muscle fibers. To compare more accurately changes in these responses, an in vitro fluid electrode technique was employed to record the action potential of whole muscle simultaneously with tension during fatigue induced by nerve stimulation in the rat extensor digitorum longus (EDL), soleus, and diaphragm muscles. In each muscle, tension declined from the start of stimulation, while action potential amplitude initially increased slightly and then declined most rapidly in EDL, more slowly in diaphragm, and most slowly in soleus. Direct stimulation of the fatigued muscle produced the greatest increase in tension in EDL, next in diaphragm, and least in soleus. These results indicate that while failure of excitation-contraction coupling or of the contractile mechanism is the initial cause of fatigue in all the muscles studied, and remains the predominant cause throughout in the soleus muscle, failure of neuromuscular transmission plays an important role in fatigue after the first 15 seconds in EDL, and to a lesser extent, after the first 90 seconds in diaphragm.

Skeletal muscle fatigue and physical endurance of young and old mice.

In order to evaluate the role played by muscular and extramuscular factors in the development of fatigue in old age, the time course of fatigue in isolated skeletal muscles and spontaneous motor activity and endurance of whole animals were monitored using young (3-6 months) and old (34-36 months) CF57BL/6J mice. The isolated extensor digitorum longus (EDL) and soleus muscles from old mice had smaller (P < 0.05) mass and developed lower (P < 0.02) maximal tetanic tension at 100-Hz stimulation than the muscles of young mice. During stimulation at 30 Hz every 2.5 s, a 50% decline in original tetanic tension occurred by 109 s in young EDL and 129 s in old EDL, but by 482 s in young soleus and 1134 s (projected) in old soleus, indicating more (P < 0.05) resistance to fatigue in old than young soleus. However, the old mice showed significantly fewer (P < 0.002) spontaneous ambulatory movements than the young mice. On a treadmill with a belt speed of 10 m/min at an inclination of 0 degrees, the old mice could only run for 22 min compared to 39 min ran by young mice (P < 0.02). They took more rest periods (P< 0.02) than the young mice. In a quantitative swimming monitor, the old mice swam for a shorter (P < 0.05) time than young mice (20.4 min compared to 28.6 min). Integrated swimming activity at 20 min was smaller (P < 0.05) in old mice than in young mice (413 g/s compared to 628 g/s). Hence increased fatigue in old age is not caused by impairment of processes within the muscles, but by impairment of central or extramuscular processes.

Aging effects on contractility of longitudinal and circular detrusor and trigone of rat bladder.

PURPOSE: Aging is associated with bladder dysfunction, including difficult voiding and urinary leakage. Voiding involves reduction in the bladder lumen in all dimensions brought about by contraction of the meshwork of longitudinal, circular and oblique layers of detrusor smooth muscles. Most in vitro physiological studies of the effects of aging on bladder function used the longitudinal detrusor. To understand the region specific effects of aging on bladder function the contractile responses of longitudinal and circular detrusor, and trigone segments of the bladder from young and old rats were monitored. MATERIALS AND METHODS: These studies were performed using male Fisher 344 rats 6 months (young) and 27 months (old) old obtained through the National Institute on Aging. Each rat was anesthetized and the bladder was isolated. From each bladder a strip of longitudinal detrusor, circular detrusor and trigone was isolated and mounted in an in vitro multi-muscle chamber containing normal physiological solution at 37C. Isometric contractions of the 3 bladder strips were monitored after electrical field stimulation, 120 mM. potassium and 1 to 1,000 microM. bethanechol using a digital oscilloscope. RESULTS: In longitudinal detrusor from old rats there was no significant difference in the contractions evoked by electrical stimulation or high potassium but there was a significant reduction in contractions evoked by bethanechol compared with the responses of longitudinal detrusor from young rats. In circular detrusor from old rats there was a significant increase in contractions evoked by electrical stimulation and a slight increase in contractions produced by high potassium but no significant change in contractions evoked by bethanechol compared with the responses of circular detrusor from young rats. In trigone from old rats there was a significant decrease in contractions evoked by electrical stimulation, high potassium and bethanechol compared with young trigone. CONCLUSIONS: The reduction in contractions evoked by bethanechol suggests an age related reduction in muscarinic receptors in the longitudinal detrusor of aged rats. An increase in contractions evoked by electrical stimulation without a change in contractions evoked by bethanechol suggests a decrease in compliance caused by an increase in collagen in the circular detrusor of aged rats. A general decline in all contractile responses, including those evoked by high potassium, suggests reduced membrane depolarization in the trigone of aged rats. The effect of aging is specific to different regions and functional components of the bladder, probably due to changes in muscarinic receptors, collagen and depolarization.

Function of M3 muscarinic receptors in the rat urinary bladder following partial outlet obstruction.

PURPOSE: Partial outlet obstruction of the rat urinary bladder leads to hypertrophy and alteration in contractility of the detrusor muscle involving changes in muscarinic receptors. m3 muscarinic receptor subtype has been known to play a predominant role in contractility of normal urinary bladder. The purpose of the present study was to assess the role of m3 receptors in contractility of the obstructed bladder. MATERIALS AND METHODS: In male rats, partial outlet obstruction of the urinary bladder was performed by surgically tying a 6-0 suture around the bladder neck, reducing the diameter of it by 2/3 of the original size. Four weeks after the surgery, the bladders were removed and thin strips were microdissected. Similarly, bladder strips from age matched unoperated normal rats were obtained. Sets of four strips from four normal or four obstructed rats were mounted in an in vitro multi-muscle chamber containing normal physiological solution at 37C. The tension responses evoked by optimal electrical field stimulation at 1, 10, 30, 50, and 100 Hz, and the contracture responses evoked by 120 mM potassium and 0.01 to 300.0 microM carbachol were recorded using a Nicolet digital oscilloscope. Similar responses were recorded in different sets of four strips following exposure to 10 and 100 nM 4-DAMP, which is a muscarinic antagonist with a high affinity for m3 and m1 receptor subtypes. RESULTS: The obstructed bladders showed 119% increase in weight. In control physiological solution, the obstructed bladder strips did not show significant difference in electrically-evoked tension or carbachol contractures, but showed significantly lower potassium contractures compared with normal bladder strips. 4-DAMP at 10 to 100 nM significantly reduced the electrically evoked tension responses by about the same degree in normal and obstructed bladders, without affecting the potassium contractures. It significantly increased the EC50 values for carbachol contractures in normal bladder, and to a significantly lesser extent in obstructed bladder. Schild plots using the Hill transformed EC50 values showed that the pA2 value for 4-DAMP was not significantly different in normal and obstructed bladders. CONCLUSIONS: Significantly smaller potassium contracture in the obstructed bladder indicates that depolarizability of the detrusor muscle membrane, and consequently the activity of voltage-gated Ca2+ channels may be reduced in the detrusor after partial outlet obstruction. Lack of a significant difference in the effect of 4-DAMP on the electrically evoked tension responses and in the pA2 values for 4-DAMP assessed by carbachol contractures, in normal and obstructed bladder strips, indicates that m3 muscarinic receptors still play a predominant role in causing detrusor contractility in the obstructed bladder, as in the normal bladder.

Responses of intercostal muscle biopsies from normal subjects and patients with myasthenia gravis.

In order to evaluate the mechanisms of weakness in muscles of patients with myasthenia gravis (MG), intercostal muscle biopsies were obtained from 9 normal subjects and 6 MG patients, and the compound muscle action potential (AP) and tension responses to nerve and muscle stimulation, and contracture responses on exposure to caffeine, were monitored in vitro. In normal muscle, on stimulation of the nerve or muscle at 30 to 100 Hz, the AP responses showed decrement in amplitude, one-third of which was attributable to failure of neuromuscular transmission and two-thirds to failure of muscle membrane excitation. On stimulation at 1 to 5 Hz, the AP responses showed very little decrement, while the contractile responses showed significant fade in tension, due to failure of E-C coupling or contractility. In muscle from patients with generalized MG, stimulation of the nerve at all frequencies (1 to 100 Hz) caused much greater decrement in APs and fade in tension responses than in normal muscle, due mainly to failure of neuromuscular transmission. However, at 100 Hz, 40% of the decrement in APs was due to failure of muscle membrane excitation, and at 1 to 5 Hz, 40% of the fade in tension was due to failure of E-C coupling or contractility, as in normal muscle. On direct stimulation the contraction and half-relaxation times were slower and the tetanic tension was smaller than in normal muscle, especially in the MG patient with thymoma. Caffeine-induced contractures were smaller in MG muscle than in normal muscle. These results indicate that while the weakness of MG muscle is due mainly to failure of neuromuscular transmission, it is also partly due to reduced E-C coupling or contractility.