Twitch Amplitude Research Articles

Myosin light chain phosphorylation during staircase in fatigued skeletal muscle.

It has been reported that the peak of the staircase or the enhanced tension response during low frequency stimulation is delayed in fatigued fast muscle. Our purpose was to determine if the rate and extent of regulatory myosin light chain (P-LC) phosphorylation, a molecular mechanism associated with the positive staircase, are also altered by fatigue. The staircase contractile response, muscle metabolites and phosphate incorporation by the P-LC were assessed at 0, 5, 10 or 20 s of 10-Hz stimulation, in either non-fatigued (control) or fatigued (10 Hz for 5 min, followed by 20 min of recovery) rat gastrocnemius muscle in situ. The concentration of adenosine triphosphate (ATP) in fatigued muscles, 21 +/- 0.9 mmol.kg-1 (dry weight) was significantly lower (P < 0.05) than in the control muscles, 26.1 +/- 1.5 mmol.kg-1. In both groups, ATP content was significantly lower after 20 s of 10 Hz stimulation. The P-LC phosphate content (in mol phosphate.mol-1 P-LC) was 0.10, 0.38, 0.60 and 0.72 after 0, 5, 10 or 20 s of 10 Hz stimulation in control muscles, but only 0.03, 0.08, 0.11 and 0.19 at these times in fatigued muscles. Although the absolute magnitude of tension potentiation was attenuated in proportion to the depressed twitch amplitude, these surprisingly low levels of phosphorylation were associated with 0, 48, 79 and 86% potentiation of the developed tension at these times in contrast with 0, 71, 87 and 49% potentiation in control muscles. These data demonstrate that while the rate and extent of phosphate incorporation is depressed in fatigued muscle, tension potentiation is still evident.(ABSTRACT TRUNCATED AT 250 WORDS)

Potentiatiof MPTP by 4-phenylpyridine on the neuromuscular blockade in mouse phrenic nerve-diaphragm

Potentiation by 4-phenylpyridine (a MAO-B inhibitor) on the neuromuscular blocking action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied in mouse phrenic nerve-diahragm. MPTP blocked nerve-evoked twitches in a concentration (1–200 μM)-dependent manner. 4-Phenylpyridine, but not pargyline or tranylcypromine potentiated this inhibitory effect of MPTP. Pretreatment with 50 μM 4-phenylpyridine, reduced IC 50 (concentration for 50% inhibition of twitch amplitude) values of MPTP from 53 to 18 μM and d-tubocurarine from 0.7 to 0.3 μM, respectively. 4-Phenylpyridine also enhanced the inhibitory action of MPTP and d-tubocurarine on acetylcholine (0.1 mM)-induced contracture of the denervated mouse diaphragm. The twitch inhibition induced by α-bungarotoxin and the specific binding of [ 125I]α-bungarotoxin to the mouse diaphragm were potentiated by 4-phenylpyridine but the inhibitory action of MPTP and d-tubocurarine on [ 125I]α-bungarotoxin binding were not significantly changed by pretreatment with 4-phenylpyridine. Electrophysiological studies revealed that the inhibitory actions of MPTP and d-tubocurarine on the amplitudes of m.e.p.ps and e.p.ps were augmented by 4-phenylpyridine. These indicate that 4-phenylpyridine enhanced the neuromuscular blocking action of the MPTP and d-tubocurarine at the postsynaptic nicotinic acetylcholine receptors. The implication of this finding is that the possible application of 4-phenylpyridine in the MPTP-induced Parkinson's disease is limited by its potentiation on the neuromuscular blocking action of MPTP.

Beta 1-adrenoceptor stimulation and beta 2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2+, and Ca2+ current in single rat ventricular cells.

The effects of beta 2- and beta 1-adrenoceptor (beta 2AR and beta 1AR, respectively) agonists on the cytosolic Ca2+ (Cai) transient (indexed by the transient increase in indo-1 fluorescence ratio after excitation), twitch amplitude (measured via photodiode array), membrane potential, and L-type sarcolemmal Ca2+ current (ICa, measured by whole-cell patch electrode) were assessed in single rat ventricular myocytes. The selective beta 2AR agonist Zinterol increased the amplitudes of both the Cai transient and twitch in a concentration-dependent manner. Similar results were obtained when beta 2ARs were stimulated with isoproterenol in the presence of the selective beta 1AR antagonist CGP 20712A. beta 1AR stimulation induced by norepinephrine increased twitch amplitude to about the same extent as did beta 2AR stimulation. However, several striking differences between response to beta 1AR and beta 2AR stimulation were observed. beta 1AR stimulation had the potent effect of abbreviating the time course of the contraction and Cai transient, and beta 2AR stimulation did not reduce the time course of the Cai transient and had only a minor effect on the twitch duration. For a given increase in twitch amplitude, beta 1AR stimulation caused a greater increase in Cai transient, suggesting a diminished Cai-myofilament interaction. beta 1AR, but not beta 2AR, stimulation evoked spontaneous Cai oscillations, increased the diastolic indo fluorescence level, and caused a decline in resting cell length. beta 1AR and beta 2AR also differed in their effects on ICa. Whereas both beta 1AR and beta 2AR stimulation increased the peak ICa amplitude, beta 2AR stimulation markedly prolonged the ICa inactivation time. Accordingly, beta 2AR stimulation prolonged the action potential duration to a greater extent than did beta 1AR stimulation. 8-(4-Chlorophenylthio)cAMP mimicked the effects of beta 1AR stimulation by norepinephrine but not those due to beta 2AR stimulation. These results clearly indicate that both beta 2ARs and beta 1ARs functionally coexist in rat ventricular myocytes but that stimulation of these receptor subtypes elicits qualitatively different cell responses at the levels of ionic channels, the myofilaments, and sarcoplasmic reticulum.

Stimulation of Opioid receptors on Cardiac Ventricular Myocytes reduces L Type Ca 2+ Channel Current

Recent studies have indicated that opioid peptide receptors are present on cardiac ventricular cells and that Leucine enkephalin (LE), a naturally occurring δ opioid peptide receptor agonist, leads to marked reductions in twitch amplitude and in the cytosolic Ca 2+ transient (Ca i) of single adult rat ventricular myocytes. The specific mechanism by which Ca i is reduced by LE have not been fully elucidated. Specifically, it is unknown whether LE affects the Ca 2+ current ( I Ca) of L type Ca 2+ channels. In the present study we determined the effect of LE on I Ca of individual cardiac ventricular cells freshly isolated from adult rats. LE (10 -8M) decreased the amplitude of I Ca by 40% (during regular whole cell voltage clamp depolarizations to 0 mV at 0.5 Hz at 23°C from a holding potential of -40 mV). The relative magnitude of this effect increased with the magnitude of the test potential from -20 to +50 mV. I Ca inactivation was also prolonged by LE. These effects of LE on I Ca were abolished by Naloxone (NAL), an opioid receptor antagonist. Thus, the effects of the opioid peptide, LE, to decrease the Ca i transient and contraction amplitudes in individual cardiac ventricular cells, are, in part, mediated by an LE induced reduction in I Ca .

Modulation of the jaw jerk reflex by the sympathetic nervous system.

The effect of sympathetic stimulation on the jaw jerk reflex has been studied in precollicular decerebrate rabbits. This reflex was elicited by a downward mandibular movement applied to the lower jaw through a servo controlled puller. Unilateral stimulation of the cervical sympathetic nerve at 10/s consistently induced a decrease in the JJR, i.e. a marked reduction of the EMG activity in the ipsilateral masseter muscle, accompanied by a 30-40% decrease in the reflexly developed force. In these trials EMG of the contralateral muscle, recorded as control, was not significantly affected. Bilateral stimulation of cervical sympathetic nerve strongly reduced or suppressed the EMG activity in both sides and produced a parallel decrease in the developed force which reached values ranging from 12.5% to 37.0% of controls (with an average of 28.9% +/- 8.9, S.D.). The effect of sympathetic stimulation was also tested on the contraction of the masseter muscle elicited by direct electrical stimulation. Sympathetic activation induced a modest increase in both amplitude and duration of muscle twitch, thus showing that the reduction in the reflex response can not be attributed to an action exerted by the adrenergic mediator on the muscular contraction. All these effects were almost completely abolished by the blockade of alpha-adrenergic receptors. They were proved not to be secondary to the sympathetically-induced vasomotor changes. Therefore the marked JJR reduction produced by activation of the sympathetic nervous system is suggested to be due to the sympathetically-induced decrease in neuromuscular spindle sensitivity to muscle length changes, previously reported.

Enantiomeric dissection of the effects of the inotropic agent, EMD 53998, in single cardiac myocytes

The effects of the thiadiazinone derivative, 5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydrochinolin-6-yl]-6-met hyl-3,6- dihydro-2H-1,3,4-thiadiazin-2-on (EMD 53998), and of its (+)EMD 57033 and (-)EMD 57439 enantiomers, were tested on the contractile properties and cytosolic [Ca2+] ([Ca2+]i) transients of single intact guinea pig cardiac myocytes. Cells were loaded with the ester form of the fluorescent probe, indo-1, and bathed in a N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid-buffered solution at 25 degrees C (1 mM of CaCl2, 1 Hz stimulation rate). All three substances exerted a pronounced increase in twitch amplitude: the maximal effect of the racemate (380% of control value) was approximately the sum of the effects of its two enantiomers (186 and 236% of control value for the (+)- and (-)-enantiomer, respectively). The [Ca2+]i transient, measured as the 410-to-490 nm indo-1 fluorescence ratio transient after excitation, was increased by the racemate and its (-)-enantiomer (172 and 152% of control value, respectively), but was not increased by the (+)-enantiomer. The racemate and the (-)-enantiomer, but not the (+)-enantiomer, markedly reduced the contraction duration and [Ca2+]i transient duration. In unstimulated cells resting length was significantly reduced by the (+)-enantiomer, and this was accompanied by a decrease in indo-1 fluorescence; the (-)-enantiomer had no effect on either parameter. In the presence of 2,3 butanedione monoxime (BDM), which markedly reduces twitch amplitude by inhibiting cross-bridge mechanics, addition of the (+)-enantiomer restored the twitch contraction to above the pre-BDM level without augmenting the [Ca2+]i transient. In contrast, the (-)-enantiomer failed to reverse the BDM-induced contractile depression, even though it caused a significant increase of the [Ca2+]i transient. Thus, in intact cells the positive inotropic effect of EMD 53998 is due to specific properties of its enantiomers: the (-)-enantiomer has adenosine 3',5'-cyclic monophosphate-like effects (increase in amplitude and reduction of [Ca2+]i transient and contraction durations), whereas the (+)-enantiomer enhances the myofilament-Ca2+ interaction.

Reversal by suramin of neuromuscular block produced by pancuronium in the anaesthetized rat

1. Rats were anaesthetized with sodium pentobarbitone and maximal twitches of a tibialis anterior muscle were evoked by stimulation of the motor nerve. 2. Suramin, injected intravenously in a series of cumulative bolus doses, each 15 mg kg-1, completely reversed a 90% depression of twitches maintained by a continuous intravenous infusion of pancuronium. The cumulated dose necessary to restore twitches to 50% of their control amplitude was 35 mg kg-1. Suramin did not modify a similar degree of block produced by suxamethonium, nor did it affect the amplitude of control maximal twitches, even in cumulative doses up to 150 mg kg-1. 3. The effects of bolus doses of suramin (85 mg kg-1), neostigmine (0.03 mg kg-1) and 4-aminopyridine (1.2 mg kg-1), calculated to restore pancuronium-blocked twitches to 95% of control amplitude, were compared. Suramin produced the most rapid reversal (1.1 +/- 0.5 min), but its duration of action was the shortest (9.4 +/- 1.6 min). Suramin was without effect on heart rate or blood pressure in the doses used. 4. The results showed that suramin reversed neuromuscular block produced by nondepolarizing blocking drug, pancuronium, but was without effect on a block produced by the depolarizing blocking drug, suxamethonium. Its short duration of action suggests that suramin would probably not be of value clinically as a reversal agent. However, it is possible that it might serve as a starter compound for the synthesis and development of a new class of reversal agents for use in anaesthetic practice.

Strength training alters contractile properties of the triceps brachii in men aged 65-78 years.

Voluntary and electrically evoked contractile properties were studied in the triceps brachii following a 24-week dynamic strength training program in ten men aged 65-78 years. Eight men of a similar age were control subjects. A resistance overload program was undertaken three times per week with subjects performing four sets of six to eight repetitions at 80% of their one repetition on maximum (1RM). Maximum voluntary contraction (MVC) and contractile properties were measured at 0, 12, and 24 weeks in the exercise group and at 0 and 24 weeks in the controls. The 1RM was used to assess dynamic strength at 0 and 24 weeks in the exercise group. Contractile measures consisted of supramaximal isometric twitch and post-activation twitch parameters. Muscle size was estimated from anthropometric measurements. Compared with the control group, the exercise group MVC increased by about 20% and time to peak tension was slowed by about 11%. Also in the exercise group the peak rate of torque development of the potentiated twitch was reduced by about 10%. Twitch potentiation was substantial in both groups (about 140%) and unaffected by training. The 1 RM increased by about 30%, and there was a non-significant positive change of 8.6% in the muscle plus bone cross-sectional area in the exercise group. The results show that the force generating capacity of the triceps brachii in these men can be significantly improved for up to 24 weeks using concentric overload training. Furthermore, the finding of slowed twitch properties and no change in peak twitch amplitude substantiate and extend the limited data currently available on intrinsic contractile changes in the elderly.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium current-dependent staircase in rat myotubes and myoballs developing in culture

Calcium current and contraction were simultaneously recorded in whole-cell patch-clamped rat skeletal muscle cells grown in primary culture. Repetitive depolarizations at low frequency, which elicited calcium currents, led to a staircase response, characterized by the progressive increase of both twitch amplitude and activation rate. It was sometimes possible to elicit a staircase response in 2 or 3 day old postfusion myotubes which did not or weakly contract initially. The staircase response was dependent on calcium entry through calcium channels, since it was reversed when calcium current was depressed by means of inorganic calcium blockers or depolarization to large positive potential. The entry of calcium was also necessary to allow the development of a staircase response following caffeine-induced contractures which partly emptied the intracellular stores of calcium. These features are consistent with the idea that calcium currents allow the initial loading of intracellular calcium stores and, in later stages, serve to replenish and maintain them constant.

Swallowing and upper esophageal sphincter contraction with transcranial magnetic-induced electrical stimulation.

This study in three dogs explores the effect of magnetically induced electrical stimulation of the brain to induce swallowing and produce contraction of the upper esophageal sphincter (UES). Single stimuli were delivered at intervals from 15 s to 3 min. Studies were performed with and without perfusion of fluid into the pharynx and upper esophagus. Results showed that magnetic stimulation produced a twitch contraction of the UES when stimulus intensity was above a threshold that varied between 14 and 20% of the stimulator output. Increasing stimulus intensity progressively increased twitch amplitude. Magnetic stimulation also induced swallowing, above a stimulus threshold similar to that for induction of the UES twitch contraction. Fluid perfusion augmented the ability of the magnetic stimulus to induce swallowing. We concluded that a magnetically induced single electrical stimulus of the cerebral cortex produces UES contraction and induces swallowing. The effect on swallowing is facilitated by sensory stimulation of the pharynx. This technique holds the potential for further study of 1) motor and sensory neural mechanisms involved in the control of swallowing and 2) the assessment and management of oropharyngeal dysphagia in humans.

Scorpion toxins targeted against the sarcoplasmic reticulum Ca(2+)-release channel of skeletal and cardiac muscle.

We report the purification of two peptides, called "imperatoxin inhibitor" and "imperatoxin activator," from the venom of the scorpion Pandinus imperator targeted against ryanodine receptor Ca(2+)-release channels. Imperatoxin inhibitor has a M(r) of approximately 10,500, inhibits [3H]ryanodine binding to skeletal and cardiac sarcoplasmic reticulum with an ED50 of approximately 10 nM, and blocks openings of skeletal and cardiac Ca(2+)-release channels incorporated into planar bilayers. In whole-cell recordings of cardiac myocytes, imperatoxin inhibitor decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the sarcoplasmic reticulum. Imperatoxin activator has a M(r) of approximately 8700, stimulates [3H]ryanodine binding in skeletal but not cardiac sarcoplasmic reticulum with an ED50 of approximately 6 nM, and activates skeletal but not cardiac Ca(2+)-release channels. These ligands may serve to selectively "turn on" or "turn off" ryanodine receptors in fragmented systems and whole cells.

Intraoperative monitoring of tibialis anterior muscle motor evoked responses to transcranial electrical stimulation during partial neuromuscular blockade.

We studied the feasibility of recording motor evoked responses to transcranial electrical stimulation (tce-MERs) during partial neuromuscular blockade (NMB). In 11 patients, compound muscle action potentials were recorded from the tibialis anterior muscle in response to transcranial electrical stimulation during various levels of vecuronium-induced NMB. The level of NMB was assessed by accelerometry of the adductor pollicis muscle after train-of-four stimulation of the ulnar nerve. The compound muscle action potential was also recorded from the tibialis anterior muscle after direct stimulation of the peroneal nerve (M-response) as an alternative means of assessing the degree of NMB. In all patients, tce-MERs could be recorded reliably during anesthesia with N2O and a continuous infusion of sufentanil (0.5 micrograms.kg-1.h-1). An intact train-of-four was present in all patients, and the amplitude of the first twitch was recorded and designated as the control value. Before administration of vecuronium, the M-response amplitude was 9.6 +/- 3.6 (mean +/- SD) mV, and the tce-MER amplitude was 1.21 +/- 0.66 mV. Although administration of vecuronium (0.05 mg/kg) resulted in loss of the mechanical adductor pollicis response in 8 of the 11 patients, the M-response and the tce-MER remained recordable. Subsequently, during an infusion of vecuronium, adjusted to maintain one or two mechanical responses to train-of-four stimulation, the average M-response to peroneal nerve stimulation was 5.2 +/- 2.5 mV (53% of the control value), and tce-MER amplitude was 0.59 +/- 0.36 mV (59% of the control value).(ABSTRACT TRUNCATED AT 250 WORDS)

The rat external urethral sphincter an in vitro model to evaluate the activity of drugs on the smooth and striated components of the urinary bladder outlet

Electrical field stimulation (EFS, 2.5 Hz, 60 V, 1 ms, trains of 5 sec every 5 min) of the rat external urethral sphincter (EUS) produced contractile responses characterized by a “slow” tonic contraction on which was superimposed a series of phasic “twitch-like” contractions. Both responses were abolished by tetrodotoxin (0.6 μM), and their amplitude was significantly lower in samples taken from denervated (15 days before) sphincters. The tonic contraction showed duration, voltage, and frequency dependency, whereas the twitches were markedly duration dependent. No correlation was found between the amplitude of the tonic and that of the twitch-like contractions. Phentolamine (3 μM) reduced by 95% the amplitude of the tonic contraction produced by maximal parameters, whereas it did not affect the twitches. On the other hand, hexamethonium (10 μM) was ineffective on both components. Dantrolene (10 μM) inhibited the twitch response, whereas it did not influence the tonic component. Preincubation with d-tubocurarine (0.2 mM) or succinylcholine (2 mM) significantly inhibited the amplitude of twitches produced by EFS (0.1 Hz, 60 V) up to a duration of 50 μsec. Stimulus width higher than 50 μsec, resulted in twitches that were resistant to neuromuscular blocking agents but sensitive to dantrolene (10 μM). Our results indicate that the rat external urethral sphincter is a reliable and easy “in vitro” model for studying the activity of drugs capable of interfering with the nerve-mediated activity of the striated and smooth muscle portion of the urinary bladder outlet.

Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes.

alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.

α-Conotoxin GI produces tetanic fade at the rat neuromuscular junction

The ability of the marine snail toxin, α-conotoxin GI, to produce blockade of singly evoked twitches and to produce tetanic and train-of-four fade has been determined in the isolated rat hemidiaphragm preparation. Results were compared to those obtained with a reversible (vecuronium) and an irreversible (α-bungarotoxin) nicotinic acetylcholine antagonist and have been interpreted in terms of relative effects on post- and prejunctional nicotinic acetylcholine receptors at the neuromuscular junction. α-Conotoxin GI (0.5–2 μM) produced a concentration-dependent, readily reversible, decrease in the peak amplitude of single twitches and 50 Hz tetani, and an increase in tetanic and train-of-four fade. α-Conotoxin GI was consistently 2–3-fold more potent than vecuronium with respect to all of the measured tension parameters. Both α-conotoxin GI and vecuronium were approximately 2-fold more potent in producing tetanic fade and in blocking tetanic contractions than in blocking single twitches. In contrast to both α-conotoxin GI and vecuronium, α-bungarotoxin (0.13 μM) reduced the peak amplitude of both single twitches and 50 Hz tetani to the same extent without the appearance of a large degree of tetanic or train-of-four fade. Based on a comparison of the in vitro time course of neuromuscular block and of the relative effects of vecuronium, α-conotoxin GI and α-bungarotoxin on twitches, tetani and trains-of-four, we conclude that α-conotoxin GI has both pre- and postjunctional activity at the neuromuscular junction. In this respect, α-conotoxin GI resembles the clinically used competitive neuromuscular blocking drugs rather than the irreversible snake α-neurotoxins.

Contribution of sarcolemmal sodium-calcium exchange and intracellular calcium release to force development in isolated canine ventricular muscle.

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.

Studies on curare-like action of 2,2',2''-tripyridine in the mouse phrenic nerve-diaphragm.

1. The curare-like action of 2,2',2''-tripyridine (a synthetic by-product of the herbicide, paraquat) was studied in mouse phrenic nerve-diaphragm preparation. The inhibition by 2,2',2''-tripyridine of nerve-evoked twitches was dependent on the concentration, ranging from 1 to 100 microM, which had no significant effect on the twitch amplitudes evoked by direct muscle stimulation. 2. The twitch inhibition by 2,2',2''-tripyridine was reversible and could be antagonized by anticholinesterase agents such as neostigmine, physostigmine as well as ecothiophate. 3. Pretreatment with either 0.7 microM (+)-tubocurarine or 2.2 microM succinylcholine shifted the concentration-inhibition curve of 2,2',2''-tripyridine to the left. 4. 2,2'2''-Tripyridine inhibited not only acetylcholine-induced contracture of the denervated mouse diaphragm but also that of the chick biventer cervicis muscle. Like (+)-tubocurarine, 2,2',2''-tripyridine protected the twitches from the inhibition by alpha-bungarotoxin and also specifically inhibited the binding of [125I]-alpha-bungarotoxin to the mouse diaphragm. All of these findings indicate that 2,2',2''-tripyridine possesses curare-like action and inhibits the muscle contractions through binding to postsynaptic acetylcholine receptors. 5. The postsynaptic inhibition exhibited by 2,2',2''-tripyridine was also implicated in the tetanic fade, a decrease in the amplitude of miniature endplate potential (m.e.p.p.) and endplate potential (e.p.p.). 6. The clinical implication of these findings is that 2,2',2''-tripyridine may be involved in the cause of respiratory failure in paraquat-intoxicated workers since 2,2',2''-tripyridine is a by-product of paraquat synthesis.

Effects of subacute pyridostigmine administration on mammalian skeletal muscle function

The subacute effects of pyridostigmine bromide were investigated on the contractile properties of rat extensor digitorum longus (EDL) and diaphragm muscles. The cholinesterase inhibitor was delivered via subcutaneously implanted osmotic minipumps (Alzet) at 9 micrograms h-1 (low dose) or 60 micrograms h-1 (high dose). Animals receiving high-dose pyridostigmine pumps exhibited marked alterations in muscle properties within the first day of exposure that persisted for the remaining 13 days. With 0.1 Hz stimulation, EDL twitch tensions of treated animals were elevated relative to control. Repetitive stimulation at frequencies greater than 1 Hz led a use-dependent depression in the amplitude of successive twitches during the train. Recovery from pyridostigmine was essentially complete by 1 day of withdrawal. Rats implanted with low-dose pyridostigmine pumps showed little or no alteration of in vivo twitch tensions during the entire 14 days of treatment. Diaphragm and EDL muscles excised from pyridostigmine-treated rats and tested in vitro showed no significant alterations in twitch and tetanic tensions and displayed the same sensitivity as muscles of control animals to subsequent pyridostigmine exposures. In the presence of atropine, subacutely administered pyridostigmine protected rats from two LD50 doses of the irreversible cholinesterase inhibitor, soman. In the absence of atropine, the LD50 of soman was not altered by subacute pyridostigmine treatment.

Contractile properties and susceptibility to exercise-induced damage of normal and mdx mouse tibialis anterior muscle.

1. The functional properties of tibialis anterior muscles of normal adult (C57BL/10) and age-matched dystrophin-deficient (C57BL/10 mdx) mice have been investigated in situ. Comparisons were made between tibialis anterior muscle strength, rates of force development and relaxation, force-frequency responses and fatiguability. Subjecting mdx and C57 muscles to a regimen of eccentric exercise allowed the hypothesis to be tested that dystrophin-deficient muscles are more susceptible to exercise-induced muscle damage. 2. mdx muscles were, on average, 30% stronger than C57 muscles and almost 80% heavier, but both had similar muscle lengths. Thus, although mdx muscles were stronger in absolute terms, their estimated force per unit cross-sectional area was significantly less than that of C57 muscles. 3. The force-frequency relationships of C57 and mdx muscles differed in that whilst, at 40 Hz, the former developed 70% of the force developed at 100 Hz, the latter developed only 55% of the maximal force. Twitch force was normal in mdx muscles, but contraction time was shortened, and the consequent reduction in fusion frequency probably explains the force-frequency differences observed between the two groups. 4. mdx muscles were less fatiguable than normal muscles when stimulated repeatedly at a frequency of 40 Hz. It is possible that the lower relative force at 40 Hz in mdx muscles entailed a lower energy demand and thus a slower rate of fatigue than seen in normal muscles. 5. Eccentrically exercised C57 muscles showed a large loss of maximal force for up to 12 days after exercise. Maximal force loss occurred 3 days after exercise (55% of non-exercised tibialis anterior muscle), which also corresponded with the period of greatest fibre necrosis. C57 muscles showed a significantly reduced 40 Hz/100 Hz force-frequency ratio at 1 and 3 days after exercise. This was primarily due to a reduced twitch amplitude rather than to a change in the time course of the twitch. It is unlikely, therefore, that the altered contractile characteristics of mdx muscle were a result of the presence of damaged but otherwise normal fibres. 6. C57 and mdx tibialis anterior muscles displayed similar degrees of force loss after exercise. Furthermore, the rate of recovery after the nadir of force loss was very similar for the two groups. By 12 days after exercise, force recovered to 76% and 80% of control in C57 and mdx muscles, respectively. Our findings do not support the hypothesis that dystrophin-deficient muscle is more susceptible to exercise-induced muscle damage.

Effect of U-50,488H on the contractile response of cardiomyopathic hamster ventricular myocytes

We examined the effects of a selective κ opioid receptor agonist (U-50,488H) on the contractile properties of single ventricular myocytes from 127 day old control (F1B) and cardiomyopathic (BIO 14.6) hamsters. Myocytes in bicarbonate buffered solution with 1.5 mM [Ca 2+] were electrically stimulated with field electrodes in the bath. Length changes were monitored via myocyte edge tracking. Twitch amplitude and the velocity of cell shortening were less in the cardiomyopathic hamster myocytes than in age-matched hamsters (P≤0.05). There was a concentration-dependent effect of U-50,488H (0.1–20 μM) to decrease twitch amplitude and shortening velocity in both control and cardiomyopathic myocytes (P≤0.001). In cells loaded with the Ca 2+ indicator indo-1 the negative inotropic action of U-50,488H was associated with a decreased indo-1 fluorescence transient amplitude. There was no difference in the negative inotropic effect of U-50,488H on control and cardiomyopathic cells. Thus, the CM hamster does not demonstrate a different contractile response to U-50,488H.